bluuebunny/crossref_metadata_2025 huggingface dataset:
Retracted: Characterization of enhanced antibacterial effects of novel silver nanoparticles
Authors: Shrivastava Siddhartha , Bera Tanmay , Roy Arnab , Singh Gajendra , Ramachandrarao P , Dash Debabrata
Published: May 2007

In the present study, we report the preparation of silver nanoparticles in the range of 10–15 nm with increased stability and enhanced anti-bacterial potency. The morphology of the nanoparticles was characterized by transmission electron microscopy. The antibacterial effect of silver nanoparticles used in this study was found to be far more potent than that described in the earlier reports. This effect was dose dependent and was more pronounced against gram-negative bacteria than gram-positive organisms. Although bacterial cell lysis could be one of the reasons for the observed antibacterial property, nanoparticles also modulated the phosphotyrosine profile of putative bacterial peptides, which could thus affect bacterial signal transduction and inhibit the growth of the organisms.
Epitope Tagging of Legume Root Nodule Extensin Modifies Protein Structure and Crosslinking in Cell Walls of Transformed Tobacco Leaves
Authors: Gucciardo Sébastien , Rathbun Elizabeth A. , Shanks Michael , Jenkyns Susan , Mak Lora , Durrant Marcus C. , Brewin Nicholas J.
Published: May 2007

Root nodule extensins (RNEs) are highly glycosylated plant glycoproteins localized in the extracellular matrix of legume tissues and in the lumen of Rhizobium-induced infection threads. In pea and other legumes, a family of genes encode glycoproteins of different overall length but with the same basic composition. The predicted polypeptide sequence reveals repeating and alternating motifs characteristic of extensins and arabinogalactan proteins. In order to monitor the behavior of individual RNE gene products in the plant extracellular matrix, the coding sequence of PsRNE1 from Pisum sativum was expressed in insect cells and in tobacco leaves. RNE products extracted from tobacco tissues were of high molecular weight (in excess of 80 kDa), indicating extensive glycosylation similar to that in pea tissues. Epitope-tagged derivatives of PsRNE1 could be localized in cell walls. However, the introduction of epitope tags at the C-terminus of RNE altered the behavior of RNE in the extracellular matrix, apparently preventing intermolecular crosslinking of RNE molecules and their covalent association with other cell wall components. These observations are discussed in the light of a computational model for the RNE glycoprotein that is consistent with an extended rod-like structure. It is proposed that RNE can undergo three classes of tyrosine-based crosslinking. Intramolecular crosslinking of vicinal Tyr residues is rod stiffening, end-to-end linkage is rod lengthening, and side-to-side intermolecular crosslinking is rod bundling. The control of these interconversions could have important implications for the biomechanics of infection thread growth.
An avrPto/avrPtoB Mutant of Pseudomonas syringae pv. tomato DC3000 Does Not Elicit Pto-Mediated Resistance and Is Less Virulent on Tomato
Authors: Lin Nai-Chun , Martin Gregory B.
Published: May 2007

AvrPto and AvrPtoB are type III effector proteins expressed by Pseudomonas syringae pv. tomato strain DC3000, a pathogen of both tomato and Arabidopsis spp. Each effector physically interacts with the tomato Pto kinase and elicits a hypersensitive response when expressed in tomato leaves containing Pto. An avrPto deletion mutant of DC3000 previously was shown to retain avirulence activity on Pto-expressing tomato plants. We developed an avrPtoB deletion mutant of DC3000 and found that it also retains Pto-specific avirulence on tomato. These observations suggested that avrPto and avrPtoB both contribute to avirulence. To test this hypothesis, we developed an ΔavrPtoΔavrPtoB double mutant in DC3000. This double mutant was able to cause disease on a Pto-expressing tomato line. Thus, avrPto and avrPtoB are the only avirulence genes in DC3000 that elicit Pto-mediated defense responses in tomato. When inoculated onto susceptible tomato leaves and compared with wild-type DC3000, the mutants DC3000ΔavrPto and DC3000ΔavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000ΔavrPtoΔavrPtoB caused even less severe disease symptoms than the single mutants and grew more slowly than them on susceptible leaves. Our results indicate that AvrPto and AvrPtoB have phenotypically redundant avirulence activity on Pto-expressing tomato and additive virulence activities on susceptible tomato plants.
Proteome Analysis of Programmed Cell Death and Defense Signaling Using the Rice Lesion Mimic Mutant cdr2
Authors: Tsunezuka Hajime , Fujiwara Masayuki , Kawasaki Tsutomu , Shimamoto Ko
Published: May 2007

