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RESEARCH PRODUCT

Impact of Plasmopara viticola on starch metabolism and stomachal functions in grapevine leaves

subject

description

The grapevine downy mildew affects leaves and young berries and can affect the harvest quality and quantity. The causal agent is the obligate biotroph oomycete Plasmopara viticola that induces severe physiological alterations in infected leaves. One is the abnormal accumulation of starch in oil spots, the characteristic symptoms of the disease. Another is a deregulation of stomatal movements. Stomata, natural openings on the leaf surface allowing gas exchanges between the plant and the atmosphere, stay abnormally open during the night and do no longer close upon water stress or ABA treatment in infected leaves. This thesis is divided into two chapters that aim to i) explain the origin and mechanism of the abnormal starch accumulation and ii) isolate and identify the compound responsible for the stomatal deregulation. The modifications of starch metabolism in infected leaves were first studied by transcriptomic analysis allowing to identify transcriptional modifications of genes involved in starch metabolism. After the validation of two reference genes, a qRT-PCR analysis was performed in order to verify the expression alterations of some of these genes and the corresponding enzymatic activities were measured. The quantification of soluble sugars and starch, and the measurement of photosynthetic activity were included in the analysis and their alterations could be correlated with the infection. Altogether, the obtained results hint towards an increase of starch synthesis via an increase in AGPase activity, that, combined with a decrease of its degradation by modification of amylase activities, could be responsible for the observed starch accumulation in oil spots. Concurrently, a source-sink transition is apparent in infected leaves by decrease of photosynthetic activity and increase of a cell wall invertase activity. The second part of this thesis dealt with a soluble molecule of plant or pathogen origin that is secreted into the apoplast during infection and could be responsible for the stomatal deregulation. The apoplastic wash fluids from infected leaves increase tha stomatal aperture in the dark in grapevine epidermal peels and counteract the ABA-induced stomatal closure, thus mimicking the deregulation observed on whole leaves. The active compound seems to be a stable protein of > 50kDa with a glycosylation that is essentiel for its activity. After separation of the fluids by size exclusion chromatography, two fractions were compared : one active on stomatal aperture and an inactive one. 9 grapevine proteins could be identified in the active sample by mass spectrometry analysis, but further analyses are needed to purify and identify the one responsible for the stomatal deregulation.