bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNAs Generated from Individual Epidermal Cells Reveal that Differential Gene Expression Predicting Subsequent Resistance or Susceptibility to Rust Fungal Infection Occurs Prior to the Fungus Entering the Cell Lumen
Authors: Mould Michael J. R. , Xu Tao , Barbara Mary , Iscove Norman N. , Heath Michèle C.
Published: May 2007

As the cowpea rust fungus penetrates the wall of a cowpea epidermal cell, resistant and susceptible plants exhibit different ultrastructural and cytochemical changes within the epidermal protoplast. To examine plant gene expression at this stage of infection, cytoplasm was extracted from individual inoculated or uninoculated epidermal cells before the fungal penetration peg reached the cell lumen. Initial differential colony hybridization screening of an expressed sequence tag library constructed from globally amplified cDNAs generated from the inoculated resistant cells resulted in 80 clones (out of 835) with a differential hybridization pattern. Further slot-blot screening and screening of the amplified cDNAs generated from inoculated or uninoculated, resistant or susceptible cells revealed 28 separate genes, mostly with no matching sequences in the databases, that were up-regulated in response to the growth of the fungus through the wall of resistant or susceptible cells. Five genes, including those coding for β- and α-tubulin, were found to be down-regulated specifically in inoculated, susceptible cells, and five were specifically up-regulated in inoculated, resistant cells, including a PR-10 homolog and a phenylalanine ammonialyase gene. Probing the amplified cDNAs from each cell type for the expression of cell death-related genes revealed that an LLS1 homolog (vuLLS1), cloned from cowpea, was up-regulated by infection in both resistant and susceptible cells and that a homolog of HSR203J was differentially up-regulated in resistant cells. These data show that changes in gene expression predicting the subsequent expression of susceptibility or hypersensitive resistance to fungal infection occur prior to the fungus entering the cell lumen.
Expression ofMsLEC1Transgenes in Alfalfa Plants Causes Symbiotic Abnormalities
Authors: Brill Laurence M. , Fujishige Nancy A. , Hackworth Cheryl A. , Hirsch Ann M.
Published: May 2007

Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021. MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely. MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants. The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems. A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips.
Tagging Quantitative Trait Loci for Maturity-Corrected Late Blight Resistance in Tetraploid Potato with PCR-Based Candidate Gene Markers
Authors: Bormann Christina Angelika , Rickert Andreas Marcus , Castillo Ruiz Rosa Angela , Paal Jürgen , Lübeck Jens , Strahwald Josef , Buhr Karsten , Gebhardt Christiane
Published: May 2007

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.
Overexpression ofNtERF5, a New Member of the Tobacco Ethylene Response Transcription Factor Family Enhances Resistance toTobacco mosaic virus
Authors: Fischer Ute , Dröge-Laser Wolfgang
Published: May 2007

A new member of the tobacco (Nicotiana tabacum) AP2/ERF (ethylene response factor) transcription factor family, designated NtERF5, has been isolated by yeast one-hybrid screening. In vitro, recombinant NtERF5 protein weakly binds GCC box cis-elements, which mediate pathogen-regulated transcription of several PR (pathogenesis related) genes. NtERF5 transcription is transiently activated by wounding, by infection with the bacterial pathogen Pseudomonas syringae, as well as by inoculation with Tobacco mosaic virus (TMV). In contrast, NtERF5 transcription is not enhanced after application of salicylic acid, jasmonic acid, or ethylene. Constitutive overexpression of NtERF5 (ERF5-Oex) under control of the 35S promoter results in no visible alterations in plant growth or enhanced resistance to Pseudomonas infection. Furthermore, no constitutive expression of PR genes has been observed. In contrast, ERF5-Oex plants show enhanced resistance to TMV with reference to reduced size of local hypersensitive-response lesions and impaired systemic spread of the virus. Since, in TMV-infected ERF5-Oex plants, the viral RNA accumulates only up to 10 to 30% of the wild-type level, we suggest that NtERF5-regulated gene expression is controlling resistance to viral propagation. Previous research has demonstrated that overexpression of ERF genes enhances resistance to bacterial and fungal pathogens. Here, we provide further evidence that resistance to viral infection can be engineered by overexpression of ERF transcription factors.
Defense Responses of Fusarium oxysporum to 2,4-Diacetylphloroglucinol, a Broad-Spectrum Antibiotic Produced by Pseudomonas fluorescens
Authors: Schouten Alexander , van den Berg Grardy , Edel-Hermann Véronique , Steinberg Christian , Gautheron Nadine , Alabouvette Claude , de Vos C. H. (Ric) , Lemanceau Philippe , Raaijmakers Jos M.
Published: May 2007

