Functional study of lipoxygenase-mediated resistance against Erysiphe necator in grapevine

Grapevine powdery mildew, caused by the fungus Erysiphe necator, is a devastating disease with a worldwide occurrence. Commercial Vitis vinifera cultivars are highly susceptible to the pathogen, while several wild Vitis spp. demonstrate a high level of resistance. Understanding the resistance mechanism will contribute to breeding programs aiming at introducing resistance factors to commercial varieties. Lipoxygenase (LOX) genes play important role in plant defense through peroxidation of lipids leading to the production of many compounds, including volatiles, involved in signalling and response or with antifungal activities. In this work the role of LOX in host resistance against E. necator was investigated in silico and in planta. Phylogenetic analysis with a maximum likelihood estimation approach of grapevine and well-characterized Arabidopsis homologs showed the separation of 9-LOX and 13-LOX and several paralogous grapevine LOXs were found tandemly localized. Moreover, several putative chloroplast transit peptides were predicted. In addition, gene network analysis performed with OneGenE identified many defence-related genes as associated with LOX isoforms. A controlled E. necator infection experiment was carried out using V. vinifera cultivar Teroldego (S) and NY_39 (R) genotype and LOXs gene expression patterns were analysed. Microscopy observation of infected leaves showed successful penetration and hyphal growth of the pathogen in the (S) variety at 24 hours post inoculation (hpi) while extensive hyphal growth was observed at 72 hpi. Conversely, pathogen establishment and growth were hampered in the (R) genotype with an evident accumulation of reactive oxygen species (ROS) at pathogen penetration site. Grapevine VviLOX2 and VviLOX12 were significantly upregulated at 12hpi in the infected leaves of (R) genotype, while upregulation of VviLOX7 and VviLOX9 occurred at 48hpi; VviLOX13 was upregulated in infected leaves of the (S) genotype at 24 hpi. Efforts are underway to over-express and knock-out (CRISPR/Cas9) candidate LOXs in the (S) background for in-depth functional characterization.