bluuebunny/crossref_metadata_2025 huggingface dataset:
The Erwinia amylovora avrRpt2EA Gene Contributes to Virulence on Pear and AvrRpt2EA Is Recognized by Arabidopsis RPS2 When Expressed in Pseudomonas syringae
Authors: Zhao Youfu , He Sheng-Yang , Sundin George W.
Published: May 2007

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene,avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical ‘hrp box,’ which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabi-dopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P. syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.
Cyclic Lipopeptide Production by Plant-Associated Pseudomonas spp.: Diversity, Activity, Biosynthesis, and Regulation
Authors: Raaijmakers Jos M. , de Bruijn Irene , de Kock Maarten J. D.
Published: May 2007

Cyclic lipopeptides (CLPs) are versatile molecules produced by a variety of bacterial genera, including plant-associated Pseudomonas spp. CLPs are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain. CLPs are very diverse both structurally and in terms of their biological activity. The structural diversity is due to differences in the length and composition of the fatty acid tail and to variations in the number, type, and configuration of the amino acids in the peptide moiety. CLPs have received considerable attention for their antimicrobial, cytotoxic, and surfactant properties. For plant-pathogenic Pseudomonas spp., CLPs constitute important virulence factors, and pore formation, followed by cell lysis, is their main mode of action. For the antagonistic Pseudomonas sp., CLPs play a key role in antimicrobial activity, motility, and biofilm formation. CLPs are produced via nonribosomal synthesis on large, multifunctional peptide synthetases. Both the structural organization of the CLP synthetic templates and the presence of specific domains and signature sequences within peptide synthetase genes will be described for both pathogenic and antagonistic Pseudomonas spp. Finally, the role of various genes and regulatory mechanisms in CLP production by Pseudomonas spp., including two-component regulation and quorum sensing, will be discussed in detail.
Potential Involvement of a Cucumber Homolog of Phloem Protein 1 in the Long-Distance Movement of Cucumber mosaic virus Particles
Authors: Requena A. , Simón-Buela L. , Salcedo G. , García-Arenal F.
Published: May 2007

The systemic movement of Cucumber mosaic virus (CMV) in cucumber plants was analyzed. The structure that is translocated and its putative interactions with phloem components were analyzed in phloem exudate (PE) samples, which reflect sieve tubes stream composition. Rate zonal centrifugation and electron-microscopy analyses of PE from CMV-infected plants showed that CMV moves through sieve tubes as virus particles. Gel overlay assays revealed that CMV particles interact with a PE protein, p48. The amino-acid sequence of several tryptic peptides of p48 was determined. Partial amino-acid sequence of p48 showed it was a cucumber homolog of phloem protein 1 (PP1) from pumpkin, with which p48 also shares several chemical properties. PP1 from pumpkin has plasmodes-mata-gating ability and translocates in sieve tubes. Encapsidated CMV RNA in PE samples from infected plants was less accessible to digestion by RNase A than RNA in purified CMV particles, a property that was reconstituted by the in vitro interaction of purified CMV particles and protein p48. These results indicate that the interaction with p48 modifies CMV particle structure and suggest that CMV particles interact with the cucumber homolog of PP1 during translocation in the sieve tubes.
Mutations in the lrpE Gene of Ralstonia solanacearum Affects Hrp Pili Production and Virulence
Authors: Murata Yukio , Tamura Naoyuki , Nakaho Kazuhiro , Mukaihara Takafumi
Published: May 2007

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the ΔlrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the ΔlrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the ΔlrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.
RMo1 Confers Blast Resistance in Barley and Is Located within the Complex of Resistance Genes Containing Mla, a Powdery Mildew Resistance Gene
Authors: Inukai Tsuyoshi , Vales M. Isabel , Hori Kiyosumi , Sato Kazuhiro , Hayes Patrick M.
Published: May 2007

Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54–20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53–33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53–33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.
MGOS: A Resource for Studying Magnaporthe grisea and Oryza sativa Interactions
Authors: Soderlund Carol , Haller Karl , Pampanwar Vishal , Ebbole Daniel , Farman Mark , Orbach Marc J. , Wang Guo-liang , Wing Rod , Xu Jin-Rong , Brown Doug , Mitchell Thomas , Dean Ralph
Published: May 2007

