Abstract:
A mutant gene coding for pectate lyase and homologs thereof is provided, which when incorporated in transgenic plants effect an increased level disease resistance in such plants. Also is provided the polypeptide sequence for the pectate lyase of the present invention. Methods of obtaining the mutant gene, producing transgenic plants which include the nucleotide sequence for the mutant gene and producing improved disease resistance in a crop of such transgenic plants are also provided.

Description:
This application claims the benefit of U.S. Provisional Application No. 60/159,566, filed Oct. 15, 1999. 

   This invention relates to a mutant gene coding for pectate lyase and homologs thereof and the use of such mutant genes in producing transgenic plants having improved disease resistance. The invention described herein was made in the course of work under grant number DE-FG02-94ER20133 and grant number DE-FG02-97ER20133 from the U.S. Department of Energy and grant number F32 GM 19499 of the National Institute of Health. The U.S. government may retain certain rights in this invention. 

   BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   2. Description of the Related Art 
   Pectate lyases, along with polygalacturonases and pectin lyases are enzymes that degrade pectin, a component of plant cell walls. Pectins are thought to act like a cement that holds adjacent cells together. Degradation of pectin plays a role in several normal developmental processes including: fruit ripening, pollen maturation, pollen tube growth and cell expansion and growth. Degradation of pectin is also a hallmark of several soft-rot diseases caused by both bacteria and fungi. Not surprisingly, pectate lyases are important virulence factors produced by several soft-rot pathogens. Therefore, the role of pathogen-produced pectate lyases in plant-pathogen interactions has been well studied. In particular, the pectate lyases (eels) of the soft-rotting Erwinia spp. have been well characterized. See Barras, F., F. Van Gijsegem, and A. K. Chatteijee 1 ). Pels break the α-1,4-glycosidic linkages in pectate by β-elimination producing unsaturated products. Pels also require Ca ++  for activity and function best at high pH, 8-9. PelA through PelE act as endo-pectate lyases with their final degradation products being short oligomers of galacturonic acid (di- to dodecamers) whose average length varies between Pels. 
   Plants also contain numerous genes homologous to pectate lyases. To date, two plant genes have been shown to have Pel activity when expressed in  E. coli  or yeast cells. One gene is primarily expressed in ripening strawberries and the other is expressed in a cultures of zinnia mesophyll cells that are differentiating into tracheary elements (See Domingo, C., et al. 2 . Also see Medina Escobar, N., et al. 3 ). Pel activity was also demonstrated in cultures of zinnia cells in response to auxin or traceary element inducing media (See Domingo, C., et al. 2 ). 
   In addition, Many pectate lyase-like genes are also highly expressed in mature pollen and are potent human allergens. Pet activity was demonstrated for a major pollen allergen of Japanese cedar, Cry j I (Taniguchi, Y., et al, Cryj I 4 ). In contrast, researchers were unable to demonstrate Pel activity of Lat59 or Lat56, two tomato pollen-specific pectate lyase-like gene, expressed in a baculovirus expression system (Dircks, L. K., G. U. Y. Vancanneyt, and S. McCormick 5 ). Therefore, it is possible that at least some of the Pel homologs found in plants may have activities other that degrading polygalacturonic acid and may utilize different pectic substrates. 
   SUMMARY OF THE INVENTION 
   The present invention discloses methods of creating disease resistant plant lines by using antisense, co-suppression or any other method to genetically engineer the loss, or partial loss, of activity of pectate lyase homologs into any plant. The inventors have surprisingly discovered that a plant-produced pectate lyase homolog is necessary for the compatible interaction between Arabidopsis and the obligate pathogens  Erysiphe cichoracearum  and  E. orontii . The present invention identifies four putative loss of function mutations in a gene, PMR6, with homology to pectate lyases and demonstrated that the mutants are highly resistant to  E. cichoracearum  and  E. orontii.  In addition, we envision that another mutant, pmr5, is resistant to powdery mildew due to a mutation in another pectate lyase homolog that contained on BAC AB009050 (SEQ ID Nos: 7 and 8). Several host defenses are also constitutively activated in these mutants, suggesting a possible mechanism for the resistance. In addition, the mutant plants are more compact and have altered leaf morphology. 
