Добірка наукової літератури з теми "Avirulence factors"

To understand why the Pvr4 resistance of pepper against Potyvirus spp. remained durable in field conditions while virulent Potato virus Y (PVY) variants could be selected in the laboratory, we studied the molecular mechanisms which generated these variants and the consequences on viral fitness. Using a reverse genetics approach with an infectious cDNA clone of PVY, we found that the region coding for the NIb protein (RNA-dependent RNA polymerase) of PVY was the avirulence factor corresponding to Pvr4 and that a single nonsynonymous nucleotide substitution in that region, an adenosine to guanosine substitution at position 8,424 of the PVY genome (A8424G), was sufficient for virulence. This substitution imposed a high competitiveness cost to the virus against an avirulent PVY variant in plants devoid of Pvr4. In addition, during serial passages in susceptible pepper plants, the only observed possibility of the virulent mutant to increase its fitness was through the G8424A reversion, strengthening the high durability potential of the Pvr4 resistance. This is in accordance with the fact that the NIb protein is one of the most constrained proteins expressed by the PVY genome and, more generally, by Potyvirus spp., and with a previously developed model predicting the durability of virus resistances as a function of the evolutionary constraint applied on corresponding avirulence factors.

The avrXa10 gene of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, is a member of the avrBs3 avirulence gene family and directs the elicitation of resistance in a gene-for-gene manner on rice lines carrying the resistance gene Xa10. The carboxyl (C) terminus of AvrXa10 has a previously undescribed domain that is structurally similar to the acidic activation domain of many eukaryotic transcription factors in addition to three nuclear localization signal (NLS) sequences. Removal of the C-terminal 38 codons containing the putative activation domain, but retaining the NLS sequences, was concomitant with the loss of avirulence activity. The C-terminal coding regions of avrBs3 and avrXa7 can be replaced by the corresponding region of avrXa10, and the genes retained specificity for the resistance genes Bs3 in pepper and Xa7 in rice, respectively. The avrBs3 and avrXa7 avirulence activities of the hybrid genes were also lost upon removal of the terminal 38 codons. When fused to the coding sequence of the Gal4 DNA binding domain, AvrXa10 activated transcription in yeast and Arabidopsis thaliana. Removal of the carboxyl region severely reduced transcriptional activation. AvrXa10 would have to be localized to the host cell nucleus to function autonomously in transcriptional activation. Consistent with this requirement, mutations in all three NLS sequences of avrXa10 caused a loss in avirulence activity. The findings demonstrate the requirement of the C terminus for AvrXa10 function and the potential for the members of this family of avirulence gene products to enter the host nucleus and alter host transcription.

In the plant immune system, according to the ‘gene-for-gene’ model, a resistance (R) gene product in the plant specifically surveils a corresponding effector protein functioning as an avirulence (Avr) gene product. This system differs from other plant–pathogen interaction systems, in which plant R genes recognize a single type of gene or gene family because almost all virus genes with distinct structures and functions can also interact with R genes as Avr determinants. Thus, research conducted on viral Avr-R systems can provide a novel understanding of Avr and R gene product interactions and identify mechanisms that enable rapid co-evolution of plants and phytopathogens. In this review, we intend to provide a brief overview of virus-encoded proteins and their roles in triggering plant resistance, and we also summarize current progress in understanding plant resistance against virus Avr genes. Moreover, we present applications of Avr gene-mediated phenotyping in R gene identification and screening of segregating populations during breeding processes.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an ‘atypical’ conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation.