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.
Mannitol 1-Phosphate Metabolism Is Required for Sporulation in Planta of the Wheat Pathogen Stagonospora nodorum
Authors: Solomon Peter S. , Tan Kar-Chun , Oliver Richard P.
Published: May 2007

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wildtype strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.
Characterization of Susceptibility and Resistance Responses to Potato Cyst Nematode (Globodera spp.) Infection of Tomato Lines in the Absence and Presence of the Broad-Spectrum Nematode Resistance Hero Gene
Authors: Sobczak Miroslaw , Avrova Anna , Jupowicz Justyna , Phillips Mark S. , Ernst Karin , Kumar Amar
Published: May 2007

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.
The Plant Gene CCD1 Selectively Blocks Cell Death During the Hypersensitive Response to Cauliflower Mosaic Virus Infection
Authors: Cawly John , Cole Anthony B. , Király Lóránt , Qiu Wenping , Schoelz James E.
Published: May 2007

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.
Proposed Guidelines for a Unified Nomenclature and Phylogenetic Analysis of Type III Hop Effector Proteins in the Plant Pathogen Pseudomonas syringae
Authors: Lindeberg Magdalen , Stavrinides John , Chang Jeffrey H. , Alfano James R. , Collmer Alan , Dangl Jeffery L. , Greenberg Jean T. , Mansfield John W. , Guttman David S.
Published: May 2007

Pathovars of Pseudomonas syringae interact with their plant hosts via the action of Hrp outer protein (Hop) effector proteins, injected into plant cells by the type III secretion system (TTSS). Recent availability of complete genome sequences for a number of P. syringae pathovars has led to a significant increase in the rate of effector discovery. However, lack of a systematic nomenclature has resulted in multiple names being assigned to the same Hop, unrelated Hops designated by the same alphabetic character, and failure of name choices to reflect consistent standards of experimental confirmation or phylogenetic relatedness. Therefore, specific experimental and bioinformatic criteria are proposed for proteins to be designated as Hops. A generic Hop name structure, HopXY#pv strain, also is proposed, wherein family membership is indicated by the alphabetic characters, subgroup membership numerically, and source pathovar and strain in subscript. Guidelines are provided for phylogenetic characterization and name selection for Hops that are novel, related to previously characterized Hops, chimeras, pseudogenes, truncations, or nonexpressed alleles. Phylogenetic analyses of previously characterized Hops are described, the results of which have been used to guide their integration into the proposed nomenclature.
Oligonucleotide Microarray Analysis of the SalA Regulon Controlling Phytotoxin Production by Pseudomonas syringae pv. syringae
Authors: Lu Shi-En , Wang Nian , Wang Jianlin , Chen Z. Jeffrey , Gross Dennis C.
Published: May 2007

