Patent Publication Number: US-2010125919-A1

Title: Methods and compositions for modulating the mirna pathway

Description:
RELATED APPLICATIONS 
     This application claims benefit of U.S. application(s) Ser. Nos. 60/881,443 and 60/881,434 both filed 18 Jan. 2007 which are incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     Compositions and methods for conferring broad spectrum pathogen resistance, against plant and animal pathogens. 
     BACKGROUND OF THE INVENTION 
     In recent years, there has been an ever increasing appreciation of the complexity and pleiotropic effects of gene silencing and components of the gene silencing machinery. From effects observed initially via transgene suppression of endogenous gene expression in petunia plants, has emerged an understanding of a penumbra of effects in plants and animals spanning antiviral defense and control of transposons through RNA-directed DNA methylation. 
     Small RNA, Dicers and Argonautes: the Biochemical Core of RNA Silencing 
     “RNA silencing” refers collectively to diverse RNA-based processes that all result in sequence-specific inhibition of gene expression, either at the transcriptional, mRNA stability or translational levels. Those processes share three biochemical features: (i) formation of double-stranded (ds)RNA, (ii) processing of dsRNA to small (s) 20-26 nt dsRNAs with staggered ends, and (iii) inhibitory action of a selected sRNA strand within effector complexes acting on partially or fully complementary RNA/DNA. While several mechanisms can generate dsRNA, the sRNA processing and effector steps have a common biochemical core. sRNAs are produced by RNAseIII-type enzymes called Dicers 1  with distinctive dsRNA binding, RNA helicase, RNAseIII and PAZ (Piwi/Argonaute/Zwille) domains. One of the two sRNA strands joins effector complexes called RISCs (RNA-induced silencing complex) that invariably contain a member of the Argonaute (Ago) protein family. Agos have an sRNA binding PAZ domain and also contain a PIWI domain providing endonucleolytic (‘slicer’) activity to those RISCs programmed to cleave target RNAs 2, 3 . In fact, sRNA-loaded human Ago2 alone constitutes a cleavage-competent RISC in vitro, but many additional proteins may be functional components of RISCs in vivo 4 . 
     Here, we review recent evidence that several pathways built over the Dicer-Ago core execute a diverse set of sRNA-directed biological functions in higher plants. These include regulation of endogenous gene expression, transposon taming, viral defense and heterochromatin formation. Our focus is primarily on plants because they exhibit a nearly full spectrum of known RNA silencing effects, but similarities and differences with other organisms are also discussed. 
     Exogenously Triggered RNA Silencing Pathways Resulting in Transcript Cleavage dsRNA-Producing Transgenes and IR-PTGS: Useful, but Mysterious 
     Post-transcriptional gene silencing (PTGS) was discovered in transgenic Petunia as loss of both transgene (in either sense or antisense configuration) and homologous endogenous gene expression 5 . The transgene loci often produced dsRNA because they formed arrays with complex integration patterns 6, 7 . Accordingly, PTGS efficacy was greatly enhanced by simultaneous sense and antisense expression 8  or by direct production of long dsRNA from inverted-repeat (IR) transgenes 9 . The latter process, IR-PTGS, currently forms the basis of experimental RNAi in plants, and involves at least two distinct sRNA classes termed short interfering (si)RNAs. 21 nt siRNAs are believed to guide mRNA cleavage, while 24 nt siRNAs may exclusively mediate chromatin modifications 10, 11 . Both siRNA classes accumulate as populations along the entire sequence of IR transcripts. Although widely used as a research tool, IR-PTGS remains one of the least understood plant RNA silencing processes ( FIG. 1A ).  FIG. 1A  shows how an inverted repeat (IR) transgene construct, typically employed for RNAi in plants, produces double-stranded (ds) transcripts with perfectly complementary arms. Two distinct Dicer-like (DCL) enzymes process the ds transcripts. DCL3 most likely produces siRNAs of the 24 nt size class, which may direct DNA/histone modification at homologous loci (see  FIG. 3 ) and appear dispensable for RNA cleavage. DCL4 is likely the preferred enzyme for production of 21 nt-long siRNAs from the dsRNA. One siRNA strand incorporates into AGO1-loaded RISC to guide endonucleolytic cleavage of homologous RNA, leading to its degradation. Both siRNA species are protected from degradation by addition of methyl groups at the 3′ termini of each RNA strand, by the methyl-transferase HEN1. 
     Hence, until recently, no mutant defective in this pathway had been recovered, despite considerable efforts in several laboratories. One likely explanation is that the high dsRNA levels produced in IR-PTGS promote the activities of different Dicers and RISCs, which would normally act in distinct pathways, to redundantly mediate silencing. Recent analyses of combinatorial Dicer knockouts in  Arabidopsis  support this idea 12, 13 . Nonetheless, Dicer-like 4 (DCL4) seems a preferred enzyme for IR-PTGS because it was specifically required for 21 nt siRNA accumulation and silencing from a moderately expressed, phloem-specific IR transgene 14 . DCL2 might also be involved in RNAi, because it processes some endogenous DCL4 substrates into 22 nt-long siRNAs in the absence of DCL4 12, 13 , although it remains unclear if those molecules can functionally substitute for the 21 nt siRNA products of DCL4. 
     S-PTGS and Transitive Silencing: Enter RDR 
     There are several examples in which single-copy transgene insertions producing sense transcripts trigger PTGS. This pathway, sense (S)-PTGS, has been dissected using  Arabidopsis  forward-genetic screens that provided insights into how dsRNA is produced ( FIG. 1B ). These screens converged on the identification of the RNA-dependent RNA polymerase RDR6, one of six putative  Arabidopsis  RDRs 15, 16 . RDR6 is thought to recognize and to use as templates certain transgene transcripts with aberrant features that include lack of 5′ capping. For instance, mutation of  Arabidopsis  XRN4, a 5′-3′ exonuclease that degrades uncapped mRNAs, enhanced accumulation of uncapped transgene mRNAs. This favored their conversion into dsRNA by RDR6 and the subsequent degradation of all transgene transcripts through the S-PTGS pathway 17 . RDR6 most likely synthesizes complementary strands from its RNA templates, resulting in dsRNA production, because a missense mutation in the GDD motif, essential for the catalytic activity of all characterized RDRs, is sufficient to alleviate S-PTGS 16 . 
     Although the Dicer producing siRNAs from RDR6 products remains to be formally identified, S-PTGS siRNA accumulation in  Arabidopsis  requires the coiled-coil protein of unknown function SGS3 16 , the RNAseD exonuclease WEX 18 , the sRNA-specific methyl transferase HEN1 19  and the putative RNA helicase SDE3 20  ( FIG. 1B ). In  FIG. 1B , the pathway is shown as being elicited by RNAs with aberrant features, although there might be alternative triggers. The RNA aberrations could include lack of a poly-A tail or lack of 5′ capping. The latter would normally lead to RNA degradation through the activity of the 5′-3′ exonuclease XRN4. Lack of XRN4 would promote accumulation of uncapped mRNA, thereby triggering their conversion into dsRNA by the combined action of RDR6, SGS3, SDE3 and, possibly, WEX. The resulting dsRNA is then processed by a DCL, most likely DCL4 (see text), producing siRNAs that are exclusively of the 21 nt size class and methylated by HEN1. These molecules can engage into two sets of reactions. First, they can be used as primers by RDR6 to reinforce production of dsRNA from single-stranded templates through a phenomenon known as ‘transitivity’ (see  FIG. 2 ). They can also incorporate into AGO1-loaded RISC to guide sequence-specific cleavage of homologous RNA. The resulting cleavage products could be perceived as aberrant RNAs and, thus, could promote further production of dsRNA, resulting in an amplified reaction. 
     Unlike RDR6, SDE3 is not stringently required for transgene silencing, and so could accessorily resolve the secondary structures found in RDR templates 20 . Accordingly, an SDE3 homologue is part of the  Schizosaccharomyces pombe  RDR complex 21 . SDE3 could also act at other RNA silencing steps because the homologous protein Armitage is required for RISC assembly in  Drosophila , an organism deprived of RDR genes 22 . WEX is related to the exonuclease domain of mut-7, required for transposon silencing and RNAi in  C. elegans  but its role in S-PTGS remains elusive 23 . HEN1-catalyzed methylation of free hydroxy termini protects  Arabidopsis  sRNAs, including S-PTGS siRNAs, from oligo-uridylation, a modification promoting their instability (see the miRNA section of the background below) 24 . 
     In one S-PTGS mutant screen, an extensive allelic series of ago1 was recovered, arguing that among the 10  Arabidopsis  AGO paralogs, AGO1 is specifically involved in this pathway 25, 26 . Even weak ago1 alleles completely lost S-PTGS siRNAs, initially suggesting a role for AGO1 in siRNA production rather than action 26 . However, since AGO1 is now recognized as a slicer activity of the plant miRNA- and siRNA-loaded RISCs, loss of siRNAs in ago1 may also result from their poor incorporation into RISC, enhancing their turnover. Nevertheless, a role for AGO1 in siRNA production—possibly linked to RDR6-dependent dsRNA synthesis—cannot be excluded because some ago1 mutants defective in S-PTGS siRNA accumulation show no defects in IR-PTGS 27 . 
     RDR6, and perhaps other S-PTGS components, is also involved in the related silencing phenomenon, transitivity 28, 29 . Transitivity is the “transition” of primary siRNAs (corresponding to a sequence interval of a targeted RNA) to secondary siRNAs targeting regions outside the initial interval ( FIG. 2 ).  FIG. 2  shows how in transitive RNA silencing, a dsRNA source of primary siRNAs promotes production of secondary siRNAs both 5′ and 3′ of the initially targeted interval of a transcript. Production of 5′ secondary siRNAs (case 1) can be explained by RDR6/SGS3/SDE3-dependent complementary strand synthesis that is primed by one of the primary siRNAs. Production of 3′ secondary siRNAs (case 2) cannot be explained by a primed reaction, and it is possible that RNA fragments resulting from primary siRNA-directed transcript cleavage are recognized as aberrant, thereby initiating dsRNA synthesis as in S-PTGS. The 5′ and 3′ reactions should not be considered mutually exclusive, as siRNAs produced in (2) could prime further dsRNA synthesis according to the scheme depicted in (1). DCL4 is shown as putatively involved in 5′ and 3′ secondary siRNA biogenesis. Unlike primary siRNAs (which can be 21 nt and 24 nt in size), secondary siRNA are exclusively of the 21 nt size class. It remains unclear whether 24 nt primary siRNAs can trigger transitive RNA silencing. 
     In plants, this transition may occur both 5′ and 3′ to the primary interval, possibly reflecting primer-dependent and primer-independent RDR6 activities. Transitivity serves as a siRNA amplification mechanism that also accounts for extensive movement of silencing throughout transgenic plants 30 . Secondary siRNAs are exclusively of the 21 nt size class 30 . Thus, given that S-PTGS siRNAs seem to accumulate as 21 nt species 32 , that DCL4 produces the 21 nt siRNAs from IR transcripts 14 , and that DCL4 and RDR6 activities are coupled for 21 nt trans-acting siRNA biogenesis (see below), it is tempting to speculate that DCL4 is also the preferred Dicer for siRNA production in both S-PTGS and transitivity ( FIGS. 1B ,  2 ). 
     What would be the biological function of an amplified and non-cell autonomous pathway based on 21 nt siRNAs? At least one answer is antiviral defense. Virus-derived 21 nt siRNAs accumulate in infected cells 31  and plants compromised for RDR6 function are hypersusceptible to several viruses 16, 32 . An RDR-amplified response primed by viral siRNAs (transitivity) and/or elicited by viral-derived aberrant RNAs (S-PTGS pathway) would ensure that the silencing machinery keeps pace with the pathogen&#39;s high replication rates. The systemic nature of the response would immunize cells that are about to be infected, resulting, in some cases, in viral exclusion. Consistent with this idea, the meristems of  Nicotiana benthamiana  with compromised RDR6 activity became invaded by several viruses, whereas those tissues are normally immune to infection 33 . 
     Endogenous RNA Silencing Pathways Involved in Post-Transcriptional Regulation MicroRNAs 
     In plants, miRNAs are produced as single-stranded, 20-24 nt sRNA species, excised from endogenous non-coding transcripts with extensive fold-back structure. miRNAs act in trans on cellular target transcripts to induce their degradation via cleavage, or to attenuate protein production ( FIG. 1C ) 34 .  FIG. 1C  shows how primary (pri) miRNA transcripts with fold-back structures are products of RNA polymerase II (Pol II). The position of the mature miRNA is boxed. The combined nuclear action of DCL1, HYL1 and HEN1 produces a mature, methylated miRNA. Upon nuclear export, possibly mediated by the  Arabidopsis  exportin 5 homolog HASTY, the mature miRNA incorporates into AGO1-loaded RISC to promote two possible sets of reactions that are not mutually exclusive. A first reaction would lead to endonucleolytic cleavage of homologous RNA, as directed by 21 nt siRNAs. This would result in a poly-urydilated 5′ cleavage fragment—a modification that might promote its rapid turnover—and a more stable 3′ fragment that could be degraded by the XRN4 exonuclease. The scheme also accommodates the possibility that mature miRNAs could have sequence-specific effects in the nucleus (see text). Those nuclear activities include RNA cleavage (upon incorporation into a putative nuclear RISC) as well as DNA methylation. 
     Currently, approximately 100  Arabidopsis  MIRNA genes falling into 25 distinct families have been identified 35 , but many more are likely to exist. miRNAs have important biological roles in plant and animal development, as evidenced by the strong developmental defects of several miRNA overexpression and loss-of-function mutants 34 . For instance, key regulatory elements of the plant response to the hormone auxin, which specifies organ shape and the axes of the plant body, are controlled by miRNAs 36, 37 . miRNAs also regulate accumulation of transcription factors (TFs) involved in floral organ identity/number 38, 39 , leaf shape 40 , abaxial/adaxial leaf asymmetry 41, 42 , and lateral root formation 43 . In addition, DCL1 and AGO1, involved in the miRNA pathway, are themselves regulated by miRNAs 44, 45 . Nonetheless, plant miRNAs with validated targets involved in primary and secondary metabolism have been identified 36, 46 , indicating that their roles are not confined to developmental regulations. miRNAs might, indeed, have broad implications in plant physiology and environmental adaptation. 
     miRNA Transcription and Biogenesis 
     Most plant and animal miRNA genes reside between protein coding genes or within introns 47 . Most are likely to be independent transcription units and their expression patterns often show exquisite tissue- or even cell-type specificity, in agreement with a role in patterning and maintenance of differentiated cell states 48, 49 . Nonetheless, transcription factors or post-transcriptional mechanisms that specify plant miRNA gene expression remain unknown. Many human primary miRNA transcripts (pri-miRNAs) are synthesized by RNA polymerase II (Pol II), because pri-miRNAs have typical Pol II 5′ caps and poly-A tails, their synthesis is inhibited by PolII-inhibiting drugs, and PolII is found at their promoters in vivo 50 . Similar, though less extensive, evidence also points to PolII as the major polymerase producing plant pri-miRNAs 35 . 
     Upon transcription, mammalian pri-miRNAs are processed via a well-defined biosynthetic pathway. The RNAseIII Drosha and its essential cofactor DGCR8/Pasha—both constituents of the nuclear Microprocessor complex—catalyze initial cuts at the basis of pri-miRNAs stem-loop to produce pre-miRNAs. Pre-miRNAs are processed by Dicer into mature miRNAs upon Exportin-5-dependent nuclear export 51 . Plants have no direct equivalent of Microprocessor. In  Arabidopsis , miRNA biosynthesis depends specifically upon DCL1 52, 53 , which is required for the nuclear stepwise processing of pri-miRNAs. Whether DCL1 itself catalyzes all of the reactions involved is uncertain 54 . The plant exportin-5 homolog HASTY is involved in miRNA biogenesis 55 , but its exact role is not as clear as in mammals where the Microprocessor pre-miRNA product is an experimentally verified cargo 56 . hasty mutants exhibit decreased accumulation of some, albeit not all, miRNAs in both nuclear and cytoplasmic fractions 55 . These observations support the existence of HASTY-independent miRNA export systems and question whether miRNAs or miRNA-containing complexes are even direct cargoes of HASTY. 
     In plants and animals, Dicer processing occurs in association with specific dsRNA-binding proteins. First observed with the Dcr2-R2D2 complex required for RISC loading in the  Drosophila  RNAi pathway 57 , this has now also been found for the Dcr1-Loqs complex involved in the  Drosophila  miRNA pathway 58 , and Dicer-TRBP as well as Dicer-PACT in human cells 59, 60 . DCL1-HYL1 constitutes a similar complex that acts in pri-miRNA processing in the  Arabidopsis  miRNA pathway (FIG.  1 C). 61-64  In all cases, Dicer produces a duplex between the mature miRNA (miR) and its complementary strand (miR*) 65 . The miR strand is generally least stably base-paired at its 5′-end and is, consequently, loaded as the guide strand into RISC, whereas the miR* strand is degraded 66  ( FIG. 1C ). In the  Drosophila  RNAi pathway, R2D2 acts as a thermodynamic asymmetry sensor of siRNA duplexes, and Logs, TRBP, PACT and HYL1 could possibly perform similar roles. 
     HEN1 is an S-adenosyl methionine (SAM)-binding methyl transferase that methylates the 2′ hydroxy termini of miR/miR* duplexes, a reaction apparently specific to the plant kingdom 67, 68 . Methylation protects miRNAs from activities that uridylate and degrade plant sRNAs from the 3′end 24 , but it is not required for RISC-dependent miRNA-guided cleavage in  Arabidopsis  extracts{Qi, 2005 #5064}. All known classes of plant sRNAs are methylated by HEN1 24 , but this modification seems to impact differentially on sRNA stability, perhaps reflecting variable interactions between HEN1 and distinct protein complexes or distinct sRNA populations. For example, the viral silencing suppressor Hc-Pro prevents methylation of virus derived siRNAs, but not of miRNAs 69  and several hen1 mutant alleles exist, in which accumulation of miRNA, but not of S-PTGS siRNAs, is impaired 19 . 
     Plant miRNA Activities 
     Most identified plant miRNAs have near-perfect complementarity to their targets and promote their cleavage. This is followed by oligo-uridylation and rapid degradation of the 5′-cleavage fragment 70 , and slower degradation of the 3′-cleavage fragment mediated, at least in some cases, by XRN4 71  ( FIG. 1C ). Animal miRNAs generally exhibit imperfect complementarity and repress protein production from intact target mRNAs. However, it is possible that the action of both plant and animal miRNAs results from a combination of both processes, whose respective contributions probably vary depending on the extent of the miRNA:target complementarity. Although the RISC(s) acting in the plant miRNA pathway remain ill defined, AGO1 associates with miRNAs and miRNA targets are cleaved in vitro by immuno-affinity-purified AGO1. Thus, in plants, the same Argonaute appears to function as a Slicer for both miRNA- and siRNA-loaded RISCs, contrasting with the situations in  Drosophila  and  C. elegans.  Plant RISC components other than AGO1 await identification and it may well be that several alternative RISCs exist, given the number of AGO-like genes in  Arabidopsis.    
     Mature plant miRNAs are detected in both nuclear and cytosolic cell fractions 55 . Likewise, RISC programmed with the let-7 miRNA can be immuno-purified from nuclear human cell fractions 72 , indicating that plant and animal miRNAs may have nuclear functions ( FIG. 1C ). These may include RNA cleavage, as suggested by the intron-targeting activity of the plant miR173 73 , but could also comprise modifications of homologous DNA 74 . Thus, in  Arabidopsis , miR165 recognition of the spliced PHB transcript apparently directs cis-methylation on the PHB template DNA. This methylation is enigmatic, however, as it occurs several kb downstream of the miRNA binding site 74 . It is conceivable that miRNA-induced cleavage of the nascent PHB transcript triggers dsRNA formation initiated at the 3′end of the transcript through a primer-independent RDR activity with moderate processivity. The resulting production of siRNA would thus be confined to the 3′end and could mediate DNA methylation according to the schemes discussed in a further section of this review. Intriguingly, some, albeit few, siRNAs corresponding to downstream parts of several miRNA targets have been detected in  Arabidopsis , although none were directly complementary to the methylated PHB sequence 75 . Direct miRNA-guided DNA methylation in cis and/or trans has also been suggested from the observation that some 21 nt miRNAs of  Arabidopsis  accumulate as a second, 24 nt species at specific developmental stages 65 . 
     Transacting siRNAs: Mixing Up miRNA and siRNA Actions 
     Transacting (ta) siRNAs are a recently discovered class of plant endogenous sRNAs. They derive from non-coding, single-stranded transcripts, the pri-tasiRNAs, which are converted into dsRNA by RDR6-SGS3, giving rise to siRNAs produced as discrete species in a specific 21 nt phase 76, 77  ( FIG. 1D ).  FIG. 1D  shows how primary (pri) trans-acting siRNA transcripts are non-coding RNAs devoid of extensive fold-back structures. A miRNA incorporated into AGO1-loaded RISC guides endonucleoytic cleavage of the pri-tasiRNA. This cut generates two cleavage fragments, one of which acts as an RDR6 template, leading to the production of dsRNA. DCL4 initiates processing exclusively from the dsRNA ends corresponding to the initial miRNA cut site, to produce phased tasiRNAs that are methylated by HEN1. tasiRNA subsequently guide cleavage of homologous mRNAs, once incorporated into AGO1-loaded RISC. The colored reactions depicted in the inlay illustrate the importance of the initial miRNA-directed cut in determining the appropriate phase for tasiRNAs (1). Incorrect phasing (2) would result in the production of off-target small RNAs. 
     The RDR6-SGS3 involvement is reminiscent of siRNA biogenesis in S-PTGS, but the genetic requirements of those pathways are not identical, because tasiRNA accumulation is normal in the hypomorphic ago1-27 mutant and in mutants defective in SDE3 and WEX 76 . Much like plant miRNAs, mature tasiRNAs guide cleavage and degradation of homologous, cellular transcripts. To date, tasiRNA generating loci (TAS1-3) have been only identified in  Arabidopsis   73 , but they are likely to exist in other plant species and possibly in other organisms that contain RDRs such as  C. elegans  or  N. crassa.    
     tasiRNA production involves an interesting mix of miRNA action and the siRNA biogenesis machinery. Pri-tasiRNAs contain a binding site for a miRNA that guides cleavage at a defined point. The initial miRNA-guided cut has two important consequences. First, it triggers RDR6-mediated transitivity on the pri-tasiRNA cleavage products, allowing dsRNA production either 5′ or 3′ of the cleavage site 73 . Second, it provides a well-defined dsRNA terminus crucial for the accuracy of a phased dicing reaction, performed by DCL4, which produces mature tasiRNAs ( FIG. 1D ). 
     What is the biological role of tasiRNAs? rdr6, sgs3, and dcl4 all exhibit accelerated juvenile-to-adult phase transition 12, 13, 77, 78 , indicating that tasiRNAs could regulate this trait. The tasiRNA targets include two auxin response factor (ARF) TFs and a family of pentatricopeptide repeat proteins, although there is no evidence for the involvement of the only functionally characterized target (ARF3/ETTIN) in juvenile-to-adult phase transition 79 , nor were heterochronic defects noticed in insertion mutants disrupting the TAS1 or TAS2 loci 76, 78 . Mutants in AGO7/ZIPPY display a similar phase transition defect 80 , suggesting that AGO7 could be part of a specific tasiRNA-programmed RISC, although tasiRNAs do co-immunoprecipitate with AGO1 to form a cleavage competent RISC{Qi, 2005 #5064}. 
     Natural Antisense Transcript siRNAs 
     An example has been recently described in which a pair of neighboring genes on opposite DNA strands (cis-antisense genes) gives rise to a single siRNA species from the overlapping region of their transcripts 81 . This 24 nt siRNA species—dubbed natural antisense transcript siRNA (nat-siRNA)—guides cleavage of one of the two parent transcripts, and is produced in a unique pathway involving DCL2, RDR6, SGS3 and the atypical DNA dependent RNA polymerase-like subunit NRPD1a (see the discussion of chromatin targeted RNA silencing pathways below). nat-siRNA guided cleavage triggers production of a series of secondary, phased 21 nt siRNAs, a reaction similar to tasiRNA biogenesis except that the Dicer involved is DCL1. The role of secondary nat-siRNAs is currently unclear, but primary nat-siRNA-guided cleavage contributes to stress adaptation, and, given the large number of cis antisense gene pairs in plant and other genomes 82, 83 , this isolated example may reflect a widespread mechanism of gene regulation. 
     Chromatin Targeted RNA Silencing Pathways 
     In addition to acting on RNA, siRNAs can guide formation of transcriptionally silent heterochromatin in fungi, animals and plants. Plant heterochromatin is characterized by two sets of modifications: methylation of cytosines and of specific histone lysine residues (histone 3 Lys9 (H3K9) and histone 3 Lys27 (H3K27) in  Arabidopsis ) 84 . In some organisms, these modifications act as assembly platforms for proteins promoting chromatin condensation.  Arabidopsis  cytosine methyl-transferases include the closely homologous DRM1/2 required for all de novo DNA methylation, MET1 required for replicative maintenance of methylation at CG sites, and CMT3 required for maintenance at CNG and asymmetrical CNN sites (reviewed in 85, 86 ). Histone methyl-transferases involved in H3K9 and H3K27 methylation belong to the group of Su(Var)3-9 homologues and include KYP/SUVH4 and SUVH2 in  Arabidopsis   87 . 
     In several organisms, siRNAs corresponding to a number of endogenous silent loci, including retrotransposons, 5S rDNA and centromeric repeats, have been found 85 . They are referred to as cis-acting siRNAs (casiRNAs) because they promote DNA/histone modifications at the loci that generate them. In plants, casiRNAs are methylated by HEN1 and are predominantly 24 nt in size. Their accumulation is specifically dependent upon DCL3 and, in many instances, upon RDR2. casiRNA accumulation also requires an isoform (containing subunits NRPD1a and NRPD2) of a plant-specific and putative DNA-dependent RNA polymerase, termed PolIV 88-90 . PolIV may act as a silencing-specific RNA polymerase that produces transcripts to be converted into siRNAs by the actions of RDR2 and DCL3. However, many aspects of PolIV silencing-related activities remain obscure. Hence, it is uncertain whether PolIV even possesses RNA polymerase activity. Additionally, a distinct PolIV isoform with subunits NRPD1b and NRPD2 is required for methylation directed by IR-derived siRNAs with transgene promoter homology, suggesting that the action of PolIV complexes may not be confined to siRNA biogenesis 91 . Finally, the requirement of NRPD1a for nat-siRNA accumulation in the presence of both antisense mRNAs (produced by PolII) suggests that PolIV may have silencing-related functions independent of DNA-dependent RNA polymerase activity 81 . Other factors involved in IR-derived siRNA-directed promoter methylation include the chromatin remodelling factor DRD1 92  and the putative histone deacetylase HDA6 93  whose activity may be required to provide free histone lysines for methylation by KYP/SUVH enzymes ( FIG. 3 ). It is currently uncertain whether DRD1 and HDA6 are also implicated in silencing of endogenous loci. 24 nt siRNAs may act in a RISC-like complex, perhaps akin to the RNA-induced transcriptional silencing complex, RITS, characterized in fission yeast 94 . This complex could contain AGO4 because ago4 mutants have phenotypes overlapping with those of rdr2, dcl3, nrpd1a and nrpd2 11 . At loci affected by the above mutations, CNG and particularly CNN methylation is strongly reduced, whereas loss of CG methylation is less pronounced, consistent with the observation that MET1-dependent promoter CG methylation could be maintained in the absence of a viral-encoded RNA trigger of TGS 95 . 
     DNA itself or nascent transcripts are both possible targets of casiRNAs ( FIGS. 3A and 3B , respectively). In  FIG. 3A , nascent polII/polIII transcript is cleaved through the action of siRNA-programmed AGO4, resulting in a truncated RNA that is converted into dsRNA by the action of RDR2. The dsRNA is then processed by DCL3 into 24 nt siRNAs that direct further cleavage of nascent transcripts and may possibly guide sequential activities of histone deacetylases (e.g., HDA6), histone methyl transferases (e.g., KYP, SUVH2) and/or DNA methyl-transferases (CMT3/DRM). It is unclear whether histone modification precedes DNA methylation or not. The process might also involve siRNA-directed chromatin remodeling factors such as DRD1. The positions of PolIVa and PolIVb in those reactions are currently ill defined. 
     In  FIG. 3B , the same effectors are involved but, in this scenario, RDR2 uses nascent transcripts as templates and siRNA-loaded AGO4 is recruited to guide chromatin modifications rather than RNA cleavage. 
     In the  S. pombe  heterochromatic RNAi pathway resulting in H3K9 (but not cytosine) methylation, target transcription by PolII is required for siRNA action, and Ago1 associates with nascent transcripts 96 . siRNA directed histone methylation of the human EF1A promoter was also dependent on active PolII transcription 97 . However, direct siRNA-DNA base-pairing cannot be excluded. For instance, in experiments involving virus derived promoter directed siRNAs, the methylated DNA interval on targeted promoters matched the primary siRNA source and did not extend any further into transcribed regions 95 . If siRNAs indeed interact directly with DNA, how does the double helix become available for siRNA pairing? PolIV could facilitate this access, for instance by moving along the DNA with associated helicases. The precise molecular mechanisms underlying sequence-specific recruitment of cytosine and histone methyl-transferases to silent loci also remains elusive, as associations between sRNA and such enzymes have been reported in only one single case, in human cells 97 . In fact, a self-sustaining loop in which siRNA production and DNA/histone methylation are mutually dependent appears to exist at endogenous silent loci, raising the possibility that production of chromatin-directed siRNAs in vivo might even be a consequence, rather than a cause, of DNA/histone methylation ( FIG. 3 ). 
     The RDR2/DCL3/NRPD1/AGO4 pathway has clear roles in transposon taming and maintenance of genome integrity in plants, because loss of casiRNA caused by mutations in the above factors reactivates transposon activity. This pathway may also maintain heterochromatin at centromeric repeats, which appears mandatory for accurate chromosome segregation in  S. pombe   98 . The 24 nt siRNA-generating machinery may also act to silence protein-coding genes. For example, expression of the key negative regulator of flowering FLC is maintained at a low level in an early-flowering  Arabidopsis  ecotype due the presence of an intronic transposon that causes repressive chromatin modifications through the action of an NRPD1a/AGO4-dependent pathway 99 . Nevertheless, several additional mechanisms, not necessarily mediated by siRNAs, account for epigenetic regulation of gene expression in plants. For example, in  Arabidopsis , mutation of the chromatin-remodeling factor DDM1 has much broader consequences on chromatin silencing than any known single mutant in the RNA silencing machinery 100, 101 . In addition, gene regulation by polycomb-like proteins in  Arabidopsis  has not been linked to RNA silencing 102 . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Overview of proteins with roles in  Arabidopsis  small RNA pathways. 
               