   A primary object of the present invention is to use any plant gene with homology to pectate lyase to increase pathogen resistance and alter plant morphology in any plant species. If the pmr5 phenotype is due to a mutation in the pectate lyase homolog on BAC MDF20, then we contemplate that loss of pectate lyase activity in general, not necessarily limited to genes with high homology to PMR6, can result in disease resistance. The notion that more distantly related pectate lyases may confer disease resistance is further supported by the fact that MDF20 and PMR6 are not extremely closely related to one another (FIG.  8 ). In fact, they are about as closely related to nine other Arabidopsis homologs as they are to each other. 
   Specifically, the present invention can employ any technology (i.e. mutagenesis, antisense, co-suppression) necessary to decrease the activity of the Pel homologs in plants. It is expected that the nucleotide sequence used for any given species will be an endogenous Ped homolog, however, using exogenous Pel homologs may also be effective in generating resistance. An important component of this system is the promoter used to express the antisense/cosuppression construct. The present invention can employ any of the promoters discussed herein or any other promoter sequence that controls expression of the construct in tissues that are normally infected by a pathogen as part of our antisense/cosuppression constructs. 
   Constitutive promoters such as: CaMV 35s and nptII, should decrease Pel activity throughout the plant and result in a phenotype similar to pmr5 and pmr6. However, for some applications, the dwarf phenotype and altered leaf morphology may not be desirable. It may be possible to separate disease resistance from the pleiotropic phenotypes by using epidermal specific (i.e. chalcone synthase promoter) or pathogen inducible promoters (i.e. PR1 promoter). In theory, these constructs should only inactivate Pel activity where/when it is necessary to achieve disease resistance rather than the global inactivation presumably necessary for the pleiotropic phenotypes. It may also be useful to employ other promoters with different expression patterns. 
   The present invention also contemplates that resistant plants can be made by expressing proteins that inhibit the enzymatic activity of the PMR proteins. 
   The present invention further contemplates that the identification of the PMR genes as disease resistance genes also opens new possibilities for rational design of chemicals that inhibit the growth of pathogens. The present invention discloses that a chemical that inhibits the action of either of the PMR proteins or related proteins will cause fungal disease resistance. Thus, it is contemplated that using known techniques the PMR genes can be used to design rational screens for such chemical compounds. As is known in the art this could be accomplished by expressing the genes in a host such as a bacterium or yeast, so that adequate amounts of largely pure protein could be produced. The protein could then be assayed for enzymatic activity in high throughput screens in the presence and absence of possible inhibitory compounds. 
   The present invention also contemplates that the PMR genes could be used to produce protein for structural studies by techniques such as Xray crystallography. Once the three dimensional structure is determined it will be possible to design inhibitors of the enzyme by those skilled in the art of rational drug design. 
   The same techniques applied to create resistant plants can also be applied to alter plant morphology. The small size, compact growth, increased branching and cupped leaves seen in pmr5 and pmr6 may be useful traits for some agricultural and horticultural applications. For example, many, horticultural crops have been selected for compact growth habit and increased branching. In addition, many dwarf varieties of vegetable crops and fruit crops are in use today. By transgenically decreasing Pel activity it may be possible to create a well branched, compact dwarf and resistant line simultaneously. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS.  1 A- 1 - 1 B- 3  shows the phenotype of pmr5 and pmr6. (A) Plants were inoculated with  E. cichoracearum  eight days prior to being photographed. Representative leaves were detached and photographed. Note the extensive fungal growth on Columbia. (B) Mature rosettes just prior to bolting, approximately 28 days old. 
       FIGS. 2A and 2B  shows the quantification of  E. chichoracearum  growth on pmr5 and pmr6. (A) Hyphal length per colony at various times after inoculation. Note that all mutants support some hyphal growth. (B) Mean conidiophores per colony at six days post inoculation. Error bars are +/−SD based on at least 15 colonies. 
     FIGS.  3 A- 1 - 3 B- 3  shows a cytological description of pmr5 and pmr6. (A) Uninfected leaves were stained with trypan blue to highlight dead cells. Note that dead cells are clustered along veins. (B) Accumulation of autoflourescent compounds in uninfected pmr5 and pmr6 leaves follows the same pattern as dead cells. 