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.
Genetic Suppressors of the Lotus japonicus har1-1 Hypernodulation Phenotype
Authors: Murray Jeremy , Karas Bogumil , Ross Loretta , Brachmann Andreas , Wagg Cameron , Geil Ryan , Perry Jillian , Nowakowski Katarzyna , MacGillivary Mandy , Held Mark , Stougaard Jens , Peterson Larry , Parniske Martin , Szczyglowski Krzysztof
Published: May 2007

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.
Yeast Increases Resistance in Arabidopsis Against Pseudomonas syringae and Botrytis cinerea by Salicylic Acid-Dependent as Well as -Independent Mechanisms
Authors: Raacke Ines C. , von Rad Uta , Mueller Martin J. , Berger Susanne
Published: May 2007

Cell-wall and glucopeptide components of yeast have been reported to exhibit elicitor activity. The mode of action of defense activation by yeast is not known so far. In this study, we used the model plant Arabidopsis to investigate the activation of defense responses by yeast, the effect on resistance against different pathogens, and the mode of action. Treatment of Arabidopsis plants with an autoclaved yeast suspension induced the expression of systemic acquired resistance-related genes and accumulation of the phytoalexin camalexin. Symptom development and bacterial growth after infection with a virulent strain of the pathogen Pseudomonas syringae was reduced in yeast-pretreated plants. No protection was detectable in mutants affected in the salicylate pathway, while mutants in the jasmonate or camalexin pathway were protected by yeast, indicating that the salicylate pathway is necessary for the yeast-induced resistance against P. syringae. Yeast also reduced symptom development after challenge with Botrytis cinerea. This protection was detectable in all mutants tested, indicating that it is independent of the salicylate, jasmonate, and camalexin pathway.
Closing the Circle on the Discovery of Genes Encoding Hrp Regulon Members and Type III Secretion System Effectors in the Genomes of Three Model Pseudomonas syringae Strains
Authors: Lindeberg Magdalen , Cartinhour Samuel , Myers Christopher R. , Schechter Lisa M. , Schneider David J. , Collmer Alan
Published: May 2007

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.
The Genomic Dynamics and Evolutionary Mechanism of the Pi2/9 Locus in Rice
Authors: Zhou Bo , Dolan Maureen , Sakai Hajime , Wang Guo-Liang
Published: May 2007

The Pi2/9 locus contains at least four resistance specificities to Magnaporthe grisea and belongs to a gene complex comprised of multiple genes that encode highly homologous nucleotide binding site (NBS) and leucine rich repeat (LRR) proteins. To investigate the genetic events involved in the evolution of the Pi2/9 locus, we analyzed the Pi2/9 locus at the inter- and intralocus levels in five rice cultivars. The NBS-LRR genes in the five cultivars belong to the same phylogenetic clade among rice NBS-LRR genes, and all have a phase-2 intron at the N-terminus. However, the paralogs within each haplotype show a significant sequence divergence and their N-terminal intron and 5′ regulatory regions are very different. On the contrary, the orthologs from different haplotypes are highly similar, indicating an obvious orthologous relationship has been maintained during the evolution of the Pi2/9 locus. These results suggest that sequence diversification in the 5′ regulatory regions and N-terminal introns of the paralogs may have led to suppression of meiotic recombination between the paralogs within each haplotype, facilitating the maintenance of the orthologous relationship among rice cultivars. Our observations provide valuable insight into the genomic dynamics and evolutionary mechanism of an NBS-LRR resistance-gene complex in rice.
The Role of Pectate Lyase and the Jasmonic Acid Defense Response in Pseudomonas viridiflava Virulence
Authors: Jakob Katrin , Kniskern Joel M. , Bergelson Joy
Published: May 2007

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coi1-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coi1-1 relative to Col-0, indicating that P. viridi-flava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.
Characterization of the Interaction Between the Bacterial Wilt Pathogen Ralstonia solanacearum and the Model Legume Plant Medicago truncatula
Authors: Vailleau Fabienne , Sartorel Elodie , Jardinaud Marie-Françoise , Chardon Fabien , Genin Stéphane , Huguet Thierry , Gentzbittel Laurent , Petitprez Michel
Published: May 2007

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solana-cearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 × 105 in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.
Ptr ToxA Interacts with a Chloroplast-Localized Protein
Authors: Manning Viola A. , Hardison Linda K. , Ciuffetti Lynda M.
Published: May 2007

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteina-ceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.
Symbiosis-Induced Cascade Regulation of the Mesorhizobium loti R7A VirB/D4 Type IV Secretion System
Authors: Hubber Andree M. , Sullivan John T. , Ronson Clive W.
Published: May 2007