            
           
           
               
               
               
               
               
            
               
                 Protein 
                 Domains and motifs 
                 Biochemical activity 
                 Pathway 
                 Ref. 
               
               
                   
               
               
                 DCL1 
                 RNase III 
                 miRNA synthesis 
                 miRNA 
                 
                   55, 85 
                 
               
               
                   
                 dsRNA bd 
                   
                 nat-siRNA 
               
               
                   
                 DEAD-box helicase 
               
               
                   
                 PAZ 
               
               
                   
                 DUF283 (unknown function) 
               
               
                 HYL1 
                 dsRNA bd 
                 dsRNA bd 
                 miRNA 
                 
                   61, 62 
                 
               
               
                 HST 
                 RanGTP bd 
                 Putative exportin 
                 miRNA 
                 
                   58 
                 
               
               
                 AGO1 
                 PAZ 
                 siRNA Slicer 
                 miRNA 
               
               
                   
                 Piwi 
                 miRNA Slicer 
                 S-PTGS 
               
               
                   
                   
                   
                 tasiRNA 
               
               
                   
                   
                   
                 Chromatin (?) 
               
               
                 HEN1 
                 dsRNA bd 
                 sRNA methyl transferase 
                 All sRNA 
                 
                   19, 24, 53, 
                 
               
               
                   
                 Lupus La RNA bd 
                   
                 pathways 
                 
                   67 
                 
               
               
                   
                 S-adenosyl bd 
               
               
                 RDR6 
                 RdRP-specific GDD 
                 RNA-dependent RNA 
                 S-PTGS 
                 
                   15, 16, 29, 
                 
               
               
                   
                   
                 polymerase 
                 Transitivity 
                 
                   30, 73, 76, 
                 
               
               
                   
                   
                   
                 tasiRNA 
                 
                   85 
                 
               
               
                   
                   
                   
                 nat-siRNA 
               
               
                 SGS3 
                 Coiled-coil 
                 Unknown 
                 S-PTGS 
                 
                   16, 76, 85 
                 
               
               
                   
                 Putative Zn II -bd 
                   
                 Transitivity 
               
               
                   
                   
                   
                 tasiRNA 
               
               
                   
                   
                   
                 nat-siRNA 
               
               
                 DCL4 
                 RNase III 
                 21nt siRNA synthesis 
                 tasiRNA 
                 
                   12-14 
                 
               
               
                   
                 dsRNA bd 
                   
                 IR-PTGS 
               
               
                   
                 Helicase 
                   
                 S-PTGS? 
               
               
                   
                 PAZ 
               
               
                 WEX 
                 3′-5′ exonuclease 
                 Putative 3′-5′ exonuclease 
                 S-PTGS 
                 
                   18 
                 
               
               
                 SDE3 
                 DEAD box 
                 Putative RNA helicase 
                 S-PTGS 
                 
                   20, 30 
                 
               
               
                   
                 Helicase 
                   
                 Transitivity 
               
               
                 DCL2 
                 RNaseIII 
                 22/24 nt siRNA synthesis 
                 nat-siRNA 
                 
                   85 
                 
               
               
                   
                 dsRNA bd 
               
               
                   
                 PAZ 
               
               
                 DCL3 
                 RNase III 
                 24nt siRNA synthesis 
                 Chromatin 
               
               
                   
                 DEAD box helicase 
               
               
                   
                 PAZ 
               
               
                 RDR2 
                 RdRP 
                 Putative RNA dependent 
                 Chromatin 
               
               
                   
                   
                 RNA polymerase 
               
               
                 AGO4 
                 PAZ 
                 Unclear 
                 Chromatin 
               
               
                   
                 Piwi 
               
               
                 NRPD1a 
                 RNA polymerase 
                 Putative DNA dependent 
                 Chromatin 
                 
                   85, 88-91 
                 
               
               
                   
                   
                 RNA polymerase 
                 nat-siRNA 
               
               
                 NRPD1b 
                 RNA polymerase 
                 Putative DNA dependent 
                 Chromatin 
                 
                   89, 91 
                 
               
               
                   
                   
                 RNA polymerase 
               
               
                 NRPD2 
                 RNA polymerase 
                 Putative DNA dependent 
                 Chromatin 
                 
                   88-91 
                 
               
               
                   
                   
                 RNA polymerase 
               
               
                 HDA6 
                 Histone deacetylase 
                 Putative histone deacetylase 
                 Chromatin 
                 
                   93 
                 
               
               
                 DRD1 
                 SNF2-related DNA and ATP bd 
                 Putative chromatin 
                 Chromatin 
                 
                   92 
                 
               
               
                   
                 Helicase 
                 remodeling 
               
               
                 CMT3 
                 Cytosine DNA methyl transf. 
                 Cytosine DNA methyl 
                 Chromatin 
                 
                   88 
                 
               
               
                   
                 Chromodomain 
                 transferase 
               
               
                   
                 Bromo-adjacent domain 
               
               
                 DRM1/2 
                 Cytosine DNA methyl transf. 
                 Cytosine DNA methyl 
                 Chromatin 
                 
                   88 
                 
               
               
                   
                   
                 transferase 
               
               
                 MET1 
                 Cytosine DNA methyl transf. 
                 Cytosine DNA methyl 
                 Chromatin 
                 
                   88 
                 
               
               
                   
                 Bromo-adjacent domain 
                 transferase 
               
               
                 KYP 
                 SET domain 
                 H3K9 methyl transferase 
                 Chromatin 
                 
                   87 
                 
               
               
                   
                 Zn II -bd pre-SET domain 
               
               
                   
                 Post-SET domain 
               
               
                   
                 YDG domain 
               
               
                   
                 EF-hand 
               
               
                 SUVH2 
                 SET domain 
                 H3K9 methyl transferase 
                 Chromatin 
                 
                   103  
                 
               
               
                   
                 Zn II -bd pre-SET domain 
               
               
                   
                 YDG domain 
               
               
                   
               
            
           
         
       
     