A collection of 76 plant-pathogenic and 41 saprophytic Fusarium oxysporum strains was screened for sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum antibiotic produced by multiple strains of antagonistic Pseudomonas fluorescens. Approximately 17% of the F. oxysporum strains were relatively tolerant to high 2,4-DAPG concentrations. Tolerance to 2,4-DAPG did not correlate with the geographic origin of the strains, formae speciales, intergenic spacer (IGS) group, or fusaric acid production levels. Biochemical analysis showed that 18 of 20 tolerant F. oxysporum strains were capable of metabolizing 2,4-DAPG. For two tolerant strains, analysis by mass spectrometry indicated that deacetylation of 2,4-DAPG to the less fungitoxic derivatives monoacetylphloroglucinol and phloroglucinol is among the initial mechanisms of 2,4-DAPG degradation. Production of fusaric acid, a known inhibitor of 2,4-DAPG biosynthesis in P. fluorescens, differed considerably among both 2,4-DAPG-sensitive and -tolerant F. oxysporum strains, indicating that fusaric acid production may be as important for 2,4-DAPG-sensitive as for -tolerant F. oxysporum strains. Whether 2,4-DAPG triggers fusaric acid production was studied for six F. oxysporum strains; 2,4-DAPG had no significant effect on fusaric acid production in four strains. In two strains, however, sublethal concentrations of 2,4-DAPG either enhanced or significantly decreased fusaric acid production. The implications of 2,4-DAPG degradation, the distribution of this trait within F. oxysporum and other plant-pathogenic fungi, and the consequences for the efficacy of biological control are discussed.
Transcriptional Regulation of Components of the Type III Secretion System and Effectors in Pseudomonas syringae pv. phaseolicola
Authors: Thwaites R. , Spanu P. D. , Panopoulos N. J. , Stevens C. , Mansfield J. W.
Published: May 2007

Quantitative real-time polymerase chain reaction was used with specific TaqMan probes to examine transcription of selected hrp and effector genes in Pseudomonas syringae pv. phaseolicola strains 1448A (race 6) and 1449B (race 7). Transcripts examined were from genes encoding the regulators hrpR and hrpL, core structural components of the type III secretion system (TTSS) hrcC, hrcJ, hrcN, hrcU, and hrpA; the first open-reading frame of each hrp operon, including hrpF, hrpJ, hrpP, and hrpY; and also secreted effectors hrpZ, avrPphE, avrPphF, and virPphA. All genes were induced by incubation in a minimal medium and showed patterns of expression indicating regulation by HrpRS and HrpL. Basal mRNA levels and the timing of accumulation of transcripts after induction differed significantly, suggesting the operation of additional regulatory elements. However, no clear transcriptional hierarchy emerged to explain the ordered construction of the TTSS. Quantitative analysis confirmed that the rates and levels of transcript accumulation within the first 2 h after inoculation were considerably higher in planta than in vitro, and indicated that plant cell wall contact may enhance transcription of TTSS and effector genes in P. syringae pv. phaseolicola. The low-abundance hrcU mRNA had a half-life of 16.5 min, whereas other transcripts had half-lives between 3 and 8 min.
NopB, a Soybean Cultivar-Specificity Protein from Sinorhizobium fredii USDA257, Is a Type III Secreted Protein
Authors: Lorio Julio C. , Kim Won Seok , Krishnan Hari B.
Published: May 2007