The salA gene is a key regulatory element for syringomycin production by Pseudomonas syringae pv. syringae and encodes a member of the LuxR regulatory protein family. Previous studies revealed that salA, a member of the GacS/GacA signal transduction system, was required for bacterial virulence, syringomycin production, and expression of the syrB1 synthetase gene. To define the SalA regulon, the spotted oligonucleotide microarray was constructed using gene-specific 70-mer oligonucleotides of all open reading frames (ORFs) predicted in the syringomycin (syr) and syringopeptin (syp) gene clusters along with representative genes important to bacterial virulence, growth, and survival. The microarray containing 95 oligos was used to analyze transcriptional changes in a salA mutant (B301DSL07) and its wild-type strain, B301D. Expression of 16 genes was significantly higher (> twofold) in B301D than in the salA mutant; the maximum change in expression was 15-fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion, and regulation of syringomycin and syringopeptin. The positive regulatory effect of SalA on transcription of sypA, syrB1, syrC, and sylD was verified by reporter fusions or real-time polymerase chain reaction analysis. None of the genes or ORFs was significantly down-regulated by the salA gene. These results demonstrated that a subgenomic oligonucleotide microarray is a powerful tool for defining the SalA regulon and its relationship to other genes important to plant pathogenesis.
OsLSD1, a Rice Zinc Finger Protein, Regulates Programmed Cell Death and Callus Differentiation
Authors: Wang Lijuan , Pei Zhongyou , Tian Yingchuan , He Chaozu
Published: May 2007

The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc finger domains and have antagonistic effects on plant programmed cell death (PCD). In this study, a rice (Oryza sativa) functional homolog of LSD1, designated OsLSD1, was identified. The expression of OsLSD1 was light-induced or dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus 35S promoter accelerated callus differentiation in transformed rice tissues and increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice plants exhibited lesion mimic phenotype, increased expression of PR-1mRNA, and an accelerated hypersensitive response when inoculated with avirulent isolates of blast fungus. Both sense and antisense transgenic rice plants conferred significantly enhanced resistance against a virulent isolate of blast fungus. Moreover, ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum) enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin. OsLSD1 green fluorescent protein fusion protein was located in the nucleus of tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating plant PCD, whereas it plays a positive role in callus differentiation.
Microarray Analysis of the Gene Expression Profile Induced by the Endophytic Plant Growth-Promoting Rhizobacteria, Pseudomonas fluorescens FPT9601-T5 in Arabidopsis
Authors: Wang Yanqing , Ohara Yumiko , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki
Published: May 2007

Pseudomonasz fluorescens FPT9601-T5 was originally identified as an endophytic plant growth-promoting rhizobacteria (PGPR) on tomato. To perform a molecular dissecttion of physiological and biochemical changes occurring in the host triggered by P. fluorescens FPT9601-T5 colonization, the model plant Arabidopsis was used in this study. Root colonization of Arabidopsis with P. fluorescens FPT9601-T5 promoted plant growth later than three weeks after inoculation and partially suppressed disease symptoms caused by Pseudomonas syringae pv. tomato DC3000, indicating that P. fluorescens FPT9601-T5 acted as a PGPR on Arabidopsis. To obtain a global view on transcript modification during the Arabidopsis-FPT9601-T5 interaction, we performed microarray analysis using Affymetrix Genechip probe arrays representing approximately 22,800 genes. The results showed that 95 and 105 genes were up- or down-regulated, respectively, more than twofold in FPT9601-T5-treated Arabidopsis plants as compared with control plants. Those up-regulated included genes involved in metabolism, signal transduction, and stress response. Noteworthy, upon FPT9601-T5 colonization, putative auxin-regulated genes and nodulin-like genes were up-regulated, and some ethylene-responsive genes were down-regulated. Our results suggest that P.fluorescens FPT9601-T5 triggered plant responses in a manner similar to known PGPR and, at least in some aspects, to rhizobia.
Viral-Like Symptoms Induced by the Ectopic Expression of the p23 Gene of Citrus tristeza virus Are Citrus Specific and Do Not Correlate with the Pathogenicity of the Virus Strain
Authors: Fagoaga Carmen , López Carmelo , Moreno Pedro , Navarro Luis , Flores Ricardo , Peña Leandro
Published: May 2007

Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour orange compared with Mexican lime also was observed in nontransgenic plants inoculated with CTV, suggesting that minimal p23 levels cause deleterious effects in the first two species. Conversely, transgenic expression of p23 in CTV nonhost Nicotiana spp. led to accumulation of p23 without phenotypic aberrations, indicating that p23 interferes with plant development only in citrus species and relatives.
Autoactive Alleles of the Flax L6 Rust Resistance Gene Induce Non-Race-Specific Rust Resistance Associated with the Hypersensitive Response
Authors: Howles Paul , Lawrence Greg , Finnegan Jean , McFadden Helen , Ayliffe Michael , Dodds Peter , Ellis Jeff
Published: May 2007

L6 is a nucleotide binding site-leucine rich repeat (NBS-LRR) gene that confers race-specific resistance in flax (Linum usitatissimum) to strains of flax rust (Melampsora lini) that carry avirulence alleles of the AvrL567 gene but not to rust strains that carry only the virulence allele. Several mutant and recombinant forms of L6 were made that altered either the methionine-histidine-aspartate (MHD) motif conserved in the NBS domain of resistance proteins or exchanged the short domain C-terminal to the LRR region that is highly variable among L allele products. In transgenic flax some of these alleles are autoactive; they cause a gene dosage-dependent dwarf phenotype and constitutive expression of genes that are markers for the plant defense response. Their effects and penetrance ranged from extreme to mild in their degree of plant stunting, survival, and reproduction. Dwarf plants were also resistant to flax rust strains virulent to wild-type L6 plants, and this nonspecific resistance was associated with a hypersensitive response (HR) at the site of rust infection. The strongest autoactive allele, expressed in Arabidopsis from an ethanol-inducible promoter, gave rise to plant death dependent on the enhanced disease susceptibility 1 (EDS1) gene, which indicates that the mutant flax (Linaceae) L6 gene can signal cell death through a defined disease-resistance pathway in a different plant family (Brassicaceae).
Global Patterns of Gene Regulation Associated with the Development of Ectomycorrhiza Between Birch (Betula pendula Roth.) and Paxillus involutus (Batsch) Fr.
Authors: Le Quéré Antoine , Wright Derek P. , Söderström Bengt , Tunlid Anders , Johansson Tomas
Published: May 2007

The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct morphological and developmental stages, such as preinfection and adhesion, mantle, and Hartig net formation. The global pattern of gene expression during these stages in the birch (Betula pendula)-Paxillus involutus ECM association was analyzed using cDNA microarrays. In comparison with nonsymbiotic conditions, 251 fungal (from a total of 1,075) and 138 plant (1,074 in total) genes were found to be differentially regulated during the ECM development. For instance, during mantle and Hartig net development, there were several plant genes upregulated that are normally involved in defense responses during pathogenic fungal challenges. These responses were, at later stages of ECM development, found to be repressed. Other birch genes that showed differential regulation involved several homologs that usually are implicated in water permeability (aquaporins) and water stress tolerance (dehydrins). Among fungal genes differentially upregulated during stages of mantle and Hartig net formation were homologs putatively involved in mitochondrial respiration. In fully developed ECM tissue, there was an upregulation of fungal genes related to protein synthesis and the cytoskeleton assembly machinery. This study highlights complex molecular interactions between two symbionts during the development of an ECM association.
Characterization of theXanthomonas campestrispv.campestrisLipopolysaccharide Substructures Essential for Elicitation of an Oxidative Burst in Tobacco Cells
Authors: Braun Sebastian G. , Meyer Andreas , Holst Otto , Pühler Alfred , Niehaus Karsten
Published: May 2007