Abstract We have identified genes for pathogenicity toward rice (Oryza sativa) and genes for virulence toward specific rice cultivars in the plant pathogenic fungus Magnaporthe grisea. A genetic cross was conducted between the weeping lovegrass (Eragrostis curvula) pathogen 4091-5-8, a highly fertile, hermaphroditic laboratory strain, and the rice pathogen O-135, a poorly fertile, female-sterile field isolate that infects weeping lovegrass as well as rice. A six-generation backcrossing scheme was then undertaken with the rice pathogen as the recurrent parent. One goal of these crosses was to generate rice pathogenic progeny with the high fertility characteristic of strain 4091-5-8, which would permit rigorous genetic analysis of rice pathogens. Therefore, progeny strains to be used as parents for backcross generations were chosen only on the basis of fertility. The ratios of pathogenic to nonpathogenic (and virulent to avirulent) progeny through the backcross generations suggested that the starting parent strains differ in two types of genes that control the ability to infect rice. First, they differ by polygenic factors that determine the extent of lesion development achieved by those progeny that infect rice. These genes do not appear to play a role in infection of weeping lovegrass because both parents and all progeny infect weeping lovegrass. Second, the parents differ by simple Mendelian determinants, "avirulence genes," that govern virulence toward specific rice cultivars in all-or-none fashion. Several crosses confirm the segregation of three unlinked avirulence genes, Avr 1-CO39, Avr 1-M201 and Avr1-YAMO, alleles of which determine avirulence on rice cultivars CO39, M201, and Yashiro-mochi, respectively. Interestingly, avirulence alleles of Avr1-CO39, Avr1-M201 and Avr1-YAMO were inherited from the parent strain 4091-5-8, which is a nonpathogen of rice. Middle repetitive DNA sequences ("MGR sequences"), present in approximately 40-50 copies in the genome of the rice pathogen parent, and in very low copy number in the genome of the nonpathogen of rice, were used as physical markers to monitor restoration of the rice pathogen genetic background during introgression of fertility. The introgression of highest levels of fertility into the most successful rice pathogen progeny was incomplete by the sixth generation, perhaps a consequence of genetic linkage between genes for fertility and genes for rice pathogenicity. One chromosomal DNA segment with MGR sequence homology appeared to be linked to the gene Avr1-CO39. Finally, many of the crosses described in this paper exhibited a characteristic common to many crosses involving M. grisea rice pathogen field isolates.(ABSTRACT TRUNCATED AT 400 WORDS)

The future prevalence and virulence of SARS-CoV-2 is uncertain. Some emerging pathogens become avirulent as populations approach herd immunity. Although not all viruses follow this path, the fact that the seasonal coronaviruses are benign gives some hope. We develop a general mathematical model to predict when the interplay among three factors, correlation of severity in consecutive infections, population heterogeneity in susceptibility due to age, and reduced severity due to partial immunity, will promote avirulence as SARS-CoV-2 becomes endemic. Each of these components has the potential to limit severe, high-shedding cases over time under the right circumstances, but in combination they can rapidly reduce the frequency of more severe and infectious manifestation of disease over a wide range of conditions. As more reinfections are captured in data over the next several years, these models will help to test if COVID-19 severity is beginning to attenuate in the ways our model predicts, and to predict the disease.

The Xanthomonas campestris pv. vesicatoria avirulence gene avrRxv specifies resistance on the tomato line Hawaii 7998 by interacting with three nondominant plant resistance genes. AvrRxv molecular properties that impinge on its avirulence activity were characterized and transcriptional changes caused by AvrRxv expression in resistant tomato plants were extensively examined. AvrRxv localized predominantly to the cytoplasm and possibly in association with plasma and nuclear membranes in both resistant and susceptible tomato plants. The AvrRxv cysteine protease catalytic core was found to be essential for host recognition, because introduction of mutations in this domain affected the ability of AvrRxv to elicit a hypersensitive response and the inhibition of bacterial growth in resistant plants. In addition, expression profiles were analyzed for approximately 8,600 tomato genes in resistant plants challenged with X. campestris pv. vesicatoria strains expressing wild-type AvrRxv or a catalytic core AvrRxv mutant. In all, 420 genes were identified as differentially modulated by the expression of a functional AvrRxv, including over 15 functional classes of proteins and a large number of transcription factors and signaling components. Findings of this study allow the development of new hypotheses about the molecular basis of recognition between AvrRxv and the corresponding resistance proteins, and set the stage for the dissection of signaling and cellular responses triggered in tomato plants by this avirulence factor.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

