bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Plasmid Encoded Virulence Region pat-1 of Phytopathogenic Clavibacter michiganensis subsp. michiganensis
Authors: Dreier Jens , Meletzus Dietmar , Eichenlaub Rudolf
Published: May 2007

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence. The region of plasmid pCM2 encoding the pathogenicity locus pat-1 was mapped by deletion analysis and complementation studies to a 1.5-kb BglII/SmaI DNA fragment. Introduction of the pat-1 region into endophytic, plasmid-free isolates of C. michiganensis subsp. michiganensis converted these bacteria into virulent pathogens. Based on the nucleotide sequence of the pat-1 region, an open reading frame (ORF1) can be predicted, coding for a protein of 280 amino acids and 29.7 kDa with homology to serine proteases. Introduction of a frame-shift mutation in ORF1 leads to a loss of the pathogenic phenotype. Northern (RNA) hybridizations identified an 1.5-knt transcript of the pat-1 structural gene. The site of transcription initiation was mapped by primer extension and a typical -10/-35 region was located with significant homology to the consensus Escherichia coli σ70 and Bacillus subtilis σ43 promoters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem repeats preceded by a run of 14 guanosine residues. DNA sequences homologous to pat-1rep were isolated and characterized from four virulent C. michiganensis subsp. mich-iganensis strains exhibiting a high extent of structural conservation. The deletion of this repetitive sequence reduced virulence significantly but did not lead to a complete loss of the virulence phenotype.
Discrete Regions of the Sensor Protein VirA Determine the Strain-Specific Ability of Agrobacterium to Agroinfect Maize
Authors: Heath J. D. , Boulton M. I. , Raineri D. M. , Doty S. L. , Mushegian A. R. , Charles T. C. , Davies J. W. , Nester E. W.
Published: May 2007

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348, to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.
A New Medicago truncatula Line with Superior in Vitro Regeneration, Transformation, and Symbiotic Properties Isolated Through Cell Culture Selection
Authors: Hoffmann Beate , Trinh Toan Hanh , Leung Jeffrey , Kondorosi Adam , Kondorosi Eva
Published: May 2007

The understanding of how leguminous plants establish symbiosis with rhizobia is limited by the lack of genetic system in most legume plants. Here we propose a Medicago truncatula line suitable for genetic analysis of Medicago-Rhizobium meliloti symbiosis because of its high regeneration capacity and broad R. meliloti strain specificity. This line has been isolated by extensive in vitro screening of explants from several ecotypes of M. truncatula that are known to be autogamous and have small diploid genomes. One such derivative identified from ecotype 108-1 has gained the capacity to be readily regenerated in vitro. The derivative, called R108-1, like its parent, can establish effective symbiosis with several widely studied R. meliloti strains (Rm41, Rm2011, Rm1021, F51, and GR4). Importantly, the nodulation characteristics of the R108-1 line were not compromised by the in vitro selection procedure. We have further established the conditions for efficient transformation of R108-1 by co-cultivation with Agrobacterium tumefaciens. Thus, this in vitro-derived plant line has most of the favorable attributes, otherwise rare among legumes, of a complementary model plant system for investigating symbiosis at the genetic and the molecular levels.
Lipopeptide Phytotoxins Produced by Pseudomonas syringae pv. syringae: Comparison of the Biosurfactant and Ion Channel-Forming Activities of Syringopeptin and Syringomycin
Authors: Hutchison Michael L. , Gross Dennis C.
Published: May 2007

The phytopathogenic bacterium Pseudomonas syringae pv. syringae produces two classes of necrosis-inducing lipodepsipeptide toxins commonly referred to as the syringomycins and syringopeptins. Members of the syringomycin class are pore-forming cytotoxins that act by promoting passive transmembrane ion flux. In this study, we test the hypothesis that syringopeptin forms SP22A and SP22B likewise function as pore-forming cytotoxins and are similar in activity to syringomycin in artificial and plant membranes. Correspondingly, syringopeptin increased the conductance of black-lipid membranes in a manner indicative of ion channel formation. In tobacco protoplast assays, syringopeptin forms SP22A and SP22B were equivalent in activity causing lysis of protoplasts and measurable 45Ca2+ influx at a threshold concentration of 50 ng/ml. A mixture of three forms of syringomycin did not show cytotoxic activity appreciably different from that of SP22A or SP22B in tobacco protoplast assays. Both forms of syringopeptin also displayed potent biosurfactant properties demonstrated by lowering of the interfacial tension of high-pressure liquid chromatography-grade water to 36 and 34.5 nm/m, respectively; the critical micellar concentration was 0.8 mg/ml for both forms of toxin. These results demonstrate that both classes of pore-forming lipodepsipeptides secreted by P. syringae pv. syringae are cytotoxic to plant cells at nanomolar concentrations and cause necrosis by forming ion channels that are freely permeable to divalent cations.
Response of Symbiotic Endomycorrhizal Fungi to Estrogens and Antiestrogens
Authors: Poulin Marie-Josée , Simard Jacques , Catford Jean-Guy , Librie Fernand , Piché Yves
Published: May 2007