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NolB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.
Induction and Spatial Organization of Polyamine Biosynthesis During Nodule Development in Lotus japonicus
Authors: Flemetakis Emmanouil , Efrose Rodica C. , Desbrosses Guilhem , Dimou Maria , Delis Costas , Aivalakis Georgios , Udvardi Michael K. , Katinakis Panagiotis
Published: May 2007

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L. japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an α-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.
Apoplastic Extracts from a Transgenic Wheat Line Exhibiting Lesion-Mimic Phenotype Have Multiple Pathogenesis-Related Proteins That Are Antifungal
Authors: Anand Ajith , Lei Zhentian , Sumner Lloyd W. , Mysore Kirankumar S. , Arakane Yasuyuki , Bockus William W. , Muthukrishnan Subbaratnam
Published: May 2007

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic β-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of β-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two β-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.
Induction of Phytohormones and Differential Gene Expression in Citrus Flowers Infected by the Fungus Colletotrichum acutatum
Authors: Lahey Katherine A. , Yuan Rongcai , Burns Jacqueline K. , Ueng Peter P. , Timmer L. W. , Chung Kuang-Ren
Published: May 2007

Colletotrichum acutatum infects citrus petals and induces premature fruit drop and the formation of persistent calyces. The accumulation of hormones and other growth regulators, and differential gene expression in affected flowers and young fruit, was examined following fungal infection. Ethylene evolution increased threefold and indole-3-acetic acid (IAA) accumulation was as much as 140 times. Abscisic acid (ABA) levels showed no significant response. After infection, both trans- and cis-12-oxo-phytodienoic acid increased 8- to 10-fold. No significant difference of trans-jasmonic acid (JA) was observed in citrus flower petals or pistils. However, a fivefold increase of cis-JA was detected. The amount of salicylic acid (SA) was elevated twofold in affected petals, but not in pistils. Northern blot analyses revealed that the genes encoding ACC oxidase or ACC synthase, and 12-oxo-phytodienoic acid (12-oxo-PDA) reductase, were highly expressed in affected flowers. The genes encoding auxin-related proteins also were upregulated. Application of 2-(4-chlorophenoxy)-2-methyl-propionic acid (clofibrate; a putative auxin inhibitor), 2,3,5-triiodobenzolic acid (an auxin transport inhibitor), or SA after inoculation significantly decreased the accumulation of the gene transcripts of auxin-responsive, GH3-like protein and 12-oxo-PDA reductase, but resulted in higher percentages of young fruit retention. The results indicate that imbalance of IAA, ethylene, and JA in C. acutatum-infected flowers may be involved in symptom development and young fruit drop.
Identification and Characterization of a Well-Defined Series of Coronatine Biosynthetic Mutants of Pseudomonas syringae pv. tomato DC3000
Authors: Brooks David M. , Hernández-Guzmán Gustavo , Kloek Andrew P. , Alarcón-Chaidez Francisco , Sreedharan Aswathy , Rangaswamy Vidhya , Peñaloza-Vázquez Alejandro , Bender Carol L. , Kunkel Barbara N.
Published: May 2007

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.
A NovelArabidopsis-ColletotrichumPathosystem for the Molecular Dissection of Plant-Fungal Interactions
Authors: O'Connell Richard , Herbert Corentin , Sreenivasaprasad Surapareddy , Khatib Moustafa , Esquerré-Tugayé Marie-Thérèse , Dumas Bernard
Published: May 2007

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25°C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15°C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.
A Rhizobium leguminosarum Lipopolysaccharide Lipid-A Mutant Induces Nitrogen-Fixing Nodules with Delayed and Defective Bacteroid Formation
Authors: Vedam Vinata , Haynes Janine G. , Kannenberg Elmar L. , Carlson Russell W. , Sherrier D. Janine
Published: May 2007

Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum bv. viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region. The exact function and importance of this residue, however, is not known. In this work, a previously constructed mutant, Rhizobium leguminosarum bv. viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype. While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion. Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were frequently enclosed in a single symbiosome membrane, a feature not observed with parent bacteroids. In addition, the mutant nodules were delayed in the onset of nitrogenase production and showed reduced nitrogenase throughout the testing period. These results imply that the lack of 27OHC28:0 in the lipid A in mutant bacteroids results in altered membrane properties that are essential for the development of normal bacteroids.
Multiple Pathways Regulate the Induction of Genes During Zoosporogenesis in Phytophthora infestans
Authors: Tani Shuji , Yatzkan Einat , Judelson Howard S.
Published: May 2007

Zoospores are a critical component of the disease cycles of most oomycete pathogens. To better understand this stage, genes induced during zoosporogenesis were identified from Phytophthora infestans, the potato late blight pathogen. Using cDNA arrays representing 2,600 genes expressed during zoosporogenesis, 69 genes showing >fourfold increases in mRNA levels were identified, of which 22 exhibited >100-fold induction. Included were putative protein kinases, transcription factors, ion channels, and other regulators. The expression of 15 genes was characterized in detail using zoosporogenesis time courses, other developmental stages, different temperature regimes, and tissue treated with signaling inhibitors. The latter were of interest because zoosporogenesis is known to be cold induced and inhibited by calcium channel blockers such as verapamil; moreover, in this study, inhibitors of phospholipase C (U-73122) and inositol trisphosphate receptor-gated calcium channels (2-aminoethoxydiphenyl borate) also were shown to block zoosporogenesis. The results indicated that the cytoplasmic and transcriptional changes occurring during zoosporogenesis are regulated by several pathways. For example, verapamil inhibited zoosporogenesis but not the up-regulation of most genes; the induction of some genes required while others were independent of calcium or phospholipid signaling; and, although most genes were induced in sporangia at 10°C but not 24°C, one was induced at both temperatures.
Salicylic Acid Is Part of theMi-1-Mediated Defense Response to Root-Knot Nematode in Tomato
Authors: Branch Craig , Hwang Chin-Feng , Navarre Duroy A. , Williamson Valerie M.
Published: May 2007

The Mi-1 gene of tomato confers resistance against three species of root-knot nematode in tomato (Lycopersicon esculentum). Transformation of tomato carrying Mi-1 with a construct expressing NahG, which encodes salicylate hydroxylase, a bacterial enzyme that degrades salicylic acid (SA) to catechol, results in partial loss of resistance to root-knot nematodes. Exogenous SA was toxic to roots expressing NahG but not to control roots. This toxicity is most likely due to the production of catechol from SA, and we report here that 100 μM catechol is toxic to tomato roots. Benzothiadiazole, a SA analog, completely restores nematode resistance in Mi-1 roots transformed with NahG but does not confer resistance to susceptible tomato roots. The localized cell death produced by transient expression in Nicotiana benthamiana of Mi-DS4, a constitutively lethal chimera of Mi-1 with one of its homologs, was prevented by coexpression of NahG. These results indicate that SA is an important component of the signaling that leads to nematode resistance and the associated hypersensitive response.
Feedback Control of theArabidopsisHypersensitive Response
Authors: Zhang Chu , Gutsche Annie Tang , Shapiro Allan D.
Published: May 2007

