bluuebunny/crossref_metadata_2025 huggingface dataset:
TIP, A Novel Host Factor Linking Callose Degradation with the Cell-to-Cell Movement ofPotato virus X
Authors: Fridborg Ingela , Grainger Jef , Page Anthony , Coleman Mark , Findlay Kim , Angell Susan
Published: May 2007

The cell-to-cell movement of Potato virus X (PVX) requires four virus-encoded proteins, the triple gene block (TGB) proteins (TGB25K, TGB12K, and TGB8K) and the coat protein. TGB12K increases the plasmodesmal size exclusion limit (SEL) and may, therefore, interact directly with components of the cell wall or with plant proteins associated with bringing about this change. A yeast two-hybrid screen using TGB12K as bait identified three TGB12K-interacting proteins (TIP1, TIP2, and TIP3). All three TIPs interacted specifically with TGB12K but not with TGB25K or TGB8K. Similarly, all three TIPs interacted with β-1,3-glucanase, the enzyme that may regulate plasmodesmal SEL through callose degradation. Sequence analyses revealed that the TIPs encode very similar proteins and that TIP1 corresponds to the tobacco ankyrin repeat-containing protein HBP1. A TIP1::GFP fusion protein localized to the cytoplasm. Coexpression of this fusion protein with TGB12K induced cellular changes manifested as deposits of additional cytoplasm at the cell periphery. This work reports a direct link between a viral movement protein required to increase plasmodesmal SEL and a host factor that has been implicated as a key regulator of plasmodesmal SEL. We propose that the TIPs are susceptibility factors that modulate the plasmodesmal SEL.
Dynamics of the VIGS-Mediated Chimeric Silencing of the Nicotiana benthamiana ChlH Gene and of the Tobacco Mosaic Virus Vector
Authors: Hiriart Jean-Baptiste , Aro Eva-Mari , Lehto Kirsi
Published: May 2007

The ChlH gene, encoding for the H subunit of the magnesium chelatase enzyme, was silenced in Nicotiana bentahamiana plants by virus-induced gene silencing (VIGS), using tobacco mosaic virus (TMV) expression vector. Strong silencing of the ChlH target gene was initiated only in the apical tissues, in which the endogenous transcription level of the target gene and the level of TMV vector RNA were both very high. The virus vector was also targeted by VIGS, and its suppression was correlated with the silencing of the ChlH mRNA. In the apical tissues, the suppression of both the virus vector and the ChlH mRNA led to a reduction of the silencing pressure and, consequently, to partial recovery of the new growth from the silencing. As the virus vector and the target mRNA levels increased, silencing was reestablished. The feedback regulation system, caused by the transient increase and reduction in levels of the virus vector and ChlH mRNA, led to a fluctuation of the silenced and recovered phenotypes in the plant apex. This TMV-vector mediated silencing system differed from previously analyzed VIGS systems; although the TMV vector was initially targeted by the silencing system, it was not permanently suppressed, indicating that, in this system, TMV was able to effectively escape post-transcriptional gene silencing.
Analysis of the Involvement of an Inducible Arabidopsis RNA-Dependent RNA Polymerase in Antiviral Defense
Authors: Yu Diqiu , Fan Baofang , MacFarlane Stuart A. , Chen Zhixiang
Published: May 2007

RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a to-bamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.
Isolation of a Premycorrhizal Infection (pmi2) Mutant of Tomato, Resistant to Arbuscular Mycorrhizal Fungal Colonization
Authors: David-Schwartz Rakefet , Gadkar Vijay , Wininger Smadar , Bendov Roza , Galili Gad , Levy Avraham A. , Kapulnik Yoram
Published: May 2007

Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc¯ plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc¯ phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc¯ phenotype response.
Overexpression of a Brassica nigra cDNA Gives Enhanced Resistance to Leptosphaeria maculans in B. napus
Authors: Wretblad Sofia , Bohman Svante , Dixelius Christina
Published: May 2007