Plant flavonoids reported previously to act as molecular signals in the arbuscular mycorrhizal (AM) symbiosis are known to bind to estrogen receptors and to exert estrogenic effects on mammalian cells. To further investigate the estrogen-like properties of flavonoids the present study examined whether estrogen and antiestrogens have fla-vonoid-related functions in AM fungi. Bioassays were performed in a monoaxenic system with the AM fungi Giga-spora margarita and Glomus intraradices. The plant flavo-noids quercetin and biochanin A stimulate hyphal growth of G. margarita and G. intraradices, respectively. The stimulatory activity of biochanin A studied at concentrations ranging from 0.01 to 10.0 μM shows an estimated EC50 value of 3.26 μM. The present results show that 17β-estradiol and biochanin A exert similar stimulatory effects in G. intraradices. The agonist effect of biochanin A was efficiently suppressed by the new antiestrogen EM-652, which is also consistent with the possible presence of estrogen-binding sites in AM fungi.
Salicylate-Independent Lesion Formation in Arabidopsis lsd Mutants
Authors: Hunt Michelle D. , Delaney Terrence P. , Dietrich Robert A. , Weymann Kris B. , Dangl Jeffery L. , Ryals John A.
Published: May 2007

In many interactions of plants with pathogens, the primary host defense reaction is accompanied by plant cell death at the site of infection. The resulting lesions are correlated with the establishment of an inducible resistance in plants called systemic acquired resistance (SAR), for which salicylic acid (SA) accumulation is a critical signaling event in Arabidopsis and tobacco. In Arabidopsis, the lesions simulating disease (lsd) mutants spontaneously develop lesions in the absence of pathogen infection. Furthermore, lsd mutants express SAR marker genes when lesions are present and are resistant to the same spectrum of pathogens as plants activated for SAR by necrogenic pathogen infection. To assess the epistatic relationship between SA accumulation and cell death, transgenic Arabidopsis unable to accumulate SA due to the expression of the salicylate hydroxylase (nahG) gene were used in crosses with the dominant mutants lsd2 or lsd4. Progeny from the crosses were inhibited for SAR gene expression and disease resistance. However, these progeny retained the spontaneous cell death phenotype similar to siblings not expressing nahG. Because lesions form in the absence of SA accumulation for lsd2 and lsd4, a model is suggested in which lesion formation in these two mutants is determined prior to SA accumulation in SAR signal transduction. By contrast, the loss of SAR gene expression and disease resistance in nahG-expressing lsd mutants indicates that these traits are dependent upon SA accumulation in the SAR signal transduction pathway.
Synthetic Peptide Combinatorial Libraries: A Method for the Identification of Bioactive Peptides Against Phytopathogenic Fungi
Authors: Reed Jerry D. , Edwards David L. , Gonzalez Carlos F.
Published: May 2007

Synthetic combinatorial libraries were evaluated with an iterative process to identify a hexapeptide with broad-spectrum activity against selected phytopathogenic fungi. A D-amino acid hexapeptide (FRLKFH) and pentapeptide (FRLHF) exhibited activity against Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani (anastomosis group 1), Ceratocystis fagacearum, and Pythium ultimum. The peptides showed no hemolytic or mutagenic activity. Fluorescent microscopy studies with a membrane impermeant dye indicated that fungal cytoplasmic membranes were compromised rapidly and that the nuclear membrane was also affected.
Mutation of the Tobacco Mosaic Tobamovirus 126-and 183-kDa Proteins: Effects on Phloem-Dependent Virus Accumulation and Synthesis of Viral Proteins
Authors: Derrick Peter M. , Carter Shelly A. , Nelson Richard S.
Published: May 2007

The masked and U1 strains of tobacco mosaic tobamovirus differ in symptom phenotype and in phloem-dependent accumulation in tobacco. The symptom phenotype is determined by eight amino acids in the 126- and 183-kDa proteins that differ between the two strains. In this study, slow phloem-dependent accumulation of the masked strain was shown to be determined by these same eight amino acids, but some symptomatically severe mutants altered at specific positions within the eight amino acids were inefficient in phloem-dependent accumulation. Therefore, the appearance of severe symptoms does not require rapid phloem-dependent accumulation. There was no consistent relationship between the accumulation of virus coat protein, movement protein, or 126- and 183-kDa protein in inoculated protoplasts and the efficiency of phloem-dependent accumulation in stem tissue. Therefore, the difference in phloem-dependent accumulation between the masked strain, its mutants, and the U1 strain in most instances resulted from the functional competence of the 126- and/or 183-kDa proteins or a host response to their change and not from their quantities or the quantities or functional competence of the movement proteins or coat proteins.
The Presence of hrp Genes on the Pathogenicity-Associated Plasmid of the Tumorigenic Bacterium Erwinia herbicola pv. gypsophilae
Authors: Nizan Roni , Barash Isaac , Valinsky Lea , Lichter Amnon , Manulis Shulamit
Published: May 2007