The lipopolysaccharides (LPS) of gram-negative bacteria are essential for perception of pathogens by animals and plants. To identify the LPS substructure or substructures recognized by plants, we isolated water-phase (w)LPS from different Xanthomonas campestris pv. campestris mutants and analyzed their sugar content and ability to elicit an oxidative burst in tobacco cell cultures. The different wLPS species are characterized by lacking repetitive subunits of the O-antigen, the complete O-antigen, or even most of the core region. Because loss of lipid A would be lethal to bacteria, pure lipid A was obtained from X. campestris pv. campestris wild-type wLPS by chemical hydrolysis. The elicitation experiments with tobacco cell cultures revealed that LPS detection is dependent on the bioavailability of the amphiphilic wLPS, which can form micelles in an aqueous environment. By adding deoxycholate to prevent micelle formation, all of the tested wLPS species showed elicitation capability, whereas the lipid A alone was not able to trigger an oxidative burst or calcium transients in tobacco cell cultures. These results suggest that the LPS substructure recognized by tobacco cells is localized in the inner core region of the LPS, consisting of glucose, galacturonic acid, and 3-deoxy-d-manno-oct-2-ulosonic acids. Although lipid A alone seems to be insufficient to induce an oxidative burst in tobacco cell cultures, it cannot be ruled out that lipid A or the glucosamine backbone may be important in combination with the inner core structures.
Visualization of Interactions Between a Pathogenic and a BeneficialFusariumStrain During Biocontrol of Tomato Foot and Root Rot
Authors: Bolwerk Annouschka , Lagopodi Anastasia L. , Lugtenberg Ben J. J. , Bloemberg Guido V.
Published: May 2007

The soilborne fungus Fusarium oxysporum f. sp. radicislycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50- fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.
GuaB Activity Is Required in Rhizobium tropici During the Early Stages of Nodulation of Determinate Nodules but Is Dispensable for the Sinorhizobium meliloti-Alfalfa Symbiotic Interaction
Authors: Collavino Mónica , Riccillo Pablo M. , Grasso Daniel H. , Crespi Martín , Aguilar O. Mario
Published: May 2007

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate thetiming of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.
A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells
Authors: Douchkov D. , Nowara D. , Zierold U. , Schweizer P.
Published: May 2007

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE proteinin three types of durable, race-nonspecific resistance was observed.
Expression of a Truncated Form of Ribosomal Protein L3 Confers Resistance to Pokeweed Antiviral Protein and the Fusarium Mycotoxin Deoxynivalenol
Authors: Di Rong , Tumer Nilgun E.
Published: May 2007

The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the α–sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3δ) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3δ suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3δ, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.
Fundamental Contribution of β-Oxidation to Polyketide Mycotoxin Production In Planta
Authors: Maggio-Hall Lori A. , Wilson Richard A. , Keller Nancy P.
Published: May 2007

Seed contamination with polyketide mycotoxins, including aflatoxin (AF) and sterigmatocystin (ST) produced by Aspergillus spp., is an agricultural, economic, and medical issue worldwide. Acetyl-CoA, the fundamental building block of all known fungal polyketides, is generated by a large number of biochemical pathways, including β-oxidation of fatty acids and glycolysis of sugars. We present several lines of evidence to support a major role for seed fatty acids in formation of AF and ST in A. flavus, A. parasiticus, and A. nidulans. Aspergillus strains exhibiting canonical signs of oleic acid-induced peroxisome proliferation, including increased catalase activity, β-oxidation gene expression, and peroxisomal clustering, also exhibited a marked increase in toxin gene expression and biosynthesis. Furthermore, microscopic observations showed that the ST and AF precursor norsolorinic acid accumulated in peroxisomes of all three Aspergilli. While a peroxisomal β-oxidation mutation eliminated oleic acid-induced increases in ST in A. nidulans, a mitochondrial β-oxidation mutation played a larger role in eliminating ST formation on oatmeal medium and on live corn kernels, implicating a fundamental role for both peroxisomal and mitochondrial β-oxidation in toxin production.
Role of an In Planta-Expressed Xylanase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice
Authors: Rajeshwari R. , Jha Gopaljee , Sonti Ramesh V.
Published: May 2007

