Two tobacco transgenic lines over-expressing grapevine polygalacturonase-inhibiting protein (Vvpgip1) vs. WT tobacco under normal growth conditions
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ABSTRACT: Polygalacturonase-inhibiting proteins (PGIPs) in plant cell walls inhibit fungal endopolygalacturonases (ePGs). Their action directly limits the effective ingress of the pathogen and is thought to lead to an up-regulated defense response. We previously showed that, PGIP activity, ePG inhibition and decreased Botrytis cinerea susceptibility were correlated in tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1. In order to evaluate the base-line impact of PGIP over-expression, the transcriptome and hormone profiles of two transgenic lines with characterized resistance phenotypes were investigated in the absence of pathogen infection.
Project description:Polygalacturonase-inhibiting proteins (PGIPs) in plant cell walls inhibit fungal endopolygalacturonases (ePGs). Their action directly limits the effective ingress of the pathogen and is thought to lead to an up-regulated defense response. We previously showed that, PGIP activity, ePG inhibition and decreased Botrytis cinerea susceptibility were correlated in tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1. In order to evaluate the base-line impact of PGIP over-expression, the transcriptome and hormone profiles of two transgenic lines with characterized resistance phenotypes were investigated in the absence of pathogen infection. Three microarray slides in total were hybridized: two slides using Vvpgip1 line 37 and WT, with a dye swap included to account for dye bias; and one slide using Vvpgip1 line 45 as test and WT as reference.
Project description:White grape (Vitis vinifera cv. Furmint) berry samples subjected to natural noble rot were collected in a vineyard in Mád, Hungary (Tokaj wine region). Raw data include grapevine and Botrytis cinerea sequence reads.
Project description:Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infectious process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus development. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes up-regulated during infection of MB are enriched in functional categories related to necrotrophy such as degradation of plant cell wall, proteolysis, membrane transport, reactive oxygen species generation and detoxification. Quantitative-PCR on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but stops at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathways switch during berry ripening. In response to B. cinerea infection, VB activated a burst of reactive oxygen species (ROS), the salicylate (SA)-dependent defense pathway, the synthesis of the resveratrol phytoalexin and cell-wall strengthening. In opposite, infected MB activated the jasmonate (JA)-dependent pathway which does not stop the fungal necrotrophic process. Grapevine berries at veraison (VB) and harvesting stages (MB) were inoculated with Botrytis cinerea B05-10 and samples were taken at 24h and 48h post-inoculation. An additional uninfected control sample taken at 0h post-inoculation was included in the experimental design. 3 replicates per sample were performed. The total-RNA samples were labeled and used for hybridization on NimbleGen 12plex Vitis vinifera gene expression array.
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:Infection by the pathogen grape powdery mildew (Erysiphe necator) causes changes in the transcriptome of its susceptible host Vitis vinifera. Infection triggers the host to synthesize the signaling molecule salicylic acid (SA) which regulates the expression of a broad range of defense-related plant genes. In addition, it is hypothesized that E. necator directly modulates gene expression in V. vinifera via the haustorial complex. This microarray experiment was designed to dissect host transcriptome changes triggered directly by E. necator infection and indirectly through the SA response. We accomplished this by conducting two separate global leaf transcriptome analyses using the Vitis Affymetrix GeneChip platform: in one, we compared the leaves with fully established PM colonies to healthy reference leaves, in another, we compared healthy leaves with artificially elevated SA levels to healthy reference leaves. Overlaying host transcriptome changes from these two experiments enabled us to glean out V. vinifera genes that modulate their expression in response E. necator in an SA-independent manner.
Project description:we analyzed pathogen-induced changes in the transcriptome of Vitis vinifera ‘Cabernet sauvignon’ and Vitis aestivalis ‘Norton’ by conducting a large-scale study to measure transcript abundance at 0, 4, 8, 12, 24, and 48 hours post-treatment in conidiospore- and mock-inoculated leaves using Affymetrix GeneChip Vitis vinifera Genome Array Keywords: time course
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:Purpose: Microarray technologies provide a unique opportunity to deeply investigate the molecular mechanisms involved in plant-pathogen interaction. Botrytis cinerea, is the agent of grapevine grey mould, but in yet uncharacterized environmental conditions, a latent infection can occur determining favourable metabolic and physico-chemical berry modifications which possibly contribute to the typical aromas of “passito” wines (“noble rot”). The present project aims at the identification of the grapevine responses to B. cinerea during fungal colonization in the latent form, in comparison with control berries. Methods: A total of 150 untreated berries were sampled as time 0 of the experiment. Moreover, 300 healthy berries have been artificially inoculated one by one with B. cinerea by injecting conidia under berry skin, in controlled conditions, reproducing an early stage (T1) and a late stage (T2) of noble rot pourri plein. Control samples (300 berries) have been inoculated with water and sampled at the same time of infected berries. The microarray experiments on T0 and healthy or infected samples in biological triplicate resulted in 15 samples to be analyzed (Agilent-048771 4x44K Grape all custom microarray chip; Agilent Technologies, Santa Clara, CA, USA). Conclusions: This work identified important molecular mechanisms involved in Botrytis cinerea colonization of grapevine berries during the noble rot infection.
Project description:Purpose: High throughput sequencing technologies provide a unique opportunity to deeply investigate the molecular mechanisms involved in plant-pathogen interaction. Botrytis cinerea, is the agent of grapevine grey mould, but in yet uncharacterized environmental conditions, a latent infection can occur determining favourable metabolic and physico-chemical berry modifications which possibly contribute to the typical aromas of “passito” wines (“noble rot”). The present project aims at the identification of the genes deployed by B. cinerea during grape berries colonization in the latent form, in comparison with the saprophytic growth in vitro. Methods: A total of 300 healthy berries have been artificially inoculated one by one with B. cinerea by injecting conidia under berry skin, in controlled conditions, reproducing the pourri plein stage of noble rot. Control samples (300 berries) have been inoculated with water. The saprophytic growth was obtained in liquid nutrient medium in laboratory flasks, and the mycelium collected by filtration. The RNA-sequencing experiments on healthy or infected samples in biological triplicate resulted in 27 data sets to be analyzed (Illumina NextSeq500 paired-end sequencing; 533.779.730 total reads, 150 Gb of data). Conclusions: This work identified important molecular mechanisms involved in Botrytis cinerea colonization of grapevine berries during the noble rot infection.