The plant hypersensitive response (HR) to avirulent bacterial pathogens results from programmed cell death of plant cells in the infected region. Ion leakage and changes in signaling components associated with HR progression were measured. These studies compared Arabidopsis mutants affecting feedback loops with wild-type plants, with timepoints taken hourly. In response to Pseudomonas syringae pv. tomato DC3000·avrB, npr1-2 mutant plants showed increased ion leakage relative to wild-type plants. Hydrogen peroxide accumulation was similar to that in wild type, but salicylic acid accumulation was reduced at some timepoints. With DC3000·avrRpt2, similar trends were seen. In response to DC3000·avrB, ndr1-1 mutant plants showed more ion leakage than wild-type or npr1-2 plants. Hydrogen peroxide accumulation was delayed by approximately 1 h and reached half the level seen with wild-type plants. Salicylic acid accumulation was similar to npr1-2 mutant plants. With DC3000·avrRpt2, ndr1-1 mutant plants showed no ion leakage, no hydrogen peroxide accumulation, and minimal salicylic acid accumulation. Results with a ndr1-1 and npr1-2 double mutant were similar to ndr1-1. A model consistent with these data is presented, in which one positive and two negative regulatory circuits control HR progression. Understanding this circuitry will facilitate HR manipulation for enhanced disease resistance.
The Barley Apoptosis Suppressor Homologue Bax Inhibitor-1 Compromises Nonhost Penetration Resistance of Barley to the Inappropriate Pathogen Blumeria graminis f. sp. tritici
Authors: Eichmann Ruth , Schultheiss Holger , Kogel Karl-Heinz , Hückelhoven Ralph
Published: May 2007

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, over-expression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.
Ptr ToxA Requires Multiple Motifs for Complete Activity
Authors: Manning Viola A. , Andrie Rachael M. , Trippe Aaron F. , Ciuffetti Lynda M.
Published: May 2007

Ptr ToxA was the first proteinaceous necrosis-inducing toxin identified and cloned from the wheat pathogen, Pyrenophora tritici-repentis. How this protein causes necrosis in sensitive wheat cultivars is not known. In an effort to understand the structural features of Ptr ToxA required for induction of necrosis, we employed a combination of site-directed mutagenesis and peptide inhibition studies. Mutagenesis was carried out on conserved motifs within the active domain of Ptr ToxA. Proteins with mutations of potential casein kinase 2 phosphorylation sites but not protein kinase C phosphorylation sites have significantly reduced activity. Additionally, mutations in a region with high homology to amino acids surrounding and including the RGD cell attachment motif of vitronectin result in proteins with significantly less activity than Ptr ToxA. The importance of the vitronectin-like motif was confirmed by a decrease of Ptr ToxA-induced activity when coinfiltrated with peptides corresponding to amino acids within this motif. Reduction in Ptr ToxA activity by competition with mutant proteins demonstrates the necessity of multiple motifs for Ptr ToxA activity.
Arabidopsis DND2, a Second Cyclic Nucleotide-Gated Ion Channel Gene for Which Mutation Causes the “Defense, No Death” Phenotype
Authors: Jurkowski Grace I. , Smith Roger K. , Yu I-ching , Ham Jong Hyun , Sharma Shashi B. , Klessig Daniel F. , Fengler Kevin A. , Bent Andrew F.
Published: May 2007

A previous mutant screen identified Arabidopsis dnd1 and dnd2 “defense, no death” mutants, which exhibit loss of hypersensitive response (HR) cell death without loss of gene-for-gene resistance. The dnd1 phenotype is caused by mutation of the gene encoding cyclic nucleotide-gated (CNG) ion channel AtCNGC2. This study characterizes dnd2 plants. Even in the presence of high titers of Pseudomonas syringae expressing avrRpt2, most leaf mesophyll cells in the dnd2 mutant exhibited no HR. These plants retained strong RPS2-, RPM1-, or RPS4-mediated restriction of P. syringae pathogen growth. Mutant dnd2 plants also exhibited enhanced broad-spectrum resistance against virulent P. syringae and constitutively elevated levels of salicylic acid, and pathogenesis-related (PR) gene expression. Unlike the wild type, dnd2 plants responding to virulent and avirulent P. syringae exhibited elevated expression of both salicylatedependent PR-1 and jasmonate and ethylene—dependent PDF1.2. Introduction of nahG+ (salicylate hydroxylase) into the dnd2 background, which removes salicylic acid and causes other defense alterations, eliminated constitutive disease resistance and PR gene expression but only weakly impacted the HR¯ phenotype. Map-based cloning revealed that dnd2 phenotypes are caused by mutation of a second CNG ion channel gene, AtCNGC4. Hence, loss of either of two functionally nonredundant CNG ion channels can cause dnd phenotypes. The dnd mutants provide a unique genetic background for dissection of defense signaling.
RaxH/RaxR: A Two-Component Regulatory System in Xanthomonas oryzae pv. oryzae Required for AvrXa21 Activity
Authors: Burdman Saul , Shen Yuwei , Lee Sang-Won , Xue Qinzhong , Ronald Pamela
Published: May 2007