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. We demonstrated earlier that the type II secretion system (T2S) is important for virulence of X. oryzae pv. oryzae and that several proteins, including a xylanase, are secreted through this system. In this study, the xynB gene encoding for the secreted xylanase was cloned as a 6.9-kb Eco RI fragment (pRR7) that also included a paralog called xynA. As in X. oryzae pv. oryzae, xynA and xynB are adjacent to each other in X. axonopodis pv. citri, whereas only the xynA homolog is present in X. campestris pv. campestris. Mutations in xynB but not xynA affect secreted xylanase activity. Western blot analysis using anti-XynB antibodies on exudates from infected rice leaves indicated that this xylanase is expressed during in planta growth. Another T2S-secreted protein was identified to be a lipase/esterase (LipA) based on the sequence tags obtained by tandem mass spectrometry analysis and biochemical assays. Mutations in either xynB or lipA partially affected virulence. However, a lipA-xynB double mutant was significantly reduced for virulence, and the pRR7 clone containing an intact xynB gene could complement the virulence-deficient phenotype of the lipA-xynB mutant. Our results suggest that there is functional redundancy among the T2S secreted proteins of X. oryzae pv. oryzae in promoting virulence on rice.
Bioinformatics Correctly Identifies Many Type III Secretion Substrates in the Plant Pathogen Pseudomonas syringae and the Biocontrol Isolate P. fluorescens SBW25
Authors: Vinatzer Boris A. , Jelenska Joanna , Greenberg Jean T.
Published: May 2007

The plant pathogen Pseudomonas syringae causes disease by secreting a potentially large set of virulence proteins called effectors directly into host cells, their environment, or both, using a type III secretion system (T3SS). Most P.syringae effectors have a common upstream element called the hrp box, and their N-terminal regions have amino acids biases, features that permit their bioinformatic prediction. One of the most prominent biases is a positive serine bias. We previously used the truncated AvrRpt281–255 effector containing a serine-rich stretch from amino acids 81 to 100 as a T3SS reporter. Region 81 to 100 of this reporter does not contribute to the secretion or translocation of AvrRpt2 or to putative effector protein chimeras. Rather, the serine-rich region from the N-terminus of AvrRpt2 is important for protein accumulation in bacteria. Most of the N-terminal region (amino acids 15 to 100) is not essential for secretion in culture or delivery to plants. However, portions of this sequence may increase the efficiency of AvrRpt2 secretion, delivery to plants, or both. Two effectors previously identified with the AvrRpt281–255 reporter were secreted in culture independently of AvrRpt2, validating the use of the C terminus of AvrRpt2 as a T3SS reporter. Finally, using the reduced AvrRpt2101–255 reporter, we confirmed seven predicted effectors from P. syringae pv. tomato DC3000, four from P. syringae pv. syringae B728a, and two from P. fluorescens SBW25.
Bacterial Type Two Secretion System Secreted Proteins: Double-Edged Swords for Plant Pathogens
Authors: Jha Gopaljee , Rajeshwari R. , Sonti Ramesh V.
Published: May 2007

The type two secretion system (T2S) is important for virulence of a number of gram-negative bacterial plant pathogens. Most of the T2S-secreted proteins that have been characterized to date are involved in degrading different components of plant cell walls. Functional redundancy appears to exist among T2S-secreted proteins because significant effects on virulence are observed only in strains in which multiple secreted proteins are mutated. Several T2S-secreted proteins have been shown to induce plant defense responses, including hypersensitive response-like reactions. Bacterial pathogens can suppress these defense responses, and recent results indicate that suppression is mediated through the type three secretion system.
Signal Signature and Transcriptome Changes of Arabidopsis During Pathogen and Insect Attack
Authors: De Vos Martin , Van Oosten Vivian R. , Van Poecke Remco M. P. , Van Pelt Johan A. , Pozo Maria J. , Mueller Martin J. , Buchala Antony J. , Métraux Jean-Pierre , Van Loon L. C. , Dicke Marcel , Pieterse Corné M. J.
Published: May 2007