The pathogenicity-associated plasmid (pPATH) of Erwinia herbicola pv. gypsophilae (Ehg), which is present only in pathogenic strains, contains a gene cluster encoding indole-3-acetic acid and cytokinin biosynthesis. The transposon-reporter Tn3-Spice was used to generate nonpathogenic mutants on two overlapping cosmids, pLA150 and pLA352, of the pPATH. A cluster of such mutations, which spanned 16 kb, mapped approximately 15 kb from the gene cluster involved in phytohormone biosynthesis. Non-pathogenic mutants also failed to elicit the hypersensitive reaction (HR) on tobacco. Pathogenicity and HR were restored concomitantly to these mutants by in trans complementation with wild-type Ehg DNA. A 3.8-kb HindIII DNA fragment that complemented the hrp mutants was sequenced and six complete and two partial open reading frames (ORFs) were identified. Comparison of the deduced amino acid sequences of the eight ORFs showed striking homology and co-linearity with hrp genes of E. amylovora as well as with other plant and mammalian pathogenic bacterial genes encoding proteins of the type III secretion system. Limited DNA sequencing at various sites on the remaining 11-kb region of pLA352 also showed high identity to Hrp proteins of E. amylovora, E. stewartii, and Pseudomonas syringae. These results suggest that hrp genes are mandatory for gall formation by E. herbicola pv. gypsophilae.
Deletion of Two Endo-β-1,4-Xylanase Genes Reveals Additional Isozymes Secreted by the Rice Blast Fungus
Authors: Wu Sheng-Cheng , Ham Kyung-Sik , Darvill Alan G. , Albersheim Peter
Published: May 2007

Fungal pathogens secrete hydrolases during infection of plant tissues capable of fragmenting the primary cell wall polysaccharides of the host. Magnaporthe grisea, the fungal pathogen that causes blast disease of graminaceous monocots, secretes two distinct endo-β-1,4-D-xylanases when grown on xylan-rich rice cell walls as the carbon source. We have previously reported the cloning of the genes encoding these two xylanases, XYL1 and XYL2 (formerly XYN22 and XYN33, respectively; see S.-C. Wu, S. Kauffmann, A. G. Darvill, and P. Albersheim, Mol. Plant-Microbe Interact. 8:506–514, 1995). We now present three M. grisea mutants created by selective deletion of XYL1 and/or XYL2. The xyl1 mutant grows as well as the parent in culture medium when rice cell walls or xylan is the sole carbon source. Under the same conditions, the xyl2 mutant grows slightly slower than the parent, whereas the xyl1/xyl2 double mutant exhibits a 50% reduction in accumulation of total mycelial mass. Under conditions idealized for infection, all three mutants infect host plants as efficiently as the parent, indicating neither XYL1 nor XYL2 is required by M. grisea for infection. Endoxylanase assays showed that, at the stationary stage of growth in culture when the accumulation of total xylanase activity is at its maximum, the xyl1 mutant retains ≈ 88%, xyl2, 39%, and xyl1/xyl2, 19% of the endoxylanase activity of the parent. Partial protein purification of xylanases secreted by the xyl1/xyl2 double mutant revealed four distinct endoxylanase activities. One of the xylanases, XYL3, is present in the culture filtrate of both the parent and the mutant strains, and, like XYL1, has been identified as a member of the Family G xylanases. The other three xylanases are not found in the culture filtrates of the parent or of the xyl1 mutant. Thus, M. grisea appears capable of secreting additional xylanases and does so when XYL2, a member of the Family F glycanases, has been eliminated.
Identification of the TMV Replicase Sequence That Activates the N Gene-Mediated Hypersensitive Response
Authors: Padgett Hal S. , Watanabe Yuichiro , Beachy Roger N.
Published: May 2007

The N gene-mediated hypersensitive response (HR) in tobacco provides a high degree of resistance against most tobamoviruses by halting the progress of infection at the site of inoculation. A previous report indicated a role for the 126/183-kDa replicase in induction of the HR in tobacco containing the N gene (H. S. Padgett and R. N. Beachy, Plant Cell 5:577-586, 1993). Chimeric virus genomes were constructed in which the genes encoding the 126/183-kDa proteins of the HR-eliciting pathogen, tobacco mosaic virus (TMV), and the resistance breaking tobamovirus, Ob, were exchanged. Inoculation of the chimeric viruses to leaves of Nicotiana tabacum cv. Xanthi NN confirmed that either the replicase protein of TMV or its mRNA was responsible for induction of HR. An expression vector based on the Ob virus was used to express fragments of various replicase genes. With this approach, it was determined that the HR is caused by a portion of the replicase protein extending from amino acid 692 to 1116. Consistent with this result, Ob mutants that induce the HR on NN tobacco were found to carry mutations within the same portion of the replicase gene. The N gene-mediated HR is inactive at high temperatures, yet these mutants were able to overcome the HR at significantly lower temperatures than could TMV, indicating that the temperature sensitivity of the N gene response is manifested at the level of interaction between the virus and the defense response mechanism.
Tobamovirus Coat Proteins: Elicitors of the Hypersensitive Response inSolanum melongena(Eggplant)
Authors: Dardick Christopher D. , Culver James N.
Published: May 2007