L’utilisation de variétés résistantes est un levier majeur dans la lutte contre les maladies des plantes, mais l’adaptation des populations d’agents pathogènes limite leur durabilité. L’agent du mildiou de la vigne, l’oomycète Plasmopara viticola, s’est ainsi montré capable de contourner rapidement plusieurs facteurs de résistance récemment déployés en Europe. Les résistances de la vigne au mildiou sont majeures mais partielles, ce qui pose la question des mécanismes de virulence du pathogène dans une interaction phénotypiquement quantitative. Une approche de cartographie de QTL a été mise en œuvre pour identifier les déterminants génétiques de l’adaptation du mildiou à trois facteurs de résistances de la vigne : Rpv3.1, Rpv10 et Rpv12. Deux croisements ont été réalisés entre des souches de mildiou aux profils de virulence contrastés. Ces descendants ont été génotypés par le séquençage ciblé de 5263 SNPs. La construction de cartes génétiques de haute densité a permis de réaliser un pseudo-assemblage du génome au niveau chromosomique (2n=34). Certains descendants sont porteurs d’anomalies caryotypiques (aneuploïdies, triploïdies) qui proviennent quasi-exclusivement du gamète mâle via plusieurs mécanismes (gamètes diploïdes, dispermie). Grâce au phénotypage de l’interaction entre ces descendances et différents cultivars de vigne (sporulation, nécrose), un QTL majeur a été détecté pour chacune des virulences étudiées. Un locus candidat pour AvrRpv12 a été identifié et contient plusieurs gènes d’effecteurs putatifs RXLR absents ou non-fonctionnels dans les allèles virulents. Ce contournement est conforme à une relation gène-pour-gène dans laquelle la virulence est récessive. Dans le cas de Rpv10, un déterminisme génétique atypique a été mis en évidence. Ce contournement, à la fois partiel et dominant, suggère fortement un mécanisme suppresseur d’avirulence. Le QTL détecté correspond à un intervalle de 537 kb peu recombinant et très riche en gènes de protéines sécrétées. Un assemblage diploïde de la souche parente a révélé d’importants réarrangements structuraux et une variation du répertoire d’effecteurs putatifs dans l’haplotype associé au contournement. L’étude d’une population issue de rétrocroisement a confirmé la dominance de cet allèle suppresseur d’avirulence. L’analyse de la structure génétique d’un panel d’isolats suggère plusieurs événements indépendants en ce qui concerne le contournement de Rpv12. En revanche, la virulence vis-à-vis de Rpv10 a probablement une origine unique liée à l’introduction récente d’un fond génétique extra-européen. La position du locus AvrRpv3.1, précédemment identifié par GWAS, a pu être confirmée. Le séquençage d’une centaine d’isolats de mildiou révèle une grande diversité d’allèles de contournement de Rpv3.1 en Europe. Cette diversité est peut-être liée à la diffusion ancienne de cépages hybrides porteurs de Rpv3.1 avant que leurs surfaces ne se réduisent fortement au milieu du XXème siècle. Un outil moléculaire a été mis au point pour suivre la présence-absence des effecteurs AvrRpv3.1 par qPCR. Cet outil permet d’envisager une surveillance haut-débit des populations de mildiou. L’ensemble de ces résultats améliorent notre compréhension des mécanismes d’adaptation de P. viticola aux résistances de la vigne. Ils ouvrent la voie à la caractérisation fonctionnelle de nouveaux effecteurs d’oomycète. Enfin, le suivi de la dynamique évolutive des gènes impliqués permettra de mieux concevoir les stratégies de déploiement des vignes résistantes