       FIG. 4  shows a timecourse of PR1 mRNA expression. Plants were heavily inoculated with  E. cichoracearum  and samples were harvested at 0, 1, 3 and 5 days post inoculation. Total RNA was prepared and the levels of PR1 mRNA determined by northern blotting, normalized to rRNA levels and plotted. Note the elevated PR1 level of pmr6 on day 0. 
     FIGS.  5 A- 1 -C show sequences relating to PMR6.  FIG. 5A  shows the genomic sequence including upstream and downstream regions (SEQ ID NO: 1).  FIG. 5B  shows a partial cDNA sequence (SEQ ID NO: 2).  FIG. 5C  shows predicted amino acid sequence for pmr6 (SEQ ID NO: 3). 
       FIG. 6  shows sequence alterations in the pmr6 alleles of the PMR6 gene. pmr6-1 has aspartic acid at 140 instead of glycine. The G to A transition in pmr6-2 alters the splice junction resulting in a 1 bp deletion in the mature message. This frameshift alters the carboxy terminal 85 amino acids of PMR6. 
     FIGS.  7 A- 1 -C show sequences relating to a protion of BAC MDF20 with homology to pectate lyase.  FIG. 7A  shows the genomic sequence including upstream and downstream regions (SEQ ID NO: 4).  FIG. 7B  shows the predicted cDNA sequence (SEQ ID NO: 5).  FIG. 7C  shows the predicted amino acid (SEQ ID NO: 5). 
       FIG. 8  shows a phylogram, with bootstrap values, of plant genes with homology to pectate lyase. Pectate lyase activity has been demonstrated for the genes in bold. PMR6 and MDF20 are marked by arrows. 
   

   DETAILED DESCRIPTION OF THE INVENTION 
   The role of pectate lyases in plant-pathogen interactions has focused on the degradation of plant tissues by pathogen secreted Pels. These Pels macerate plant tissue and facilitate further pathogen growth. At the same time, Pel activity produces poly galacturonic acid oligomers that serve as potent elicitors of plant defenses (reviewed in (Vancutsem, P. and J. Messiaen 12 ). Thus, Pel activity allows plants to sense and respond to pathogen attack. In contrast, plant Pels have not previously been shown to be involved in plant-pathogen interactions. 
   The present invention discloses the surprising discovery that a plant gene with homology to pectate lyase is required for powdery mildew susceptibility. This seems counterintuitive because Pel activity should produce polygalacturonic acid oligomers that could then elicit host defenses. The inventors, therefore, ask why do mutations in a putative Pel gene result in resistant plants? The inventors have formed the following non-limiting theories in response to that question. One possibility is that PMR6 is involved in recycling pectin and when its activity is lost polygalacturonic acid oligomers, that would normally be degraded by PMR6, accumulate and stimulate host defenses. This interpretation is consistent with the constitutive accumulation of autoflourescent compounds and callose, the spontaneous formation microlesions and the basal levels of PR1 expression sometimes seen in the mutants. In this scenario, pmr6 plants are primed to respond to pathogen attack. 
   Another possibility involves the active suppression of host defenses by di- and trimers of galacturonic acid. This phenomenon, has recently been observed in wheat leaves infiltrated with digested pectin products (Moerschbacher, B. M., et al. 13 ). In this scenario, in a compatible interaction  E. cichoracearum  would rely on PMR6 activity to produce di- and trimers of galacturonic that, in turn, would suppress host defenses. Ironically, host defenses are elicited by longer oligomers of galacturonic acid. However, this scenario does not fit the constitutive expression of host defenses observed in the mutants. This entire scenario is very speculative and based on a single report of inhibition of resistance by di- and trimers. Therefore, we favor the pectin-recycling scenario at this time. 
   The vegetative phenotype of pmr5 and pmr6, compact growth habit, and cupped leaves, may result directly from the loss of Pel activity or may be indirect results of stress or possibly hormonal activity of polygalacturonic acid oligomers. We favor a fairly direct explanation: Cupped leaves could be the result of a failure of Pel activity to sufficiently loosen cell-cell attachments to allow normal cell expansion and growth Similarly, short petioles could be the result of petiole cells that fail to fully expand. Without regard to the non-limiting theories proposed, the inventors have discovered and herein discloses the surprising discovery that a plant gene with homology to pectate lyase is required for powdery mildew susceptibility. Further, the inventors have discovered the necessary nucleotide sequences and methods to employ to use any plant gene with homology to pectate lyase to increase pathogen resistance and alter plant morphology in any plant species. Other discoveries of the present invention are detailed below and in the claims of the present application 
   The present invention discloses that a plant gene having homology to pectate lyase can effectively increase pathogen resistance. 