Using a polymerase chain reaction-based strategy, we have isolated a cDNA sequence from Brassica nigra, denoted Lm1, which significantly improves blackleg resistance when expressed in transgenic oilseed rape, B. napus. Lm1 was shown to map to locations on linkage groups 3 and 8 in the Brassica B-genome previously linked to both cotyledon, adult leaf, and stem resistance. B. napus plants transformed with Lm1 under the control of a constitutive promoter exhibited broad spectrum resistance to all L. maculans isolates tested, but enhanced resistance to Alternaria brassicae and Verticillium longisporum was not observed. A transcript corresponding to the cDNA size was induced in B. nigra 12 h after fungal challenge from a level of weak constitutive expression previous to inoculation. The Lm1 sequence bears no resemblance to previously characterized plant resistance genes but has two predicted transmembrane motifs. Several sequences with high homology to Lm1 were found in the databases. Lm1 appears to be a member of a larger group of related genes present in a variety of plant species. Most of them have unknown functions, but homology between Lm1 and the nodule inception gene of Lotus japonicus suggests an intriguing link between defense-related and symbiotic pathways.
Extracellular Proteins Involved in Soybean Cultivar-Specific Nodulation Are Associated with Pilus-Like Surface Appendages and Exported by a Type III Protein Secretion System in Sinorhizobium fredii USDA257
Authors: Krishnan Hari B. , Lorio Julio , Kim Won Seok , Jiang Guoqiao , Kim Kil Yong , DeBoer Margreet , Pueppke Steven G.
Published: May 2007

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.
Cytological and Molecular Analysis of the Hordeum vulgare-Puccinia triticina Nonhost Interaction
Authors: Neu Christina , Keller Beat , Feuillet Catherine
Published: May 2007

Cultivated barley, Hordeum vulgare L., is considered to be a nonhost or intermediate host species for the wheat leaf rust fungus Puccinia triticina. Here, we have investigated, at the microscopic and molecular levels, the reaction of barley cultivars to wheat leaf rust infection. In the nonhost resistant cultivar Cebada Capa, abortion of fungal growth occurred at both pre- and posthaustorial stages, suggesting that defense genes are expressed throughout the development of the inappropriate fungus during the nonhost resistance reaction. In the two barley lines L94 and Bowman, a low level of prehaustorial resistance to P. triticina was observed and susceptibility was comparable to that of wheat control plants. Suppression subtractive hybridization was used to identify genes that are differentially expressed during the nonhost resistance reaction in Cebada Capa as well as during the successful establishment of the inappropriate wheat leaf rust fungus in L94. Northern analysis indicated that two candidate genes, including a barley ortholog of the rice resistance gene Xa21, are putatively involved in non-host and non-race-specific resistance reactions. In addition, a new gene that is specifically induced during the successful development of the inappropriate fungus P. triticina in barley has been identified.
GacS Sensor Domains Pertinent to the Regulation of Exoproduct Formation and to the Biocontrol Potential of Pseudomonas fluorescens CHA0
Authors: Zuber Sophie , Carruthers Fiona , Keel Christoph , Mattart Alexandre , Blumer Caroline , Pessi Gabriella , Gigot-Bonnefoy Cécile , Schnider-Keel Ursula , Heeb Stephan , Reimmann Cornelia , Haas Dieter
Published: May 2007

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extra-cellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS¯), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.
Organ-Specificity in a Plant Disease Is Determined Independently of R Gene Signaling
Authors: Hermanns Monika , Slusarenko Alan J. , Schlaich Nikolaus L.
Published: May 2007

The molecular basis of organ specificity in plant diseases is little characterized. Downy mildew of Arabidopsis caused by the oomycete Hyaloperonospora parasitica (formerly Peronospora parasitica) is characteristically a leaf disease. Resistant host genotypes recognize the pathogen in a gene-for-gene dependent manner and respond with the production of H2O2 and the execution of a genetically programmed hypersensitive cell death (HR). We inoculated the roots of Arabidopsis genotypes Col-0, Ws-0, and Wei-0 with the NOCO and WELA races of the pathogen and compared the responses with those observed in leaves. Combinations of incompatible genotypes of host and pathogen showed the expected responses of an oxidative burst and the HR in leaves, but surprisingly, roots showed no signs of active defense and appeared completely susceptible to all the H. parasitica isolates tested. Reverse transcrip tase-polymerase chain reaction showed that the R gene RPP1, which mediates resistance in leaves of accession Ws-0 to the H. parasitica isolate NOCO, was expressed in leaves as well as in roots. Similarly, NDR1 and EDS1, two components of R gene-mediated signaling pathways, are also expressed in both tissues. To our knowledge, it has not been previously demonstrated that expression of R genes and downstream components of the signaling cascade are not sufficient for the induction of avirulence gene-mediated defense mechanisms in roots.
The Sclerotinia sclerotiorum pac1 Gene Is Required for Sclerotial Development and Virulence
Authors: Rollins Jeffrey A.
Published: May 2007