The Mesorhizobium loti R7A symbiosis island contains genes encoding a VirB/D4 type IV secretion system (T4SS) similar to that of Agrobacterium tumefaciens. This system has host-dependent effects on symbiosis that probably are due to translocation of two effector proteins, Msi059 and Msi061, into host cells. Here we report that, as in A. tumefaciens, the M. loti vir genes are transcriptionally regulated by a VirA/VirG two-component regulatory system. A virGN54D mutant gene of M. loti caused constitutive expression of lacZ reporter gene fusions to virB1, virD4, msi059, and msi061. Expression of these gene fusions also was activated by a NodD gene product from Rhizobium leguminosarum in the presence of the inducer naringenin, as was a virA∷lacZ fusion. This activation was dependent on a nod box present 851 bp upstream of virA, and a mutant with the nod box deleted formed effective nodules on Leucaena leucocephala, the same symbiotic phenotype as other M.loti vir mutants. In contrast, the wild-type strain formed small, empty nodules whereas a nodD1 mutant was completely Nod¯. These results indicate that the M. loti vir genes are induced in a symbiosis-specific manner that involves a two-tiered regulatory cascade, and that the vir effectors act after Nod factor during infection thread formation.
ABA Is Required for Leptosphaeria maculans Resistance via ABI1- and ABI4-Dependent Signaling
Authors: Kaliff Maria , Staal Jens , Myrenås Mattias , Dixelius Christina
Published: May 2007

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLM1Col pathway. ABA-deficient and -insensitive mutants in Ler-0 background (aba1-3 and abi1-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abi1-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abi1-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. β-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1Col)pad3 and rlm1Lerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1Col·ABI1, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1Col expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABI1 activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.
The HopX (AvrPphE) Family of Pseudomonas syringae Type III Effectors Require a Catalytic Triad and a Novel N-Terminal Domain for Function
Authors: Nimchuk Zachary L. , Fisher Emily J. , Desveaux Darrell , Chang Jeffery H. , Dangl Jeffery L.
Published: May 2007

Many gram-negative plant pathogenic bacteria employ type III secretion systems to deliver effector proteins directly into the host cell during infection. On susceptible hosts, type III effectors aid pathogen growth by manipulating host defense pathways. On resistant hosts, some effectors can activate specific host disease resistance (R) genes, leading to generation of rapid and effective immune responses. The biochemical basis of these processes is poorly understood. The HopX (AvrPphE) family is a widespread type III effector among phytopathogenic bacteria. We determined that HopX family members are modular proteins composed of a conserved putative cysteine-based catalytic triad and a conserved potential target/cofactor interaction domain. HopX is soluble in host cells. Putative catalytic triad residues are required for avirulence activity on resistant bean hosts and for the generation of a cell-death response in specific Arabidopsis genotypes. The putative target/cofactor interaction domain is also required for these activities. Our data suggest that specific interaction with and modification of a cytosolic host target drives HopX recognition in resistant hosts and may contribute to virulence in susceptible hosts. Surprisingly, the Legionella pneumophila genome was found to contain a protein with similarity to HopX in sequence and domain arrangement, suggesting that these proteins might also contribute to animal pathogenesis and could be delivered to plant and animal hosts by diverse secretion systems.
A Polygalacturonase-Inhibiting Protein from Grapevine Reduces the Symptoms of the Endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana Leaves Without Any Evidence for In Vitro Interaction
Authors: Joubert Dirk A. , Kars Ilona , Wagemakers Lia , Bergmann Carl , Kemp Gabré , Vivier Melané A. , van Kan Jan A. L.
Published: May 2007

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgip1 gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.
The Iturin and Fengycin Families of Lipopeptides Are Key Factors in Antagonism of Bacillus subtilis Toward Podosphaera fusca
Authors: Romero Diego , de Vicente Antonio , Rakotoaly Rivo H. , Dufour Samuel E. , Veening Jan-Willem , Arrebola Eva , Cazorla Francisco M. , Kuipers Oscar P. , Paquot Michel , Pérez-García Alejandro
Published: May 2007

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopep-tides have a major role in the antagonism of B. subtilis toward P. fusca.
Mir1 is Highly Upregulated and Localized to Nuclei During Infectious Hyphal Growth in the Rice Blast Fungus
Authors: Li Lei , Ding Sheng-Li , Sharon Amir , Orbach Marc , Xu Jin-Rong
Published: May 2007

