Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Dissection of the molecular mechanisms underlying the phenomenon of “priming” of defence responses in Arabidopsis thaliana

Pathogens have a significant impact on crop yield, and microbial diseases are mostly controlled by genetic or chemical means. Breeding of resistant crops is nowadays based on the use of monogenic resistances, that are effective but narrow-ranged and not durable. The use of elicitors, natural compounds able to increase the natural ability of plants to counteract infections, might be an environmentally sustainable way to provide protection against a wide range of pathogens. However, the molecular mechanisms underlying elicitor-induced resistance (EIR) are currently not well defined, as most studies on plant immunity focused on responses triggered within minutes/hours after treatment, whereas EIR is observed days after elicitation. Previous work indicates that two elicitors, oligogalacturonides (OGs) and flg22, induce in Arabidopsis thaliana an increased resistance to the fungal pathogen Botrytis cinerea that is independent of the defense-related hormones jasmonic acid (JA) and salicylic acid (SA) but requires the production of the indolic phytoalexin camalexin. We have therefore analyzed the long-term effects of OGs and flg22 in Arabidopsis and investigated the molecular basis of these effects. We found that both elicitors induce a durable resistance against B. cinerea that is transgenerationally transmitted without significant fitness cost. These observations suggested that elicitor treatments prime plants to respond more efficiently to subsequent infections. A reverse genetics approach was adopted to characterize genes that, according to previous microarray experiments, are induced by elicitors at 24 h of treatment and are therefore potentially involved in EIR. Insertional mutants impaired in two of these genes, encoding the mitogen-activated protein (MAP) kinase MPK11 and the APETALA2/ETHYLENE-RESPONSIVE FACTOR RAP2.6, showed altered defense responses regulated by different hormonal pathways. Notably, flg22 pre-treatments prime wild-type, but not mpk11 mutant seedlings to accumulate increased transcript levels of PAD3, a gene required for camalexin production, when subsequently treated with chitin, suggesting that MPK11 is involved in long-term response to elicitors. Based on these promising results, we have performed a whole-genome RNAseq analysis of plants treated for 24 h with elicitors as well as plants primed with OGs or flg22 and subsequently inoculated with B. cinerea. After 24 h of elicitation, plants do not show significantly increased expression of well-known defense marker genes but display altered transcript levels of genes involved in photosynthesis, plastid movement and biogenesis, as well as in growth and response to abiotic stress. Notably, elicitation with both elicitors reduced the expression of MYC2, encoding a transcription factor that modulate JA responses and down-regulates production of indolic metabolites. Consistently, plants primed with elicitors and inoculated with B. cinerea showed major changes in the biosynthesis of antimicrobial compounds known to mediate resistance to this fungus, i.e. indole glucosinolates and camalexin. These results suggest that plants treated with elicitors can respond more efficiently to subsequent infection by priming production of antimicrobial compounds, and this might be due to altered expression of key regulators of defense responses, like MYC2. Moreover, trans-generational effects of elicitation indicate that epigenetic changes in these genes, as well as in genes encoding enzymes mediating the biosynthesis of antimicrobial metabolites, might contribute to EIR. These results could provide the basis to further studies that will clarify the priming phenomenon and identify potential targets for the improvement of crop resistance.