Solanum melongena (eggplant) exhibits a hypersensitive response (HR) when infected with tobacco mosaic tobamovirus (TMV). In contrast, a TMV mutant unable to express coat protein (CP) did not elicit the HR, while a potexvirus vector engineered to express TMV CP did elicit the eggplant HR. The CPs of U2 and odontoglossum ringspot tobamoviruses also elicited the HR. However, the HR was not elicited by the CP of cucumber green mottle mosaic tobamovirus. Taken together, these findings demonstrate that specific tobamovirus CPs function as elicitors of the eggplant HR.
Spatial Distribution of Acidic Chitinases and Their Messenger RNAs in Tobacco Plants Infected with Cherry Leaf Roll Virus
Authors: Balsalobre J. M. , Más P. , Sánchez-Pina M. A. , Pallás V.
Published: May 2007

Cherry leaf roll virus (CLRV) infection in Nicotiana tabacum cv. Xanthi nc plants induces the expression of, among others, basic and acidic chitinases at 3 d.p.i. (days postinoculation) and 8 d.p.i. in inoculated and uninoculated, systemically infected leaves, respectively. The spatial distribution of the acidictobacco chitinases (PR-P and PR-Q) mRNAs in CLRV-infected tobacco leaves has been studied by tissue printing and Northern (RNA) blot analysis. The mRNA was preferentially detected in and around the concentric necrotic rings caused by the viral infection. In addition, the accumulation pattern of the proteins was very similar although a basal signal was detected in nonsymptomatic tissue in uninoculated, systemically infected leaves. These results indicate a positive association of acidic chitinase gene expression with the necrotic tissue, where the virus is mainly localized, in the CLRV-tobacco interaction.
Induction of Extracellular Matrix Glycoproteins in Brassica Petioles by Wounding and in Response to Xanthomonas campestris
Authors: Davies Huw A. , Daniels Michael J. , Dow J. Maxwell
Published: May 2007

A panel of monoclonal antibodies that recognize plant extracellular matrix glycoproteins previously implicated in plant-microbe interactions was used to study the effects of pathogen inoculation and wounding on glycoproteins in petioles of Brassica campestris. The panel of monoclonals comprised two sets: JIM11, JIM12, and JIM20 recognize epitopes carried on hydroxyproline-rich glycoproteins (HRGPs) (M. Smallwood, A. Beven, N. Donovan, S. J. Neill, J. Peart, K. Roberts, and J. P. Knox, Plant J. 5:237–246, 1994); MAC204 and MAC265 recognize glycoproteins of the Rhizobium infection thread (K. A. VandenBosch, D. J. Bradley, S. Perotto, G. W. Butcher, and N. J. Brewin, EMBO J. 8:335–342, 1989). Wounding or inoculation of petioles with avirulent strains of pathovars of Xanthomonas campestris induced the synthesis of two new groups of antigens: gp160 ran as a smear on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with apparent molecular mass from 120 to 200 kDa and was recognized by JIM20 and MAC204; gpS remained in the stacking gel on SDS-PAGE and was recognized by JIM11, JIM20, and MAC204. The response to virulent strains of pathovars of X. campestris was either less pronounced or absent. gpS comprised several components that were resolved by cation-exchange chromatography. Some of these components were characterized as extensin-like HRGPs. The level of induction of the gpS group of antigens by virulent strains was not altered by mutation of a number of genes required for basic pathogenicity or by heat-killing the bacteria.
ThephtELocus in the Phaseolotoxin Gene Cluster Has ORFs with Homologies to Genes Encoding Amino Acid Transferases, the AraC Family of Transcriptional Factors, and Fatty Acid Desaturases
Authors: Zhang Yuan Xin , Patil Suresh S.
Published: May 2007

A cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, contains eight (phtA through phtH) complementation groups (Y. X. Zhang, K. B. Rowley, and S. S. Patil, J. Bacteriol., 175:6451–6458, 1993). In this study, sequencing of the region encompassing the phtE locus revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction. Reverse transcription-polymerase chain reaction suggested that the phtE locus is transcribed as one large (6.4 kb) transcript, indicating that the ORFs constitute an operon. Primer extension analysis showed that the transcript begins at a T, located 31 bp upstream of the ATG codon of ORF1. Comparison of the sequences of the putative ORFs with the sequences of known genes revealed that ORF3, encoding a protein containing 395 amino acids, has 55% similarity to the acetylornithine aminotransferase gene from Escherichia coli, and the ornithine aminotransferase genes from other organisms. A lysine residue that is a binding site for pyridoxal phosphate and an arginine residue that is a binding site for the α-carboxylate group of the substrate are conserved in ORF3. These data suggest that ORF3 encodes a protein involved in the biosynthesis of ornithine, a constituent of phaseolotoxin. ORF5, encoding a peptide of 378 amino acid residues, possesses a helix-turn-helix motif at the C-terminal end that is characteristic of the AraC family of transcriptional factors, and there is a possible leucine zipper at the N-terminal end of this peptide. ORF6, encoding a protein of 327 amino acids, has about 40% similarity with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803 and considerable similarity with fatty acid desaturase genes from other organisms. ORF6 and desA show very similar hydropathy profiles and both contain a copper binding signature. Computer searches did not discover significant homologies in the data base for the other ORFs, but hydropathy analysis showed that all of them contain one to several hydrophobic domains, suggesting that the gene products of these ORFs may be membrane associated.
Cloning and Characterization of a cDNA Encoding an Elicitor of Phytophthora parasitica var. nicotianae That Shows Cellulose-Binding and Lectin-Like Activities
Authors: Mateos François Villalba , Rickauer Martina , Esquerré-Tugayé Marie-Thérèse
Published: May 2007