   Related nucleotide or amino acid sequences are found when there is similarity or identity of sequence and this may be determined by comparison of sequence information, nucleotide or amino acid, or through hybridization between a gene endcoding pectate lysase probe and a candidate source (e.g., Southern or Northern blots, genomic or cDNA libraries). Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered in determining sequence similarity. 
   Typically, a nucleotide sequence may show as little as 80% sequence identity, and more preferably at least 90% sequence identity, between the target sequence and the pectate lysase polynucleotide excluding any deletions or additions which may be present, and still be considered related. Nucleotide sequence identity may be at least 95% and, most preferably, nucleotide sequence identity is at least 98%. Amino acid sequences are considered to be related with as little as 90% sequence identity between the two polypeptides; however, 95% or greater sequence identity is preferred and 98% or greater sequence identity is most preferred. 
   Pectate lysase. Thus, the use of complex mathematical algorithms is not required because amino acid sequences can be aligned without introducing many gaps. But such algorithms are known in the art, and implemented using default parameters in commercial software packages provided by DNASTAR, Genetics Computer Group, Hitachi Genetics Systems, and Oxford Molecular Group (formerly Intelligenetics). See Doolittle,  Of URFS and ORFS , University Science Books, 1986; Gribskov and Devereux,  Sequence Analysis Primer , Stockton Press, 1991; and references cited therein. Percentage identity between a pair of sequences may be calculated by the algorithm implemented in the BESTFIT computer program (Smith and Waterman, J. Mol. Biol., 147, 195-197, 1981; Pearson, Genomics, 11, 635-650, 1991). Another algorithm that calculates sequence divergence has been adapted for rapid database searching and implemented in the BLAST computer program (Altschul et al., Nucl. Acids Res., 25, 3389-3402, 1997). 
   Conservative amino acid substitutions, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn, may also be considered when making comparisons because the chemical similarity of these pairs of amino acid residues would be expected to result in functional equivalency. Amino acid substitutions that are expected to conserve the biological function of the native pectate lysase polypeptide would conserve chemical attributes of the substituted amino acid residues such as hydrophobicity, hydrophilicity, side-chain charge, or size. Functional equivalency or conservation of biological function may be evaluated by methods for structural determination and bioassay as disclosed herein. Thus, amino acid sequences are considered to be related with as little as 90% sequence similarity between the two polypeptides; however, 95% or greater sequence similarity is preferred and 98% or greater sequence similarity is most preferred. 
   The present invention is more fully defined by reference to the following examples, which are provided only for demonstration of the inventive concept of the present invention and are not intended to further limit the scope of the invention further than that of the claims provided herewith. 
   EXAMPLES 
   Materials and Methods 
   Plant lines and growth conditions:  Arabidopsis thaliana  ecotype Columbia (Col) and Wassilewskija (WS) were used in this study as indicated. Adult plants were grown in ProMix HP (Premier Horticulture Inc., Red Hill, Pa.) and fertilized once with one tsp/gal of Peter&#39;s Professional fertilizer (20-20-20) at the first watering after germination. Plants to be infected with powdery mildew were grown in a growth chamber at 22° C. with 14 hours of illumination per day or in greenhouses with supplemental lighting to increase daylength to 14 hr when necessary. Seeds were mutagenized with methane-sulfonic acid ethyl ester (EMS) as described or by transformation with Agrobacterium as described (Bechtold, N., J. Ellis, and G. Pelletier 6 ). 
   Squash, variety Kuta (Park Seed Co.) and  Capsella bursera - pastoris  (gift from F. Ausabel and L. Reuber) were used as a host for the production of  Erysiphe cichoracearum  and  E. orontii  inoculum, respectively. These alternate hosts were used rather than Arabidopsis because they have larger leaves than Arabidopsis. Another advantage to using Capselia rather than Arabidopsis is that Capsella plants are very slow to bolt allowing us to maintain cultures for months on the same plant. 