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, one of the most serious diseases in rice. X. oryzae pv. oryzae Philippine race 6 (PR6) strains are unable to establish infection in rice lines expressing the resistance gene Xa21. Although the pathogen-associated molecule that triggers the Xa21-mediated defense response (AvrXa21) is unknown, six rax (required for AvrXa21 activity) genes encoding proteins involved in sulfur metabolism and Type I secretion were recently identified. Here, we report on the identification of two additional rax genes, raxR and raxH, which encode a response regulator and a histidine protein kinase of two-component regulatory systems, respectively. Null mutants of PR6 strain PXO99 that are impaired in either raxR, raxH, or both cause lesions significantly longer and grow to significantly higher levels than does the wild-type strain in Xa21-rice leaves. Both raxR and raxH mutants are complemented to wild-type levels of AvrXa21 activity by introduction of expression vectors carrying raxR and raxH, respectively. These null mutants do not affect AvrXa7 and AvrXa10 activities, as observed in inoculation experiments with Xa7- and Xa10-rice lines. Western blot and raxR/gfp promoter-reporter analyses confirmed RaxR expression in X. oryzae pv. oryzae. The results of promoter-reporter studies also suggest that the previously identified raxSTAB operon is a target for RaxH/RaxR regulation. Characterization of the RaxH/RaxR system provides new opportunities for understanding the specificity of the X. oryzae pv. oryzae-Xa21 interaction and may contribute to the identification of AvrXa21.
The Expression of MaEXP1, a Melilotus alba Expansin Gene, Is Upregulated During the Sweetclover-Sinorhizobium meliloti Interaction
Authors: Giordano Walter , Hirsch Ann M.
Published: May 2007

Expansins are a highly conserved group of cell wall-localized proteins that appear to mediate changes in cell wall plasticity during cell expansion or differentiation. The accumulation of expansin protein or the mRNA for specific expansin gene family members has been correlated with the growth of various plant organs. Because expansin proteins are closely associated with plant cell wall expansion, and as part of a larger study to determine the role of different gene products in the legume-Rhizobium spp. symbiosis, we investigated whether a Melilotus alba (white sweetclover) expansin gene is expressed during nodule development. A cDNA fragment encoding an expansin gene (EXP) was isolated from Sinorhizobium meliloti-inoculated sweetclover root RNA by reverse-transcriptase polymerase chain reaction using degenerate primers, and a full-length sweetclover expansin sequence (MaEXP1) was obtained using 5′ and 3′rapid amplification of cDNA end cloning. The predicted amino acid of the sweetclover expansin is highly conserved with the various α-expansins in the GenBank database. MaEXP1 contains a series of eight cysteines and four tryptophans that are conserved in the α-expansin protein family. Northern analysis and whole-mount in situ hybridization analyses indicate that MaEXP1 mRNA expression is enhanced in roots within hours after inoculation with S. meliloti and in nodules. Western and immunolocalization studies using a cucumber expansin antibody demonstrated that a cross-reacting protein accumulated in the expanding cells of the nodule.
Flagellin Is Not a Major Defense Elicitor in Ralstonia solanacearum Cells or Extracts Applied to Arabidopsis thaliana
Authors: Pfund Christine , Tans-Kersten Julie , Dunning F. Mark , Alonso Jose M. , Ecker Joseph R. , Allen Caitilyn , Bent Andrew F.
Published: May 2007