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and F. occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.
Genome-Wide Analysis of Gene Expression in Ralstonia solanacearum Reveals That the hrpB Gene Acts as a Regulatory Switch Controlling Multiple Virulence Pathways
Authors: Occhialini Alessandra , Cunnac Sébastien , Reymond Nancie , Genin Stéphane , Boucher Christian
Published: May 2007

A 70-mer oligonucleotide-based DNA microarray covering 5,074 of the 5,120 predicted genes from Ralstonia so-lanacearum has been generated and used to investigate the repertoire of genes that are under the control of the transcription activator HrpB, which governs pathogenicity in this plant pathogenic bacterium. This study identified 143 hrpB up-regulated genes and 50 hrpB down-regulated genes. In addition to extending the repertoire of type III effector proteins with 26 new candidates, this work demonstrates that the hrpB regulon extends beyond type III secretion system—related functions to include a number of genes governing chemotaxy, biosynthesis or catabolism of various low-molecular-weight chemical compounds, and siderophore production and uptake. The presence of several transcripttional regulators and a cluster of genes predicted to encode the synthesis of an acyl-homoserine lactone together with the absence of a consensus hrpII box in the promoter of a significant proportion of the hrpB-regulated genes suggest that, for some genes, hrpB regulation might be indirect. Altogether, the data indicate that hrpB acts as a master regulatory gene governing a physiological swing associated with the shift from saprophytic to parasitic life.
An Iron Uptake Operon Required for Proper Nodule Development in the Bradyrhizobium japonicum-Soybean Symbiosis
Authors: Benson Heather P. , Boncompagni Eric , Guerinot Mary Lou
Published: May 2007

Rhizobia live in the soil or enter into a nitrogen-fixing symbiosis with a suitable host plant. Each environment presents different challenges with respect to iron acquisition. The soybean symbiont Bradyrhizobium japonicum 61A152 can utilize a variety of siderophores (Fe[III]-specific ligands). Purification of iron-regulated outer membrane proteins had previously allowed the cloning of a gene, fegA, from B. ja-ponicum 61A152, whose predicted protein shares significant amino acid similarity with known TonB-dependent siderophore receptors. Here, we show that fegA is in an operon with a gene, fegB, that is predicted to encode an inner membrane protein. Characterization of fegAB and fegB mutants shows that both fegA and fegB are required for utilization of the siderophore ferrichrome. Whereas the fegB mutant forms a normal symbiosis, the fegAB mutant has a dramatic phenotype in planta. Six weeks after inoculation with a fegAB strain, soybean nodules do not contain leghemoglobin and do not fix nitrogen. Infected cells contain few symbiosomes and are filled with vesicles. As ferrichrome is a fungal siderophore not likely to be available in nodules, the symbiotic defect suggests that the fegAB operon is serving a different function in planta, possibly one involved in signaling between the two partners.
Suppression of Root Nodule Formation by Artificial Expression of the TrEnodDR1 (Coat Protein of White clover cryptic virus 1) Gene in Lotus japonicus
Authors: Nakatsukasa-Akune Mitsumi , Yamashita Kenji , Shimoda Yoshikazu , Uchiumi Toshiki , Abe Mikiko , Aoki Toshio , Kamizawa Ayumi , Ayabe Shin-ichi , Higashi Shiro , Suzuki Akihiro
Published: May 2007