   Growth and maintenance of powdery mildews: Inoculum of  Erysiphe cichoracearum  was prepared by touch-inoculating three-week-old squash plants with infected squash leaves. The inoculum was ready for use after 10-12 days of growth. Arabidopsis plants were inoculated by placing approximately four foot high cardboard settling towers over 1 or 2 flats and gently tapping 1-4 squash leaves over the top of the settling towers. For measuring fungal growth it was important to have colonies originating from widely spaced single conidia. Therefore, the squash leaves were tapped over a 3-41/33 Nitex nylon monofilament screen (Sefar America Inc., Kansas City, Mo.) and the resulting powder was brushed back and forth to allow single conidia to fall through. After allowing spores to settle for about 15 minutes, the plants were placed in a dew chamber for 45-60 minutes as described (Adam, L. and S. C. Somerville 7 ). Upon removal from the dew chamber the plants were placed in a growth chamber matching the chamber they were grown in prior to inoculation. If the plants were grown in the greenhouse prior to inoculation they were placed in a phytocell, a completely enclosed greenhouse with precise temperature control, after inoculation. The phytocell typically maintained 85-100% relative humidity. 
     E. orontii  was maintained on Capsella by simply placing uninfected plants in a growth chamber with infected plants and allowing them to be inoculated by air movement in the growth chamber. Capsella plants grown in this fashion could supply inoculum for several months as new leaves continually grow and become infected. Arabidopsis plants were inoculated by placing a 65 cm tall inoculation tower over a 13.5 cm pot and tapping 4-6 heavily infected Capsella leaves over the top of the tower. After allowing five minutes for the spores to settle the pot was placed directly into the growth chamber. 
   Isolation of mutants: Mutagenized seeds were sown on flats and placed at 4° for 2-4 days then moved to a greenhouse. Approximately two weeks after germination plants were thinned to approximately one plant per six cm 2 . When the plants had at least four true leaves, after 17-21 days of growth, the plants were inoculated with powdery mildew. One week later the plants were screened and plants with reduced powdery mildew growth were retained. 
   Genetic mapping: Mutants were crossed with WS ecotype plants to create F 2  mapping populations. F 2  seeds was planted as described for screening and DNA was extracted from resistant plants, homozygous mutant, as described (Edwards, K, C. Johnstone, and C. Thompson 8 ) PCR-based markers, SSLP and CAPS, polymorphic between Col and WS were used to map the mutations based on co-segregation of markers with the mutation. 
   Northern blot analysis: To evaluate the correlation between defense gene expression and powdery mildew resistance, the levels of PR1 mRNA after infection was determined. Plants were heavily inoculated with  E. cichoracearum  and 15 plants were harvested for each point at 0,1,3 and 5 days after inoculation. Plants were frozen in liquid nitrogen and ground using mortar and pestle. Total RNA was prepared using TRIZOL reagent following manufacturers instructions (Gibco). Ten ug of RNA was separated using glyoxal gel electrophoresis and blotted to nylon membrane. Blots were also hybridized to a rDNA probe to control for loading. Signals were visualized and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) 
   Microscopy: To visualize fungal hyphae, infected leaves were cleared in 95% ethanol until completely white and then stained with 250 ug/ml trypan blue in a 1:1:1 solution of lactic acid: glycerol: water for 15 minutes, rinsed in lactic acid: glycerol: water solution and mounted with 50% glycerol. Individual colonies were photographed at 50-100×magnification using a digital camera (Pixara) attached to a Leica microscope. Total hyphal length was measured using NIH image software. 
   To visualize microscopic lesions, leaves were stained using a modification of a previously described procedure (Bowling, S. A., et al. 9 ). Leaves were placed in a 1:1:1:1 solution of phenol, lactic acid, glycerol, water plus 250 ug/ml trypan blue. Samples were then placed in a bell jar and a vacuum was applied and slowly released twice. Next the tubes containing the leaves and stain were placed in a boiling water bath for two minutes and then allowed to cool for at least one hour. The leaves were destained in the 1:1:1:1 solution used for staining, without trypan blue, for at least two hours. The samples were mounted in 50% glycerol and examined under bright field illumination. 
   To visualize callose, leaves were cleared in 95% ethanol and then placed in 0.02% analine blue in 150 mM potassium phosphate dibasic, pH&gt;9, and allowed to stain for at least on hour. Leaves were then mounted in 50% glycerol and examined for fluorescence as described (Adam, L. and S. C. Somerville 7 ). 