Rice blast, caused by Magnaporthe grisea, is a devastating disease of rice throughout the world. Many recent molecular studies have focused on the early infection stages, but our knowledge about molecular events at the infectious hyphae stage is limited. In this study, 750 hygromycin-resistant transformants were isolated by transforming M. grisea Guy11 with a promoterless enhanced green fluorescent protein (EGFP) construct. In one of the transformants, L1320, EGFP signals were observed in the nuclei of infectious hyphae. The transforming vector was inserted in a predicted gene named MIR1 and resulted in a Mir11–107-EGFP fusion. Mir1 is a low-complexity protein with no known protein domain and has no homolog in GenBank or other sequenced fungal genomes. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis and expression assays of MIR1-EGFP fusion constructs indicated that the expression of MIR1 was highly induced during plant infection. Deletion analyses identified a 458-bp region that was sufficient for the MIR1 promoter activity. Further characterization revealed that a 96-bp sequence was essential for the enhanced in planta expression. MIR1 is an M. grisea-specific gene that is highly conserved among the field isolates belonging to the M. grisea species complex. The mir1 mutants had no obvious defects in appressorial penetration and rice infection. When overexpressed with the RP27 promoter, nuclear localization of the Mir1-EGFP fusion was observed in conidia and vegetative hyphae. These data suggest that the expression but not the nuclear localization of MIR1 is specific to infectious hyphae and that reporter genes based on MIR1 may be suitable for monitoring infectious growth in M. grisea.
Heterochromatin-Like Regions as Ecological Niches for Avirulence Genes in the Leptosphaeria maculans Genome: Map-Based Cloning of AvrLm6
Authors: Fudal I. , Ross S. , Gout L. , Blaise F. , Kuhn M. L. , Eckert M. R. , Cattolico L. , Bernard-Samain S. , Balesdent M. H. , Rouxel T.
Published: May 2007

Map-based cloning of avirulence genes of the AvrLm1-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLm1, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5′ and 3′ sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.
Functional Analysis of Lipid Metabolism in Magnaporthe grisea Reveals a Requirement for Peroxisomal Fatty Acid β-Oxidation During Appressorium-Mediated Plant Infection
Authors: Wang Zheng-Yi , Soanes Darren M. , Kershaw Michael J. , Talbot Nicholas J.
Published: May 2007

The rice blast fungus Magnaporthe grisea infects plants by means of specialized infection structures known as appressoria. Turgor generated in the appressorium provides the invasive force that allows the fungus to breach the leaf cuticle with a narrow-penetration hypha gaining entry to the underlying epidermal cell. Appressorium maturation in M. grisea involves mass transfer of lipid bodies to the developing appressorium, coupled to autophagic cell death in the conidium and rapid lipolysis at the onset of appressorial turgor generation. Here, we report identification of the principal components of lipid metabolism in M. grisea based on genome sequence analysis. We show that deletion of any of the eight putative intracellular triacylglycerol lipase-encoding genes from the fungus is insufficient to prevent plant infection, highlighting the complexity and redundancy associated with appressorial lipolysis. In contrast, we demonstrate that a peroxisomally located multifunctional, fatty acid β-oxidation enzyme is critical to appressorium physiology, and blocking peroxisomal biogenesis prevents plant infection. Taken together, our results indicate that, although triacylglycerol breakdown in the appressorium involves the concerted action of several lipases, fatty acid metabolism and consequent generation of acetyl CoA are necessary for M. grisea to complete its prepenetration phase of development and enter the host plant.
OsWRKY13 Mediates Rice Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling
Authors: Qiu Deyun , Xiao Jun , Ding Xinhua , Xiong Min , Cai Meng , Cao Yinglong , Li Xianghua , Xu Caiguo , Wang Shiping
Published: May 2007

Although 109 WRKY genes have been identified in the rice genome, the functions of most are unknown. Here, we show that OsWRKY13 plays a pivotal role in rice disease resistance. Overexpression of OsWRKY13 can enhance rice resistance to bacterial blight and fungal blast, two of the most devastating diseases of rice worldwide, at both the seedling and adult stages, and shows no influence on the fertility. This overexpression was accompanied by the activation of salicylic acid (SA) synthesis-related genes and SA-responsive genes and the suppression of jasmonic acid (JA) synthesis-related genes and JA-responsive genes. OsWRKY13 bound to the promoters of its own and at least three other genes in SA- and JA-dependent signaling pathways. Its DNA-binding activity was influenced by pathogen infection. These results suggest that OsWRKY13, as an activator of the SA-dependent pathway and a suppressor of JA-dependent pathways, mediates rice resistance by directly or indirectly regulating the expression of a subset of genes acting both upstream and downstream of SA and JA. Furthermore, OsWRKY13 will provide a transgenic tool for engineering wider-spectrum and whole-growth-stage resistance rice in breeding programs.
Twitching Motility Is Essential for Endophytic Rice Colonization by the N2-Fixing Endophyte Azoarcus sp. Strain BH72
Authors: Böhm Melanie , Hurek Thomas , Reinhold-Hurek Barbara
Published: May 2007

Azoarcus sp. strain BH72, as an endophyte of grasses, depends on successful host colonization. Type IV pili are essential for mediating the initial interaction with rice roots. In the genome sequence analysis, the pilT gene was identified, which encodes for a putative type IV pilus retraction protein. PilT of Azoarcus sp. BH72 shares high similarity to PilT of the human pathogen Pseudomonas aeruginosa PAO1 (77% amino acid sequence identity) and contains a predicted nucleotide-binding motif. To gain more insights into the role of the type IV pili in the colonization process of Azoarcus spp., we constructed an insertional mutant of pilT and a deletion mutant of pilA, the major structural component of the pilus structure. The pilT mutant, as the pilin deletion mutant ΔpilA, was abolished in twitching motility. Western blot analyses and electron microscopy studies demonstrated an enhanced piliation of the Azoarcus pilT mutant strain compared with the wild type, indicating that, indeed, PilT has a role in pilus retraction. Studies on rice root colonization in gnotobiotic cultures revealed that the establishment of microcolonies on the root surface was strongly reduced in the ΔpilA mutant, whereas the surface colonization was reduced by only 50% in the nontwitching pilT mutant. However, endophytic colonization of rice roots was strongly reduced in both mutants. These results demonstrate that the retractile force mediated by PilT is not essential for the bacterial colonization of the plant surface, but that twitching motility is necessary for invasion of and establishment inside the plant. Thus, a novel determinant for endophytic interactions with grasses was identified.
Evidence That the Nonstructural Protein ofTomato spotted wilt virusIs the Avirulence Determinant in the Interaction with Resistant Pepper Carrying theTswGene
Authors: Margaria P. , Ciuffo M. , Pacifico D. , Turina M.
Published: May 2007

All known pepper cultivars resistant to Tomato spotted wilt virus (TSWV) possess a single dominant resistance gene, Tsw. Recently, naturally occurring resistance-breaking (RB) TSWV strains have been identified, causing major concerns. We used a collection of such strains to identify the specific genetic determinant that allows the virus to overcome the Tsw gene in Capsicum spp. A reverse genetic approach is still not feasible for TSWV; therefore, we analyzed reassortants between wild-type (WT) and RB strains. Our results confirmed that the S RNA, which encodes both the nucleocapsid protein (N) and a nonstructural protein (NSs), carries the genetic determinant responsible for Tsw resistance breakdown. We then used full-length S RNA segments or the proteins they encode to compare the sequences of WT and related RB strains, and obtained indirect evidence that the NSs protein is the avirulence factor in question. Transient expression of NSs protein from WT and RB strains showed that they both can equally suppress post-transcriptional gene silencing (PTGS). Moreover, biological characterization of two RB strains carrying deletions in the NSs protein showed that NSs is important in maintaining TSWV infection in newly emerging leaves over time, preventing recovery. Analysis of another RB strain phenotype allowed us to conclude that local necrotic response is not sufficient for resistance in Capsicum spp. carrying the Tsw gene.
A Novel Role for Catalase B in the Maintenance of Fungal Cell-Wall Integrity During Host Invasion in the Rice Blast Fungus Magnaporthe grisea
Authors: Skamnioti Pari , Henderson Catherine , Zhang Ziguo , Robinson Zena , Gurr Sarah Jane
Published: May 2007