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.
Soilborne Wheat Mosaic Virus Movement Protein and RNA and Wheat Spindle Streak Mosaic Virus Coat Protein Accumulate Inside Resting Spores of Their Vector, Polymyxa graminis
Authors: Driskel Barbara A. , Doss Phoebe , Littlefield Larry J. , Walker Nathan R. , Verchot-Lubicz Jeanmarie
Published: May 2007

To study virus-vector interactions between Soilborne wheat mosaic virus (SBWMV) or Wheat spindle streak mosaic virus (WSSMV) and Polymyxa graminis Ledingham, P. graminis was propagated in plants grown hydroponically. P. graminis accumulated to high levels in several barley cultivars tested. Multiple developmental stages of P. graminis could be identified in infected barley roots. Accumulation of SBWMV and WSSMV inside P. graminis sporosori in the roots of soil-grown winter wheat and hydroponically grown barley was compared to determine if data obtained from plants naturally infected plants and plants infected by manual inoculation were similar. WSSMV coat protein (CP), SBWMV RNAs, SBWMV movement protein but not SBWMV CP were detected in both soil-grown winter wheat and hydroponically grown barley roots. These data are the first direct evidence that SBWMV and WSSMV are internalized by P. graminis.
Introduction of Plant and Fungal Genes into Pea (Pisum sativum L.) Hairy Roots Reduces Their Ability to Produce Pisatin and Affects Their Response to a Fungal Pathogen
Authors: Wu Qindong , VanEtten Hans D.
Published: May 2007

Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less pisatin. To evaluate the role of pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.
Changes in Gene Expression in Canola Roots Induced by ACC-Deaminase-Containing Plant-Growth-Promoting Bacteria
Authors: Hontzeas Nikos , Saleh Saleema S. , Glick Bernard R.
Published: May 2007

The technique of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) was used to study changes in gene expression over time in canola roots treated with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant-growth-promoting bacterium Enterobacter cloacae UW4 and to compare the changes with those in a mutant of E. cloacae UW4 in which the ACC deaminase structural gene acdS was replaced by homologous recombination with acdS with an intentional knockout containing a tetracycline resistance gene. Genes that were either up- or down-regulated over a three-day period in canola plants treated with wild-type or mutant bacteria were isolated, cloned, and sequenced; all appeared to have high homology with Arabidopsis thaliana genes. The upregulated genes included a cell division cycle protein 48 homolog and a eukaryotic translation initiation factor 3 subunit 7 gene homolog. The downregulated genes included one encoding a glycine-rich RNA binding protein with a function in RNA processing or binding during ethylene-induced stress, which is expressed only in roots, and another gene thought to be involved in a defense signaling pathway. All RAP-PCR results were verified using Northern blotting. These data, indicate that roots isolated from canola seeds treated with the ACC deaminase-producing E. cloacae UW4 up-regulate genes involved in cell division and proliferation but down-regulate stress genes. This data is in agreement with a model in which ACC deaminase-containing plant-growth-promoting bacteria reduce plant stress and induce root elongation and proliferation in plants, largely by lowering ethylene levels.
Phenotypic Variation of Pseudomonas brassicacearum as a Plant Root-Colonization Strategy
Authors: Achouak Wafa , Conrod Sandrine , Cohen Valérie , Heulin Thierry
Published: May 2007

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.
Transient Coexpression of Individual Genes Encoded by the Triple Gene Block of Potato mop-top virus Reveals Requirements for TGBp1 Trafficking
Authors: Zamyatnin Andrey A. , Solovyev Andrey G. , Savenkov Eugene I. , Germundsson Anna , Sandgren Maria , Valkonen Jari P. T. , Morozov Sergey Y.
Published: May 2007

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the “triple gene block” (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBp1 to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.