     Disease Resistance in Plants and Animals 
     There is extensive evidence that the plant RNAi pathway plays essential roles in antiviral defense 104 . Double-stranded RNA derived from viral genomes is diced into siRNAs by the redundant activities of both DCL4 (the major antiviral Dicer) and DCL2 (a surrogate of DCL4) 105 . These siRNAs then incorporate into RISC (the RNA Induced Silencing Complex) to mediate slicing of viral transcripts and thereby reduce the overall viral load in plant cells 105 . AGO1 is a likely effector protein of the siRNA loaded RISC, although other AGO paralogs might also be involved 106 . A cell-to-cell and long distance signal for RNA silencing also accounts for the systemic spread of the antiviral innate immune response throughout plants 104 . As a counter-defensive strategy, viruses encode suppressor proteins that are targeted against key processors and effectors of antiviral silencing. For instance, the P19 protein of tombusviruses sequesters siRNAs and prevents their use by RISC 107 , the 2b protein of Cucumber mosaic virus physically interacts with AGO1 and inhibits its cleavage activity 106 , and the P38 protein of Turnip crinckle virus strongly inhibits DCL4 activity 105 . DCL3 (producing heterochromatic siRNAs) and DCL1 (producing miRNAs) do not appear to have a significant impact on plant virus accumulation. 
     Apart from antiviral defense, there is currently scant information available on the role of small RNA pathways in defense against other types of pathogens including bacteria and fungi, which account for major yield losses worldwide. In plants, fungal and bacterial resistance has been most thoroughly studied in the context of race-specific interactions, in which a specific resistance (R) protein protects the plant against a particular pathogen&#39;s race 108 . This highly specific recognition leads to activation of defense responses and local cell death referred to as ‘hypersensitive response’ (HR). A well-characterized example of HR elicitation through race-specific interaction is provided by the  Arabidopsis  RPS2 gene that confers resistance to  Pseudomonas syringae  pv. tomato strain DC3000 (Pst DC3000) producing the corresponding AvrRpt2 elicitor protein. The presence of both RPS2 and AvrRpt2 components leads to resistance, whereas the absence of either component leads to disease 108 . 
     Beside the race-specific interaction is a basal defense mechanism that plays a pivotal role in “non-host resistance”, which accounts for the fact that most plants and animals are resistant to most pathogens. Basal defense relies on both constitutive and inducible responses. The inducible basal defense response is triggered upon perception of general elicitors known as ‘pathogen-associated molecular patterns’ (PAMPs). One such PAMP is a conserved 22 amino acid motif (flg-22) of the bacterial flagellin 109 , which is recognized in several plant species, including  A. thaliana.  Perception of flg-22 triggers an innate immune response in plants that elevates resistance to the virulent Pst DC3000 strain 110 . As a counter-defensive strategy, bacterial pathogens have evolved to suppress basal defense responses by injecting TTS-proteins, refereed to as ‘effectors’ 111 . These bacterial effectors are, therefore, virulence factors: their lack causes a loss of disease symptoms and a general inability of the pathogen to proliferate on leaves. 
     miRNAs and the Basal Defense Response 
     We have recently shown that miR393, a plant canonical and conserved miRNA, is rapidly induced by flg-22, leading to the repression of the entire signaling cascade that normally orchestrates the response to the phytohormone auxin 112 . This report is Navarro, L., et al.,  Science  (2006) 312:436-439, incorporated herein by reference. The resulting repression of auxin-signaling restricts bacterial growth, implicating auxin in disease susceptibility, and miRNA-mediated suppression of auxin-signaling in disease resistance. We hypothesized that miR393 was not an isolated example and that a large set of miRNAs may act as positive regulators of the plant defense response to pathogens. 
     DISCLOSURE OF THE INVENTION 
     A specific spectrum of plant and animal miRNAs confer enhanced resistance to virulent pathogens. We show that plants deficient in miRNA accumulation are hyper-susceptible to non-virulent bacterial pathogens. As a corollary, we show that virulent bacteria have evolved strategies to suppress the miRNA pathway in plants, for example, by using some of the injected type-III secreted (TTS) proteins. 
     In one aspect, the invention is directed to a method for modulating the miRNA pathway in plants and animals which comprises introduction into a plant or animal a nucleic acid construct comprising a constitutive or pathogen responsive promoter operatively linked to a pathogen associated molecular pattern (PAMP)-responsive pri-miRNA sequence or a sequence which encodes a component involved in miRNA biogenesis or activity, optionally further including cis-regulatory elements within or 5′ to said promoter. In one particular embodiment, the miRNA pathway modulated is other than that of miR393. In still another embodiment, said pathway regulated includes miR393. 
     In one embodiment, such modulation of the miRNA pathway in plants and animals is performed in such a way that adverse effects on plant and animal development are avoided or minimized 
     In another embodiment, compositions and methods are provided for conferring on plants and animals enhanced pathogen resistance by selective modulation of the miRNA pathway. In one embodiment, the miRNA pathway modulated is other than miR393; in still another embodiment, the pathway includes that of miR393. 
     The invention provides methods to identifying compounds useful in the selective modulation of the miRNA pathway in plants and animals by using bacterial-derived suppressors of RNA-silencing as molecular probes. Such RNA-silencing factors likely interact directly with some of the bacterial silencing suppressors as described below. 
     The invention is also directed for identifying compounds or establishing genetic approaches to counteract bacterial suppression of RNA-silencing in both plant and animal cells. The overall approaches confer resistance to plant and animal pathogens. 
     Biochemical and genetic approaches known by those skilled in the art are used to identify additional Bss targets. These approaches include, but are not restricted to, yeast two-hybrid screen of plant cDNA libraries or biochemical pull downs using Bss tagged versions. The identified components likely interact (either physically or genetically) with, e.g., DCL1, AGO01, HEN1, SERRATE and may be further used to manipulate specifically miRNA activities, using the methods described above. Importantly, these Bss proteins are also used to inhibit miRNA function in animal cells as observed with some viral suppressor of RNA-silencing derived from plant pathogenic viruses that are also functional in animal cells. 
     Another aspect of the invention takes advantage of the ability of Bss to suppress the silencing of transgenes thereby enhancing the production of recombinant proteins using hosts in which the Bss proteins are effective. In one embodiment, such hosts are plants. The recombinant host cell or plant is modified to contain an expression system for a desired protein, such as a therapeutic, fused to a Bss protein. Standard biochemical and molecular biology techniques are employed to construct suitable expression systems and to modify host cells for the production of a desired protein. Alternatively, separate constructs for the Bss protein and the desired protein may be used and co-transformed into the same cell or organism. By virtue of the presence of the Bss protein, cellular mechanisms that would silence its expression are inhibited. Thus, the level of production is enhanced and if the desired protein is produced with a tag sequence, purification is simplified. 
     Another application employs the availability of Bss proteins to identify compounds that repress bacterial infection by screening candidate compounds for ameliorating the effects of such proteins. The identified compounds may also be useful in other applications when silencing mechanisms are desirably enhanced. In this method, compounds that experimentally counteract Bss triggered suppression of RNA silencing and restore a normal vein chlorotic phenotype are selected. Endogenous compounds which have this effect may also be identified by mutagenizing plants in which silencing has been suppressed using Bss proteins and identifying genetic changes in plants where RNA silencing has been restored. 
     Furthermore, by constitutively or conditionally enhancing the activity of the components of the miRNA pathway identified as described herein above, increased resistance to a broad spectrum of pathogens is achieved in a variety of crop species. This method also allows inducible, enhanced resistance, which is desirable because it is not, or is less, detrimental to plant development and physiology in non-infected conditions. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1D  show the post-transcriptional RNA silencing pathways in plants.  FIG. 1A  shows IR-PTGS pathway;  FIG. 1B  shows S-PTGS pathway;  FIG. 1C  shows micro (mi)RNA pathway; and  FIG. 1D  shows trans-acting (ta)siRNA pathway; and  FIG. 1D  shows trans-acting (ta)siRNA pathway. 
         FIG. 2  shows transitive RNA silencing. 
         FIGS. 3A and 3B  show chromatin-targeted RNA silencing, showing two of many non-mutually exclusive scenarios that possibly account for siRNA-directed chromatin modifications at endogenous loci. Both scenarios are based on circular and amplified schemes in which siRNA production and chromatin modification reinforce one another. 
         FIG. 4  shows over-representation of W-box elements in the AtmiR393b promoter. 
         FIG. 5  shows typical stem loop formations associated with pri-miRNA. 
         FIGS. 6A and 6B  demonstrate the susceptibility of miRNA deficient mutants to fungal and bacterial pathogens.  FIG. 6A  shows susceptibility to fungal pathogens and  FIG. 6B  to bacterial pathogens. 
         FIGS. 7A-7E  show experimental results that confirm that the miRNA pathway confers basal as well as non-host resistance to bacteria. 
         FIGS. 8A-8D  demonstrate that infection by TuMV suppresses basal as well as non-host resistance to bacteria. 
         FIGS. 9A-9C  demonstrate that the virulent strain Pst DC3000 can suppress transcriptional induction of some PAMP-responsive miRNAs. 
         FIGS. 10A-10D  provide experimental results demonstrating that Pst DC3000 encodes Type III secreted protein that suppress miRNA biosynthesis and/or activity. 
         FIGS. 11A-11F  provide experimental results demonstrating that HopT1-1 suppresses a silencing function mediated by RISC miRNA . 
         FIGS. 12A-12D  present experimental results demonstrating that overexpression of HopY1 suppresses RISC miRNA  function. 
         FIGS. 13A-13E  present experimental results showing that HopU1 suppresses miRNA and siRNA triggered translational inhibition. 
     
    
    
     MODES OF CARRYING OUT THE INVENTION 
     By exploring the elements of pathogen resistance and its relationship to silencing pathways, we have determined a number of factors and methods to enhance resistance of plants and animals to pathogen infection, to identify components critical to resistance, and to screen for compounds or agents that are helpful in enhancing resistance. For example, known components of the miRNA pathway (e.g., DCL1, AGO1, HEN1, SERRATE) and the components identified as disclosed herein above (e.g., AtGRP7, GRP8) are modified such that they become resistant to the action of Bss proteins. Engineering resistant alleles of these components is achieved according to methods known by those skilled in the art, including, but not limited to, site-directed mutagenesis of key amino acids and transgenesis, as well as Targeted Induced Local Lesions in Genomes (TILLING) of non-transgenic crop species. The method disclosed allows for natural and/or engineered resistance to Bss action and thereby confers enhanced basal defense to crop species against virulent pathogens. 
     The sequences identified in Example 1 below and listed in Table 2 that express pri-miRNA are operatively linked to suitable promoters and used to modify plants to confer resistance to virulent infection. One of these sequences, miR393 was earlier shown by us to be transcriptionally induced by flg-22. Its constitutive over-expression confers enhanced resistance to virulent Pst DC3000 (Navarro, L., et al.,  Science  (2006) 312:436-439. Additional pri-miRNA sequences thus identified are used to elevate the plant resistance to a broad spectrum of pathogens. Individual or groups of these sequences, designated patho-miRNAs, are expressed transgenically in plants using methods known by those skilled in the art, such as for the overexpression of one of the two miR393 loci Navarro, L., et al. (supra). Expression of these sequences (+40 nt upstream and downstream) is either constitutive or, preferably, is driven by promoters that are known to be broadly responsive to bacterial, fungal and viral pathogens. Examples of such promoters include, but are not restricted to, WRKY6 and PR1. The method allows inducible, enhanced resistance, which is desirable because it is not, or is less, detrimental to plant development and physiology in non-infected conditions. 
     Moreover, several of these patho-miRNAs are conserved across plant species (monocots and dicots), indicating that  Arabidopsis -derived patho-miRs will be directly effective in a large variety of crops. 
     Constructs are prepared wherein, in one embodiment, a constitutive or pathogen responsive promoter (including but not limited to, for example, the WRKY6 promoter, the PR1 promoter and the like) is operatively linked to a nucleic acid sequence which is transcribed into one or more individual patho-miR sequences to confer enhanced resistance to unrelated pathogens in various plant species, including crops. 
     Computational analyses reveal an over-representation of specific regulatory elements located within 1.5 or 2 kilobases (kb) upstream of the patho-miR stem-loop structures. Some of those regulatory elements have been previously described: as an example, the flg-22-responsive At-miR393a promoter contains 10 W-box elements, whereas the At-miR393b promoter, that is not responsive to flg-22 treatment, contains only 3 W-box elements. See  FIG. 4 .  FIG. 4  shows over-representation of the W-box element within At-miR393a promoter sequence. 1.5 Kilobase upstream of the At-miR393a stem loop structure was extracted and analyzed for the representation of W-box elements. In red in the figure and underlined in the sequence provided below is the core sequence of the W-box element. We also identified an over-representation of the characterized auxin-responsive element AuRE as well as the RY element within subsets of patho-miR promoters. In addition, we found an over-representation of novel elements referred to as Flg-22-Induced MiRNA (FIM) element. These latter represent an original source of pathogen-responsive DNA elements to be used and manipulated in conventional approaches to enhanced pathogen resistance. Such approaches include the generation of chimeric DNA segments containing multiple copies of these known or novel cis-regulatory elements. The method hereby disclosed is thus used to specifically hyper-induce the expression of protein-coding genes or non-protein-coding genes (e.g., miRNA genes) upon pathogen infections, in plants, including crops. 
     Thus, an artificial pathogen-responsive promoter carrying multiple copies of FIM elements, or alternatively a native patho-miR promoter, operatively linked to a coding sequence from plant defense genes or non-coding sequences (e.g., miRNA sequences) that play a role in disease resistance may be used to control expression for disease resistance. This approach allows the conditional and effective expression of protein-coding genes or non-protein coding genes during pathogen infections. 
     Because the miRNA pathway is the prominent RNA silencing pathway in higher vertebrates, and because mammalian cells are commonly infected by TTS-bacterial pathogens, similar mechanisms of defense/counter-defense are operational in mammals. Recent findings also highlight the key role of miRNAs in the animal innate immune response. By using miRNA sensor genes akin to those exemplified herein for  Arabidopsis , but incorporating components of human or other animal pathogenic bacteria (e.g.,  Yersinia pestis, Pseudomonas aeruginosa, Shigella flexneri ) methods and compositions that interfere with miRNA activity/biogenesis in human or animal cultured cells are tested. Individual TTS-effector proteins that account for this interference and are key virulence factors that negate the mammalian innate immune responses to life-threatening pathogens are thus identified. This technology provides a method whereby transfection of plasmids encoding individual TTS-proteins (together with their corresponding chaperone), in human cells expressing an miRNA sensor which comprises control sequences for expression of animal generators of miRNA operatively linked to a reporter sequence construct permits identification of therapeutic agents. Molecules that interfere with TTS-protein activity without damaging cellular miRNA functions are selected as candidates for further study and drug development. Cultured human cells co-expressing the miRNA sensor and bacterial TTS-proteins are subjected to high-throughput delivery of a large collection of active molecules. Those molecules suppressing miRNA sensor expression are isolated and further tested for their potential therapeutic effect as described above. 
     Bss proteins from mammalian pathogens identified through the methods described hereinabove are also useful proteins to isolate animal components involved in miRNA biogenesis and/or activity using biochemical and genetic approaches known by those skilled in the art. These approaches include, but are not restricted to, yeast two-hybrid screen of mammalian cDNA libraries, biochemical pull downs and forward genetic screens for loss of Bss function in Ethyl-methyl sulfonate (EMS)-mutagenized cells. The identified components likely interact (either physically or genetically) with, e.g., mammalian Dicer, mammalian Drosha, mammalian TBP and may be further used to manipulate specifically miRNA activities in mammals. 
     Using the high-throughput screening methods described herein above, active molecules are identified that promote or enhance the activity of known components of the mammalian miRNA pathway (e.g., mammalian Dicer, mammalian Drosha, mammalian TBP) and of the components of the mammalian miRNA pathway identified herein above. Treatments of human or other animal cells with such molecules enhance the basal defense to a broad range of pathogens. 
     By constitutively or conditionally enhancing the activity of the components of the mammalian miRNA pathway identified herein above, increased resistance to a broad spectrum of pathogens is achieved in a variety of host mammalian cells. Conditional expression is conferred by, for example, the NFκB promoter. The method hereby disclosed thus allows inducible, enhanced resistance, which is desirable because it is not, or is less, detrimental to development and physiology in non-infected conditions. The methods of this invention are applicable to all infections involving injection/delivery of factors of parasitic origin. Examples include biotrophic fungi such as  E. cichoracearum,  a Powdery mildew that over-accumulates on both hen1-1 and dcl1-9 mutants, and therefore likely secrete effector proteins to suppress RNA-silencing pathways. 
     The Bss proteins can also be used to inhibit miRNA function so as to alter physiological and developmental processes normally orchestrated by these small RNA molecules. This is achieved by fusing Bss coding sequences to tissue-specific plant promoters known by those skilled in the art (driving expression in, e.g., roots, leaves, stem, inflorescences), which may also include the patho-miR promoter elements identified herein. The method hereby disclosed thus allows tissue-specific expression of Bss proteins for the spatial and temporal control of miRNA function. 
     Bss proteins are thus used to suppress transgene silencing and allow high expression of any proteins of interest (e.g., therapeutic molecules). In one application, single or multiple constructs carrying the gene(s) of interest and a construct carrying a Bss cDNA (e.g., HopU1) fused to strong promoter are co-delivered in planta (using  Agrobacterium -mediated transient assay). This transient assay can be performed in leaves from  N. benthamiana,  in which transient assay is more efficient, or preferentially in plant cell cultures (such as  Arabidopsis  Col-0 cells). The protein(s) of interest are further purified using methods known by those skilled in the art and their biological activity tested. The use of plant cell cultures will allow the production of large amounts of recombinant proteins. Furthermore, this system will facilitate the protein purification step as the starting materials will contain reduced amounts of plant-derived components that are unwanted in protein purification (e.g., polyphenols). 
     HopN1 mutated versions (with C172S, H283A or D299A substitutions, abolished in their cysteine protease activity) will be particularly useful for this approach as these mutant versions will not be, or less be, recognized by resistant proteins (which trigger host programmed-cell death that could impact negatively on recombinant protein yields). 
     Another application is in a method for identifying compounds that counteract bacterial suppression of RNA-silencing. Bss proteins and a miRNA sensor construct (e.g., 171 sensor) or the SUC-SUL transgene (or any other silencing reporter constructs) are co-expressed stably in plants and sprayed with a library of compounds to identify molecules that counteract Bss-triggered suppression of RNA-silencing. As an example, the SUC-SUL plants expressing HopT1-1 display attenuated vein chlorotic phenotype due to the suppression of AGO1-containing RISC activity. These plants are sprayed with a battery of molecules to identify compounds that counteract Bss-triggered suppression of RNA-silencing and likewise restore a normal vein chlorotic phenotype. Such molecules are used to confer broad spectrum resistance to pathogens in various plant species including crops. 
     Alternatively, plants are mutagenized (using methods known by those skilled in the art such as EMS mutagenesis) to identify mutants in which RNA-silencing is restored. For example, SUC-SUL plants that express HopT1-1 are mutagenized and mutants that restore a normal vein chlorotic phenotype isolated. The corresponding genes are further identified using methods known by those skilled in the art (e.g., map-based cloning). Orthologs of such genes, in various plant species including crops, are identified and methods used to knock-out or knock-down these particular genes applied. 
     Because animal pathogenic bacteria also use the type-three secretion system to deliver key virulence proteins, animal pathogenic bacteria (including human pathogenic bacteria) have also evolved to suppress RNA-silencing. Such bacterial virulence genes are fused to a strong promoter (e.g., CMV) and delivered to animal cells using known methods. Levels of endogenous miRNAs as well as miRNA targets are monitored to identify proteins that suppress RNA-silencing (as performed in the plant experimental systems described above). These Bss proteins, derived from animal pathogenic bacteria, are further co-expressed in animal cells with an miRNA sensor construct. Molecules that—restore a normal expression of the miRNA sensor are further identified and used to confer enhanced resistance to bacterial pathogens. Such molecules potentially represent substitutes for antibiotics. Alternatively, the above animal cells are mutagenized using methods known by those skilled in this art and the corresponding genes identified. Methods that allow knock-down or knock-out of such genes are used to elevate resistance to bacterial pathogens. Such methods include but are not restricted to, the use of artificial siRNAs directed against endogenous repressors of Bss-triggered suppression of RNA-silencing. 
     The following are non-limiting aspects of the invention. 
     1. A method for modulating the miRNA pathway in plants and animals which comprises introduction into a plant or animal a nucleic acid construct comprising a constitutive or pathogen responsive promoter operatively linked to a miRNA encoding sequence or a sequence which encodes a miRNA effector protein, optionally further including cis-regulatory elements within or 5′ to said promoter. 
     This method may be designed so that adverse effects on plant and animal development are avoided or minimized, and may provide enhanced pathogen resistance upon expression in said plant or animal, for example, by employing inducible or conditional control sequences. 
     In this method, the effector protein is a protein set forth in Table 1, e.g., DCL1, AGO1, SERRATE, and/or HEN1. 
     2. A method for identifying compounds useful in the selective modulation of the miRNA pathway in plants and animals by monitoring expression in a plant or animal cell of a sensor transgene reporting the activity of endogenous miRNAs in response to perturbation caused by exposure of said plant or animal cell to a candidate compound. 
     3. The invention also provides a bacterial silencing suppressor, e.g., HopN1, HopH1, HopPto, HopT1, HopY1, or HopU1 or a functional derivative thereof. 
     These may be used in a method for selectively modulating miRNA expression in a cell by contacting said cell with the bacterial silencing suppressor. 
     4. A method for identifying miRNAs associated with a plant or animal response to a pathogenic elicitor. A plant or animal or plant or animal cell is exposed to a pathogenic elicitor and compared with a substantially identical plant or animal or plant or animal cell not exposed to the pathogenic elicitor. Transcriptional fluctuation of computationally predicted or experimentally validated miRNAs as primary transcripts is monitored and compared. The invention is also directed to an isolated pathogen-elicited miRNA so identified. 
     The invention is also directed to a method for conferring enhanced pathogen resistance on a plant or animal or plant or animal cell by effecting expression of a pathogen-elicited miRNA identified as above. The expression may be under the operative control of a promoter which is activated on exposure to a pathogen, e.g., the WRKY6 promoter or the PR1 promoter. 
     One or more cis-acting regulatory elements may also be provided upstream of or incorporated within said promoter and miRNA encoding sequence. The cis-acting regulatory element may be a W box, an AuxRE element, an RY element, an FIM element, multiples of these elements or combinations thereof. 
     5. A method for determining the role of a miRNA by suppressing miRNA in a cell that otherwise exhibits expression of said miRNA by contacting said cell with a bacterial suppressor of silencing (Bss). 
     6. A method for modulating the resistance in an animal to infection by a pathogen which comprises either enhancing the expression of miRNA pathway components suppressed by proteins secreted by said pathogen or increasing the resistance of miRNA pathway components to suppression by proteins secreted by said pathogen. 
     7. A method of conferring resistance in a plant to pathogens which comprises selecting plants with miRNA pathway components resistant to the action of suppressors of silencing, such as a bacterial suppressor of silencing, and plants selected by this method. 
     8. A method for specifically manipulating miRNA accumulation in order to alter physiological and developmental processes normally orchestrated by those molecules by fusing a Bss coding sequence to a tissue-specific plant promoter driving expression in roots, leaves, stem, inflorescences, optionally including patho-miR promoter elements to achieve spatial and temporal control of miRNA activity. 
     9. A method for exploiting Bss triggered suppression of RNA-silencing activity without recognition by the plants. This includes generating mutants that can still suppress the miRNA and siRNA pathways but can no longer be perceived by plant derived R genes. HopN1 mutated in the catalytic triads can be used because these mutants suppress miRNA biogenesis at the same level as does HopN1 wildtype but are not perceived by R proteins. 
     10. A method to identify components of the siRNA and/or miRNA pathway by using Bss proteins as molecular probes in both plant and animal cells and retrieving components that physically interact with such proteins. 
     11. A method to suppress miRNA function in animal cells by using Bss proteins derived from plant pathogenic bacteria such as Pst DC3000. The Bss proteins are fused to constitutive human promoters and transfected in animal cells in order to suppress miRNA function. This may be used in combination with recombinant expression of desired proteins. 
     12. A method for overexpressing recombinant proteins (e.g., human proteins with therapeutic activity) in plant cells. This will be performed by expressing Bss proteins that inhibit transgene silencing. This approach is preferentially performed in cell cultures using an  Agrobacterium  transient assay. The purified recombinant proteins are further tested for their biological activities and used as medicines. 
     13. A method for identifying molecules that promote transcription of endogenous factors involved in miRNA biogenesis of activity. One method employs constructs in which control sequences of miRNA expression are coupled to reporter sequences. Another method employs reporter constructs that are silenced by miRNA. In the first case, molecules or genetic alterations that increase expression of reporters are enhancers of miRNA expression. In the second case, the opposite is true. 
     14. A method for identifying compounds that counteract Bss-triggered suppression of RNA-silencing in either plant or animal cells. This approach comprises the use of plant or animal cells that co-express an RNA-silencing reporter construct with a Bss protein. Molecules that restore normal expression of the RNA-silencing reporter gene are identified and used to elevate resistance to bacteria in plants and animals. Such molecules may be used as antibiotics in animal cells. Alternatively, endogenous repressors of Bss-triggered suppression of RNA-silencing are identified by using mutagenesis of the said plant or animal cell lines co-expressing a RNA-silencing reporter construct and a Bss protein. 
     15. The approaches described herein are used to identify secreted proteins from parasites that could suppress RNA-silencing pathways. 
     The following examples are offered to illustrate but not to limit the invention. 
     EXAMPLES 
     The Examples show that 1) a specific spectrum of plant miRNAs confers enhanced resistance to virulent pathogens; and that 2) plants deficient in miRNA accumulation are hyper-susceptible to virulent and non-virulent bacterial pathogens and that, as a corollary; virulent bacteria must therefore have evolved strategies to suppress the miRNA pathway, for instance by using some of the injected type-III secreted (TTS) proteins. 
     All the results below were generated in the model species  Arabidopsis thaliana,  as illustrative of plants in general including crops. While the specifics of the examples that follow are provided to fully enable those skilled in the art to understand and practice this invention, to provide the best mode for practicing this invention, and to supply a thorough written description of the invention, the invention should not be construed as being limited to the specifics as outlined in these examples. 
     Example 1  
     A Specific Spectrum of Plant miRNAs is Up-Regulated by flg-22 Peptide 
     A set of primary miRNA transcripts was identified using total RNA fractions isolated from naïve or flg-22-treated plants. flg-22 Is a peptide eliciting a response to pathogen-associated molecular patterns (PAMP). Reverse complements of 60 nt long sequences located upstream of predicted and validated pre-miRNA stem loops were spotted onto an array and used as probes to profile primary miRNA transcripts upon flg-22 treatment. 
     As noted in the Background above, miRNA is generated initially from primary RNA (pri-miRNA) transcripts which are subsequently cleaved to pre-miRNA transcripts from which miRNA transcripts are formed. Because the pri-miRNA transcripts are characterized by a stem loop structure, as shown in  FIG. 5 , identification of the stem loops in predicted RNA structures permitted identification of putative locations for pri-miRNA locations in the genome. 
     The method described above was applied to wildtype plants as well as to various silencing mutants, including dcl4-1 and dcl1-9. 
     We identified 68 pri-miRNAs, that are significantly up-regulated upon flg-22 treatment in at least one genetic background (either Col-0, dcl1-9 or dcl4-1 backgrounds). Some of these primary miRNA transcripts have not yet been described in other reports indicating that many miRNAs are specifically expressed upon biotic stress treatments but not in standard growth conditions. Most of these precursors give rise to extensive fold-back structures. 
     The sequences of the 68 flg-22-induced miRNA precursors are shown in Table 2. The sequences highlighted in bold are the predicted mature miRNA sequences. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
             