Breeding for resistant varieties is one of the most efficient approach to control plant diseases, but the adaptation of pathogen populations limits their durability. The causal agent of grapevine downy mildew, the oomycete Plasmopara viticola, has demonstrated the ability to rapidly overcome several resistance factors recently deployed in Europe. Grapevine resistances to downy mildew are strong but partial, which raises the question of the mechanisms of pathogen virulence in this phenotypically quantitative interaction. A QTL mapping approach was used to identify the genetic determinants of P. viticola adaptation to three grapevine resistance factors: Rpv3.1, Rpv10 and Rpv12. Two crosses were made between downy mildew strains with contrasting virulence profiles. These progenies were genotyped by targeted sequencing of 5263 SNPs. The construction of high-density linkage maps enabled a pseudo-assembly of the genome at the chromosome level (2n=34). Some offspring carry karyotypic abnormalities (aneuploidies, triploidies) that originate almost exclusively from the male gamete, via several mechanisms (diploid gametes, dispermy). By phenotyping the interaction between these progenies and different grapevine cultivars (sporulation, necrosis), a major QTL was detected for each virulence. A candidate locus for AvrRpv12 was identified, containing several putative RXLR effector genes absent or non-functional in the virulent alleles. This resistance breakdown is consistent with a gene-for-gene relationship in which virulence is recessive. In the case of Rpv10, an atypical genetic determinism was observed. Resistance breakdown is partial and dominant, which strongly suggests an avirulence suppressor mechanism. The QTL detected corresponds to a 537 kb interval that is poorly recombinant and highly enriched in secreted protein genes. A haplotype-aware assembly of the parent strain revealed major structural rearrangements and variation in the repertoire of putative effectors in the virulent haplotype. Study of a backcross population confirms the dominance of this avirulence suppressor allele. Analysis of the genetic structure of a panel of isolates suggests several independent events with regard to Rpv12 breakdown. In contrast, virulence against Rpv10 probably has a unique origin linked to the recent introduction of a non-European genetic background. The position of the AvrRpv3.1 locus, previously identified by GWAS, was confirmed. The sequencing of around a hundred P. viticola isolates revealed a wide diversity of Rpv3.1 bypass alleles in Europe. This diversity may be linked to the long-standing distribution of hybrid grape varieties carrying Rpv3.1, before their areas were drastically reduced in the mid-twentieth century. A molecular tool has been developed to monitor the presence-absence of AvrRpv3.1 effectors by qPCR. This tool makes high-throughput monitoring of mildew populations possible. Taken together, these results improve our understanding of the mechanisms by which P. viticola adapts to grapevine resistances. They also pave the way for the functional characterization of new oomycete effectors. Finally, monitoring the evolutionary dynamics of the genes involved will inform the design of better deployment strategies for resistant grapevines

