Powdery mildew and grapevine: which genes, enzymes and metabolites in resistant and susceptible cultivars?

Tese de mestrado, Biologia dos Recursos Vegetais, Universidade de Lisboa, Faculdade de Ciências, 2020The Eurasian Vitis vinifera, one of the most cultivated horticultural crops, has its economic impact derived from the fruits which are utilized to produce mainly wine, table grapes, raisins. The process of domestication originated a superior species with desirable agronomic traits and highly adaptable to external environment but prevented a coevolution with natural biotic stresses. In result, V. vinifera is highly susceptible to several pathogens. Erysiphe necator is one of the most threatening pathogens, being the etiologic agent of powdery mildew (PM). As an obligate biotrophic fungus, E. necator differentiates specialized infection (appressorium) and feeding (haustorium) structures to invade grapevine cells and acquired resources to complete his life cycle. At these stages of infection, a complex pathogen-host interaction occurs, and the outcome is the establishment of an incompatible (resistance) or compatible (disease) interaction. In grapevine, constitutive defences will avoid adhesion, germination and penetration. Induced defences organized in a two-layered system will block the endophytic growth and infection propagation. In these, in an intricate network, hormones act as secondary messengers to trigger downstream structural and biochemical changes. Based on additional undergoing studies and previous reports transcriptome and proteome reprogramming related with signalling and hormonal metabolism occurs; our study was performed to give some clues about the phytohormone network upon E. necator infection. For that, through LC-MS/MS, was quantified the content of some hormones in non-infected and E. necator-infected leaves of the resistant hybrid Vitis rupestris × riparia cv. 101-14 Millardet et de Grasset and the susceptible V. vinifera cv. Aragonez. Quantitative real time-PCR (qPCR) was performed to analyse the expression profiling of genes putatively involved in the chosen phytohormones pathways. At constitutive levels, a higher content of salicylic acid (SA) and auxins (mainly indole acetic acid; IAA) and a specific selection of jasmonates (JAs) and abscisic acid (ABA) were present in resistant and susceptible species, respectively. Upon infection, an accumulation of these hormones in each species was noticed. Also, a putative phytohormonal network based on a blend between SA/IAA and JAs/ABA was present in resistant and susceptible species, respectively. Altogether, these results suggest a role of hormones, and particularly the balance among them, in modulating the type of interaction and a possible involvement in response to E. necator. Nevertheless, the gene expression analysis revealed a more complex regulation of hormonal pathways than in others reported plant-PM interactions. Research on grapes to understand the defences responses upon E. necator infection have been neglected. In parallel with a broader transcriptomic and metabolomic-based project, and as for leaves, it was quantified the hormonal content and analysed the gene expression of susceptible grapes of V. vinifera cv. Carignan. In these, even delayed, a putative involvement of SA and JA pathways seems to respond to E. necator infection. Together with the gene expression analysis, the study pointed a different network of phytohormones and activation of different gene isoforms among leaves and grapes in response to E. necator infection. Reports also indicate a reprogramming of metabolism upon E. necator infection. Regarding the secondary metabolism, the phenylpropanoid pathway produce a plethora of phenolic compounds and phytoalexins important in defence responses against E. necator. Through spectrophotometry, was performed a quantification of total phenolic content in leaves and grapes. Additionally, in grapes, the phenylalanine ammonia lyase (PAL) biochemical activity was determined. In both organs, no change was noticed in total phenolic content upon E. necator infection nor in PAL activity in grapes. This suggest a reprogramming of phenylpropanoid pathway in order to accumulate specific phenolic compounds. The new obtained data in leaves and grapes of grapevine upon E. necator infection, once fully dissected and validated, will be useful for breeding programmes in order to improve tolerance against this fungus