            
               
                 &gt;mirspot730 
               
               
                 TTAGATCATCATCCATGGCACTGACGCCGTTCACGGCAACTGCCGTAGAC 
               
               
                 GTTGTTGTTGCCGTG AACGGCGTGAGTGCCGTAGA TTATTGGCTTAT 
               
               
                   
               
               
                 &gt;mirspoT107/997/998 
               
               
                 TCTCGCTAGAGCTCTTCTCTCCCGGCTGTCTCCTGCTCCTGCCTAAG CGA   
               
               
                   TGGCCTGGAGAGTGCTCT AGTGGTG 
               
               
                   
               
               
                 &gt;mirspot156 
               
               
                 GAGTGATAGCCA TGGCATGGAAGAAAGTGAGA TTTGCCTCAATCGATCGT 
               
               
                 GAATCAAAACCTTTATGATTATCACTGCAAGCTTTACCTTCTTCTTAGCC 
               
               
                 ATGATTATCACTG 
               
               
                   
               
               
                 &gt;miRspoT193 
               
               
                 ATCAAGTGTGGGG TGTCGAGAGTCTTTAGATTTGG TGTGAATAATCTGAC 
               
               
                 AATTTGGATTTGAACTCTGCTTTGACATCCTGACATTAGAA 
               
               
                   
               
               
                 &gt;miRspoT894 (mi398c) = miRspot4 = 895 = 402 = 896 
               
               
                 TGGATCTCGACAGGGTTGATATGAGAACACACGAGCAATCAACGGCTATA 
               
               
                 ACGACGCTACGTCATTGTTACAGCTCTCGTTTCA TGTGTTCTCAGGTCAC   
               
               
                   CCCTG CTGAGCTCTTT 
               
               
                   
               
               
                 &gt;AtmiR398a 
               
               
                 TTCAAAGGAGTGGCATGTGAACACATATCCTATGGTTTCTTCAAATTTCC 
               
               
                 ATTGAAACCATTGAGTTT TGTGTTCTCAGGTCACCCCTT TGAATCT 
               
               
                   
               
               
                 &gt;miRspot701/703/704/706/709 
               
               
                 GTCACTGGACCGCAAGAGCATTGATAGGACTCACTCCATCTCCA ATGTCT   
               
               
                   CATGAGGGTCCATGA C 
               
               
                   
               
               
                 &gt;mirspot711/712/713/325 
               
               
                 CTGTCACTGGACCGCAAGAACATTGATAGGGCACACTCCATCTCTA ATGT   
               
               
                   CTCATGAGGGTCAATGACA C 
               
               
                   
               
               
                 &gt;mirspot326/715 
               
               
                 CTGGACCGCAAGAGCATTGATAGGGGTCACTCCATCTCCA ATGTCTCATG   
               
               
                 
                   ATGCTCCATGA 
                 
               
               
                   
               
               
                 &gt;AtmiR395f 
               
               
                 ATGTCCCCTTGAGTTCCCTTAAACGCTTCATTGTTCATACTTTGTTATCA 
               
               
                 TCTATCGATCGATCAATCAATCTGATGAACA CTGAAGTGTTTGGGGGGAC   
               
               
                   TC TAGGTGACATC 
               
               
                   
               
               
                 &gt;mirspot108 
               
               
                 TCGGGTTCT CGGGTCCGGTTCAATTCCGG TTTTTGACCCGAACCTGTTTC 
               
               
                 CGTCTTCTTCTCAACGGTTATGCTTCTGAAGTGATATCACCACTCTCTCT 
               
               
                 CGTCTGAACCTGAATTTTCAACCCGACCCGACTCCAACTT 
               
               
                   
               
               
                 &gt;mirspot110/1002/460/miR160c 
               
               
                 CGTTA TGCCTGGCTCCCTGTATGCCA CGAGTGGATACCGATTTTGGTTTT 
               
               
                 AAAATCGGCTGCCGGTGGCGTACAAGGAGTCAAGCATGACCAG 
               
               
                   
               
               
                 &gt;AtmiR393a (miRspot90) 
               
               
                 GCAACTAGAGGAAGGA TCCAAAGGGATCGCATTGATC CTAATTAAGGTGA 
               
               
                 ATTCTCCCCATATTTTCTTTATAATTGGCAAATAAATCACAAAAATTTGC 
               
               
                 TTGGTTTTGGATCATGCTATCTCTTTGGATTCATCCTTCGGTAGCT 
               
               
                   
               
               
                 &gt;miRspot255/mirspot1102 
               
               
                 GATGTGTCTATATCTTTCTCTATCCCCCACTCCAATCAATTTCAAGTTAT 
               
               
                 TATTAAATTATCTTGATTTGGTAAGAGTTAGTTTTGTAAAGTACGTAAAA 
               
               
                 TTTGAAAAACAATTAATTAAAAAATGAAGG TTGGGTGGGGAAGAGGCAGA   
               
               
                 TATGAACACGTAGTGAGGATA 
               
               
                   
               
               
                 &gt;miRspot142/1016/1018 
               
               
                 AAAGTTGTTCGTTTGCCTGTCGCTGGTTCAACGACCAAAAGTAGCGACCA 
               
               
                 GCGACCGCAATTTTTGATCGCTGAAATTTTTAGCGATCAGTCGCTGGTTT 
               
               
                 CAGCGATTAGTCGCT GCTTTTGGTCGCTGAATCCA GCGACATGCAAACGA 
               
               
                 ACAAC 
               
               
                   
               
               
                 &gt;mirspot35/23 (miR405) 
               
               
                 AATTAAATAAGTTATGGGTTGACCCAACCTATTTAACATA ATGAGTTGGG   
               
               
                   TCAACCCATAACT CATTTAATTT 
               
               
                   
               
               
                 &gt;mirspot292 
               
               
                 ATCTATAGCAGCAAAGCTTTTTTGTCATGAGAAGAAGAAGAAGAATAAGA 
               
               
                 GGTCAAAGAAGATCTCTATTCATGATGCTCCTTCTGAAGCTT TGAGAAAG   
               
               
                   CATTTTGTCGCAT ATGGGTTT 
               
               
                 &gt;miRspot300 
               
               
                 CCTATGTCTCCATACATACACATTCTCTTCAAAAC TCATTTCTTCGTCCG   
               
               
                   GTCCCC TCTTTAAATAGCGCTTCTCTCCCTTCATTCATACATACATTG 
               
               
                 ATCACATCCATGAAGAAGGAGAGGAAGAGACAAGTACAGGAGGAGGAGGA 
               
               
                 GGAGAAGGAGGTGGAGGTTGTTGTGGTGGATGCGTTGA 
               
               
                   
               
               
                 &gt;miRspot495/496/497 (At-miR396b) 
               
               
                 ATCCTGGTCATACTT TTCCACAGCTTTCTTGAACTT TCTTTTTCATTTCC 
               
               
                 ATTGTTTTTTTCTTAAACAAAAGTAAGAAGAAAAAAAACTTTAAGATTAA 
               
               
                 GCATTTTGGAAGCTCAAGAAAGCTGTGGGAAAACATGACAATTCAGGGTT 
               
               
                   
               
               
                 &gt;miRspot73/miR169a/384 
               
               
                 CAACGGAGTAGAATTGCATGAAGTGGAGTAGAGTATAAT GCAGCCAAGGA   
               
               
                   TGACTTGCCG GAACGTTGTTAACCATGCATATGAATAATGTGATGATTAA 
               
               
                 TTATGTGATGAACATATTTCTGGCAAGTTGTCCTTCGGCTACATTTTGCT 
               
               
                 CTCTTCTTCTCATGCAAACTTTCCTTG 
               
               
                   
               
               
                 &gt;miRspot375 
               
               
                 CCAAAAGTTGTTTGTTTGCCTATCGCTGATTCAGCGACAAAAATTAGCGA 
               
               
                 CAGTCGCCAGCGACTGCAATTTTTAGTCGCTGAAATTTTTAGCGATCAGT 
               
               
                 CGTTGGTTTCAGCGATTAGTTGCTGCTTTTGGT CGCTGGATCCAGCGACA   
               
               
                   TGC AAACGAACAACTTTG 
               
               
                   
               
               
                 &gt;miRspot383/miR419 
               
               
                 GAAA TTATGAATGCTGAGGATGTTG TTATTACGAGCAATGAGATGTCTTT 
               
               
                 TTTTAAAAAAAAAAATTTGGTTGCTTGCTTGCAAGAGGACATCTTAGCAT 
               
               
                 CAAATTTG 
               
               
                   
               
               
                 &gt;miRspot1213 
               
               
                 TTTTTTTCTTAGCTTTCCAATCTCTGCCTTTTCTCTGGTCTCTATATCGT 
               
               
                 CGTTTTTGCTACATTTGATTGGGAGTAGTAAAGATGAAGAGACA GATCGG   
               
               
                   ATCGGAGGAAGAGAG GAAGAAGAGAGAAATGGT 
               
               
                   
               
               
                 &gt;miRspot658/275 
               
               
                 CGCCTTCTTCCTTCCCTAGTCATTCACTCTTCTCTAACTTCGCTTTTTTT 
               
               
                 TTGGAGAGCAAAGGTGATGATGAATGCAGAGGAAGATAGT 
               
               
                   
               
               
                 &gt;miRspot441 
               
               
                 TTC GTTTGCTTGTCGCTGGCGACT GAAACCAGCGACAGCGACCAAAAGTT 
               
               
                 GTTCGTTTGCCTGTCGCTGGTTCAGCGACCAAAACTAGTGACAGTCGCCA 
               
               
                 GCGACCAGCGACCGCA 
               
               
                   
               
               
                 &gt;miRspot316 
               
               
                 TAATTTATTTGAGGGGAGAAATATT TGACACGGAAGCATAGCTCCATA TC 
               
               
                 CTTCAATGGAGGTGTGGTCCTTCAACAAAAATACCCCCCTCTTGAAACTC 
               
               
                 TGTTTCACCACACCTCCATTGAAGGACCTGAAGCTATGCTTCCTTGTCAT 
               
               
                 ATTCCTTACCATCAAATAAATGCT 
               
               
                   
               
               
                 &gt;miRspot64 
               
               
                 TTTTAGAGGTGAATCTATTTTAGAGGCATTGTGCTCCAATGGTCACTTCT 
               
               
                 AAAATAGAGTTTCCTCAAAAATAGAGGAAAAAATAGAGATGAATTGTAGA 
               
               
                 GATCTCTATTTATAGAGACAAAAAGTAAATATCTCTATTTTTTCTCTATT 
               
               
                 ATAGAGGAAACTCTATTTTAGAGGTGATCATTGGAGCACAATCCCTCCAA 
               
               
                 AATAGAATCACCTCTA 
               
               
                   
               
               
                 &gt;miRspot919 
               
               
                 TGCAGAATAAAAATGAATAGACTAGAAACAATGTAACAATGTATTTTGTG 
               
               
                 TGGTATT ttggtcttgttcagttctgtt CCC 
               
               
                   
               
               
                 &gt;miRspot418/941 IGR At1g40143-136 and IGR 
               
               
                 At1g40129-131 
               
               
                 AGGGTTTAGGGTTTAGGGTTTTGGTTTAAGGGTTTAGGGTTAAAAGTT ta   
               
               
                   tggtttagggtttacggtt TTGGGTTTGGGATTTAGGGTATAGGGGTTAG 
               
               
                 GGTAAAGAATTTATGATTTTATGTGTAGGATTGAATATAAAACTAGAACC 
               
               
                 TCAACAAGATACCGAAGAGTGGACCGAACTGTCTCACGACGTTCTAAACC 
               
               
                 CAGCTCA 
               
               
                   
               
               
                 &gt;miRspot948 IGR At1g40118-112 and IGR At1g40135- 
               
               
                 137 
               
               
                 ACTAGATGCTTTGTTTATCATTGAGCATAAGCACTAGAACCGCAACCGTA 
               
               
                 TTCCGGATGCCTAAAGTAGGATTTAGGTTTTAAAGTTTGGGATT tatggt   
               
               
                   ttagggtttaggtt TAAGGGTTTAGGGTTAACAGTTTATGGTTTAGGGTT 
               
               
                 TAGG 
               
               
                   
               
               
                 &gt;miRspot1169 IGR At3g30859-0867 
               
               
                 T ttcagaccaatgaggataggatatga TTATTGGAGTCTCTAACAGGATT 
               
               
                 TACAAGCCAAGGTGAAAATGTAGGAATTACTCGTCCACCGAGTGGGTCTT 
               
               
                 GTACGCCTCGATCATCTGATCCATCATCTGGTCCATC 
               
               
                   
               
               
                 &gt;miRspot142 IGR At1g47370-At1g47380 
               
               
                 AAGTTGTTCGTTTGCCTGTCGCTGGTTCAACGACCAAAAGTAGCGACCAG 
               
               
                 CGACCGCAATTTTTGATCGCTGAAATTTTTAGCGATCAGTCGCTGGTTTC 
               
               
                 AGCGATTAGTCGCT gcttttggtcgctgaatcca GCGACATGCAAACGAA 
               
               
                 CAACTT 
               
               
                   
               
               
                 &gt;miRspot1019 IGR At1g72880-890 
               
               
                 AAAGTTGTTCGTTTGCCTGTCGCTGGTTCAGCGACCAAAAGTAGCGACAG 
               
               
                 TCGCCAGCGATCAGCGACCGCAATTTTTGGTCGCTGAAATTTTTAGCGAT 
               
               
                 CAGTCGCTGGTTTCAGCGATTAGTCGCT gcttttggtcgctggatcca GC 
               
               
                 GACAAGCAAACGAACAACTTA 
               
               
                   
               
               
                 &gt;miRspot1020 IGR At1g10680-690 mirSpot1019 paralog 
               
               
                 AAAAGTTGTTTGTTTGCCTATCGCTGATTCAGCGACAAAAATTAGCGACA 
               
               
                 GTCGCCAGCGACTGCAATTTTTAGTCGCTGAAATTTTTAGCGATCAGTCG 
               
               
                 TTGGTTTCAGCGATTAGTTGCT gcttttggtcgctggatcca GCGACATG 
               
               
                 CAAACGAACAACTTTG 
               
               
                   
               
               