TrEnodDR1 (Trifolium repens early nodulin downregulation 1) encodes a coat protein of White clover cryptic virus 1. Its expression in white clover was down-regulated at the time when root nodules formed. We surmised that its artificial expression would interfere with root nodulation. Therefore, we investigated the effects of its artificial expression on the growth and root nodulation of Lotus japonicus (a model legume). Transformants were prepared by Agrobacterium spp.-mediated transformation. The growth of transformants was reduced and the number of root nodules per unit root length was greatly decreased relative to control. The concentration of endogenous abscisic acid (ABA), which controls nodulation, increased in plants containing TrEnodDR1. These phenotypes clearly were canceled by treatment with abamine, a specific inhibitor of ABA biosynthesis. The increase in endogenous ABA concentration explained the reduced stomatal aperture and the deformation of root hairs in response to inoculation of transgenic L. japonicus with Mesorhizobium loti. Transcriptome comparison between TrEnodDR1 transformants and control plants showed clearly enhanced expression levels of various defense response genes in transformants. These findings suggest that TrEnodDR1 suppresses nodulation by increasing the endogenous ABA concentration, perhaps by activating the plant's innate immune response. This is the first report of the suppression of nodulation by the artificial expression of a virus coat protein gene.
Aspergillus Infection Inhibits the Expression of Peanut 13S-HPODE-Forming Seed Lipoxygenases
Authors: Tsitsigiannis Dimitrios I. , Kunze Susan , Willis David K. , Feussner Ivo , Keller Nancy P.
Published: May 2007

Oxylipins recently have been implicated as signaling molecules for cross-kingdom communication in plant-pathogen interactions. Linoleic acid and its two plant lipoxygenase (LOX) oxylipin products 9- and 13-hydroperoxy fatty acids (9S- and 13S-HPODE) have been shown to have a significant effect on differentiation processes in the mycotoxigenic seed pathogens Aspergillus spp. Whereas both fatty acids promote sporulation, 9S-HPODE stimulates and 13S-HPODE inhibits mycotoxin production. Additionally, Aspergillus flavus infection of seed promotes linoleate 9-LOX expression and 9S-HPODE accumulation. Here, we describe the characterization of two peanut seed lipoxygenase alleles (PnLOX2 and PnLOX3) highly expressed in mature seed. PnLOX2 and PnLOX3 both are 13S-HPODE producers (linoleate 13-LOX) and, in contrast to previously characterized 9-LOX or mixed function LOX genes, are repressed between 5-fold and 250-fold over the course of A. flavus infection. The results of these studies suggest that 9SHPODE and 13S-HPODE molecules act as putative susceptibility and resistance factors respectively, in Aspergillus seed-aflatoxin interactions.
Expression Profiling Soybean Response toPseudomonas syringaeReveals New Defense-Related Genes and Rapid HR-Specific Downregulation of Photosynthesis
Authors: Zou Jijun , Rodriguez-Zas Sandra , Aldea Mihai , Li Min , Zhu Jin , Gonzalez Delkin O. , Vodkin Lila O. , DeLucia Evan , Clough Steven J.
Published: May 2007

Transcript profiling during susceptible (S) and hypersensitive response-associated resistance (R) interactions was determined in soybean (Glycine max). Pseudomonas syringae pv. glycinea carrying or lacking the avirulence gene avrB, was infiltrated into cultivar Williams 82. Leaf RNA was sampled at 2, 8, and 24 h postinoculation (hpi). Significant changes in transcript abundance were observed for 3,897 genes during the experiment at P ≤ 0.000005. Many of the genes showed a similar direction of increase or decrease in abundance in both the S and R responses, but the R response generally showed a significantly greater degree of differential expression. More than 25% of these responsive genes had not been previously reported as being associated with pathogen interactions, as 704 had no functional annotation and 378 had no homolog in National Center for Biotechnology Information databases. The highest number of transcriptional changes was noted at 8 hpi, including the downregulation of 94 chloroplast-associated genes specific to the R response. Photosynthetic measurements were consistent with an R-specific reduction in photosystem II operating efficiency (ΦPSII) that was apparent at 8 hpi for the R response with little effect in the S or control treatments. Imaging analyses suggest that the decreased ΦPSIIwas a result of physical damage to PSII reaction centers.