The synergistic activities of oxalic acid and endopolygalacturonases are thought to be essential for full virulence of Sclerotinia sclerotiorum and other oxalate-producing plant pathogens. Both oxalic acid production and endopolygalacturonase activity are regulated by ambient pH. Since many gene products with pH-sensitive activities are regulated by the PacC transcription factor in Aspergillus nidulans, we functionally characterized a pacC gene homolog, pac1, from S. sclerotiorum. Mutants with loss-of-function alleles of the pac1 locus were created by targeted gene replacement. In vitro mycelial growth of these pac1 mutants was normal at acidic pH, but growth was inhibited as culture medium pH was increased. Development and maturation of sclerotia in culture was also aberrant in these pac1 replacement mutants. Although oxalic acid production remained alkaline pH-responsive, the kinetics and magnitude of oxalate accumulation were dramatically altered. Additionally, maximal accumulation of endopolygalacturonase gene transcripts (pg1) was shifted to higher ambient pH. Virulence in loss-of-function pac1 mutants was dramatically reduced in infection assays with tomato and Arabidopsis. Based on these results, pac1 appears to be necessary for the appropriate regulation of physiological processes important for pathogenesis and development of S. sclerotiorum.
Comparison of Nodule Induction in Legume and Actinorhizal Symbioses: The Induction of Actinorhizal Nodules Does Not Involve ENOD40
Authors: Santi Carole , von Groll Uritza , Ribeiro Ana , Chiurazzi Maurizio , Auguy Florence , Bogusz Didier , Franche Claudine , Pawlowski Katharina
Published: May 2007

Two types of root nodule symbioses are known for higher plants, legume and actinorhizal symbioses. In legume symbioses, bacterial signal factors induce the expression of ENOD40 genes. We isolated an ENOD40 promoter from an actinorhizal plant, Casuarina glauca, and compared its expression pattern in a legume (Lotus japonicus) and an actinorhizal plant (Allocasuarina verticillata) with that of an ENOD40 promoter from the legume soybean (GmENOD402). In the actinorhizal Allocasuarina sp., CgENOD40-GUS and GmENOD40-2-GUS showed similar expression patterns in both vegetative and symbiotic development, and neither promoter was active during nodule induction. The nonsymbiotic expression pattern of CgENOD40-GUS in the legume genus Lotus resembled the nonsymbiotic expression patterns of legume ENOD40 genes however, in contrast to GmENOD40-2-GUS, CgENOD40-GUS was not active during nodule induction. The fact that only legume, not actinorhizal, ENOD40 genes are induced during legume nodule induction can be linked to the phloem unloading mechanisms established in the zones of nodule induction in the roots of both types of host plants.
The Phytoalexin-Inducible Multidrug Efflux Pump AcrAB Contributes to Virulence in the Fire Blight Pathogen, Erwinia amylovora
Authors: Burse Antje , Weingart Helge , Ullrich Matthias S.
Published: May 2007

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.
Early Induction of the Arabidopsis GSTF8 Promoter by Specific Strains of the Fungal Pathogen Rhizoctonia solani
Authors: Perl-Treves Rafael , Foley Rhonda C. , Chen Wenqiong , Singh Karam B.
Published: May 2007

The Arabidopsis glutathione S-transferase GSTF8 promoter directs root-specific responses to stress. In this study, the response of this promoter to plant infection with Rhizoctonia solani was investigated using a luciferase reporter system. Arabidopsis seedlings harboring the GSTF8:luciferase construct were monitored in vivo for bioluminescence following infection with R. solani. Although the reporter gene was induced in infected roots, the response differed markedly between R. solani strains and was not observed with aggressive strains that caused death of the seedlings. The three strains tested in detail progressed through typical stages of infection, but ZG1-1 induced the GSTF8 promoter in most seedlings, ZG3 induced it in approximately 25% of seedlings, and ZG5 caused little response. Induction of specific root segments occurred early in the infection process in root regions with very limited mycelium visible. In root segments with substantial mycelium, GSTF8 promoter activity no longer was observed. Induction by ZG1-1 also was observed in plants harboring a tetramer of the ocs element from the GSTF8 promoter, suggesting that this element helps mediate the response. Crossing GSTF8:luciferase plants with plants harboring an Nah-G construct that degrades salicylic acid did not abolish the response, indicating that the GSTF8 promoter response to R. solani may be mediated by signals other than salicylic acid.
Impact of Temperature on In Planta Expression of Genes Involved in Synthesis of the Pseudomonas syringae Phytotoxin Coronatine
Authors: Weingart Helge , Stubner Stephan , Schenk Alexander , Ullrich Matthias S.
Published: May 2007

Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P. syringae pv. glycinea PG4180, a pathogen of soybean, and P. syringae pv. tomato DC3000, a pathogen of tomato and crucifers. Previously, it was shown in vitro that the cma operon involved in COR synthesis in PG4180 is expressed in a temperature-dependent manner, with maximal rates at 18°C and low activity at 28°C. However, nothing was known about the influence of temperature on the expression of COR biosynthetic genes in planta. Therefore, transcriptional fusions of the PG4180 and DC3000 cma promoter regions to a promoterless egfp gene were constructed and expressed in both P. syringae strains. The fluorescence patterns in response to temperature during growth of a strain in vitro were consistent with its COR production and the cma transcript abundance as revealed by RNA dot blot hybridization. Quantification of fluorescence indicated that cma promoter activity was dependent on the genetic background of the host strain. Expression of cma∷egfp in PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue. In contrast, transcription of the cma operon was not significantly affected by temperature in DC3000. However, cells of DC3000 harboring the cma∷egfp fusions showed higher levels of fluorescence when recovered from infected host plants compared with cells grown in minimal medium. These results indicate that the signals for induction of COR biosynthesis differ significantly in PG4180 and DC3000.
Flavonoids, NodD1, NodD2, and Nod-Box NB15 Modulate Expression of the y4wEFG Locus That Is Required for Indole-3-Acetic Acid Synthesis in Rhizobium sp. strain NGR234
Authors: Theunis Mart , Kobayashi Hajime , Broughton William J. , Prinsen Els
Published: May 2007

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.
Localization of Superoxide Dismutases and Hydrogen Peroxide in Legume Root Nodules
Authors: Rubio Maria C. , James Euan K. , Clemente Maria R. , Bucciarelli Bruna , Fedorova Maria , Vance Carroll P. , Becana Manuel
Published: May 2007

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2 and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plant's response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SODs are a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the absence or large reduction of H2O2 in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H2O2 originates by the sequential operation of an NADPH oxidase- like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.
Overexpression of (At)NPR1 in Rice Leads to a BTH- and Environment-Induced Lesion-Mimic/Cell Death Phenotype
Authors: Fitzgerald Heather A. , Chern Maw-Sheng , Navarre Roy , Ronald Pamela C.
Published: May 2007

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype. Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wild-type plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.
Establishment of a Functional Genomics Platform for Leifsonia xyli subsp. xyli
Authors: Brumbley Stevens M. , Petrasovits Lars A. , Murphy Rachel M. , Nagel Roland J. , Candy Judith M. , Hermann Scott R.
Published: May 2007

Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx∷Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx∷Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis.
Host-Specific Generation and Maintenance of Tomato bushy stunt virus Defective Interfering RNAs
Authors: Omarov Rustem T. , Rezende Jorge A. M. , Scholthof Herman B.
Published: May 2007

The accumulation of Tomato bushy stunt virus (TBSV) defective interfering RNAs (DIs) has been observed in several species of plants, but the involvement of host-specific processes and the functional role of DIs are still poorly understood. In this study, the accumulation of DIs was compared after several passages of TBSV through Nicotiana benthamiana and pepper (Capsicum annuum). As anticipated, passages of wild-type TBSV through N. benthamiana resulted in the accumulation of significant levels of TBSV DIs, which caused symptom attenuation and prevented the plants from lethal necrosis. On the contrary, TBSV infection of pepper plants caused severe local and systemic chlorosis, but continuous virus passages did not result in detectable levels of DIs accumulation. In addition, the inoculation of pepper plants with a mixture of helper virus and DI either from in vitro generated transcripts or from infected N. benthamiana did not yield DI in upper pepper leaves. Our cumulative results suggest that complex host-specific determinants play an important role in TBSV DI generation and their subsequent maintenance and accumulation.
The typA Gene is Required for Stress Adaptation as Well as for Symbiosis of Sinorhizobium meliloti 1021 with Certain Medicago truncatula Lines
Authors: Kiss Ernö , Huguet Thierry , Poinsot Véréna , Batut Jacques
Published: May 2007

In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.
Nitric Oxide and Reactive Oxygen Species Do Not Elicit Hypersensitive Cell Death but Induce Apoptosis in the Adjacent Cells During the Defense Response of Oat
Authors: Tada Yasuomi , Mori Tomoyo , Shinogi Takeshi , Yao Nan , Takahashi Satsuki , Betsuyaku Shigeyuki , Sakamoto Masaru , Park Pyoyun , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki
Published: May 2007

Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2probe DCF demonstrated that NO and H2O2were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2by interacting with O2-in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2-but not H2O2are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.
Mutations in the Pseudomonas syringae avrRpt2 gene That Dissociate Its Virulence and Avirulence Activities Lead to Decreased Efficiency in AvrRpt2-Induced Disappearance of RIN4
Authors: Lim Melisa T. S. , Kunkel Barbara N.
Published: May 2007

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.
Proline Accumulation and AtP5CS2 Gene Activation Are Induced by Plant-Pathogen Incompatible Interactions in Arabidopsis
Authors: Fabro Georgina , Kovács Izabella , Pavet Valeria , Szabados László , Alvarez María E.
Published: May 2007

Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant—pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.
CPTF1, a CREB-Like Transcription Factor, Is Involved in the Oxidative Stress Response in the Phytopathogen Claviceps purpurea and Modulates ROS Level in Its Host Secale cereale
Authors: Nathues Eva , Joshi Suchitra , Tenberge Klaus B. , von den Driesch Marcell , Oeser Birgitt , Bäumer Nicole , Mihlan Martina , Tudzynski Paul
Published: May 2007

CPTF1, a transcription factor with significant homology to ATF/CREB bZIP factors, was identified during an expressed sequence tag (EST) analysis of in planta-expressed genes of the phytopathogen Claviceps purpurea. Using a gene-replacement approach, deletion mutants of cptf1 were created. Expression studies in axenic culture showed that the H2O2-inducible gene cpcat1 (encoding a secreted catalase) had a reduced basal expression level and no longer responded to oxidative stress in the Δcptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Δcptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in Δcptf1-infected rye epidermal tissues, indicating that the plant tissue displayed an oxidative burst-like reaction, an event not detected in wild-type infections. These data indicate that CPTF1 is involved not only in oxidative stress response in the fungus but also in modulation of the plant's defense reactions.
Albicidin Pathotoxin Produced byXanthomonas albilineansIs Encoded by Three Large PKS and NRPS Genes Present in a Gene Cluster Also Containing Several Putative Modifying, Regulatory, and Resistance Genes
Authors: Royer Monique , Costet Laurent , Vivien Eric , Bes Martine , Cousin Arnaud , Damais Anne , Pieretti Isabelle , Savin Alexandre , Megessier Sandrine , Viard Magali , Frutos Roger , Gabriel Dean W. , Rott Philippe C.
Published: May 2007

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS—NRPS gene cluster for toxin production in the genus Xanthomonas.
The R3 Resistance to Phytophthora infestans in Potato is Conferred by Two Closely Linked R Genes with Distinct Specificities
Authors: Huang Sanwen , Vleeshouwers Vivianne G. A. A. , Werij Jeroen S. , Hutten Ronald C. B. , van Eck Herman J. , Visser Richard G. F. , Jacobsen Evert
Published: May 2007

The R3 locus of potato (Solanum tuberosum L.) confers full resistance to avirulent isolates of Phytophthora infestans, the causal agent of late blight. R3 resides in the distal part of chromosome 11 and segregates in a potato mapping population, from which a well-saturated amplified fragment length polymorphism map is available. Using a population of 1,748 plants, we constructed a high-resolution genetic map at the R3 locus. Using the combination of fine mapping and accurate disease testing with specific P. infestans isolates, we detected that the R3 locus is composed of two genes with distinct specificities. The two genes R3a and R3b are 0.4 cM apart and have both been introgressed from S. demissum, the ‘donor’ species of most characterized race-specific R genes to P. infestans. A natural recombinant between R3a and R3b was discovered in one accession of S. demissum. The synteny between the R3 locus and the tomato I2 locus is discussed.
Expression of Cyanobacterial Genes Involved in Heterocyst Differentiation and Dinitrogen Fixation Along a Plant Symbiosis Development Profile
Authors: Wang Chun-Mei , Ekman Martin , Bergman Birgitta
Published: May 2007