                 &gt;miRspot953 IGR At1g40113-115 941/418 and 948 
               
               
                 paralog 
               
               
                 CCGGATTCCGGAAGCTTAAAAGTATAATTTAGGTTTTAAAGTTTGGTATC 
               
               
                 TATTGTTTAGGGTTTAGGTTTAAGGGTTTAGGGTTCAGAGTT tatggttt   
               
               
                   agggtttacggtt CCGG 
               
               
                   
               
               
                 &gt;miRspot607 IGR At5g23220-23230 
               
               
                 ACTCTTTAAATTGGTAGATTCAAGTTTGATTTCAACAATTCTGGGTGTTG 
               
               
                 CAACGAATTTGATAGAAAATTTGGTAATTTAAAGG 
               
               
                   
               
               
                 &gt;miRspot650 IGR At5g33250-251 
               
               
                 GGTTTGCATTGCATATTTCTAAAACAAAGCAAAAAAAAAACAATGTCCGC 
               
               
                 CAGCTCGGGATCGATCGTTCCCGTTCTAGCAGACGATTTTACTTCGTGGA 
               
               
                 TGAGTTTT ggatcgatcgatcccgaactgg GGAACATTTTTTTTTTTGGC 
               
               
                 TTTGTTTCAGAAATATGCAATGCAAACA 
               
               
                   
               
               
                 &gt;miRspot1018 IGR At1g36990-7000 paralog of miRspot 
               
               
                 1019/1020 
               
               
                 AAGTTGTTCGTTTGCTTGTCGCTGGTTCAGCGATCAAAAGTAGCGACAGT 
               
               
                 CGCCAGGGACCAGCGACCGTAATTTTTTGTCGTTAAAATTTTTAGCGATT 
               
               
                 AGTCGCT gcttttggtcgctgaatcca GCGACATGCAAACGAACAACTT 
               
               
                   
               
               
                 &gt;miRspot1147 IGR At2g07687-784 
               
               
                 TT gggaggatgccggggtgtgc TAGTAAGCAAATGGGAAGTTGATCCGAT 
               
               
                 CTTAAGTAGCCCAGGATCCATCCCAGG 
               
               
                   
               
               
                 &gt;miRspot1204 IGR At5g06250-6260 
               
               
                 AGAATTGAAGATGCATGGAATGGTGTGTGGGAAAGGCAAAGCACCATGAC 
               
               
                 TTCACAAGTTGCGTGAGGGCAAAGTATCTATTTTGGGTGAAACCATTTTG 
               
               
                 CCCTCTCAGCCGTTGGATCTCTTTCTTCCTTCA tcatcattccgtcatcc   
               
               
                   tctt TGTTC 
               
               
                   
               
               
                 &gt;miRspot1208 At1g34844 
               
               
                 AGTTGTGTCTCTTGA gtaggaggacccattggggt TACGGATGATGAGAG 
               
               
                 AGAGATCCATGGTGCATTCCAAACCAGGGTATCAGCTCCAGAACCAATCG 
               
               
                 ATCTTCCTAGTTGGGACTAGCA 
               
               
                   
               
               
                 &gt;miRspot1213 IGR At5g67411-420 
               
               
                 CGAGTCTTTGAGTTGAGTTGAGTCGCCGTCG ggtgaagcgaggttgttga   
               
               
                   g CACCCAAATGATCTGTTGAGCCAACGTGGCGTCGTTTGATTCGATGGCG 
               
               
                 TTTGCGCAATGGAGGAGAAGCTGCTCCATGCAGTTAGCATCACCGCTAAG 
               
               
                 AGATTTG 
               
               
                   
               
               
                 &gt;miRspot199 IGR At1g480090-095 
               
               
                 TCTCTTAACTTTGATGAAACCTAGGCAATTGTCTCTTAGTTAAGAGATAA 
               
               
                   ttggtcttggtttcaccaaatt TAAGAGA 
               
               
                   
               
               
                 &gt;miRspot205 IGR At2g01940-950 
               
               
                 ATCTCTCTCTCTCGTTTTCATCATTTGTGCTAACACGCAGAGAGGTTTGC 
               
               
                 AGATTCTGCAGC tatgtttgtcacataaagagagg TGGAGAGAGAGAGAA 
               
               
                   
               
               
                 &gt;miRspot258 At4g13900 pseudogene 
               
               
                 GAACTATCCTGGGTTTGAATCTGAGTGGTTTGTGGTATTGGACCTTCAAG 
               
               
                 CCTGTTGTAAGAGAAGTTCATCCGCGCTAGAAATGTGAGTTCCCCGAGCT 
               
               
                 CTCCTGGGATACTGCCGGATAATCTGTTTTGAGATAGATCCAATGA ttgg   
               
               
                   agattgctcaagtttgat AGAGATGGTGG 
               
               
                   
               
               
                 &gt;miRspot298 IGR At1g01470-80 
               
               
                 AGGAGGATTTGAGTTTTTGACATTCAGACGATAAAAATTATGAACTAGGT 
               
               
                 CTAGTCACGTGGTCGACGCGTGAGAGTTTCCGGCGTGAACTGCAAGTAAA 
               
               
                 AT cacgtagagcatgtgattga CTTGACCAAAGAGTCCAAACCCACCA 
               
               
                   
               
               
                 &gt;miRspot315 IGR At2g24780-790 
               
               
                 GGGACTAAATCCGTTATCCGCGGGTATTCGAATCCGGATCCGTGATCCGA 
               
               
                 TCCGGAAAACCGAATAATTAGGTGCGACGGATCCGGA tacgagtccggcg   
               
               
                   gatctgg ATACGAGTCCG 
               
               
                   
               
               
                 &gt;miRspot1174 IGR At3g10113-116 
               
               
                 GTAGTCCGTTTGTTGTCACTTTGGTTCGTCGC gggttcgtagttttgaga   
               
               
                   gat ATCTTCGAGCTATCCCCCTACCTGGCGCGCCAACTGTTGATGCACGA 
               
               
                 ATCACACAAGTACGAAAATGGGATCTCTAGGGAAGGAAGAAGAATCTTTC 
               
               
                 TATTAATGACGAGCCCGCGACTTAGGCGAATTGGACGGATTAC 
               
               
                   
               
               
                 &gt;miRspot352 IGR At4g06613-06614 
               
               
                 TTTGGTGGACTATTTCACTGGGAAGCATTTGATTGTATC ccccaatgttg   
               
               
                   agcatttggtg GTGTTCGCCAATGTTGTGCATTTGGTGGTGTTCCCCAAT 
               
               
                 GTTGAACATTTGGTGGTGTGCCCCATTGGTGGTGTTTCCTAGGCCTGAGA 
               
               
                 TTTGTGTCCGACCGGT 
               
               
                   
               
               
                 &gt;miRspot369 IGR At5g16470-560 
               
               
                 CATATGATTGTTCGGGAACTTTACAGGCTTCTGTTAAATCTCTGTCTCTG 
               
               
                 ATTAGGCATGTTTGGTAAGCGTATCTTTTGTTTGAAGCCGTGGGGATTTG 
               
               
                   aggaagagtgaaagtttctgc AACTCATGTT 
               
               
                   
               
               
                 &gt;miRspot582 IGR At1g06550-560 
               
               
                 TAGATGGGCCTTGGGTTGCAAAGAA taagcccatatcattcagagc TTTA 
               
               
                 ATGACAGATGGGCCTTGGGTTGCAATGAATAAGCCCATCACATTCAGAGC 
               
               
                 TTTAATGGTATATGGGCCTTAGGTTGCAAAGAATAAGTCCATCA 
               
               
                   
               
               
                 &gt;miRspot155 IGR At4g11130-40 
               
               
                 GTGATGATAGGAGCAGAAGAAAGTAAGAATTGCGTTGATCAGAAAATCAA 
               
               
                 GATATCCAACTTGTGGAGGTTTTGATTCACGATGCAATTCTCACCTTCTT 
               
               
                 TCATGCCATGACCATCAC 
               
               
                   
               
               
                 &gt;miRspot194 (At-miR404) 
               
               
                 TCGAAACGAACACAAAACCTGCGGTTGCGACAGCGGCTGCGGCAACGTTG 
               
               
                 GCGGCGACGAAACGAACAACAACCTGCGGCAG tgttaccgttgccgctgc   
               
               
                   cgc AACCGCAGCCGCTGCCGC 
               
               
                   
               
               
                 &gt;miRspot1047 (At-miR404) 
               
               
                 TCGAAACGAACACAAAACCTGCGGTTGCGACAGCGGCTGCGGCAACGTTG 
               
               
                 GCGGCGACGAAACGAACAACAACCTGCGGCAG tgttaccgttgccgctgc   
               
               
                   cgc AACCGCAGCCGCTGCCGC 
               
               
                   
               
               
                 &gt;miRspot29 (At-miR405a) 
               
               
                 TCAAAATGGCTAACCCAACTCAACTCAACTCATAATCAAATGAGTTTAGG 
               
               
                 GTTAAATGAGTTATGGGTTGACCCAACCCATTTAACAAAATGAGTTGGGT 
               
               
                 CAACCCATAACTCATTTAATTTGATG 
               
               
                   
               
               
                 &gt;miRspot43 (At-miR405a) 
               
               
                 TCATGGGTAACCCAACTCAACTCAACTCATAATCAAATGAGTTTAGGGTT 
               
               
                 AAATGAGTTATGGGTTGATCCAACCCATTTAACAAAATGAGTTGGGTCAA 
               
               
                 CCCATAACTCATTTAATTTG 
               
               
                   
               
               
                 &gt;miRspot506 (At-miR416) 
               
               
                 CGAAACTGAACCCGGTTTGTACGTACGGACCGCGTCGTTGGAATCCAAAA 
               
               
                 GAACCG ggttcgtacgtacgctgttca TCG 
               
               
                   
               
               
                 &gt;miRspot854 (At-miR166f) 
               
               
                 AAGTTCAGGTGAATGATGCCTGGCTCGAGACCATTCAATCTCATGATCTC 
               
               
                 ATGATTATAACGATGATGATGATGATG tcgGaccaggcttcattcccc TC 
               
               
                 AA 
               
               
                   
               
               
                 &gt;miRspot76/92/74 (At-miR169d) 
               
               
                 GTATCATAGAGTCTTGCATGGAAAAATTAAAGAATGAGATTGAGCCAAGG 
               
               
                 ATGACTTGCCGATGTTATCAACAAATCTTAACTGATTTTGGTGTCCGGCA 
               
               
                 AGTTGACCTTGGCTCTGTTTCCTTCTTTTCTTTTCAATGTCAAACTCTAG 
               
               
                 ATAT 
               
               
                   
               
               
                 &gt;miRspot512 (At-miR172e) 
               
               
                 GTAGTCGCAGATGCAGCACCATTAAGATTCACAAGAGATGTGGTTCCCTT 
               
               
                 TGCTTTCGCCTCTCGATCCGCAGAAAAGGGTTCCTTATCGAGTG ggaatc   
               
               
                   ttgatgatgctgcat CAGCAAATAC 
               
               
                   
               
               
                 &gt;miRspot83 (A6-miR394b) 
               
               
                 CTTACAGAGATCT ttggcattctgtccacctcc TCTCTCTATATTTATGT 
               
               
                 GTAATAAGTGTACGTATCTACGGTGTGTTTCGTAAGAGGAGGTGGGCATA 
               
               
                 CTGCCAATAGAGATCTGTTAG 
               
               
                   
               
               
                 &gt;miRspot182 (A6-miR395e) 
               
               
                 ATGTTTTCTAGAGTTCCTCTGAGCACTTCATTGGAGATACAATTTTTTAT 
               
               
                 AAAATAGTTTTCTA ctgaagtgtttgggggaactc CCGGGCTGAT 
               
               
                   
               
               
                 &gt;miRspot3 (At-miR397b) 
               
               
                 TAGAAAAACATAATTGAATGCAACGCTGATATATACTTCTTTAATTAATT 
               
               
                 CAACAATGGAATAAAATAAGTAAAATTACATCAACGATGCACTCAATGAT 
               
               
                 GTTCATTCA 
               
               
                   
               
               
                 &gt;miRspot5/4 (At-miR398b) 
               
               
                 TGGATCTCGACAGGGTTGATATGAGAACACACGAGTAATCAACGGCTGTA 
               
               
                 ATGACGCTACGTCATTGTTACAGCTCTCGTTTTCA tgtgttctcaggtca   
               
               
                   cccctg CTGAGCTCTT 
               
               
                   
               
               
                 &gt;miRspot18/788 (At-miR405a) 
               
               
                 TAAATGGTTAACCCATTTAACAATTCAACCCATCAAATGAAATGAGTTAT 
               
               
                 GGGTTAGACCCAACTCATTTAACAAA atgagttgggtctaacccataact   
               
               
                 CATTTAATTATAAACTCATTTGATTATGAGTTGGGTTGGGTTGGGTTACC 
               
               
                 CATTTTGA 
               
               
                   
               
               
                 &gt;miRspot383 (At-miR419) 
               
               
                 AAAttatgaatgctgaggatgttgTTATTACGAGCAATGAGATGTCTTTT 
               
               
                 TTTAAAAAAAAAAATTTGGTTGCTTGCTTGCAAGAGGACATCTTAGCATC 
               
               
                 AAATTT 
               
               
                   
               
               
                 &gt;miRspot690 (At-miR836) 
               
               
                 ATTTCGTTTTTAAAAGTCTCCACGCATCAAAGGAAACACAGGAAAACAGA 
               
               
                 GCATTTATTTGATGGTAAGGAATATGACAAGGAAGCATAGCTTCAGGTCC 
               
               
                 TTCAATGGAGGTGTGGTGAAACAGAGTTTCAAGAGGGGGGTATTTTTGTT 
               
               
                 GAAGGACCACACCTCCATTGAAGGATATGGAGCTATGCTTCCGTGTCAAA 
               
               
                 TATTTCTCCCCTCAAATAAATTATATCTCTTCTAGTGTTTCCTTCGAT 
               
               
                   
               
               
                 &gt;miRspot1039 (At-miR842) 
               
               
                 GAGCTTCACTTTTCAATTGTCCATATTTGTTGACCTAAGAAAACATAAGT 
               
               
                 GGGATGACGGATCTGACCATGATGGTGTTTCGATCCCTGGACAATAACTA 
               
               
                 CATCATACATAAATTTCTGCAA caccatcatggtcggattca TCATCCCG 
               
               
                 CTTATAGCCTCTCTTTTCGAAAATGTTTCTGTCACCCTGAACGGTACTG 
               
               
                   
               
            
           
         
       
     
     Among the flg-22-induced pri-miRNAs, many corresponding mature miRNA sequences are conserved in maize and rice. The afore-mentioned method can also be used to identify the full spectrum of miRNAs elicited by general elicitors exhibiting pathogen-associated molecular patterns (PAMPs) such as flg-22. 
     miRNA primary transcripts suppressed by suppressor proteins such as those associated with virulent bacteria can be identified by challenging wildtype Col-0 leaves with Pst DC3000 hrcC (a Pst DC3000 bacterium that cannot inject suppressor proteins into the host cells) and virulent Pst DC3000 (which can) for 6 hours and by further selecting miRNA primary transcripts (using the above method) that are up-regulated by Pst DC3000 hrcC but not by virulent Pst DC3000 as described below. Because these pri-miRNAs are transcriptionally repressed by Pst DC3000 TTS proteins, they likely act as key components of the antibacterial defense response. The afore-mentioned method can also be used to identify the full spectrum of miRNA transcripts that are up-regulated by unrelated biotrophic and necrotrophic pathogens as well as abiotic stresses. 
     Example 2 
     Plant Mutants with Compromised miRNA Accumulation are More Susceptible to Virulent Pathogens 
     We demonstrated the involvement of the miRNA pathway in plant disease resistance, by challenging  Arabidopsis  hen1-1 and dcl1-9 mutants with a virulent Powdery mildew  Erysiphe cichoracearum  or with virulent Pst DC3000. We found that hen1-1 is hyper-susceptible to both the fungus and the bacterium, and that dcl1-9 displays enhanced susceptibility to  Erysiphe cichoracearum  and enhanced disease symptoms upon virulent Pst DC3000 infection ( FIGS. 6A , B, C). In  FIG. 6A , both hen1-1 and dcl1-9 are hyper-susceptible to Powdery mildew. Six week-old La-er, hen1-1 and dcl1-9 plants were infected with  E. cichoracearum  (UEA isolate) spores and fungal growth assessed visually 10 dpi (upper panel). Fungal growth and sporulation were also assessed microscopically after leaf staining with trypan blue at 2-3 dpi (bottom panels). 
     In  FIG. 6B , both hen1-1 and dcl1-9 display enhanced disease symptoms upon Pst DC3000 infection. La-er, hen1-1 and dcl1-9 were challenged with Pst DC3000 of 10 5  colony forming units (cfu/ml) and bacterial disease symptoms assessed visually 3 dpi. 
     As shown in  FIG. 6C , Hen1-1 displays higher Pst DC3000 titers. La-er, hen1-1 and dcl1-9 were treated as in  FIG. 6B  and bacterial growth measured 4 dpi. 
     Constitutively or conditionally enhancing the activity/expression of components involved in miRNA biogenesis or activity, such as DCL1, AGO1, SERRATE, HEN1 using the methods above increases resistance to a broad spectrum of pathogens is achieved in a variety of plants, including crop species. The method disclosed thus allows inducible, enhanced resistance, which is desirable because it is not, or is less, detrimental to plant development and physiology in non-infected conditions. 
       Arabidopsis  transgenic lines carrying 1.5 Kb upstream regions from genes involved in miRNA biogenesis and/or activity (e.g., DCL1) are fused to a reporter sequence. These transgenic lines are used to screen for chemical compounds that trigger up-regulation of reporter mRNA or protein 
     This is achieved by monitoring mRNA levels (using methods known by those skilled in the art such as Northern analysis, semi-quantitative RT-PCR analysis or quantitative RT-PCR analysis) after exposure of these transgenic lines to a library of chemical agents. Molecules that induce reporter mRNA (or protein) levels are further used to confer antibacterial and antifungal resistance in a variety of plant species including crops. 
     Example 3  
     miRNA-Deficient Plants are Susceptible to Non-Virulent Bacteria 
     The induction, by flg-22, of a subset of patho-miRs (see Example 1) suggests that the miRNA pathway plays a pivotal role in basal resistance to pathogens. We challenged the dcl1-9 and hen1-1 mutants of  Arabidopsis  with Pst DC3000 deficient in type three secreted (TTS) protein. Pst DC3000 hrcC, a strain which in wildtype  Arabidopsis  is unable to mount an effective infection, was injected into these mutants. Both mutants exhibited full disease symptoms, resembling the phenotype induced by virulent bacteria on wildtype  Arabidopsis  ( FIGS. 7A ,  7 B). 
     In  FIG. 7A , both hen1-1 and dcl1-9, but not siRNA-deficient mutants, are compromised in basal resistance. Col-0, dcl2-1, dcl3-1, dcl4-2, dcl3-1/dcl4-2, dcl2-1/dcl4-1, dcl2-1/dcl3-1/dcl4-2, rdr6-1, rdr2-1, La-er, hen1-1 and dcl1-9 were challenged with hrcC −  Pst DC3000 at 10 6  colony forming units (cfu/ml) concentration and bacterial titers measured 6 dpi. 
     In  FIG. 7B , both hen1-1 and dcl1-9 displayed disease symptoms when inoculated with hrcC −  Pst DC3000 strain. Inoculations were performed as in  FIG. 7A . and bacterial disease symptoms were assessed visually. 
     We found that induction of WRKY30 (a well-characterized basal defense marker gene) was compromised in both hen1-1 and dcl1-9 mutants challenged with Pst DC3000 HrcC −  ( FIG. 7C ), indicating a loss of basal defense response in those plants. In  FIG. 7C , the induction of the WRKY30 gene was impaired in both hen1-1 and dcl1-9 treated with hrcC Pst DC3000 strain. Inoculations were performed as in  FIG. 7A  and WRKY30 levels were analyzed by quantitative RT-PCR (qRT-PCR) on 12 hours post inoculated samples. 
     Likewise, both hen1-1 and dcl1-9 sustained infection from the non-host bacterial pathogen  Pseudomonas syringae  pv.  phaseolicola  (NPS3121) ( FIG. 7D ), which is normally virulent on beans, but not on  Arabidopsis . In  FIG. 7D , both hen1-1 and dcl1-9 are compromised in non-host resistance. La-er, hen1-1 and dcl1-9 were challenged with  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121) at 10 6  colony forming units (cfu/ml) concentration and bacterial titers measured 4 dpi. 
     WRKY30 induction was also impaired on challenge with  Pseudomonas syringae  pv.  phaseolicola  (NPS3121) ( FIG. 7E ). In  FIG. 7E , the induction of the WRKY30 gene was impaired in both hen1-1 and dcl1-9 treated with  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121). Inoculations were performed as in  FIG. 7D  and WRKY30 levels were analyzed by quantitative RT-PCR (qRT-PCR) on 12 hours post inoculated samples. Importantly, similar results were obtained with the non-pathogenic  Pseudomonas fluorescence  (data not shown). 
     We also found that TuMV virus (that encodes the viral suppressor of silencing P1-HcPro) suppresses basal resistance to non-virulent bacteria Pst DC3000 hrcC and  Pseudomonas syringae  pv.  phaseolicola  (NPS3121) suggesting that suppression of the RNA-silencing machinery might play a pivotal role in polymicrobial infection. 
       FIG. 8A  shows TuMV infection rescues bacterial disease symptoms of Pst DC3000 hrcC mutant. Wildtype Col-0 plants were treated with 10 6  cfu/ml of Pst DC3000 hrcC for 6 days (left panel) or SAP inoculated with TuMV for 7 days and further treated with 10 6  cfu/ml Pto DC3000 hrcC for another 6 days (right panel). 
       FIG. 8B  shows TuMV infection rescues bacterial growth of Pst DC3000 hrcC mutant. Wildtype Col-0 plants were treated as in  FIG. 8A  and bacterial titers were measured 6 dpi. 
       FIG. 8C  shows TuMV infection rescues bacterial disease symptoms of the non-host  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121). Wildtype Col-0 plants were treated with 10 6  cfu/ml of  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121) for 4 days (left panel) or SAP inoculated with TuMV for 7 days and further treated with 10 6  cfu/ml  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121) for another 4 days (right panel). 
       FIG. 8D  shows TuMV infection rescues bacterial growth of  Pseudomonas syrinage  pv.  phaseolicola  (NPS3121). Wildtype Col-0 plants were treated as in  FIG. 8C  and bacterial titers were measured 4 dpi. 
     Example 4 
     Bacterial Silencing Proteins Suppress miRNA Identification of Bacterial Silencing Suppressors (Bss) 
     We also conclude from the results of Example 3 that pathogens, such as viruses and virulent bacteria, must have evolved strategies to suppress components of the silencing machinery. 
     To test this hypothesis we first investigated whether virulent Pst DC3000 could suppress the transcription of PAMP-responsive miRNAs. We measured the impact of Pto DC3000 effector (TTS protein) delivery on pri-miRNA expression. Virulent Pto DC3000, or its non-virulent counterpart Pto DC3000 hrcC, were inoculated onto  Arabidopsis  Col-0 plants and the levels of several pri-miRNAs induced by Pto DC3000 hrcC (referred to as PAMP-responsive) were then monitored over a 6 hour timecourse. In virulent Pto DC3000-treated plants, induction of the PAMP-responsive pri-miR393a/b and pri-miR396b was significantly suppressed at 6 hour post inoculation (hpi), as was the induction of the basal defense markers WRKY30 and Flagellin Receptor Kinase 1 (FRK1) used as internal controls ( FIG. 9A ).  FIG. 9A  shows induction of PAMP-responsive miRNAs is suppressed by virulent Pst DC3000. Wildtype Col-0 leaves were inoculated with Pst DC3000 hrcC at 2×10 7  cfu/ml concentration and the levels of PAMP-responsive primary miRNA transcripts were monitored by semi-quantitative RT-PCR over a 6 hours timecourse. Pri-miR166a was used as a negative control in this experiment. By contrast, the levels of the PAMP-insensitive pri-miR166 and pri-miR173 remained unchanged. 
     We then used the previously described miR393a-p::eGFP and miR393b-p::eGFP transgenic lines, which report miR393 transcriptional activity. At 6 hpi, Pto DC3000 hrcC caused an increase in eGFP mRNA levels in both transgenic lines, indicating the presence of PAMP-responsive elements upstream of MIR393a and MIR393b ( FIG. 9B ).  FIG. 9B  shows Pst DC3000 suppresses miR393a/b induction at the transcriptional level. Transgenic lines expressing either miR393a-p::eGFP or miR393b-p::eGFP transgenes were treated as in  FIG. 9A  for 6 hours and the levels of eGFP transcript were monitored by RT-qPCR. The receptor-like kinase FRK1 was used as a positive control in this experiment. However, this induction was compromised by Pto DC3000, as was the induction of the FRK1 control. Because the two bacteria differ in their ability to deliver TTS effectors into host cells, these results suggest that some injected bacterial proteins suppress specifically the transcriptional activation of pri-miR393a/b and perhaps of other PAMP-responsive miRNAs. 
     To identify such bacterial proteins, we delivered individual Pst DC3000 effectors (driven by the strong 35S promoter) in efr1 mutant leaves, a mutant in which  Agrobacterium  transient assay is facilitated, and monitored PAMP-responsive pri-miRNA levels. Delivery of AvrPtoB significantly reduces pri-miR393a/b and pri-miR396b levels with no significant effect on the PAMP-insensitive pri-miR166a ( FIG. 9C ).  FIG. 9C  shows AvrPtoB suppresses PAMP-responsive pri-miRNAs accumulation. Efr1 mutant plants were Agro-infiltrated at a OD of 0.4 with 35S::GUSintron, 35S:AvrPtoB and pri-miRNA levels monitored by semi-quantitative RT-PCR analysis. Thus, AvrPtoB potentially inhibits PAMP-responsive miRNA expression at the transcriptional level. 
     We further tested whether the overall miRNA pathway could be affected by Pst DC3000 effectors. For this purpose, we generated Col-0 transgenic plants expressing a sensor construct depicting miR171 activity. miR171, when expressed, silences a GFP reporter construct. Such transgenic plants constitutively express a GFP reporter gene in which a miR171 target site is added in the 3′UTR of the GFP gene ( FIG. 10A ). The miRNA target site was modified in order to avoid RDR6-triggered transgene transitivity as previously described. Because miR171 is well expressed in leaves, no GFP signal can be detected in standard growth conditions. Leaves from the miR171 sensor lines were challenged with the virulent Pst DC3000 and the non-virulent TTS-deficient Pst DC3000 hrcC, and GFP levels monitored over a timecourse experiment. We found that virulent Pst DC3000, but not Pst DC3000 hrcC, restored GFP gene expression at 24 hours post inoculation ( FIG. 10A ).  FIG. 10A  shows how Pst DC3000 restores GFP expression in the miR171 sensor lines. Schematic representation of the miR171 sensor construct (upper panel). Leaves from miR171 sensor lines were inoculated with mock (MgCl 2 ) (left panel), hrcC −  Pst DC3000 (middle panel) and virulent Pst DC3000 (right panel). Pictures were taken 30 hours post inoculation (hpi). A concentration of 10 5  colony forming units (cfu/ml) was used in this assay. This result indicates that some TTS proteins act as suppressors of the miRNA pathway either by inhibiting the miRNA biogenesis and/or the miRNA activity. 
     Example 5 
     Identification of Proteins that Suppress miRNA Pathways 
     To identify such bacterial factors, 23 individual TTS effector genes (driven by the strong 35S promoter) were transiently delivered in  Arabidopsis  efr1 mutant leaves ( FIG. 10B ) ( FIG. 10B  shows accumulation of both miR173 and tas255 is partially impaired in leaves expressing hopN1, H1 and Pto. The 23 constructs were transiently delivered in  Arabidopsis  leaves using  Agrobacterium  transformation and a low molecular weight Northern analysis was performed using oligo probes complementary to miR173 and tas255 small RNAs.) The levels of the endogenous miR173 and Tas255 (which relies on miR173 activity for its biogenesis) were monitored. We found that three of those proteins (the cysteine protease HopN1, HopH1, and AvrPto) caused a significant decrease in the steady-state levels of miR173 and Tas255 ( FIG. 10C ).  FIG. 10C  shows accumulation of miR173 and tas255 is drastically impaired in leaves co-expressing hopN1 and hopH1. A transient delivery of hopN1 and hopH1 overexpressor contructs were performed as described in  FIG. 10B  Northern analysis was done as in  FIG. 10B . Moreover, these effects were additive upon co-delivery of HopN1 and HopH1 indicating that these proteins interfere, directly or indirectly, with distinct host factors involved in RNA-silencing. Both HopN1 wildtype and mutant versions, the latter being altered in the predicted cysteine catalytic triad (C172S, H283A and D299A, rescued GFP expression when delivered in transgenic efr1 plants expressing miR171 sensor construct described above (data not shown). All the HopN1 mutant versions were stable in planta as assayed by Western analysis (data not shown). 
       FIG. 10D  shows HopN1-triggered suppression of RNA-silencing does not require cysteine protease activity. Efr1 mutant plants expressing the miR171 sensor construct were agro-infiltrated (OD=0.4) with 35S::GUSintron, 35S::hopN1-HA, 35S::HopN1 C172S, 35S::HopN1 H283A and the levels of GFP analyzed visually under UV. This indicates that the cysteine protease activity is not required for suppression of RNA-silencing. All together, the identified TTS proteins suppress miRNA biogenesis. They are thus referred to here as ‘Bacterial silencing suppressors’ (Bss). 
     The nucleotide sequences (coding sequences) from the Bss proteins that suppress miRNA biogenesis are as follows: 
     
       
         
           
               
            
               
                 &gt;AvrPtoB coding sequence: 
               
               
                 ATGGCGGGTATCAATAGAGCGGGACCATCGGGCGCTTATTTTGTTGGCCA 
               
               
                   
               
               
                 CACAGACCCCGAGCCAGTATCGGGGCAAGCACACGGATCCGGCAGCGGCG 
               
               
                   
               
               
                 CCAGCTCCTCGAACAGTCCGCAGGTTCAGCCGCGACCCTCGAATACTCCC 
               
               
                   
               
               
                 CCGTCGAACGCGCCCGCACCGCCGCCAACCGGACGTGAGAGGCTTTCACG 
               
               
                   
               
               
                 ATCCACGGCGCTGTCGCGCCAAACCAGGGAGTGGCTGGAGCAGGGTATGC 
               
               
                   
               
               
                 CTACAGCGGAGGATGCCAGCGTGCGTCGTAGGCCACAGGTGACTGCCGAT 
               
               
                   
               
               
                 GCCGCAACGCCGCGTGCAGAGGCAAGACGCACGCCGGAGGCAACTGCCGA 
               
               
                   
               
               
                 TGCCAGCGCACCGCGTAGAGGGGCGGTTGCACACGCCAACAGTATCGTTC 
               
               
                   
               
               
                 AGCAATTGGTCAGTGAGGGCGCTGATATTTCGCATACTCGTAACATGCTC 
               
               
                   
               
               
                 CGCAATGCAATGAATGGCGACGCAGTCGCTTTTTCTCGAGTAGAACAGAA 
               
               
                   
               
               
                 CATATTTCGCCAGCATTTCCCGAACATGCCCATGCATGGAATCAGCCGAG 
               
               
                   
               
               
                 ATTCGGAACTCGCTATCGAGCTCCGTGGGGCGCTTCGTCGAGCGGTTCAC 
               
               
                   
               
               
                 CAACAGGCGGCGTCAGCGCCAGTGAGGTCGCCCACGCCAACACCGGCCAG 
               
               
                   
               
               
                 CCCTGCGGCATCATCATCGGGCAGCAGTCAGCGTTCTTTATTTGGACGGT 
               
               
                   
               
               
                 TTGCCCGTTTGATGGCGCCAAACCAGGGACGGTCGTCGAACACTGCCGCC 
               
               
                   
               
               
                 TCTCAGACGCCGGTCGACAGGAGCCCGCCACGCGTCAACCAAAGACCCAT 
               
               
                   
               
               
                 ACGCGTCGACAGGGCTGCGATGCGTAATCGTGGCAATGACGAGGCGGACG 
               
               
                   
               
               
                 CCGCGCTGCGGGGGTTAGTACAACAGGGGGTCAATTTAGAGCACCTGCGC 
               
               
                   
               
               
                 ACGGCCCTTGAAAGACATGTAATGCAGCGCCTCCCTATCCCCCTCGATAT 
               
               
                   
               
               
                 AGGCAGCGCGTTGCAGAATGTGGGAATTAACCCAAGTATCGACTTGGGGG 
               
               
                   
               
               
                 AAAGCCTTGTGCAACATCCCCTGCTGAATTTGAATGTAGCGTTGAATCGC 
               
               
                   
               
               
                 ATGCTGGGGCTGCGTCCCAGCGCTGAAAGAGCGCCTCGTCCAGCCGTCCC 
               
               
                   
               
               
                 CGTGGCTCCCGCGACCGCCTCCAGGCGACCGGATGGTACGCGTGCAACAC 
               
               
                   
               
               
                 GATTGCGGGTGATGCCGGAGCGGGAGGATTACGAAAATAATGTGGCTTAT 
               
               
                   
               
               
                 GGAGTGCGCTTGCTTAACCTGAACCCGGGGGTGGGGGTAAGGCAGGCTGT 
               
               
                   
               
               
                 TGCGGCCTTTGTAACCGACCGGGCTGAGCGGCCAGCAGTGGTGGCTAATA 
               
               
                   
               
               
                 TCCGGGCAGCCCTGGACCCTATCGCGTCACAATTCAGTCAGCTGCGCACA 
               
               
                   
               
               
                 ATTTCGAAGGCCGATGCTGAATCTGAAGAGCTGGGTTTTAAGGATGCGGC 
               
               
                   
               
               
                 AGATCATCACACGGATGACGTGACGCACTGTCTTTTTGGCGGAGAATTGT 
               
               
                   
               
               
                 CGCTGAGTAATCCGGATCAGCAGGTGATCGGTTTGGCGGGTAATCCGACG 
               
               
                   
               
               
                 GACACGTCGCAGCCTTACAGCCAAGAGGGAAATAAGGACCTGGCGTTCAT 
               
               
                   
               
               
                 GGATATGAAAAAACTTGCCCAATTCCTCGCAGGCAAGCCTGAGCATCCGA 
               
               
                   
               
               
                 TGACCAGAGAAACGCTTAACGCCGAAAATATCGCCAAGTATGCTTTTAGA 
               
               
                   
               
               
                 ATAGTCCCCTGA 
               
               
                   
               
               
                 &gt;HopH1 coding sequence: 
               
               
                 ATGATCACTCCGTCTCGATATCCAGGCATCTATATCGCCCCCCTCAGTAA 
               
               
                   
               
               
                 CGAACCGACAGCAGCTCACACATTTAAAGAACAAGCAGAGGAAGCACTTG 
               
               
                   
               
               
                 ACCATATCAGCGCCGCACCCTCTGGCGATAAGCTATTGCGAAAAATATCC 
               
               
                   
               
               
                 ACTCTTGCCAGTCAAAAAGATAGAAAAGTCACGCTAAAAGAGATTGAAAT 
               
               
                   
               
               
                 AAATAACCAGTGTTATACCGAGCTGTTCTGAGCAGGAGGCAACTGGAAAA 
               
               
                   
               
               
                 GTACGAACCAGAAAACTTTAACGAGAACCGGCACATTGCATCACAGCTAT 
               
               
                   
               
               
                 CACGAAAGGGGACCTTTACCAAAGGTGAAGGAAGCAACGCGATTATTGGC 
               
               
                   
               
               
                 TGGTCACCAGACAAAGCAAGCATACGCTTAAATCAGAATGGCTCACCGTT 
               
               
                   
               
               
                 ACACCTTGGAATGGATAACGACGACAAAATCACGACCCTAGCTCATGAGC 
               
               
                   
               
               
                 TCGTTCATGCTCGACATGTGTTAGGTGGCAGCTCCTTAGCGGATGGCGGA 
               
               
                   
               
               
                 GATCGCTATAATCCACGTACGGGATCTGGCAAAGAGGAACTTAGGGCCGT 
               
               
                   
               
               
                 TGGATTAGATAAGTACCGCTATTCACTTACAAAAAAACCGTCAGAGAACT 
               
               
                   
               
               
                 CCATCCGAGCTGAACACGGCCTGCCTCTGCGCATGAAGTACAGGGCACAT 
               
               
                   
               
               
                 CAATAG 
               
               
                   
               
               
                 &gt;HopN1 coding sequence: 
               
               
                 ATGTATATCCAGCAATCTGGCGCCCAATCAGGGGTTGCCGCTAAGACGCA 
               
               
                   
               
               
                 ACACGATAAGCCCTCGTCATTGTCCGGACTCGCCCCCGGTTCGTCGGATG 
               
               
                   
               
               
                 CGTTCGCCCGTTTTCATCCCGAAAAGGCGGGCGCCTTTGTCCCATTGGAG 
               
               
                   
               
               
                 GGGCATGAAGaGGTCTTTTTCGATGCGCGCTCTTCCTTTTCGTCGGTCGA 
               
               
                   
               
               
                 TGCCGCTGATCTTCCCAGTCCCGAGCAGGTACAACCCCAGCTTCATTCGT 
               
               
                   
               
               
                 TGCGTACCCTGCTACCGGATCTGATGGTCTCTATCGCCTCATTACGTGAC 
               
               
                   
               
               
                 GGCGCCACGCAATACATCAAGACCAGAATCAAGGCTATGGCGGACAACAG 
               
               
                   
               
               
                 CATAGGCGCGACTGCGAACATCGAAGCCAAAAGAAAGATTGCCCAAGAGC 
               
               
                   
               
               
                 ACGGCTGTCAGCTTGTCCACCCGTTTCACCAGAGCAAATTTCTATTTGAA 
               
               
                   
               
               
                 AAAACTATCGATGATAGAGCGTTTGCTGCTGATTATGGCCGCGCGGGTGG 
               
               
                   
               
               
                 CGACGGGCACGCTTGTCTGGGGCTATCAGTAAATTGGTGTCAGAGCCGTG 
               
               
                   
               
               
                 CAAAAGGGCAGTCGGATGAGGCCTTCTTTCACAAACTGGAGGACTATCAG 
               
               
                   
               
               
                 GGCGATGCATTGCTACCCAGGGTAATGGGCTTCCAGCATATCGAGCAGCA 
               
               
                   
               
               
                 GGCCTATTCAAACAAGTTGCAGAACGCAGCACCTATGCTTCTGGACACAC 
               
               
                   
               
               
                 TTCCCAAGTTGGGCATGACACTTGGAAAAGGGCTGGGCAGAGCACAGCAC 
               
               
                   
               
               
                 GCGCACTATGCGGTTGCTCTGGAAAACCTTGATCGCGATCTCAAAGCAGT 
               
               
                   
               
               
                 GTTGCAGCCCGGTAAAGACCAGATGCTTCTGTTTTTGAGTGATAGCCATG 
               
               
                   
               
               
                 CGATGGCTCTGCATCAGGACAGTCAGGGATGTCTGCATTTTTTTGATCCT 
               
               
                   
               
               
                 CTTTTTGGCGTGGTTCAGGCAGACAGCTTCAGCAACATGAGCCATTTTCT 
               
               
                   
               
               
                 TGCTGATGTGTTCAAGCGCGACGTAGGTACGCACTGGCGTGGCACGGAGC 
               
               
                   