Abstract By 1983, several laboratories had identified and isolated the etiologic agent of AIDS, the retrovirus now denoted HIV, and by 1985 nucleotide sequences derived from those early isolates were reported. This molecular information was available in time to address a flurry of speculations and allegations concerning the sudden emergence of the AIDS virus. In 1986, two avirulent strains of herpes simplex virus were discovered to have generated a lethal recombinant in vitro (Javier et al., 1986), and this quickly touched off speculation that the AIDS virus may have been similarly generated in nature or in a testtube. Such speculations contributed to a world wide climate of anxiety and created a potential for dissemination of false and misleading information. In light of this, we at the HIV Sequence Database and Analysis Project (an NIH-funded project at the Los Alamos National Laboratory), recognized the immediate need for an investigation of the molecular facts pertinent to the origin of AIDS.

The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.

The specific objectives of this BARD proposal were: Use a comparative genomics approach to identify T3Es in group I, II and III strains of A. citrulli. Determine the bacterial genes contributing to host preference. Develop mutant strains that can be used for biological control of BFB. Background to the topic: Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovoraxcitrulli, is a devastating disease that affects watermelon (Citrulluslanatus) and melon (Cucumismelo) production worldwide, including both Israel and USA. Three major groups of A. citrullistrains have been classified based on their virulence on host plants, genetics and biochemical properties. The host selection could be one of the major factors that shape A. citrullivirulence. The differences in the repertoire of type III‐ secreted effectors (T3Es) among the three A. citrulligroups could play a major role in determining host preferential association. Currently, there are only 11 A. citrulliT3Es predicted by the annotation of the genome of the group II strain, AAC00‐1. We expect that new A. citrulliT3Es can be identified by a combination of bioinformatics and experimental approaches, which may help us to further define the relationship of T3Es and host preference of A. citrulli. Implications, both scientific and agricultural: Enriching the information on virulence and avirulence functions of T3Es will contribute to the understanding of basic aspects of A. citrulli‐cucurbit interactions. In the long term, it will contribute to the development of durable BFB resistance in commercial varieties. In the short term, identifying bacterial genes that contribute to virulence and host preference will allow the engineering of A. citrullimutants that can trigger SAR in a given host. If applied as seed treatments, these should significantly improve the effectiveness and efficacy of BFB management in melon and atermelon production.

The research problem: Bacterial spot and bacterial speck diseases of tomato are causedby strains of Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv.tomato (Pst), respectively. These bacteria colonize aerial parts of the plant and causesignificant losses in tomato production worldwide. Protection against Xcv and Pst bycultural practices or chemical control has been unsuccessful and there are only limitedsources of genetic resistance to these pathogens. In previous research supported in part byBARD IS-3237-01, we extensively characterized changes in tomato gene expression uponthe onset of spot and speck disease resistance. A remarkable finding of these studies wasthe inducibility in tomato leaves by both Xcv and Pst strains of genes encodingtranscriptional activator of the GRAS family, which has not been previously linked todisease resistance. Goals: Central goals of this research were to investigate the role of GRAS genes in tomatoinnate immunity and to assess their potential use for disease control.Specific objectives were to: 1. Identify GRAS genes that are induced in tomato during thedefense response and analyze their role in disease resistance by loss-of-function experiments.2. Overexpress GRAS genes in tomato and characterize plants for possible broad-spectrumresistance. 3. Identify genes whose transcription is regulated by GRAS family. Our main achievements during this research program are in three major areas:1. Identification of tomato GRAS family members induced in defense responses andanalysis of their role in disease resistance. Genes encoding tomato GRAS family memberswere retrieved from databases and analyzed for their inducibility by Pst avirulent bacteria.Real-time RT-PCR analysis revealed that six SlGRAS transcripts are induced during theonset of disease resistance to Pst. Further expression analysis of two selected GRAS genesshowed that they accumulate in tomato plants in response to different avirulent bacteria orto the fungal elicitor EIX. In addition, eight SlGRAS genes, including the Pst-induciblefamily members, were induced by mechanical stress in part in a jasmonic acid-dependentmanner. Remarkably, SlGRAS6 gene was found to be required for tomato resistance to Pstin virus-induced gene silencing (VIGS) experiments.2. Molecular analysis of pathogen-induced GRAS transcriptional activators. In aheterologous yeast system, Pst-inducible GRAS genes were shown to have the ability toactivate transcription in agreement with their putative function of transcription factors. Inaddition, deletion analysis demonstrated that short sequences at the amino-terminus ofSlGRAS2, SlGRAS4 and SlGRAS6 are sufficient for transcriptional activation. Finally,defense-related SlGRAS proteins were found to localize to the cell nucleus. 3. Disease resistance and expression profiles of transgenic plants overexpressing SlGRASgenes. Transgenic plants overexpressing SlGRAS3 or SlGRAS6 were generated. Diseasesusceptibility tests revealed that these plants are not more resistant to Pst than wild-typeplants. Gene expression profiles of the overexpressing plants identified putative direct orindirect target genes regulated by SlGRAS3 and SlGRAS6. Scientific and agricultural significance: Our research activities established a novel linkbetween the GRAS family of transcription factors, plant disease resistance and mechanicalstress response. SlGRAS6 was found to be required for disease resistance to Pstsuggesting that this and possibly other GRAS family members are involved in thetranscriptional reprogramming that takes place during the onset of disease resistance.Their nuclear localization and transcriptional activation ability support their proposed roleas transcription factors or co-activators. However, the potential of utilizing GRAS familymembers for the improvement of plant disease resistance in agriculture has yet to bedemonstrated.