   Autoflourescent compounds were detected by clearing leaves in 95% ethanol, equilibrating them in a 1:1:1 solution of lactic acid: glycerol: water and mounting in 50% glycerol. The leaves were examined under epifluorescent illumination as described (Adam, L. and S. C. Somerville 7 ). 
   Cloning: Four alleles of pmr6 were identified in our screen, two were derived from T-DNA mutagenized populations and two were derived from EMS mutagenized populations. Plant DNA flanking the site of insertion for pmr6-3 was recovered using the genome walker kit following manufactures instructions (Clontech). The following oligonucleotides, specific for the T-DNA used in the mutagenesis, were used to amplify plant DNA flanking the left border region: LB1-AACTTGATTTGGGTGATGGTTCACGTAGTG (SEQ ID NO; 9) and LB2-GCCCTGATAGACGGTTTTTCGCCCTTTGAC (SEQ ID NO: 10); and the right border region RB1-CAATCCATCTTGTTCAATCATGCGAAACGA (SEQ ID NO: 11) and RB2-CGACTTTTGAACGCGAATAATGGTTTCTG (SEQ ID NO: 12). The PCR products obtained for both the left and right border were sequenced using BigDye cycle sequencing kit (ABI Prism) and an ABI310 sequencer. The site of the T-DNA insertion in pmr6-4 was determined by sequencing a DNA fragment amplified by PCR using one primer in the T-DNA, JV4-AGGTCTRGCGAAGGATAGTGGGATTGT (SEQ ID NO: 13), and another primer in the putative gene, 3417-ATGTATGCTATTGGTGGTAGTG (SEQ ID NO: 14). The mutations in the two EMS alleles was determined by sequencing a DNA fragment amplified with primers flanking the gene, 2026-GAGAGGCCCAATATAAAATACGAG (SEQ ID NO: 15) and 6097-ATAATCCGGACTGGAAAAACAAAC (SEQ ID NO: 16). 
   pmr5 was mapped 1.7 cM above the CAPS marker LFY3. In that same region lies the BAC MDF20 (SEQ ID NOs: 7 and 8), genbank number AB009050 and on that BAC lies a putative gene homologous to PMR6. Given the very similar phenotypes of pmr5 and pmr6, we propose that the pmr5 phenotype is due to a mutation in this pectate lyase-like gene. To test this hypothesis, the gene MDF20 will be amplified using the primers, MD3′-ATCCCGGGAATACAAGAACTTGACAGCTCC (SEQ ID NO: 17) and MD5′-TACCCGGGGAAGCAGAGATCCTC (SEQ ID NO: 18), and the resulting DNA will be transformed into pmr5 to see if it can complement the pmr5 mutation. 
   Phylogeny of pectate lyase genes: The protein or predicted protein sequences from several plant pectate lyase genes were aligned and a phylogenic tree constructed using clustalX software. The output from clustalX was displayed using TreeViewPPC software (FIG.  8 ). 
   Results 
   Isolation of mutants and genetics: Five mutants were found that are examples of the invention taught by this patent Crosses between mutants show that two complementation groups that are strongly resistant to powdery mildew are defined by this collection (FIG.  1 ). The first mutant has been designated powdery mildew resistant 5, pmr5, The remaining four mutants are allelic and have been designated pmr6-1 through pmr6-4. All mutants were backcrossed to Col plants two times prior to all characterizations, excluding mapping. Segregation of powdery mildew resistance in the F1 and F2 generations indicated that all mutants are recessive, nuclear genes that segregate in a simple 3.1 Mendelian fashion ( See Table 1). In addition to powdery mildew resistance, both pmr5 and pmr6 are smaller than wild-type, more compact (shorter petioles) and have leaves cupped up rather than down as in wild-type (FIG.  1 ). In addition, pmr5 seems to be somewhat more branched than wild-type. 