               
               
                 AACGTCTGCAACTGAGCGAAATGGTGCCCAGAGCAGACTTTCACTTGCGA 
               
               
                   
               
               
                 TAA 
               
               
                   
               
               
                 &gt;AvrPto coding sequence: 
               
               
                 ATGGGAAATATATGTGTCGGCGGATCCAGGATGGCCCATCAGGTGAACTC 
               
               
                   
               
               
                 CCCAGACCGAGTTAGTAACAACTCGGGTGACGAAGATAACGTAACGTCCA 
               
               
                   
               
               
                 GTCAACTGCTGAGCGTCAGACATCAACTTGCGGAGTCTGCTGGTGTACCA 
               
               
                   
               
               
                 AGAGATCAGCATGAATTTGTTAGTAACCAAGCACCTCAAAGCCTGAGAAA 
               
               
                   
               
               
                 TCGCTACAACAATCTTTACTCACATACGCAAAGAACACTGGATATGGCGG 
               
               
                   
               
               
                 ACATGCAGCATAGGTACATGACGGGAGCGTCAGGAATCAATCCGGGAATG 
               
               
                   
               
               
                 CTGCCACATGAGAATGTGGACGATATGCGTAGCGCTATAACTGATTGGAG 
               
               
                   
               
               
                 TGACATGCGCGAAGCTCTGCAGTACGCAATGGGTATCCATGCCGACATCC 
               
               
                   
               
               
                 CACCGTCTCCAGAGCGATTTGTTGCGACTATGAACCCGAACGGATCAATT 
               
               
                   
               
               
                 CGAATGTCAACACTTTCTCCTAGCCCGTACCGTAACTGGCAATGA 
               
            
           
         
       
     
     The amino acid sequences from the Bss proteins that suppress miRNA biogenesis are as follows: 
     
       
         
           
               
            
               
                 &gt;HopPtoB amino acid sequence: 
               
               
                 MAGINRAGPSGAYFVGHTDPEPVSGQAHGSGSGASSSNSPQVQPRPSNTP 
               
               
                   
               
               
                 PSNAPAPPPTGRERLSRSTALSRQTREWLEQGMPTAEDASVRRRPQVTAD 
               
               
                   
               
               
                 AATPRAEARRTPEATADASAPRRGAVAHANSIVQQLVSEGADISHTRNML 
               
               
                   
               
               
                 RNAMNGDAVAFSRVEQNIFRQHFPNMPMHGISRDSELAIELRGALRRAVH 
               
               
                   
               
               
                 QQAASAPVRSPTPTPASPAASSSGSSQRSLFGRFARLMAPNQGRSSNTAA 
               
               
                   
               
               
                 SQTPVDRSPPRVNQRPIRVDRAAMRNRGNDEADAALRGLVQQGVNLEHLR 
               
               
                   
               
               
                 TALERHVMQRLPIPLDIGSALQNVGINPSIDLGESLVQHPLLNLNVALNR 
               
               
                   
               
               
                 MLGLRPSAERAPRPAVPVAPATASRRPDGTRATRLRVMPEREDYENNVAY 
               
               
                   
               
               
                 GVRLLNLNPGVGVRQAVAAFVTDRAERPAVVANIRAALDPIASQFSQLRT 
               
               
                   
               
               
                 ISKADAESEELGFKDAADHHTDDVTHCLFGGELSLSNPDQQVIGLAGNPT 
               
               
                   
               
               
                 DTSQPYSQEGNKDLAFMDMKKLAQFLAGKPFEPMTRETLNAENIAKYAFR 
               
               
                   
               
               
                 IVP 
               
               
                   
               
               
                 &gt;HopH1 amino acid sequence: 
               
               
                 MITPSRYPGIYIAPLSNEPTAAHTFKEQAEEALDHISAAPSGDKLLRKIS 
               
               
                   
               
               
                 TLASQKDRKVTLKEIEINNQCYTEAVLSRRQLEKYEPENFNENRHIASQL 
               
               
                   
               
               
                 SRKGTFTKGEGSNAIIGWSPDKASTRLNQNGSPLHLGMDNDDKITTLAHE 
               
               
                   
               
               
                 LVHARHVLGGSSLADGGDRYNPRTGSGKEELRAVGLDKYRYSLTKKPSEN 
               
               
                   
               
               
                 SIRAEHGLPLRMKYRAHQ 
               
               
                   
               
               
                 &gt;HopN1 amino acid sequence: 
               
               
                 MYTQQSGAQSGVAAKTQEDKPSSLSGLAPGSSDAFARFHPEKAGAFVPLE 
               
               
                   
               
               
                 GHEEVFFDARSSFSSVDAADLPSPEQVQPQLHSLRTLLPDLMVSIASLRD 
               
               
                   
               
               
                 GATQYIKTRIKAMADNSIGATANIEAKRKIAQEHGCQLVHPFHQSKFLFE 
               
               
                   
               
               
                 KTIDDRAFAADYGRAGGDGHACLGLSVNWCQSRAKGQSDEAFFHKLEDYQ 
               
               
                   
               
               
                 GDALLPRVMGFQHIEQQAYSNKLQNAAPMLLDTLPKLGMTLGKGLGRAQH 
               
               
                   
               
               
                 AHYAVALENLDRDLKAVLQPGKDQMLLFLSDSHAMALHQDSQGCLHFFDP 
               
               
                   
               
               
                 LFGVVQADSFSNMSHFLADVFKRDVGTHWRGTEQRLQLSEMVPRADFHLR 
               
               
                   
               
               
                 &gt;AvrPto amino acid sequence: 
               
               
                 MGNICVGGSRMAHQVNSPDRVSNNSGDEDNVTSSQLLSVRHQLAESAGVP 
               
               
                   
               
               
                 RDQHEFVSNQAPQSLRNRYNNLYSHTQRTLDMADMQHRYMTGASGINPGM 
               
               
                   
               
               
                 LPHENVDDMRSAITDWSDMREALQYAMGIHADIPPSPERFVATMNPNGSI 
               
               
                   
               
               
                 RMSTLSPSPYRNWQ 
               
            
           
         
       
     
     Example 6  
     Identification of Proteins that Suppress AGO1-Containing RISC 
     We then investigated whether any Pst DC3000 effectors could also suppress the AGO1-containing RISC function as observed with some viral-derived suppressors of RNA-silencing. The same set of 23 Pto DC3000 effectors was further tested for possible interference with miRNA activities. Transient expression of one or more HopT1-1 did not affect miR834 accumulation but dramatically increased the levels of its cognate target, the COP-interacting protein 4 (CIP4) ( FIG. 11A ). By contrast, neither miR834 nor CIP4 levels were altered upon transient delivery of the unrelated Pto DC3000 effectors HopC1, -X1 ( FIG. 11A ). 
       FIG. 11A  shows overexpression of hopT1-1 promotes the accumulation of miR834 target and has no significant effect on miR834 steady state levels. Efr1 mutant leaves were agro-infiltrated with 35S::hopT1-1 construct (OD=0.4) and miR834 as well as miR834 target (COP-interacting protein 4, CIP4) levels were monitored 3 days post infiltration by Northern analysis (Upper panel) and Western analysis (Bottom panel), respectively. 
     These results suggested that HopT1-1 acts downstream of miRNA biogenesis, potentially by inhibiting the AGO1-directed RISC, which is recruited by most plant miRNAs. 
     To test this hypothesis further, we used the SUC-SUL (SS) reporter line 120  in which phloem-specific expression of an inverted-repeat transgene triggers non-cell-autonomous RNAi of the endogenous SULPHUR (SUL) transcript, resulting in a chlorotic phenotype that expands beyond the vasculature ( FIG. 11B ).  FIG. 11B  shows a weak allele of ago1 suppresses artificial silencing with no significant effect on the steady state levels of 21-24 nt siRNAs. Ago1-12 mutation suppresses artificial silencing triggered by an artificial hairpin, targeting the endogene sulphure (SUL), and driven by the phloem specific promoter (SUC2). Ago1-12 suppresses the vein chlorotic phenotype triggered by SUC-SUL transgene (Upper panel). Ago1-12 mutation does not significantly interfere with the accumulation of 21-24 nt siRNA as assayed by Northern analysis (bottom panel). Of the 21 nt (DCL4-dependent) and 24 nt (DCL3-dependent) SUL siRNA species, only the former is required for RNAi in a strict AGO1-dependent manner. 
     The 35S::HopT1-1 construct was transformed into SUC-SUL, and two independent T2 lines were selected, showing moderate (#4) or strong (#22) HopT1-1 mRNA accumulation ( FIG. 11C ).  FIG. 11C  shows overexpression of HopT1-1 suppresses the SUC-SUL phenotype with no significant effect on neither 21-24 nt siRNA nor endogenous miRNA accumulation. Suppression of the vein chlorotic phenotype trigger by the SUC-SUL transgene (Upper panel). Low molecular weight Northern on the 21-24 SUL siRNA (middle and right panel), RT-PCR on HopT1 mRNA (middle and left panel), LMW Northern on a subset of endogenous miRNAs (Bottom panel). Both lines exhibited little or no cholorosis and accumulated higher SUL mRNA levels. However, accumulation of SUL siRNAs remained unaltered mimicking the effects of the ago1-12 mutation in SUC-SUL plants. Also as in ago1-12 mutants, the levels of several canonical miRNAs were normal in HopT1-1-expressing lines, despite higher accumulation of their target transcripts. 
       FIG. 11D  shows schematic representation depicting the expected effect of a perturbation of the RISC miRNA  function.  FIG. 11E  shows overexpression of hopT1-1 promotes the accumulation of miR834 target (CIP4) with no effect on miR834 accumulation. Western analysis results using an anti-CIP4 antibody.  FIG. 11F  shows overexpression of hopT1-1 promotes the accumulation of miRNA targets. A subset of miRNA targets are more elevated in two independent transgenic lines overexpressing hopT1-1 (Left panel). The same subset of miRNA targets are more elevated in ago1-11 and ago1-12 mutants (Right panel). Collectively these results indicate that HopT1-1 likely interferes with the function of the AGO1-RISC, resulting in suppression of miRNA, as well siRNA activities. 
     We obtained similar results when we overexpressed hopY1 effector in the silencing reporter line SUC-SUL ( FIG. 12 ), except that a slight increase in the levels of 21-24 nt SUL siRNA as well as endogenous miRNAs was observed in this particular case. HopY1 overexpressor lines displayed a significant increase in the accumulation of several miRNA targets ( FIG. 12C ).  FIG. 12C  shows overexpression of hopY1 slightly increases the accumulation of some endogenous miRNAs. However, despite its effect on miRNA activity, HopY1 did not induce drastic developmental alteration ( FIG. 12A ).  FIG. 12A  shows overexpression of hopY1 reduces the vein chlorotic phenotype triggered by the SUC-SUL transgene.  FIG. 12B  shows overexpression of hopY1 slightly increases the accumulation of 21-24 nt SUL siRNAs.  FIG. 12D  shows overexpression of hopY1 promotes the accumulation of a subset of miRNA targets as well as SUL mRNA. 
     The nucleotide sequences encoding the Bss proteins that interfere with the RISC miRNA  function are as follows: 
     
       
         
           
               
            
               
                 &gt;HopT1-1 coding sequence: 
               
               
                 ATGATGAAAACAGTCAGCAATCACTCGATACCCAGTACAAATCTCGTCGT 
               
               
                   
               
               
                 GGATGCGGGAACGGAAACTTCGGCGCAGAAATCCCAGCCGGTTTGCAGCG 
               
               
                   
               
               
                 AAATCCAGCGTAACAGCAAGATCGAAAAAGCAGTCATCGAACACATTGCC 
               
               
                   
               
               
                 GACCACCCGGCAGCGAAAATGACAATAAGCGCGCTGGTTGACACGTTGAC 
               
               
                   
               
               
                 AGACGTTTTTGTCAGGGCTCATGGGGAGGTTAAGGGGTGGGCCGAAATCG 
               
               
                   
               
               
                 TCCAGGCAGTCTCTCGCCCTCATGACAGTAATCGACACGGCAGTGGAGTG 
               
               
                   
               
               
                 CTCAGCCCGCGCTTTGATGTAATGGGGAGTGTTGGTTGGAATGCGGCAGC 
               
               
                   
               
               
                 TATCCGGGCCACCAGTCGCGTCGGGACGCTTCGAGAGAAAGGTACACTGT 
               
               
                   
               
               
                 TCACTAACCTTATGCTCAGTAACAACTTTAAACATTTGCTTAAACGAGTG 
               
               
                   
               
               
                 GTTAACGATCCAGCCTTGCAGCAAAAGCTCGACGGTGGGTTAGACCTCAA 
               
               
                   
               
               
                 CTATCTGAAGGCTTGTGAAGGCGATCTTTATGTCATGTCAGGGTGGGCTG 
               
               
                   
               
               
                 CACGGGCTAGCGAAAGTCGTGAACAAATTGGCAAAGCCCGGTATGAAACG 
               
               
                   
               
               
                 GCATCAAATCTTAGCCAGACGCTGATCAGTGCACGTGAGTTGGCTTTTCA 
               
               
                   
               
               
                 TCGTCACAATCCGGTTAATCATCCGTCTGCCCAAACGAAAGTGGGCTTCG 
               
               
                   
               
               
                 ATAAGGGTTTGCCTGAGGAATCTGATCTGCAGGTTCTGAGAGGCCATGGC 
               
               
                   
               
               
                 AGCAGTGTATGGAGTGTAAAACCGGGCAGCGATTTCGCAAAGCGTGCTGA 
               
               
                   
               
               
                 AGTTTCTGGAAAGCCTATTATCGCCGGCCCGTCCGGTACCGCTTCGCGCA 
               
               
                   
               
               
                 TGGTCGCTGTTGCGCGTTTTCTGGCACCGGCTTGTTTGAAAAGCCTGGGT 
               
               
                   
               
               
                 ATTGAGAGTGAGCAGAACCTGAAAGAGCTTGTGCGGTATGCCTGCTATGC 
               
               
                   
               
               
                 CTATTTCGGTCAGGACAGCCACCATTCGATGCTTGAAGTGAATCTTGGTG 
               
               
                   
               
               
                 TCGCTTCCCATGGAATGCCGGAACAATGGGACGACACGCTTTATAACGAG 
               
               
                   
               
               
                 CCTTTCAGTAATTCAATTAAAGGTCGCGGGTTTGGTATAGACAATCTCGC 
               
               
                   
               
               
                 GCATAGGCAAGTCGTCAGGCAGGCGGCTCAAAAGTCATGA 
               
               
                   
               
               
                 &gt;HopY1 coding sequence: 
               
               
                 ATGAACATTACGCCGCTCACGTCAGCCGCGGGCAAGGGCTCGTCCGCACA 
               
               
                   
               
               
                 AGGCACAGACAAAATTTCCATTCCCAACTCCACGCGCATGATCAATGCCG 
               
               
                   
               
               
                 CTTCAATCAAGTGGTTGAATAAGGTGCGTAGCGCCATCAGTGACCACATC 
               
               
                   
               
               
                 CGCACCAGCATCGAGAAAGGGAAACTGTTCGAGCTCGCCTCCTTGGGCAG 
               
               
                   
               
               
                 CAACATGTTCGGTGTCCCGGCTCTTTCAGCGCGCCCCTCGACGCTCCAAC 
               
               
                   
               
               
                 CTGTGTTGGCGTTTGAGGCTGACCCCAATCACGACCTGAACCTTGTCAGG 
               
               
                   
               
               
                 GTCTATATGCAGGACAGCGCCGGCAAGCTCACTCCCTGGGACCCGACGCC 
               
               
                   
               
               
                 CAACGCGGTCACGACGACGTCGAATCCATCAGAGCCTGATGCGCAGAGCG 
               
               
                   
               
               
                 ATACGGCTTCGTCATCATTACCTCGGCGGCCTCCCGCAGGCTCGGTGCTG 
               
               
                   
               
               
                 AGTTTGCTGGGCATTGCGCTGGATCACGCGCAACGCCACAGTCCTCGCGC 
               
               
                   
               
               
                 GGACAGGTCTGCCAAGGGACGACCTGGCCGAGAGGAGAGGAACGGGGCAA 
               
               
                   
               
               
                 GGTTCAATGCCAAGCAAACAAAGCCGACAGAGGCTGAAGCCTACGGTGAT 
               
               
                   
               
               
                 CATCAGACACCCAATCCTGATTTGCACAGGCAAAAAGAGACAGCTCAACG 
               
               
                   
               
               
                 CGTTGCTGAAAGCATCAACAGCATGCGAGAGCAGCAAAATGGAATGCAAC 
               
               
                   
               
               
                 GCGCCGAAGGGCTTCTCAGAGCCAAAGAAGCGTTGCAAGCTCGGGAAGCC 
               
               
                   
               
               
                 GCGCGCAAGCAGCTTCTGGACGTGCTCGAGGCCATCCAGGCTGGCCGTGA 
               
               
                   
               
               
                 AGACTCCACCGACAAGAAGATCAGCGCCACTGAAAAGAACGCCACGGGCA 
               
               
                   
               
               
                 TCAACTACCAGTGA 
               
            
           
         
       
     
     The amino acid sequences from the Bss proteins that interfere with the RISC miRNA  function are as follows: 
     
       
         
           
               
            
               
                 &gt;HopT1-1 amino acid sequence: 
               
               
                 MMKTVSNHSIPSTNLVVDAGTETSAQKSQPVCSEIQRNSKIEKAVIEHIA 
               
               
                   
               
               
                 DHPAAKMTISALVDTLTDVFVRAHGEVKGWAEIVQAVSRPHDSNRHGSGV 
               
               
                   
               
               
                 LSPRFDVMGSVGWNAAAIRATSRVGTLREKGTLFTNLMLSNNFKHLLKRV 
               
               
                   
               
               
                 VNDPALQQKLDGGLDLNYLKACEGDLYVMSGWAARASESREQIGKARYET 
               
               
                   
               
               
                 ASNLSQTLISARELAFHRHNPVNHPSAQTKVGFDKGLPEESDLQVLRGHG 
               
               
                   
               
               
                 SSVWSVKPGSDFAKRAEVSGKPIIAGPSGTASRMVAVARFLAPACLKSLG 
               
               
                   
               
               
                 IESEQNLKELVRYACYAYFGQDSHHSMLEVNLGVASHGMPEQWDDTLYNE 
               
               
                   
               
               
                 PFSNSIKGRGFGIDNLAHRQVVRQAAQKS 
               
               
                   
               
               
                 &gt;HopY1 amino acid sequence: 
               
               
                 MNITPLTSAAGKGSSAQGTDKISIPNSTRMINAASIKWLNKVRSAISDHI 
               
               
                   
               
               
                 RTSIEKGKLFELASLGSNMFGVPALSARPSTLQPVLAFEADPNHDLNLVR 
               
               
                   
               
               
                 VYMQDSAGKLTPWDPTPNAVTTTSNPSEPDAQSDTASSSLPRRPPAGSVL 
               
               
                   
               
               
                 SLLGIALDHAQRHSPRADRSAKGRPGREERNGARFNAKQTKPTEAEAYGD 
               
               
                   
               
               
                 HQTPNPDLHRQKETAQRVAESINSMREQQNGMQRAEGLLRAKEALQAREA 
               
               
                   
               
               
                 ARKQLLDVLEAIQAGREDSTDKKISATEKNATGINYQ 
               
            
           
         
       
