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Unveiling grapevine-Plasmopara viticola dynamics through a combined analysis of leaf and apoplast proteome modulation at 6 hours of interaction

ABSTRACT: Plant apoplast is the first hub of plant-pathogen communication. Indeed, during interaction, pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. Within this first contact, a defence response is triggered by the plant involving several extra and intracellular proteins. In grapevine, little is known about the communication between cells and apoplast in the context of plant-pathogen interactions, also apoplast dynamics, particularly considering the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on 6 hours after Plasmopara viticola inoculation to evaluated grapevine proteome modulation both in the apoplastic fluid (APF) and total leaf tissue. Plasmopara viticola secretome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Also, our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in ‘Regent’ total leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication between grapevine and defence activation leading to the successful establishment of an incompatible interaction

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Vitis Vinifera (grape)

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Céline LECLERCQ

LAB HEAD: Celiine LECLERCQ

PROVIDER: PXD030508 | Pride | 2022-11-09

REPOSITORIES: Pride

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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. The whole experiment was carried out in triplicate, with two time points (5 and 7 days post-inoculation) and three cell types (pathogen-proximal, pathogen-distal and uninfected control), totalling 18 microarrays. Transgenic Arabidopsis thaliana Col-0 with the transgene pDMR6:GFP was used for all experiments. Seedling populations were inoculated with 30,000-60,000 spores/ml of Hyaloperonospora arabidopsis strain Noks1 at 7 days old, and overground tissue sampled at 5 and 7 days post-inoculation. Protoplasts were generated from these samples according to Grønlund et al. 2012 JOVE, and sorted by fluorescence activated cell sorting into a GFP-positive (pathogen-proximal) and GFP-negative (pathogen-distal) population. As a control, uninfected seedlings were also sampled at 12 and 14 days old (equivalent age of 5 and 7 days post-inoculation), protoplasts generated and sorted to yield a GFP-negative population of uninfected control cells. Gene expression was analysed using NimbleGen 12 x 135K microarrays, designed for the TAIR10 genome. Data was normalised using the Robust Multichip Averaging algorithm.

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Unveiling grapevine-Plasmopara viticola dynamics through a combined analysis of leaf and apoplast proteome modulation at 6 hours of interaction

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