Our project focused on genes for high affinity iron acquisition in Cochliobolus heterostrophus, a necrotrophic pathogen of maize, and their intertwined relationship to oxidative stress status and virulence of the fungus on the host. An intriguing question was why mutants lacking the nonribosomal peptide synthetase (NRPS) gene (NPS6) responsible for synthesis of the extracellular siderophore, coprogen, are sensitive to oxidative stress. Our overall objective was to understand the mechanistic connection between iron stress and oxidative stress as related to virulence of a plant pathogen to its host. The first objective was to examine the interface where small molecule peptide and reactive oxygen species (ROS) mechanisms overlap. The second objective was to determine if the molecular explanation for common function is common signal transduction pathways. These pathways, built around sensor kinases, response regulators, and transcription factors may link sequestering of iron, production of antioxidants, resistance to oxidative stress, and virulence. We tested these hypotheses by genetic manipulation of the pathogen, virulence assays on the host plant, and by following the expression of key fungal genes. An addition to the original program, made in the first year, was to develop, for fungi, a genetically encoded indicator of redox state based on the commercially available Gfp-based probe pHyper, designed for animal cell biology. We implemented several tools including a genetically encoded indicator of redox state, a procedure to grow iron-depleted plants, and constructed a number of new mutants in regulatory genes. Lack of the major Fe acquisition pathways results in an almost completely avirulent phenotype, showing how critical Fe acquisition is for the pathogen to cause disease. Mutants in conserved signaling pathways have normal ability to regulate NPS6 in response to Fe levels, as do mutants in Lae1 and Vel1, two master regulators of gene expression. Vel1 mutants are sensitive to oxidative stress, and the reason may be underexpression of a catalase gene. In nps6 mutants, CAT3 is also underexpressed, perhaps explaining the sensitivity to oxidative stress. We constructed a deletion mutant for the Fe sensor-regulator SreA and found that it is required for down regulation of NPS6 under Fe-replete conditions. Lack of SreA, though, did not make the fungus over-sensitive to ROS, though the mutant had a slow growth rate. This suggests that overproduction of siderophore under Fe-replete conditions is not very damaging. On the other hand, increasing Fe levels protected nps6 mutants from inhibition by ROS, implying that Fe-catalyzed Fenton reactions are not the main factor in its sensitivity to ROS. We have made some progress in understanding why siderophore mutants are sensitive to oxidative stress, and in doing so, defined some novel regulatory relationships. Catalase genes, which are not directly related to siderophore biosynthesis, are underexpressed in nps6 mutants, suggesting that the siderophore product (with or without bound Fe) may act as a signal. Siderophores, therefore, could be a target for intervention in the field, either by supplying an incorrect signal or blocking a signal normally provided during infection. We already know that nps6 mutants cause smaller lesions and have difficulty establishing invasive growth in the host. Lae1 and Vel1 are the first factors shown to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field. Mutants in genes encoding NADPH oxidase (Nox) are compromised in development and virulence. Indeed the triple mutant, which should lack all Nox activity, was nearly avirulent. Again, gene expression experiments provided us with initial evidence that superoxide produced by the fungus may be most important as a signal. Blocking oxidant production by the pathogen may be a way to protect the plant host, in interactions with necrotrophs such as C. heterostrophus which seem to thrive in an oxidant environment.