Phytophthora parasitica var. nicotianae produces a 34-kDa glycoprotein elicitor (CBEL) that is localized in the cell wall. A cDNA encoding the protein moiety of this elicitor was cloned and characterized. The deduced amino acid sequence consisted of two direct repeats of a cysteine-rich domain, joined by a Thr/Pro-rich region. Although having no general homology with published sequences, the positions of the cysteine residues in the two repeats show a conserved pattern, similar to that of the cellulose-binding domain of fungal glycanases. CBEL did not possess hydrolytic activity on a variety of glycans, but bound to fibrous cellulose and plant cell walls. In addition, it exerted a lectin-like hemagglutinating activity. Infiltration of tobacco leaves (cultivar 46–8) with this molecule elicited necrosis and defense gene expression at 150 nM. Elicitor pre-treatment of this tobacco cultivar resulted in protection against subsequent inoculation with an otherwise virulent race of P. parasitica var. nicotianae. All these biological activities were exerted within a low concentration range. This is the first report that a fungal elicitor exhibits cellulose-binding and lectin-like activities. The possible implications of such a multifunctional elicitor in plant-microbe interactions are discussed.
G Protein α Subunit Genes Control Growth, Development, and Pathogenicity of Magnaporthe grisea
Authors: Liu Shaohua , Dean Ralph A.
Published: May 2007

Three G protein α subunit genes have been cloned and characterized from Magnaporthe grisea: magA is very similar to CPG-2 of Cryphonectria parasitica; magB is virtually identical to CPG-1 of Cryphonectria parasitica, to gna1 of Neurospora crassa, and to fadA of Emericella nidulans; and magC is most similar to gna2 of Neurospora crassa. Homologous recombination resulting in targeted deletion of magA had no effect on vegetative growth, conidiation, or appressorium formation. Deletion of magC reduced conidiation, but did not affect vegetative growth or appressorium formation. However, disruption of magB significantly reduced vegetative growth, conidiation, and appressorium formation. magB¯ transformants, unlike magA¯ and magC¯ transformants, exhibited a reduced ability to infect and colonize susceptible rice leaves. G protein α subunit genes are required for M. grisea mating. magB¯ transformants failed to form perithecia, whereas magA¯ and magC¯ transformants did not produce mature asci. These results suggest that G protein α subunit genes are involved in signal transduction pathways in M. grisea that control vegetative growth, conidiation, conidium attachment, appressorium formation, mating, and pathogenicity.
The Isolation and Mapping of Disease Resistance Gene Analogs in Maize
Authors: Collins N. C. , Webb C. A. , Seah S. , Ellis J. G. , Hulbert S. H. , Pryor A.
Published: May 2007

Many of the plant disease resistance genes that have been isolated encode proteins with a putative nucleotide binding site and leucine-rich repeats (NBS-LRR resistance genes). Oligonucleotide primers based on conserved motifs in and around the NBS of known NBS-LRR resistance proteins were used to amplify sequences from maize genomic DNA by polymerase chain reaction (PCR). Eleven classes of non-cross-hybridizing sequences were obtained that had predicted products with high levels of amino acid identity to NBS-LRR resistance proteins. These maize resistance gene analogs (RGAs) and one RGA clone obtained previously from wheat were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect co-segregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes.
A Model for the Catabolism of Rhizopine in Rhizobium leguminosarum Involves a Ferredoxin Oxygenase Complex and the Inositol Degradative Pathway
Authors: Bahar Masoud , de Majnik John , Wexler Margaret , Fry Judith , Poole Philip S. , Murphy Peter J.
Published: May 2007

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.
Biocontrol by Phenazine-1-carboxamide-Producing Pseudomonas chlororaphis PCL1391 of Tomato Root Rot Caused by Fusarium oxysporum f. sp. radicis-lycopersici
Authors: Chin-A-Woeng Thomas F. C. , Bloemberg Guido V. , van der Bij Arjan J. , van der Drift Koen M. G. M. , Schripsema Jan , Kroon Bernadette , Scheffer Rudy J. , Keel Christoph , Bakker Peter A. H. M. , Tichy Hans-Volker , de Bruijn Frans J. , Thomas-Oates Jane E. , Lugtenberg Ben J. J.
Published: May 2007

Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)-producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.
Interpopulation Signaling via N-Acyl-Homoserine Lactones among Bacteria in the Wheat Rhizosphere
Authors: Pierson Elizabeth A. , Wood Derek W. , Cannon Jeffrey A. , Blachere Francoise M. , Pierson Leland S.
Published: May 2007

The biological control bacterium Pseudomonas aureofaciens 30-84 utilizes an N-acyl-homoserine lactone (AHL) signal molecule to control phenazine antibiotic production in the wheat rhizosphere (D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). In this study, we demonstrate that naturally co-existing, non-isogenic bacterial populations interact with strain 30-84 at the level of gene expression via the exchange of diffusible signals on wheat roots. Wheat plants from three geographic locations were used to generate a random library of 700 rhizobacterial isolates. Roughly 8% of the isolates from each location restored phenazine gene expression to an AHL-deficient strain of 30-84 in vitro. Five of these isolates were further tested for their ability to influence gene expression of an AHL-deficient reporter of strain 30-84 on wheat roots. All five, isolated from different geographic locations, restored phenazine gene expression by the reporter to wild-type levels. This suggests that in vitro assays can identify bacterial isolates with the potential to influence phenazine expression in strain 30-84 via AHLs on wheat roots. The occurrence of such strains in all fields sampled suggests that AHL-mediated communication is a common occurrence in the wheat rhizosphere.
The Maize Lethal Leaf Spot 1 Mutant Has Elevated Resistance to Fungal Infection at the Leaf Epidermis
Authors: Simmons Carl , Hantke Sabine , Grant Susan , Johal Gurmukh S. , Briggs Steven P.
Published: May 2007

The maize lethal leaf spot 1 (lls1) mutant exhibits enhanced resistance to fungal pathogens. The lls1 resistance to Cochliobolus heterostrophus has two components: (i) lesion number is reduced 40% relative to wild type; and (ii) the lesions that do form often do not contain viable fungus. This lesion sterility is dependent upon leaf maturity and light, whereas reduced lesion number is not. The lls1 lesions express pathogenesis-related proteins at high levels, so lesion sterility likely results from activation of defense systems and necrosis. Reduced lesion number is correlated with a reduction of C. heterostrophus spore germination, hyphal growth, and haustoria formation on the leaf epidermis. The rust pathogen Puccinia sorghi has reduced pustule formation on lls1, and its germination and growth are also slowed on the epidermis. However, after entering the mesophyll through stomata, P. sorghi can form pustules on lls1, and even green islands within necrotic lls1 lesions. In situ mRNA hybridization shows that Lls1 is predominantly expressed in the leaf epidermis, coincident with the site of resistance in the mutant.
Molecular Characterization of pssCDE Genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD Mutant Is Affected in Exopolysaccharide Synthesis and Endocytosis of Bacteria
Authors: Król Jaroslaw , Wielbo Jerzy , Mazur Andrzej , Kopcinska Joanna , Lotocka Barbara , Golinowski Wladyslaw , Skorupska Anna
Published: May 2007

We have identified the three genes pssCDE in Rhizobium leguminosarum bv. trifolii TA1. Even though they were almost identical to earlier identified pssCDE genes of R. leguminosarum, they differed in gene lengths and gene overlaps. The predicted gene products of pssCDE genes shared significant homology to prokaryotic glycosyl transferases involved in exopolysaccharide synthesis. The Tn5 insertion in pssD created the nonmucoid mutant that induced non-nitrogen-fixing nodules. The microscopic analysis of the nodules, induced on Trifolium pratense by the pssD133 mutant, showed abnormally enlarged infection threads densely packed with bacteria, which were released from the infection threads in an unusual way. The symbiosomes were observed very rarely and the nodule remained almost empty. Symbiotic phenotype of the pssD133 suggested a correlation between this mutation and defective endocytosis of bacteria into nodule cells.
A Race-Specific Insertion of Transposable Element IS801inPseudomonas syringaepv.phaseolicola
Authors: González A. I. , Ruiz M. L. , Polanco C.
Published: May 2007

The isolation and cloning of a random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) band specific for Pseudomonas syringae pv. phaseolicola race 1 allowed us to design a pair of primers that amplify 1.2-kb race 1-specific and 2.7-kb race 2-specific fragments, providing a rapid method for the identification of races by standard PCR methods. Restriction analysis revealed identical endonuclease sites in both fragments but the race 2 fragment contains a 1.5-kb insertion, identified as transposable element IS801 by sequence comparison. One complete and one partial open reading frame (ORF), each with a high probability of encoding a protein, have been identified in the 1.2-kb fragment common to both race 1 and race 2 sequences. As IS801 disrupts the partial ORF in the race 2 fragment, the complete sequence of this ORF has been obtained as well as its promoter region. The possibility that it may encode an avirulence gene is discussed as well as the role of transposable elements in pathogen evolution.
Agrobacterium tumefaciens Transformation and Cotransformation Frequencies of Arabidopsis thaliana Root Explants and Tobacco Protoplasts
Authors: De Buck Sylvie , Jacobs Anni , Van Montagu Marc , Depicker Ann
Published: May 2007

