The grapevine Pectin Methylesterases gene family and its involvement in Botrytis bunch rot control

Plant Pectin Methylesterases (PMEs) represent a group of tissue-specific and developmentally regulated proteins. The gene family is involved in the plant cell wall (CW) remodelling process, by the control of the degree of cell wall pectin methylesterification. Pectin methylesterification also influence the susceptibility to pathogens as Botrytis cinerea (Bc), a necrotrophic fungus responsible of the Botrytis bunch rot in grapevine. In Botrytis, PME as well as other CW degrading enzymes have been identified as virulence factors. To further characterize the PME gene family and its role in the Botrytis bunch rot, the latest genome assembly and annotation were revised and through sequence homology search, a total of 63 PME domain containing proteins were identified, 16 more than a previous identification in grapevine. The in-silico analyses of the family by means of the Vitis gene expression database VESPUCCI as well as Aggregated Gene Co-expression Network approach (AggGCNs) allowed us to identify and enrich gene co-expression modules and build gene co-expression networks. Interestingly, one of the co-expression modules showed a high modulation in presence of Botrytis cinerea infection and particular attention was paid to it. To investigate the contribution of the genes of that module, their expression level in different organs and developmental stages from two grapevine cultivars with divergent Bc susceptibility was investigated. Furthermore, berries were artificially infected with Bc at mature stage to evaluate PME gene expression level and their possible role in the grapevine bunch rot susceptibility. The results obtained contribute to characterize the grapevine PME gene family and the role of specific members in the grapevine-Bc interaction and to select PME genes candidate to the control of Botrytis bunch rot in grapevine

New strategies for Botrytis bunch rot control for a sustainable viticulture

Grapevine (Vitis vinifera L.) is cultivated throughout the world in a wide range of climates, from temperate to tropical ones. It’s highly susceptible to fungi, being Botrytis cinerea (Bc) among the most important pathogens causing bunch rot (BR), a disease mostly observed on ripe berries, normally controlled through use of chemicals. To date, the defence against Bc is still guaranteed by an intensive use of chemical fungicides. Consequently, the development of alternative strategies for Bc control with benefits both for human health and the environment is of high importance and a priority. In the last years, the concept of “integrated defence” against Bc is increasingly widespread, using defence protocols integrating agronomic control tools with interventions based on synthetic fungicides and natural antagonists to be carried out in the periods of higher susceptibility. In this project, a molecular method for monitoring Bc load in the field was setted-up in order to quantify the colonization by Bc of vineyards of V. vinifera cv Sangiovese and Trebbiano at different locations (hilly site vs plain site) and cultivated applying different integrated defence protocols against Bc. Samples at were collected at three different developmental stages (full flowering, bunch enclosure and veraison) from different biological replicates of different thesis of treatment and molecularly analyzed calculating the colonization coefficient for each sample. The correlation between molecular data with BcBR severity and incidence data at harvest in the different thesis for the two cultivars are here presented. The molecular assay will be useful both to quantify Bc load at the early season in order to predict BcBR severity at harvest, and more in general to evaluate the effect of disease management protocols adopted on the reduction of Bc inoculum

De novo transcriptome assembly and functional analysis reveal a dihydrochalcone 3-hydroxylase(DHC3H) of wild Malus species that produces sieboldin in vivo

Sieboldin is a specialised secondary metabolite of the group of dihydrochalcones (DHC), found in high concentrations only in some wild Malus species, closely related to the domesticated apple (Malus × domestica L.). To date, the first committed step towards the biosynthesis of sieboldin remains unknown. In this study, we combined transcriptomic analysis and a de novo transcriptome assembly to identify two putative 3-hydroxylases in two wild Malus species (Malus toringo (K. Koch) Carriere syn. sieboldii Rehder, Malus micromalus Makino) whose DHC profile is dominated by sieboldin. We assessed the in vivo activity of putative candidates to produce 3-hydroxyphloretin and sieboldin by de novo production in Saccharomyces cerevisiae. We found that CYP98A proteins of wild Malus accessions (CYP98A195, M. toringo and CYP98A196, M. micromalus) were able to produce 3-hydroxyphloretin, ultimately leading to sieboldin accumulation by co-expression with PGT2. CYP98A197-198 genes of M. × domestica, however, were unable to hydroxylate phloretin in vivo. CYP98A195-196 proteins exerting 3-hydroxylase activity co-localised with an endoplasmic reticulum marker. CYP98A protein model from wild accessions showed mutations in key residues close to the ligand pocket predicted using phloretin for protein docking modelling. These mutations are located within known substrate recognition sites of cytochrome P450s, which could explain the acceptance of phloretin in CYP98A protein of wild accessions. Screening a Malus germplasm collection by HRM marker analysis for CYP98A genes identified three clusters that correspond to the alleles of domesticated and wild species. Moreover, CYP98A isoforms identified in M. toringo and M. micromalus correlate with the accumulation of sieboldin in other wild and hybrid Malus genotypes. Taken together, we provide the first evidence of an enzyme producing sieboldin in vivo that could be involved in the key hydroxylation step towards the synthesis of sieboldin in Malus specie

ESICM LIVES 2016: part two : Milan, Italy. 1-5 October 2016.

Meeting abstrac