New strategies for Botrytis bunch rot control for a sustainable viticulture

Vitis vinifera L.(Vv), the European cultivated grapevine is one of the most worldwide important crops but is highly susceptible to the necrotrophic fungus Botrytis cinerea (Bc), the causal agent of bunch rot (BR) disease. In grapevine, as well as in other fruit species, it has been described a primary infection by Bc at full bloom, followed by a quiescence period during the berry development, and the fungal egression after veraison reaching the maximum at harvest. Today, this important disease is mainly controlled by massive use of fungicides, which are applied at different developmental stages that happen to be critical during the grapevine-Bc interaction. During the contact, the fungus must overcome several barriers from the host, which protect it from the pathogen attack and might be also modulated or activated due to the presence of the pathogen itself. The cuticle and the cell wall (CW) represent the first barriers from the plant encountered by the pathogen. To successfully colonize the plant tissue, Bc possesses several virulence factors, and CW modifying enzymes (CWMEs) are part of them. On the other hand, the regulated activity of the CWMEs, expressed both by the host and the pathogen, could alter the plant CW composition and porosity, therefore facilitating, or limiting the penetration of the fungus. Among the CWMEs, Pectin methylesterases (PMEs) regulate the degree of methyl esterification of the pectin homogalacturonan, also modifying the epitopes for the activity of other CWMEs such as polygalacturonases (PGs) and pectate lyases (PLs) by whose action, pectin becomes more susceptible to degradation and the CW more accessible by the pathogen. Previous works both in Arabidopsis thaliana and in crop species identified several PME genes with an altered expression in response to pathogens. A previous report characterizing Atpme17 mutant lines has highlighted a role of AtPME17 in the resistance response to Bc in contrast with the opposite role of AtPME3, suggesting that PME genes could have a completely different action during Bc response depending on the isoform involved. In this context, the main objective of the project was to identify new strategies for Bc BR-control, and specifically i) to identify candidate genes involved in the response to Bc, whose inactivation/overexpression would lead to Bc resistant plants and ii) to set up a molecular method to monitor the Bc load in the field and therefore implement a more sustainable control of the pathogen. To further understand the effects of the Bc primary infection in grapevine flowers at CW level, two contrasting genotypes (Souvignier gris (SG) and Teroldego (TE)) in their resistance to the fungus were considered. An artificial inoculation of different biological replicates, in vase maintained, was performed at full bloom, in controlled conditions, and samples were collected at 24 hours post-inoculation (hpi) with the fungus and post-treatment with the respective control, for the following RNA-seq analysis and biochemical characterization of PME activity and CW composition in the two genotypes before and upon infection. The Bc load was estimated in the flowers using qPCR and as expected, a higher biomass of Bc was found in TE, the susceptible cultivar, than in SG, the resistant one. The analysis of CW composition, PME activity and degree of pectin methyl-esterification, both in treated and control flowers, showed significant differences between the two genotypes, in particular SG showed a significant induction of PME activity with respect to the control, evidence not present in the susceptible genotype. The RNAseq analysis on the same samples showed a total of 4800 genes modulated, out of which 3064 are only modulated in TE, 739 only in SG and a common group of 997 genes. Regardless of the cultivar, upon infection there was a total 2919 genes upregulated vs 1909 genes downregulated. A gene set enrichment analysis (GSEA) indicated several over-represented categories upon infection, including response to pathogens and biosynthesis of secondary metabolites, with a general down-regulation of those genes related to CW organization and pectin modification (CWMEs), mostly in the resistant genotype. Within the down-regulated CWMEs, Pectin methylesterase (PME) genes were found highly represented. Unlike, a larger gene set, in many cases with a higher fold-change of induction, was identified in TE respect to SG. This is the case of genes involved in the defense response and its regulation, and in the modification/reinforcement of the cell wall, therefore attesting for an initial tentative by the susceptible genotype to counteract the pathogen, although at the end without success. This was also the case of the seven VviPME genes previously highlighted by the in-silico co-expression analysis and therefore of VviPME10, the gene with the highest homology to AtPME17. Among the regulators, one WRKY factor (VviWRKY3), known to be related to defense response in grapevine mediated by stilbene synthesis, was also further characterized as putative regulator of VviPME10, whose promoter hosts more than one several predicted binding sites for VviWRKY3. Indeed, luciferase assay results indicate a significant activation of VviPME10 promoter by VviWRKY3 factor. Parallelly, the genome-wide analysis of the last structural annotation of the grapevine genome assembly allowed us to identify 62 VviPME gene members, 15 more than a previous report, and manually curate the gene structure for 39 of them. Then, to corroborate the idea of the role of the CW, and in particular of PME activity, in the grapevine response to the fungus, an in silico co-expression analysis of the 62 VviPME members, considering the publicly available RNA-seq experiments related to grapevine-Bc interactions and the RNAseq experiment conducted in this project, was performed. The analysis highlighted a group of seven genes (VviPME1, VvPME9, VviPME10, VviPME11, VviPME12, VviPME13 and VviPME54) with significant induction upon Bc infection, five of them (VviPME8, VviPME9, VviPME10, VviPME11, and VviPME54) located in the same chromosome (chr06). VviPME10 showed the highest homology and was found to be phylogenetically close to the Arabidopsis thaliana PME17 gene, suggesting being considered as its putative orthologue. Afterward, Therefore, considering the increased VviPME10 expression upon infection, and the reported effect of AtPME17 in A. thaliana, VviPME10 was selected as a potential candidate to study its role in grapevine. In this regard, two strategies were adopted, i. VviPME10 knock-out (KO) with CRISPR/Cas9 and ii. VviPME10 overexpression (OE, under CaMV35S promoter) through embryogenic callus transformation of the grapevine cultivar ‘Sugraone’ mediated by Agrobacterium tumefaciens. More than 100 embryos developed, and around 20 plantlets per transformation were analyzed to check the presence of the transgenic construct. Then, the mutation profile, in the case of KO lines, and the expression analysis of the transgene, in the case of OE lines, were carried out to select the appropriate lines to acclimatize. OE lines were also tested for VviPME10 activity. A total protein extract was obtained from the leaves of the lines, showing a higher protein activity compared to the control, and indicating the functionality of the enzyme. Unfortunately, grapevine OE lines couldn’t be analyzed for their response to Bc, while KO lines showed a significantly larger lesion area when compared to the control at 5 days post fungal inoculation (dpi). However, the effect of VviPME10 overexpression upon Bc infection was evaluated also in Nicotiana benthamiana VviPME10-OE lines, generated in parallel. At 3 dpi a significant reduction was observed in the lesion area compared to the control. These results suggest that pectin modification, mediated by VviPME10, plays an important role in the grapevine response to Bc, in particular it seems to behave more like a resistance gene than a susceptibility one. For this reason, it could be considered as a valuable target to improve resistance to Bc in susceptible grapevine varieties. Finally, a molecular method for Bc detection, based on quantitative RT-PCR assays, was set up and applied to estimate the Bc load in field conditions. Although the method allowed the successful detection of the presence of the fungus in samples at different developmental stages, from two V. vinifera cultivars, in different vineyards, the lack of environmental conditions for the development of the disease might have impaired the correlation between detection and the development of the disease. Nonetheless, the method represents a good alternative for monitoring the Bc load in the field at the early season, to predict the BcBR severity at harvest and eventually apply the disease management protocols based on the real need.