     
     Example 7 
     Identification of Proteins that Suppress miRNA Translational Inhibition 
     We finally investigated whether bacterial effector proteins could suppress miRNA-directed translational inhibition, a phenomenon well characterized in animals but also effective in plants. For this purpose, we transiently delivered the same set of Pst DC3000 effectors in the efr1 mutant leaves and screened for effectors that would enhance protein accumulation of miRNA targets with no major impact on either miRNA target mRNA levels nor mature miRNA levels. Among the subset of effectors tested, the mono-ADP ribosyltransferase HopU1 fulfilled these criteria as it induces higher miR398 target protein levels: the Superoxide dismutase 1 (CSD1) and 2 (CSD2), with no significant effect on the accumulation of CSD1/2 mRNA levels or miR398 levels (data not shown). Moreover, delivery of HopU1 in the efr1 mutant expressing the miR171 sensor construct restored a high GFP protein accumulation but did not alter GFP mRNA levels indicating that HopU1 indeed interferes with miRNA-directed translational inhibition (FIGS.  13 A/B and data not shown).  FIG. 13A  shows overexpression of HopU1 but not HopU1DD mutant version restores GFP expression in efr1 plants expressing miR171 sensor constructs. miR171 Sensor (efr1) plants were Agro-infiltrated (OD=0.4) with the 35S::GUSintron, 35S::HopU1 and 35S::HopU1DD constructs and GFP levels analyzed visually under UV.  FIG. 13B  shows Western analysis using an anti-GFP antibody. Importantly, no rescue of GFP expression nor of the endogenous miR398 target CSD2 was observed when we transiently delivered a HopU1 mutant version that abolishes its ADP-ribosyltransferase activity. This indicates that ADP-ribosyltransferase activity is required for HopU1-triggered suppression of RNA-silencing. 
     To further investigate whether HopU1 interferes with a putative siRNA-directed translational inhibition, we transformed 35S::HopU1 construct in the SUC-SUL reference line and selected T2 transgenic lines expressing high levels of hopU1 proteins. Expression of HopU1 in these stable transgenic lines diminishes slightly the 21-24 SUL siRNA levels, but did not affect SUL mRNA levels.  FIG. 13C  shows SUC-SUL plants expressing HopU1.  FIG. 13D  shows LMW Northern analysis. However, a significant reduction in the vein chlorotic phenotype was observed, which is diagnostic of higher SUL protein levels. Western analysis using an anti CSD2 antibody suggests that HopU1 additionally interferes with siRNA-directed translational inhibition.  FIG. 13E  shows qRT-PCR on SUL mRNA. 
     The nucleotide sequence encoding the Bss protein interferes with miRNA-mediated translational inhibition is as follows: 
     
       
         
           
               
            
               
                 &gt;HopU1 coding sequence: 
               
               
                 ATGAATATAAATCGACAACTGCCTGTATCAGGCTCGGAGCGATTGTTGAC 
               
               
                   
               
               
                 TCCCGACGTGGGCGTATCTCGCCAGGCTTGTTCCGAAAGGCATTATTCTA 
               
               
                   
               
               
                 CTGGACAGGATCGGCATGATTTTTACCGTTTTGCTGCCAGGCTACATGTG 
               
               
                   
               
               
                 GATGCGCAGTGTTTTGGTCTGTCAATAGACGATTTGATGGATAAGTTTTC 
               
               
                   
               
               
                 TGACAAGCACTTCAGGGCTGAGCATCCTGAATACAGGGATGTCTATCCGG 
               
               
                   
               
               
                 AGGAATGTTCTGCCATTTATATGCATACCGCTCAAGACTATTCTAGTCAC 
               
               
                   
               
               
                 CTCGTAAGGGGGGAAATAGGAACGCCGCTGTACCGAGAGGTCAATAATTA 
               
               
                   
               
               
                 TCTTCGACTTCAACATGAGAATTCTGGGCGAGAAGCTGAAATTGATAATC 
               
               
                   
               
               
                 ACGACGAAAAGCTATCGCCTCACATAAAAATGCTTTCATCTGCGCTTAAT 
               
               
                   
               
               
                 CGTTTAATGGATGTCGCCGCTTTTAGAGGAACGGTTTATAGAGGCATTCG 
               
               
                   
               
               
                 CGGTGATTTAGATACCATTGCTCGGCTCTACCATCTATTCGATACGGGCG 
               
               
                   
               
               
                 GCCGGTACGTAGAGCCCGCTTTCATGAGTACAACTCGAATAAAGGACAGT 
               
               
                   
               
               
                 GCCCAGGTGTTTGAGCCAGGCACGCCAAACAACATAGCTTTCCAGATAAG 
               
               
                   
               
               
                 CCTAAAAAGAGGCGCCGACATTTCGGGATCTTCCCAAGCGCCCTCAGAGG 
               
               
                   
               
               
                 AAGAAATCATGCTACCCATGATGAGTGAGTTCGTCATTGAACATGCATCC 
               
               
                   
               
               
                 GCTCTTTCCGAAGGAAAGCATTTATTTGTATTAAGTCAGATTTGA 
               
            
           
         
       
     
     The amino acid sequence of the Bss protein that interferes with miRNA-mediated translational inhibition is as follows: 
     
       
         
           
               
            
               
                 &gt;HopU1 amino acid sequence: 
               
               
                 MNINRQLPVSGSERLLTPDVGVSRQACSERHYSTGQDRHDFYRFAARLHV 
               
               
                   
               
               
                 DAQCFGLSIDDLMDKFSDKHFRAEHPEYRDVYPEECSAIYMHTAQDYSSH 
               
               
                   
               
               
                 LVRGEIGTPLYREVNNYLRLQHENSGREAEIDNHDEKLSPHIKMLSSALN 
               
               
                   
               
               
                 RLMDVAAFRGTVYRGIRGDLDTIARLYHLFDTGGRYVEPAFMSTTRIKDS 
               
               
                   
               
               
                 AQVFEPGTPNNIAFQISLKRGADISGSSQAPSEEEIMLPMMSEFVIEHAS 
               
               
                   
               
               
                 ALSEGKHLFVLSQI 
               
            
           
         
       
     
     Bss proteins mutated in key residues that do not perturb suppression of RNA-silencing will be particularly useful as they might not be recognized by plant resistance (R) proteins and therefore would not induce the classical R-mediated programmed cell death (which is often detrimental for the plants). Examples include versions of HopN1 that are mutated in the predicted cysteine protease catalytic triads and still retained their ability to suppress RNA-silencing ( FIG. 10C ). These mutant versions might be compromised in R gene recognition and yet still suppress RNA-silencing in different plant species. The method hereby disclosed thus allows the generation of mutated versions of Bss proteins in order to uncouple the suppression of RNA-silencing from the R-gene recognition. 
     Bss identified according to this invention are also useful proteins to identify plant and animal components involved in miRNA biogenesis and/or activity. As an example, the mono-ADP-ribosyltransferase HopU1 discussed above was recently shown to directly interact with the glycine-rich RNA-binding proteins AtGRP7 and AtGRP8. ADP-ribosylation of GRP7 by HopU1 occurs on two conserved arginine residues located in the RNA-recognition domain of GRP7 and likely perturbs its ability to bind RNA. Because HopU1 ADP-ribosyltransferase activity is required for its RNA-silencing suppression activity, we anticipate that AtGRP7 and AtGRP8 are novel silencing factors involved in miRNA- and siRNA-directed translational inhibition.  FIG. 13  shows overexpression of HopU1 but not HopU1DD mutant version restore GFP expression in efr1 plants expressing miR171 sensor constructs. Other Bss proteins will also directly interact with and perturb host components involved in RNA-silencing and can therefore be used as molecular probes to identify RNA-silencing factors or regulators thereof, in both plant and animal systems. 
     The coding sequences from the putative novel RNA-silencing components from  Arabidopsis thaliana  are as follows: 
     
       
         
           
               
            
               
                 &gt;AtGRP7 (At2g21660) coding sequence: 
               
               
                 ATGGCGTCCGGTGATGTTGAGTATCGGTGCTTCGTTGGAGGTCTAGCATG 
               
               
                   
               
               
                 GGCCACTGATGACAGAGCTCTTGAGACTGCCTTCGCTCAATACGGCGACG 
               
               
                   
               
               
                 TTATTGATTCCAAGATCATTAACGATCGTGAGACTGGAAGATCAAGGGGA 
               
               
                   
               
               
                 TTCGGATTCGTCACCTTCAAGGATGAGAAAGCCATGAAGGATGCGATTGA 
               
               
                   
               
               
                 GGGAATGAACGGACAAGATCTCGATGGCCGTAGCATCACTGTTAACGAGG 
               
               
                   
               
               
                 CTCAGTCACGAGGAAGCGGTGGCGGCGGAGGCCACCGTGGAGGTGGTGGC 
               
               
                   
               
               
                 GGTGGATACCGCAGCGGCGGTGGTGGAGGTTACTCCGGTGGAGGTGGTAG 
               
               
                   
               
               
                 CTACGGAGGTGGCGGCGGTAGACGCGAGGGTGGAGGAGGATACAGCGGCG 
               
               
                   
               
               
                 GCGGCGGCGGTTACTCCTCAAGAGGTGGTGGTGGCGGAAGCTACGGTGGT 
               
               
                   
               
               
                 GGAAGACGTGAGGGAGGAGGAGGATACGGTGGTGGTGAAGGAGGAGGTTA 
               
               
                   
               
               
                 CGGAGGAAGCGGTGGTGGTGGAGGATGGTAA 
               
               
                   
               
               
                 &gt;AtGRP8 (At4g39260) coding sequence: 
               
               
                 ATGTCTGAAGTTGAGTACCGGTGCTTTGTCGGCGGCCTTGCCTGGGCCAC 
               
               
                   
               
               
                 CAATGATGAAGATCTTCAAAGGACGTTCTCACAGTTCGGCGACGTTATCG 
               
               
                   
               
               
                 ATTCTAAGATCATTAACGACCGCGAGAGTGGAAGATCAAGGGGATTCGGA 
               
               
                   
               
               
                 TTCGTCACCTTCAAGGACGAGAAAGCCATGAGGGATGCGATTGAAGAGAT 
               
               
                   
               
               
                 GAACGGTAAAGAGCTCGATGGACGTGTCATCACCGTGAACGAGGCTCAGT 
               
               
                   
               
               
                 CGAGAGGTAGCGGCGGTGGCGGAGGAGGCCGTGGTGGAAGCGGTGGTGGT 
               
               
                   
               
               
                 TACCGCAGCGGAGGCGGTGGTGGATACTCAGGAGGCGGTGGCGGCGGATA 
               
               
                   
               
               
                 CTCAGGAGGAGGCGGTGGTGGTTACGAGAGACGTAGCGGAGGTTACGGAT 
               
               
                   
               
               
                 CTGGTGGAGGCGGTGGTGGCCGAGGATACGGTGGTGGTGGACGCCGTGAG 
               
               
                   
               
               
                 GGAGGTGGCTACGGAGGCGGTGATGGTGGAAGTTACGGAGGCGGTGGTGG 
               
               
                   
               
               
                 CGGCTGGTAA 
               
            
           
         
       
     
     The amino acid sequences from the putative novel RNA-silencing components from  Arabidopsis  are as follows: 
     
       
         
           
               
            
               
                 &gt;AtGRP7 amino acid sequence: 
               
               
                 MASGDVEYRCFVGGLAWATDDRALETAFAQYGDVIDSKIINDRETGRSRG 
               
               
                   
               
               
                 FGFVTFKDEKAMKDAIEGMNGQDLDGRSITVNEAQSRGSGGGGGHRGGGG 
               
               
                   
               
               
                 GGYRSGGGGGYSGGGGSYGGGGGRREGGGGYSGGGGGYSSRGGGGGSYGG 
               
               
                   
               
               
                 GRREGGGGYGGGEGGGYGGSGGGGGW 
               
               
                   
               
               
                 &gt;AtGRP8 amino acid sequence: 
               
               
                 MSEVEYRCFVGGLAWATNDEDLQRTFSQFGDVIDSKIINDRESGRSRGFG 
               
               
                   
               
               
                 FVTFKDEKAMRDAIEEMNGKELDGRVITVNEAQSRGSGGGGGGRGGSGGG 
               
               
                   
               
               
                 YRSGGGGGYSGGGGGGYSGGGGGGYERRSGGYGSGGGGGGRGYGGGGRRE 
               
               
                   
               
               
                 GGGYGGGDGGSYGGGGGGW 
               
            
           
         
       
     
     Homology search in animals revealed several possible orthologs of AtGRP8 in humans, suggesting that one or several of these orthologs might be involved in executing or regulating miRNA-directed functions in animals. Therefore, plant and animal Bss are also valuable tools to uncover novel RNA silencing components in a broad range of organisms. The various human proteins with homology to AtGR8 are listed below: 
     
       
         
           
               
            
               
                 &gt;HsCIRBP gi|4502847|ref|NP_001271.1|cold inducible 
               
               
                 RNA binding protein [ Homo sapiens ] 
               
               
                 MASDEGKLFVGGLSFDTNEQSLEQVFSKYGQISEVVVVKDRETQRSRGFG 
               
               
                   
               
               
                 FVTFENIDDAKDAMMAMNGKSVDGRQIRVDQAGKSSDNRSRGYRGGSAGG 
               
               
                   
               
               
                 RGFFRGGRGRGRGFSRGGGDRGYGGNRFESRSGGYGGSRDYYSSRSQSGG 
               
               
                   
               
               
                 YSDRSSGGSYRDSYDSYATHNE 
               
               
                   
               
               
                 &gt;HsCIRP CDS: 
               
               
                 ATGGCATCAGATGAAGGCAAACTTTTTGTTGGAGGGCTGAGTTTTGACAC 
               
               
                   
               
               
                 CAATGAGCAGTCGCTGGAGCAGGTCTTCTCAAAGTACGGACAGATCTCTG 
               
               
                   
               
               
                 AAGTGGTGGTTGTGAAAGACAGGGAGACCCAGAGATCTCGGGGATTTGGG 
               
               
                   
               
               
                 TTTGTCACCTTTGAGAACATTGACGACGCTAAGGATGCCATGATGGCCAT 
               
               
                   
               
               
                 GAATGGGAAGTCTGTAGATGGACGGCAGATCCGAGTAGACCAGGCAGGCA 
               
               
                   
               
               
                 AGTCGTCAGACAACCGATCCCGTGGGTACCGTGGTGGCTCTGCCGGGGGC 
               
               
                   
               
               
                 CGGGGCTTCTTCCGTGGGGGCCGAGGACGGGGCCGTGGGTTCTCTAGAGG 
               
               
                   
               
               
                 AGGAGGGGACCGAGGCTATGGGGGGAACCGGTTCGAGTCCAGGAGTGGGG 
               
               
                   
               
               
                 GCTACGGAGGCTCCAGAGACTACTATAGCAGCCGGAGTCAGAGTGGTGGC 
               
               
                   
               
               
                 TACAGTGACCGGAGCTCGGGCGGGTCCTACAGAGACAGTTACGACAGTTA 
               
               
                   
               
               
                 CGCTACACACAACGAGTAA 
               
               
                   
               
               
                 &gt;HsRBP3 gi|5803137|ref|NP_006734.1|RNA binding 
               
               
                 motif protein 3 [ Homo sapiens ] 
               
               
                 MSSEEGKLFVGGLNFNTDEQALEDEFSSFGPISEVVVVKDRETQRSRGFG 
               
               
                   
               
               
                 FITFTNPEHASVAMRAMNGESLDGRQIRVDHAGKSARGTRGGGFGAHGRG 
               
               
                   
               
               
                 RSYSRGGGDQGYGSGRYYDSRPGGYGYGYGRSRDYNGRNQGGYDRYSGGN 
               
               
                   
               
               
                 YRDNYDN 
               
               
                   
               
               
                 &gt;HsRBP3 CDS: 
               
               
                 ATGTCCTCTGAAGAAGGAAAGCTCTTCGTGGGAGGGCTCAACTTTAACAC 
               
               
                   
               
               
                 CGACGAGCAGGCACTGGAAGACCACTTCAGCAGTTTCGGACCTATCTCTG 
               
               
                   
               
               
                 AGGTGGTCGTTGTCAAGGACCGGGAGACTCAGCGGTCCAGGGGTTTTGGT 
               
               
                   
               
               
                 TTCATCACCTTCACCAACCCAGAGCATGCTTCAGTTGCCATGAGAGCCAT 
               
               
                   
               
               
                 GAACGGAGAGTCTCTGGATGGTCGTCAGATCCGTGTGGATCATGCAGGCA 
               
               
                   
               
               
                 AGTCTGCTCGGGGAACCAGAGGAGGTGGCTTTGGGGCCCATGGGCGTGGT 
               
               
                   
               
               
                 CGCAGCTACTCTAGAGGTGGTGGGGACCAGGGCTATGGGAGTGGCAGGTA 
               
               
                   
               
               
                 TTATGACAGTCGACCTGGAGGGTATGGATATGGATATGGACGTTCCAGAG 
               
               
                   
               
               
                 ACTATAATGGCAGAAACCAGGGTGGTTATGACCGCTACTCAGGAGGAAAT 
               
               
                   
               
               
                 TACAGAGACAATTATGACAACTGA 
               
               
                   
               
               
                 &gt;HsRBPX gi|89059830|ref|XP_933552.1|PREDICTED: 
               
               
                 similar to RNA binding motif protein, X-linked 
               
               
                 [ Homo sapiens ] 
               
               
                 MGEADRLGKFFIGGLNTETNKKALEAVFGKYGQIVEVHLMKDCETNKSRG 
               
               
                   
               
               
                 FAFITFERPADAKDAARDMNGKSLDGKAIKVEQATKPSFESGRRGPPPPP 
               
               
                   
               
               
                 RSRGPPRVLRGGRGGSGGTREPPSRGGHMDDWWIFHEF 
               
               
                   
               
               
                 HsRBPX CDS: 
               
               
                 ATGGGTGAAGCAGATCGCCTAGGAAAGTTTTTCATTGGTGGGCTTAATAC 
               
               
                   
               
               
                 GGAAACAAATAAGAAAGCTCTTGAAGCAGTATTTGGCAAATATGGACAAA 
               
               
                   
               
               
                 TAGTGGAAGTACACTTGATGAAAGACTGTGAAACCAACAAATCAAGAGGA 
               
               
                   
               
               
                 TTTGCTTTTATCACCTTTGAAAGACCAGCAGACGCTAAGGATGCAGCCAG 
               
               
                   
               
               
                 AGACATGAATGGAAAGTCATTAGATGGAAAAGCCATCAAGGTGGAACAAG 
               
               
                   
               
               
                 CCACCAAACCGTCATTTGAAAGTGGTAGACGTGGACCGCCTCCACCTCCA 
               
               
                   
               
               
                 AGAAGTAGAGGCCCTCCAAGAGTTCTTAGAGGTGGAAGAGGAGGAAGTGG 
               
               
                   
               
               
                 AGGAACCAGGGAACCTCCCTCACGGGGAGGACACATGGATGACTGGTGGA 
               
               
                   
               
               
                 TATTCCATGAATTTTAA 
               
            
           
         
       
     
     REFERENCES 
     1. Bernstein, E., et al. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference.  Nature  409, 363-366 
     2. Song, J. J., et al. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity.  Science  305, 1434-1437 
     3. Liu, J., et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi.  Science  305, 1437-1441 
     4. Rivas, F. V., et al. (2005) Purified Argonaute2 and an siRNA form recombinant human RISC.  Nat Struct Mol Biol  12, 340-349 
     5. Napoli, C., et al. (1990) Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans.  Plant Cell  2, 279-289 
     6. Stam, M., et al. (1997) Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats.  Plant Journal  12, 63-82 
     7. Metzlaff, M., et al. (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in petunia.  Cell  88, 845-854 
     8. Waterhouse, P. M., et al. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.  Proceedings Of The National Academy Of Sciences Of The United States Of America  95, 13959-13964 
     9. Chuang, C.-H., and Meyerowitz, E. M. (2000) Specific and heritable genetic interference by double-stranded RNA in  Arabidopsis thaliana. Proc. Natl. Acad. Sci.  USA 97, 4985-4990 
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