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.
The Medicago truncatula MtAnn1 Gene Encoding an Annexin Is Induced by Nod Factors and During the Symbiotic Interaction with Rhizobium meliloti
Authors: de Carvalho Niebel Fernanda , Lescure Nicole , Cullimore Julie V. , Gamas Pascal
Published: May 2007

Here we report the characterization of a new Nod factor-induced gene from Medicago truncatula identified by mRNA differential display. This gene, designated MtAnn1, encodes a protein homologous to the annexin family of calcium- and phospholipid-binding proteins. We further show that the MtAnn1 gene is also induced during symbiotic associations with Rhizobium meliloti, both at early stages in bacterial-inoculated roots and in nodule structures. By in situ hybridization, we demonstrate that MtAnn1 expression in nodules is mainly associated with the distal region of invasion zone II not containing infection threads, revealing MtAnn1 as a new marker gene of the pre-infection zone. Moreover, analyses of MtAnn1 expression in response to bacterial symbiotic mutants suggest that the expression of MtAnn1 during nodulation requires biologically active Nod factors and is independent of the infection process.
Induction of Cell Death in Transgenic Plants Expressing a Fungal Glucose Oxidase
Authors: Kazan Kemal , Murray Fiona R. , Goulter Ken C. , Llewellyn Danny J. , Manners John M.
Published: May 2007

Hydrogen peroxide (H2O2) has been implicated in the induction of plant defense genes and programmed cell death. Expression of a chimeric fungal glucose oxidase (GO) gene driven by a pathogen- and wound-inducible promoter was evaluated in transgenic tobacco and canola as a possible tool for engineering plant cell death and defense gene induction. Expression of this gene under the control of a peroxidase gene promoter resulted in the accumulation of relatively low levels of H2O2 in the young leaves of transgenic tobacco plants and this was not sufficient to cause any visible cell death and defense gene induction as measured by PR-1a mRNA induction. Older leaves of transgenic tobacco plants, however, exhibited visible necrotic lesions and constitutively expressed PR-1a mRNA when grown under high light conditions. Inoculation of cotyledons of control and transgenic canola with Leptosphaeria maculans resulted in rapid cotyledon senescence in the transgenic plants. Strong activators of the peroxidase promoter, i.e., wounding and inoculation of transgenic plants with Cercospora nicotianae, were not sufficient to trigger any additional visible cell death in transgenic tobacco plants, compared with controls. However, when exogenous glucose was supplied to transgenic tissue, massive cell death and PR-1a gene induction were observed in tobacco. Exogenously applied salicylic acid further increased the rate and extent of cell death. Our results suggest that efficacy of GO expression for the induction of cell death is restricted by glucose supply in the plants and are consistent with a role for salicylic acid in the potentiation of plant cell death by H2O2.
Induction of PR-1 Proteins and Potentiation of Pathogen Signals by Salicylic Acid Exhibit the Same Dose Response and Structural Specificity in Plant Cell Cultures
Authors: Xie Zhixin , Fan Baofang , Chen Zhixiang
Published: May 2007

Based on the marked difference in both dose response and structural specificity, it has been recently proposed that the induction of acquired resistance and pathogenesis-related (PR) proteins in intact plant leaves and the potentiation of pathogen signals in plant cell cultures by salicylic acid (SA) and its analogues are mediated by different SA signaling pathways initiated from different SA receptor systems. We show here, however, that the induction of PR-1 proteins, the most widely used marker of acquired resistance, by SA and its analogues in tobacco cell cultures exhibits the same dose response and structural specificity as the potentiation of pathogen signals demonstrated in soybean cell cultures. The different dose response and structural specificity for the induction of PR-1 proteins by SA between intact tobacco leaves and cell cultures appear to be caused largely by the difference in uptake and/or metabolism of these chemical inducers by different types of plant cells. These results suggest that the potentiation of pathogen signals by SA, which may contribute greatly to SA function, does not involve a different, more potent signaling pathway from the one(s) responsible for the induction of acquired resistance and PR protein accumulation.
Induced Resistance Responses in Maize
Authors: Morris Shericca W. , Vernooij Bernard , Titatarn Somkiat , Starrett Mark , Thomas Steve , Wiltse Curtis C. , Frederiksen Richard A. , Bhandhufalck Amornrut , Hulbert Scot , Uknes Scott
Published: May 2007