Asexual spores of the rice blast fungus germinate to produce a specialized and melanized infection structure, the appressorium, which is pivotal to successful plant penetration. To investigate whether Magnaporthe grisea counteracts the toxic burst of H2O2 localized beneath the site of attempted invasion, we examined the temporal expression of five candidate antioxidant genes. Of these, the putatively secreted large subunit catalase CATB gene was 600-fold up-regulated in vivo, coincident with penetration, and moderately up-regulated in vitro, in response to exogenous H2O2. Targeted gene replacement of CATB led to compromised pathogen fitness; the catB mutant displayed paler pigmentation and accelerated hyphal growth but lower biomass, poorer sporulation, fragile conidia and appressoria, and impaired melanization. The catB mutant was severely less pathogenic than Guy 11 on barley and rice, and its infectivity was further reduced on exposure to H2O2. The wild-type phenotype was restored by the reintroduction of CATB into the catB mutant. We found no evidence to support a role for CATB in detoxification of the host-derived H2O2 at the site of penetration. Instead, we demonstrated that CATB plays a part in strengthening the fungal wall, a role of particular importance during forceful entry into the host.
The MAP Kinase Kinase MKK2 Affects Disease Resistance inArabidopsis
Authors: Brader Günter , Djamei Armin , Teige Markus , Palva E. Tapio , Hirt Heribert
Published: May 2007

The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) was shown to mediate cold and salt stress responses through activation of the two MAP kinases MPK4 and MPK6. Transcriptome analysis of plants expressing constitutively active MKK2 (MKK2-EE plants) showed altered expression of genes induced by abiotic stresses but also a significant number of genes involved in defense responses. Both MPK4 and MPK6 became rapidly activated upon Pseudomonas syringae pv. tomato DC3000 infection and MKK2-EE plants showed enhanced levels of MPK4 activation. Although MKK2-EE plants shared enhanced expression of genes encoding enzymes of ethylene (ET) and jasmonic acid (JA) synthesis, ET, JA, and salicylic acid (SA) levels did not differ dramatically from those of wild-type or mkk2-null plants under ambient growth conditions. Upon P. syringae pv. tomato DC3000 infection, however, MKK2-EE plants showed reduced increases of JA and SA levels. These results indicate that MKK2 is involved in regulating hormone levels in response to pathogens. MKK2-EE plants were more resistant to infection by P. syringae pv. tomato DC3000 and Erwinia carotovora subsp. carotovora, but showed enhanced sensitivity to the fungal necrotroph Alternaria brassicicola. Our data indicate that MKK2 plays a role in abiotic stress tolerance and plant disease resistance.
The Vicia faba Leghemoglobin Gene VfLb29 Is Induced in Root Nodules and in Roots Colonized by the Arbuscular Mycorrhizal Fungus Glomus fasciculatum
Authors: Frühling Martin , Roussel Hélène , Gianinazzi-Pearson Vivienne , Pühler Alfred , Perlick Andreas M.
Published: May 2007

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.
A Gene Encoding a Protein Elicitor of Phytophthora infestans Is Down-Regulated During Infection of Potato
Authors: Kamoun Sophien , van West Pieter , de Jong Anke J. , de Groot Koen E. , Vleeshouwers Vivianne G. A. A. , Govers Francine
Published: May 2007

Most species of the genus Phytophthora produce 10-kDa extracellular protein elicitors, collectively termed elicitins. Elicitins induce hypersensitive response in a restricted number of plants, particularly in the genus Nicotiana within the Solanaceae family. A cDNA encoding INF1, the major secreted elicitin of Phytophthora infestans, a pathogen of solanaceous plants, was isolated and characterized. The expression of the corresponding inf1 gene during the disease cycle of P. infestans was analyzed. inf1 was shown to be expressed in mycelium grown in various culture media, whereas it was not expressed in sporangiospores, zoospores, cysts, and germinating cysts. In planta, during infection of potato, particularly during the biotrophic stage, expression of inf1 was down-regulated compared to in vitro. The highest levels of expression of inf1 were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The potential role of INF1 as an elicitor in interactions between P. infestans and Solanum species was investigated. Nineteen lines, representing nine solanaceous species with various levels of resistance to P. infestans, were tested for response to an Escherichia coli expressed INF1. Within the genus Solanum, resistance to P. infestans did not appear to be mediated by a defense response elicited by INF1. However, INF1 recognition could be a component of nonhost resistance of tobacco to P. infestans.
Rhizobia Modulate Root-Hair-Specific Expression of Extensin Genes
Authors: Arsenijevic-Maksimovic Ivana , Broughton William J. , Krause Andrea
Published: May 2007

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGRΔnodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGRΔnodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5′ flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.