<i>Pyrenophora teres</i> Drechs. (anamorph: <i>Drechslera teres</i> (Sacc.) Shoem.) is the causal agent of barley net blotch. Net blotch is an economically important disease commonly found throughout the barley producing regions of the world. Significant financial losses result from yield reductions, ranging from 15-35%, and decreased grain quality. Despite its prevalence, it is unclear if the P. teres-barley pathosystem follows a gene-for-gene model, and more generally, little is known about its genetic organization. Three studies were initiated to address these questions.<p>The first study investigated the genetic control of avirulence in <i>P. teres.</i> To establish an appropriate study system, a collection of ten net form (<i>P. teres f. teres</i>) and spot form (<i>P. teres f. maculata</i>) isolates were evaluated on a set of eight differential barley lines to identify two isolates with differential virulence on a specific host line. WRS 1906, exhibiting low virulence on the cultivar Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for virulence on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (Chi-square = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism (AFLP) markers closely linked to the avirulence gene (AvrHeartland). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model.<p>In the second study, five isolates of P. teres, representing both net and spot forms, were analyzed by the germ tube burst method (GTBM) and pulsed field gel electrophoresis (PFGE) to determine the species karyotype. Nine chromosomes were observed in all isolates using the GTBM and estimation of chromosome lengths varied from 0.5 to 3.0 µm. PFGE separated 7 to 8 bands depending on isolate, but analysis of bands by densitometry indicated nine chromosomes. Chromosome size ranged from 1.8 to ~6.0 Mb providing a genome size estimate of 32 to 39 Mb. Significant chromosome-length polymorphisms (CLP) were observed between isolates. These CLP did not hinder mating between mating-type compatible net form isolates. No particular CLP or individual chromosome could be associated with differences in disease symptoms observed between pathogen forms. This study provides the first karyotype of both P. teres forms and will assist genetic mapping of this pathogen.<p>A genetic linkage map of <i>P. teres f. teres</i>, was constructed in the third study using the population of 67 progeny derived from the WRS 1906  WRS 1607 cross. The map consists of 138 markers including 114 AFLPs, 21 telomere RFLPs, the mating-type (MAT) locus and an avirulence locus (AvrHeartland) controlling interaction with barley cultivar Heartland. Markers were distributed across 24 linkage groups ranging in length from 2 to 110 cM with an average marker interval of 8.5 cM. The total map length was 797 cM. A telomere-specific probe, consisting of the sequence (TTAGGC)4, was used to map 15 of the 18 telomeres. One of these telomeres mapped to within 3 cM of the AvrHeartland locus. Attempts to consolidate linkage groups by hybridizing markers to the electrophoretically separated chromosomes was unsuccessful because probes bound to multiple chromosomes, likely due to repetitive DNA within the probe. This is the first genetic map reported for this species and it will be a useful genetic tool for map-based cloning of the AvrHeartland gene tagged in this study. <p>This research has provided a number of new insights into the net blotch pathogen and provides a useful research tool in the form of a genetic map. This information lays the foundation for further genetic study of P. teres and will complement studies on barley resistance to net blotch that may potentially lead to more durable resistance.