                                                                                         TABLE 1                   Genetic analysis of powdery mildew resistant mutants.                        Disease response                        Suscep-   Resis-           Cross (female × male)   Type   Total   tible   tant   X 2                      PMR5/PMR5 × pmr5/pmr5   F1   13   13   0           PMR5/PMR5 × pmr5/pmr5   F2   98   74   22   0.22 a ;                           P &gt; 0.05       PMR6/PMR6 × pmr6/pmr6   F1   20   20   0       PMR6/PMR6 × pmr6/pmr6   F2   486   257   129   0.62 a ;                           P &gt; 0.05                    
A Chi-squared calculated for an expected 3:1, wild-type:mutant ratio
 
   The position of each locus on the Arabidopsis genetic map was determined using PCR-based CAPS and SSLP markers. pmr5 mapped to chromosome 5,1.7 cM above LFY3 based on 116 chromosomes. pmr6 was mapped to chromosome 3, 9.4 cM below R30025 and 4.2 cM above nga707 based on 96 chromosomes. 
   Timecourse of fungal growth: In order to determine if the resistance observed in the mutants was correlated with a block at a defined stage of fungal development, the growth of hyphae and level of conidiation was measured over time using trypan blue staining. The same trend was apparent for both pmr5 and pmr6 mutants. After landing on a leaf, fungal spores germinate and begin growth in a normal fashion. However, colonies on mutant plants grew slower than colonies on wild-type plants. Two days after inoculation hyphal length/colony is significantly less than wild-type (FIG.  2 ). Eventually, many colonies appear to be dead or dying (shriveled and broken hypha) on the mutants, while colonies on wild-type plants have hundreds of conidiophores (FIG.  2 ). However, on both pmr5 and pmr6plants a small subset of colonies do produce some conidiophores indicating that neither of the mutations results in a complete block of any stage of fungal development. 
   Resistance to another powdery mildew: To determine if the powdery mildew resistance observed in pmr5 and pmr6 was broad spectrum or restricted to  E. cichoracearum  the mutants were challenged with another species of powdery mildew,  E orontii.  Plants were inoculated with  E. orontii  and symptoms were scored on a disease response, DR, scale from 0=fully resistant to 4=fully susceptible 7-10 days after inoculation The average DR scores based on nine replicates were: Col 3.6, pmr5 0 and pmr6 0.1, indicating that the mutants are strongly resistant to  E. orontii.    
   Cytological characterization: To see if the accumulation of callose, autoflourescent compounds or the presence of microscopic lesions was correlated with resistance or were constitutively expressed in pmr5 or pmr6, infected leaves were examined to look for these common defense responses. At least eight leaves from several infected plants were examined with each stain at zero, one and five days post inoculation, d.p.i. Many fungal colonies were examined on each leaf. The entire experiment was repeated at least twice for each stain. 
   Trypan blue staining was used to visualized dead and dying cells, a hallmark of the hypersensitive response, a common defense employed by plants. Interestingly, both pmr5 and pmr6 had occasional clusters of what appear to be individual dead, crushed mesophyll cells closely associated with veins toward leaf tips (FIG.  3 ). Col plants did not display any microlesions prior to infection. However, there was no apparent increase in lesions on pmr5 or pmr6 leaves in response to powdery mildew infection at one or 5 d.p.i. when compared to Col. Indeed, no lesions were found to be associated with fungal colonies on pmr5, pmr6 or Col at one d.p.i. After five days, occasional dead epidermal cells beneath a small percentage of colonies were seen on all plants examined. The fact that the majority of colonies on the mutants were not associated with dead epidermal cells, but were stunted, indicates that cell death is not the primary mechanism of resistance in the mutants. 
   Another defense strategy plants employ to limit pathogen growth is the production of antimicrobial autoflourescent compounds. To determine if levels of autoflourescent compounds were increased in the mutants cleared, unstained leaves were examined under epifluorescent illumination as described (Adam, L. and S. C. Somerville 7 ). Both pmr5 and pmr6 constitutively accumulate large amount of autoflourescent compounds, again focused along veins toward the leaf tips (FIG.  3 ). As with the microlesions, the level of autoflourescent compounds did not noticeably increase after infection with powdery mildew. No autoflourescent compounds were seen in Col leaves before or after infection with powdery mildew. 
