Proteomic characterization of grapevine resistance against downy mildew activated by Trichoderma harzianum T39

The host-specific oomycete Plasmopara viticola is the causal agent of downy mildew, one of the most important grapevine diseases. In recent years, several molecules able to induce a plant-mediated resistance against P. viticola have been described. Some inducers, i.e. benzothiaziadole (BTH), activate a direct resistance involving constitutive barriers and local and systemic defences, with a high metabolic cost for the plant. On the other hand Trichoderma harzianum T39, a beneficial microorganism, primes for defence against grapevine downy mildew. This mechanism presents advantages in terms of energy costs for the plant, because defence responses are expressed upon pathogen attack and, therefore, only when they are really needed. The present study aims to understand cellular processes involved in reaction to P. viticola inoculation in plants pre-treated with the biocontrol agent T. harzianum T39 in comparison to the water-treated and BTH-treated ones. By monitoring the kinetic of intercellular colonization of P. viticola, callose and lignin deposition in grapevine cell wall and the production of reactive oxygen species, we demonstrated a priming mechanism for the post-invasion reactions in T39- and BTH-treated plants with difference in timing and magnitude of responses. Moreover, to explore the processes involved in grapevine self-defence induced by T. harzianum T39 before and after pathogen inoculation, a proteomic approach was used following an 8-plex differential mass tags iTRAQ protocol. Among proteins identified and quantified by LC-MS-MS (more than 900), 89 were significantly modulated 1 day post P. viticola inoculation in water-treated plants. Eighty-three proteins were directly modulated by T39 prior pathogen infection and 104 in T39-treated plants at 1 day post P. viticola inoculation. A general re-direction of primary and secondary metabolism, together with a reaction involving early steps of recognition (receptors and downstream signalling molecules) were induced by P. viticola within the first day of infection in water-treated plants. On the contrary a general number of proteins including hormones signalling, pathogenesis related proteins and oxidative stress-related proteins, were induced or primed for induction upon pathogen inoculation in T39-treated plants. All these results offer a greater understand of the mechanisms underlying the grapevine T39-induced resistance and illustrate the priming effect of T39 on both defence-related proteins abundance and constitutive barriers formation. Further research will be focused on a detailed functional characterization of potential elicitors of grapevine defence mechanism.