Members of the cyanobiont genus Nostoc, forming an endosymbiosis with members of the angiosperm genus Gunnera, undergo a number of characteristic phenotypic changes during the development of the symbiosis, the genetic background of which is largely unknown. Transcription patterns of genes related to heterocyst differentiation and dinitrogen fixation and corresponding protein profiles were examined, using reverse transcription-polymerase chain reaction and Western blots, along a developmental (apex to mature parts) sequence in Gunnera magellanica and G. manicata and under mimicked symbiotic conditions in a free-living Gunnera isolate (Nostoc strain 0102). The hetR gene was highly expressed and correlated positively with an increase in heterocyst frequency and with ntcA expression, whereas nifH expression was already high close to the growing apex and glnB (PII) expression decreased along the symbiotic profile. Although gene expression appeared to be regulated to a large extent in the same fashion as in free-living cyanobacteria, significant differences were apparent, such as the overexpression of both hetR and ntcA and the contrasting down-regulation of glnB, features indicating important regulatory differences between symbiotic and free-living cyanobacteria. The significance of these findings is discussed in a symbiotic context.
The HopPtoF Locus of Pseudomonas syringae pv. tomato DC3000 Encodes a Type III Chaperone and a Cognate Effector
Authors: Shan Libo , Oh Hye-sook , Chen Jianfu , Guo Ming , Zhou Jianmin , Alfano James R. , Collmer Alan , Jia Xu , Tang Xiaoyan
Published: May 2007

Type III secretion systes are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcFPto and HopFPto, that are preceded by a single hrp box promoter. We present evidence here to show that ShcFPto and HopFPto encode a type III chaperone and a cognate effector, respectively. ShcFPto interacts with and stabilizes the HopFPto protein in the bacterial cell. Translation of HopFPto starts at a rare initiation codon ATA that limits the synthesis of the HopFPto protein to a low level in bacterial cells.
The Disruption of a Gα Subunit Sheds New Light on the Pathogenicity of Stagonospora nodorum on Wheat
Authors: Solomon Peter S. , Tan Kar-Chun , Sanchez Pedro , Cooper Richard M. , Oliver Richard P.
Published: May 2007

Gna1, a gene encoding a Gα subunit, a key component of signal transduction pathways, has been cloned and characterized in the wheat pathogen Stagonospora nodorum. Analysis of Gna1 expression during infection revealed a slight decrease in transcript levels shortly after germination, after which levels steadily increased until sporulation. Inactivation of Gna1 had a pleiotropic effect on phenotype. The Gna1 mutants were less pathogenic, attributed to coinciding with a defect in direct penetration. Also, Gna1 mutants were unable to sporulate, showed an albino phenotype, and secreted one or more brown pigments into growth media. Analysis of growth medium identified tyrosine, phenylalanine, and dihydroxyphenylalanine (L-DOPA) were excreted by the gna1 strains but not by wild type. The presence of these compounds, and the insensitivity of melanization to tricyclazole suggest that S. nodorum synthesizes melanin via the L-DOPA pathway, the first fungal phytopathogen described to do so. Decreases in protease (and several other depolymerases) activities and sensitivity to osmotic stress were other phenotypes identified in the Gna1 mutants. Gna1 is the first signal transduction gene to be cloned and characterized from S. nodorum and its inactivation has uncovered several previously unknown facets of pathogenicity.
Potyviral 6K2 Protein Long-Distance Movement and Symptom-Induction Functions Are Independent and Host-Specific
Authors: Spetz Carl , Valkonen Jari P. T.
Published: May 2007

Deletion of various portions, or insertion of six histidine residues (6×His) into various positions of the membrane-bound 6K2 protein (53 amino acids) of Potato virus A (PVA, genus Potyvirus), inhibited systemic infection in Nicotiana tabacum and N. benthamiana plants. However, a spontaneous mutation (Gly2Cys) that occurred in 6K2 adjacent to the 6×His insert placed between Ser1 and Gly2 enabled systemic infection in a single N. benthamiana plant. No symptoms were observed, but virus titers were similar to the symptom-inducing wild-type (wt) PVA. N. tabacum plants were not systemically infected, albeit virus propagation was observed in inoculated protoplasts. The 6×His/Gly2Cys mutant was reconstructed in vitro and serially propagated by mechanical inoculation in N. benthamiana. Following the third passage, a novel viral mutant appeared, lacking the last four His residues of the insert, as well as the Gly2 and Thr3 of 6K2. It infected N. tabacum plants systemically, and in the systemically infected N. benthamiana leaves, vein chlorosis and mild yellowing symptoms were observed, typical of wt PVA infection. The mutant virus accumulated to titers similar to wt PVA in both hosts. These results show that the PVA 6K2 protein affects viral long-distance movement and symptom induction independently and in a host-specific manner.