   In response to penetration attempts by powdery mildews, plants reinforce the cell wall directly beneath the penetration peg by depositing new cell wall material creating a cell wall apposition called a papilla Papilla formation is thought to be an attempt by the plant to prevent invasion by the fungus. Enhanced papilla formation is correlated with the resistance of some barley cultivars and is proposed to be a mechanism for the resistance (Jorgensen, J. H. 10 ). To address the possibility that the mutants may have enhanced papilla formation, we stained infected leaves for callose, a component of the papilla, using analine blue. Col, pmr5 and pmr6 plants all produced similar papilla in response to fungal penetration attempts (data not shown). However, a small amount of callose not associated with the fungus was apparent in both mutants. Examination of uninfected leaves revealed that this callose accumulation is constitutive. Similar to the microlesions, callose deposits are concentrated along veins toward the leaf tips. However, the bright background of autoflourescent compounds made the determination of the full extent of the callose deposits impossible. 
   Kinetics of PR1: In order to determine if the resistance observed in the mutants was correlated with increases in the expression of a known defense gene northern blots were used to determine the steady state levels of MnRNA for the PR1 gene at various times after inoculation. PR1 was chosen as a marker for the SA-dependent SAR pathway (Ryals, J. A., et al. 11 ). Both pmr5 and pmr6 constitutively express variable levels of PR1 mRNA ranging from almost no expression, similar to wild-type, to levels higher than pathogen infected Col plants. One day after infection pmr5 and pmr6 typically have higher levels of PR1 than Col plants (FIG.  4 ). However, by three days after infection the levels of PR1 in both mutants are typically not as high as wild-type levels. 
   Cloning: Plant DNA flanking the T-DNA insert in pmr6-3 was isolated using the genome walker kit. A database search using the flanking DNA indicated that the region was contained on a sequenced BAC, F28P10. After the BAC was annotated the gene corresponding to PMR6 was designated F28P10.100, from nucleotide 38336 to 41968. NetGene software indicated that the insertion site lies within a predicted gene. A putative cDNA and the corresponding predicted protein sequence were pieced together from the genomic sequence (FIG.  5 ). The genomic sequence including upstream and down stream regions is shown in  FIG. 5A. A  partial cDNA sequence is shown in FIG.  5 B. The predicted amino acid sequence for PMR6 is shown in FIG.  5 C. Further BLAST searches using the predicted cDNA and protein sequences indicated that the gene had homology to the pectate lyase gene family. Also, one EST matched our tagged sequence and was obtained from the ABRC stock center. However, this clone was not full length, so a RT-PCR product was sequenced to verify the predicted message sequence. 
   To verify that the gene disruption in pmr6-3 was responsible for the powdery mildew resistant phenotype, the lesions in three independent pmr6 alleles identified by sequence analysis (FIG.  6 ). pmr6-4 contained a T-DNA insertion at nt 2868. pmr6-1 had a G to A transition that results in a substitution of aspartic acid for glycine at amino acid 140. pmr6-2 had a G to A transition at nt 2908 which results in miss-splicing of the mature message. The miss-splicing was verified by sequencing a RT-PCR product from pmr6-2. The miss-splicing results in a single base deletion causing a frameshift that alters the last 85 amino acids of the protein. 
   Arabidopsis contains at least 16 putative pectate lyase family members, one of which, on BAC MDF20, maps very close to pmr5. The sequence of a portion of BAC MDF20 with homology to pectate lyase is shown in FIG.  7 .  FIG. 7A  shows the genomic sequence including upstream and down stream regions. Coding regions are shown in CAPS.  FIG. 7B  shows the predicted cDNA sequence with the predicted open reading frame shown in CAPS.  FIG. 7C  shows the predicted amino acid sequence. Given the extremely similar phenotypes of pmr5 and pmr6 it seems likely that the pmr5 phenotype could also be due to a mutation in a pectate lyase gene. To address this hypothesis we are currently sequencing the putative gene from pmr5 plants to look for mutations and are also transforming the wild-type gene into pmr5 to see if it can complement the mutation. 
   Phylogeny of pectate lyase genes: A comparison of several plant pectate lyase and putative pectate lyase genes was conducted to see where PMR6 and the gene on MDF20 fit into the gene family. The predicted protein sequences were aligned using clustalX software and the output was visualized graphically by constructing a phylogram using TreeViewPPC software (FIG.  8 ). It appears that MDF20 and PMR6 are not very closely related, in fact, they are about as closely related to nine other Arabidopsis homologs as they are to each other. This suggests that decreasing the activity of any pectate lyase may be result in disease resistant plants. 
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