<i>Pyrenophora teres</i> Drechs. (anamorph: <i>Drechslera teres</i> (Sacc.) Shoem.) is the causal agent of barley net blotch. Net blotch is an economically important disease commonly found throughout the barley producing regions of the world. Significant financial losses result from yield reductions, ranging from 15-35%, and decreased grain quality. Despite its prevalence, it is unclear if the P. teres-barley pathosystem follows a gene-for-gene model, and more generally, little is known about its genetic organization. Three studies were initiated to address these questions.<p>The first study investigated the genetic control of avirulence in <i>P. teres.</i> To establish an appropriate study system, a collection of ten net form (<i>P. teres f. teres</i>) and spot form (<i>P. teres f. maculata</i>) isolates were evaluated on a set of eight differential barley lines to identify two isolates with differential virulence on a specific host line. WRS 1906, exhibiting low virulence on the cultivar Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for virulence on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (Chi-square = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism (AFLP) markers closely linked to the avirulence gene (AvrHeartland). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model.<p>In the second study, five isolates of P. teres, representing both net and spot forms, were analyzed by the germ tube burst method (GTBM) and pulsed field gel electrophoresis (PFGE) to determine the species karyotype. Nine chromosomes were observed in all isolates using the GTBM and estimation of chromosome lengths varied from 0.5 to 3.0 µm. PFGE separated 7 to 8 bands depending on isolate, but analysis of bands by densitometry indicated nine chromosomes. Chromosome size ranged from 1.8 to ~6.0 Mb providing a genome size estimate of 32 to 39 Mb. Significant chromosome-length polymorphisms (CLP) were observed between isolates. These CLP did not hinder mating between mating-type compatible net form isolates. No particular CLP or individual chromosome could be associated with differences in disease symptoms observed between pathogen forms. This study provides the first karyotype of both P. teres forms and will assist genetic mapping of this pathogen.<p>A genetic linkage map of <i>P. teres f. teres</i>, was constructed in the third study using the population of 67 progeny derived from the WRS 1906  WRS 1607 cross. The map consists of 138 markers including 114 AFLPs, 21 telomere RFLPs, the mating-type (MAT) locus and an avirulence locus (AvrHeartland) controlling interaction with barley cultivar Heartland. Markers were distributed across 24 linkage groups ranging in length from 2 to 110 cM with an average marker interval of 8.5 cM. The total map length was 797 cM. A telomere-specific probe, consisting of the sequence (TTAGGC)4, was used to map 15 of the 18 telomeres. One of these telomeres mapped to within 3 cM of the AvrHeartland locus. Attempts to consolidate linkage groups by hybridizing markers to the electrophoretically separated chromosomes was unsuccessful because probes bound to multiple chromosomes, likely due to repetitive DNA within the probe. This is the first genetic map reported for this species and it will be a useful genetic tool for map-based cloning of the AvrHeartland gene tagged in this study. <p>This research has provided a number of new insights into the net blotch pathogen and provides a useful research tool in the form of a genetic map. This information lays the foundation for further genetic study of P. teres and will complement studies on barley resistance to net blotch that may potentially lead to more durable resistance.

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.

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.

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.

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.

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.

Le Grapevine fanleaf virus (genre Nepovirus) est l'agent principal du court-noué de la vigne. Il induit des symptômes très variables. Ce travail présente une étude mécanistique de la symptomatologie du GFLV sur un hôte herbacé et sur l'hôte d’intérêt agronomique. Par détection de marqueurs biochimiques et moléculaires j'ai montré que le GFLV-F13 induit une réponse hypersensible (HR) sur N. occidentalis et une restriction partielle du virus. J'ai identifié puis cartographié le déterminant viral de cette HR en utilisant des réassortants, des recombinants et des variants naturels du virus. Sur vigne, sur un dispositif expérimental unique en son genre, j'ai mené une approche sans a priori d’étude transcriptomique par RNA-Seq. J'ai comparé des vignes du cépage gewurztraminer mono-infectées par une souche sévère induisant des symptômes de rabougrissement et par une souche plus modérée. 1 023 gènes sont spécifiquement dérégulés par la souche sévère parmi lesquels des gènes impliqués dans la régulation de la HR. Ce résultat permet de proposer pour la première fois qu'une HR pourrait être mise en place dans la vigne en réponse à une infection virale. / Grapevine fanleaf virus (genus Nepovirus) the causative agent of fanleaf degeneration, induces variable symptoms. This manuscript presents a mechanistic study of GFLV symptomatology on both an herbaceous model plant and an agronomically important crop plant.On N. occidentalis, I demonstrated that GFLV-F13 induces a reaction exhibiting hallmarks of a hypersensitive response (HR), partially restricting virus spread. Using reassortants, recombinants and natural variants of the virus, I could identify and map the viral determinant of this HR. On grapevine, I took advantage of a unique experimental set-up and used RNA-Seq to compare the transcriptoms of Gewurztraminer plants infected with two different GFLV strains, one of which induced stunting symptoms and the other mild symptoms. 1,023 genes among which genes involved in the regulation of HR, were specifically regulated by the more severe strain This is the first hint of a HR taking place in grapevine in response to a virus infection.

No description available.