Systemic acquired resistance (SAR) is a widely distributed plant defense system that confers broad-spectrum disease resistance and is accompanied by coordinate expression of the so-called SAR genes. This type of resistance and SAR gene expression can be mimicked with chemical inducers of resistance. Here, we report that chemical inducers of resistance are active in maize. Chemical induction increases resistance to downy mildew and activates expression of the maize PR-1 and PR-5 genes. These genes are also coordinately activated by pathogen infection and function as indicators of the defense reaction. Specifically, after pathogen infection, the PR-1 and PR-5 genes are induced more rapidly and more strongly in an incompatible than in a compatible interaction. In addition, we show that monocot lesion mimic plants also express these defense-related genes and that they have increased levels of salicylic acid after lesions develop, similar to pathogen-infected maize plants. The existence of chemically inducible disease resistance and PR-1 and PR-5 gene expression in maize indicates that maize is similar to dicots in many aspects of induced resistance. This reinforces the notion of an ancient plant-inducible defense pathway against pathogen attack that is shared between monocots and dicots.
Nodule Organogenesis and Symbiotic Mutants of the Model Legume Lotus japonicus
Authors: Szczyglowski Krzysztof , Shaw Robert S. , Wopereis Judith , Copeland Sue , Hamburger Dirk , Kasiborski Beth , Dazzo Frank B. , de Bruijn Frans J.
Published: May 2007

A detailed microscopical analysis of the morphological features that distinguish different developmental stages of nodule organogenesis in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants was performed, to provide the necessary framework for the evaluation of altered phenotypes of L. japonicus symbiotic mutants. Subsequently, chemical ethyl methanesulfonate (EMS) mutagenesis of L. japonicus was carried out. The analysis of approximately 3,000 M1 plants and their progeny yielded 20 stable L. japonicus symbiotic variants, consisting of at least 14 different symbiosis-associated loci or complementation groups. Moreover, a mutation affecting L. japonicus root development was identified that also conferred a hypernodulation response when a line carrying the corresponding allele (LjEMS102) was inoculated with rhizobia. The phenotype of the LjEMS102 line was characterized by the presence of nodule structures covering almost the entire root length (Nod++), and by a concomitant inhibition of both root and stem growth. A mutation in a single nuclear gene was shown to be responsible for both root and symbiotic phenotypes observed in the L. japonicus LjEMS102 line, suggesting that (a) common mechanism(s) regulating root development and nodule formation exists in legumes.
Identification of an Arabidopsis thaliana Mutation (vsm1) That Restricts Systemic Movement of Tobamoviruses
Authors: Lartey Robert T. , Ghoshroy Soumitra , Citovsky Vitaly
Published: May 2007

Following inoculation, many plant viruses spread locally from cell to cell until they reach the vascular system, through which they then move to other parts of the plant, resulting in systemic infection. To isolate host genes involved in systemic transport of plant viruses, ethyl methanesulfonate-mutagenized Arabidopsis thaliana plants were screened for significant delays in the systemic movement of turnip vein clearing virus (TCVC). One such mutant, designated vsm1 (virus systemic movement), was identified. Unlike the wild-type plants, vsm1 did not develop viral disease and did not allow the systemic spread of the virus. The local viral movement within the inoculated vsm1 leaves, however, was not affected. TVCV systemic movement within the vsm1 plants was likely blocked at the step of viral entry into the host plant vasculature from the infected leaf tissue. vsm1 plants also restricted the systemic movement of another tobamovirus but not of an unrelated carmovirus.
Elicitation ofRx-Mediated Resistance to PVX in Potato Does Not Require New RNA Synthesis and May Involve a Latent Hypersensitive Response
Authors: Gilbert Julie , Spillane Charles , Kavanagh Tony A. , Baulcombe David C.
Published: May 2007

The Rx gene in potato confers extreme resistance against potato virus X (PVX) that is elicited by the viral coat protein (CP). Using actinomycin D to inhibit transcription of nuclear genes, we have shown that Rx is expressed prior to virus inoculation and that induction of the resistance mechanism does not require novel gene expression or continued transcription of previously expressed genes. We have also shown that transgenic plants could not be produced carrying a CP transgene from an Rx-avirulent PVX in an Rx background. These data indicate that Rx-mediated resistance involves a mechanism that suppresses viability of plant cells.
Multicopy Vectors Carrying the Klebsiella pneumoniae nifA Gene Do Not Enhance the Nodulation Competitiveness of Sinorhizobium meliloti on Alfalfa
Authors: van Dillewijn Pieter , Martínez-Abarca Francisco , Toro Nicolás
Published: May 2007

It has been reported that Sinorhizobium meliloti strains harboring IncQ and IncP multicopy vectors containing constitutively expressed Klebsiella pneumoniae nifA exhibit an increase in nodulation competitiveness on alfalfa (J. Sanjuan and J. Olivares, Mol. Plant-Microbe Interact. 4:365–369, 1991). In our efforts to understand the mechanisms involved, in this work, we have found that the observed enhancement on nodulation competitiveness by IncQ derivatives carrying K. pneumoniae nifA was not dependent on the plasmid-borne nifA activity but on the sensitivity of nonresistant strains to the streptomycin carried over from growth cultures. Furthermore, it was also determined that the nifA of K. pneumoniae on an IncP vector does not have an effect on competitiveness.