Project description:A comparative proteomic analysis of grapevine leaves from the resistant genotype V. davidii ‘LiuBa-8’ (LB) and susceptible genotype V. vinifera ‘Pinot Noir’ (PN) at 12 hpi was conducted to understand the complex relationship between grapevine and P. viticola and the molecular mechanisms between difference resistant vitis genotypes at the early stage of infection. A total of 444 and 349 differentially expressed proteins (DEPs) were identified in LB and PN respectively at 12 hpi by iTRAQ. MapMan analysis showed that the majority of these DEPs were related to photosynthesis, metabolism, stress and redox. More up-expressed DEPs involved in photosynthesis and less down-expressed DEPs involved in metabolism contribute to the resistance in LB. PR10.2, PR10.3, HSF70.2 and HSP90.6 are proposed to be key proteins in both compatible and incompatible interactions. Accumulation H2O2 established the ROS signaling in incompatible interaction and APXs, GSTs maybe associated with the resistance of grapevine against downy mildew. Moreover, we verified four proteins to ensure the accuracy of proteome data using PRM. Overall, these data provide new insights into molecular events and provide valuable candidate proteins that could be used to illuminate molecular mechanisms underlying the incompatible and compatible interaction in early stage and eventually to be exploited to develop new protection strategy against downy mildew in grapevine.
Project description:A comparative proteomic analysis of grapevine leaves from the resistant genotype V. davidii ‘LiuBa-8’ (LB) and susceptible genotype V. vinifera ‘Pinot Noir’ (PN) at 12 hpi was conducted to understand the complex relationship between grapevine and P. viticola and the molecular mechanisms between difference resistant vitis genotypes at the early stage of infection. A total of 444 and 349 differentially expressed proteins (DEPs) were identified in LB and PN respectively at 12 hpi by iTRAQ. MapMan analysis showed that the majority of these DEPs were related to photosynthesis, metabolism, stress and redox. More up-expressed DEPs involved in photosynthesis and less down-expressed DEPs involved in metabolism contribute to the resistance in LB. PR10.2, PR10.3, HSF70.2 and HSP90.6 are proposed to be key proteins in both compatible and incompatible interactions. Accumulation H2O2 established the ROS signaling in incompatible interaction and APXs, GSTs maybe associated with the resistance of grapevine against downy mildew. Moreover, we verified four proteins to ensure the accuracy of proteome data using PRM. Overall, these data provide new insights into molecular events and provide valuable candidate proteins that could be used to illuminate molecular mechanisms underlying the incompatible and compatible interaction in early stage and eventually to be exploited to develop new protection strategy against downy mildew in grapevine.
Project description:The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infect Arabidopsis by forming feeding structures called haustoria inside the host cell. Previous transcriptome analysis revealed host genes specifically induced during infection. However, whole infected tissue-derived RNA profiling may fail to capture the key transcriptional events that may occur exclusively in haustoriated host cells where the pathogen injects virulence effectors to modulate host immunity for successful accommodation. To understand the interaction between Arabidopsis and H. arabidopsidis at the cellular level, we established a new translating ribosome affinity purification (TRAP) system applicable to pathogen-responsive promoters, enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing new insights into the Arabidopsis/downy mildew interaction. We also expect that our new TRAP system could be applicable to several stimulus-specific contexts as well as other plant–pathogen interactions.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar ‘Regent’, when compared to the susceptible cultivar ‘Trincadeira’, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera ‘Regent’ is induced after infection. This study provides the identification of several candidate genes that may be related to ‘Regent’ defense mechanisms, allowing a better understanding of this cultivar's resistance traits.
Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression.
Project description:Here we report on the identification of Arabidopsis genes that are induced during compatible but not during incompatible interactions with the downy mildew pathogen Hyaloperonospora arabidopsidis. This set of so-called compatible specific (CS) genes contrasts the large group of defence-associated genes that is differentially expressed during both compatible and incompatible interactions. From the 17 identified CS-genes, 6 belong to the ERF family of transcription factor genes, suggesting that these ERFs have a role during compatibility. The majority of CS-genes are differentially regulated in response to various forms of abiotic stress. In silico analysis of the CS-genes revealed an over-representation of DREB1A/CBF3 binding sites and EveningElement motifs in their promoter regions. The CS-ERFs are closely related to the CBF transcription factors and could potentially bind the DREB1A/CBF3 promoter elements in the CS-genes. Transcript levels of CS-genes peak at 2-3 days post inoculation, when pathogen growth is highest, and decline at later stages of infection. The induction of several CS-genes was found to be isolate-dependant. This suggests that the identified CS-genes could be the direct or indirect targets of downy mildew effector proteins that promote disease susceptibility.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar M-bM-^@M-^XRegentM-bM-^@M-^Y, when compared to the susceptible cultivar M-bM-^@M-^XTrincadeiraM-bM-^@M-^Y, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera M-bM-^@M-^XRegentM-bM-^@M-^Y is induced after infection. This study provides the identification of several candidate genes that may be related to M-bM-^@M-^XRegentM-bM-^@M-^Y defense mechanisms, allowing a better understanding of this cultivar's resistance traits. 3 time points: 0, 6 and 12 hours post inoculation by P. viticola. Two cultivars: control (Trinacedira) and test (Regent). Two biological replicates were performed at 0 hpi, and 3 biological replicates at 6 and 12hpi. At 12hpi, three technical replicates also were performed.
Project description:Here we report on the identification of Arabidopsis genes that are induced during compatible but not during incompatible interactions with the downy mildew pathogen Hyaloperonospora arabidopsidis. This set of so-called compatible specific (CS) genes contrasts the large group of defence-associated genes that is differentially expressed during both compatible and incompatible interactions. From the 17 identified CS-genes, 6 belong to the ERF family of transcription factor genes, suggesting that these ERFs have a role during compatibility. The majority of CS-genes are differentially regulated in response to various forms of abiotic stress. In silico analysis of the CS-genes revealed an over-representation of DREB1A/CBF3 binding sites and EveningElement motifs in their promoter regions. The CS-ERFs are closely related to the CBF transcription factors and could potentially bind the DREB1A/CBF3 promoter elements in the CS-genes. Transcript levels of CS-genes peak at 2-3 days post inoculation, when pathogen growth is highest, and decline at later stages of infection. The induction of several CS-genes was found to be isolate-dependant. This suggests that the identified CS-genes could be the direct or indirect targets of downy mildew effector proteins that promote disease susceptibility. Compatible interaction - Arabidopsis Ler inoculated with Cala2 compared to Ler with mock inoculation with 3 biological replicates. Each biological replicate is analysed by 4 technical dyeswapped replicates. Incompatible interaction - Arabidopsis Ler inoculated with Waco9 compared to Ler with mock inoculation with 3 biological replicates. Each biological replicate is analysed by 4 technical dyeswapped replicates.
Project description:European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. In this study, Rpv3-mediated defense responses were studied in Rpv3+ and Rpv3ˉ grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3ˉ, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses.