Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Plant immunity mechanisms triggered by PME activity

SYNOPSIS OF THE THESIS The intricate interplay between plants and microorganisms involves a sophisticated plant immune system. The first line of plant defence is exerted by plasma membrane Pattern Recognition Receptors (PRRs) serve as key components in immune orchestra, capable of detecting a diverse range of molecular patterns. These include Microbe-, Pathogen-, or endogenous -Damage Associated Molecular Patterns (MAMPs, PAMPs, or DAMPs) that triggers Pattern-Triggered Immunity (PTI). However, the deployment of immune responses often comes at the cost of growth, necessitating a delicate fine-tuning of immune pathways to limit the growth-defense trade-off. In the last years, Pectin Methylesterases (PMEs) have emerged as essential factors in plant-fungus interactions. The study delves the intricate modulation of PME activity during plant-pathogen interactions, focusing on the response to the necrotrophic fungus Botrytis cinerea. PMEs are enzymes that regulate the methylesterification of Homogalacturonan (HG), a major constituent of pectin in dicot plant cell walls. Arabidopsis harbors 66 PME isoforms, classified into two groups, Group 1 and Group 2. Pro-PMEs, belonging to Group 2, are organized as zymogens with an N-terminal pro region structural similar to PME Inhibitors (PMEIs). PME activity contributes to the immunity producing de-methylesterified negatively charged carboxyl groups to reinforce cell walls and may also favour the release and perception of DAMP-related signals immunity, such as oligogalacturonides (OGs) and methanol (MeOH) inducing defense responses, enhancing plant resistance to pathogens. However, the molecular mechanisms governing PME activation during disease remain largely unexplored. The investigation extends to the post-transcriptional control of PME activity by specific subtilisin-like serine proteases, named subtilases (SBTs), investigating their involvement as modulators of PME activity in Arabidopsis immunity. In Arabidopsis, 56 SBT isoforms are categorized into 6 subfamilies. Proteolysis of multiple host proteins, mediated by SBTs, emerges as a critical regulator in development and plant immunity. The study establishes SBT3.3 and SBT3.5 as post-transcriptional regulators of PME activity during Arabidopsis immunity against Botrytis. SBT3.3 and SBT3.5 genes are induced during various pathogen interactions and elicitor responses, particularly co-expressed with Pro-PME17, a molecular biomarker for microbial infection, during Botrytis infection. Through biochemical and reverse genetic approaches, was demonstrated that Arabidopsis mutants lacking SBT3.3 and SBT3.5 exhibit reduced PME activity induction and increased susceptibility to the fungus. In contrast, the overexpression of SBT3.3 results in higher defence related PME activity, increased Botrytis resistance, and elevated expression of defence-related genes such as WRKY33, PAD3, CYP81F2, and WAK2. The results suggest a potential positive feedback loop involving SBT3.3, PME activity, and production of active OGs to enhance defence responses. The study uncovers the secretion patterns and subcellular localization of SBT3.3 and Pro-PME17, demonstrating that SBT3.3 follows an unconventional secretion route, colocalizing with the exocyst complex, while Pro-PME17 follows a conventional secretion pathway, accumulating in the apoplast. This research sheds light on the distinct secretion pathways employed by SBT3.3 and Pro-PME17, indicating their temporal and spatial colocalization in the cell wall. Moreover, using molecular methodologies successfully generated a pGEM vector containing AtPME17 promoter, paving the way for further experiments to identify transcriptional factors activating AtPME17 during plant infection. The role of pectin de-methylesterification in FERONIA-mediated Rapid Alkalinization Factor (RALF) sensing is also explored. The research contributes to understanding the role of PME in intricate FER signaling pathways involved in development and plant immune responses. The study reveals that plants with genetically modified PME activity retain the capacity to sense RALF peptides through FERONIA, suggesting that RALF-induced signaling through FER is independent of PME activity. Furthermore, the study investigates the involvement of PMEs in the resistance of Vitis vinifera to B. cinerea. Different grapevine varieties exhibit varying resistance to the fungus. The research highlighted that the resistant variety Souvigner Gris showed a significant induction of defense-related PME activity. The results suggest a possible role of PME activity in varietal resistance to gray mold in grapevines, offering insights for potential strategies to enhance vineyard efficiency through genome editing tools to reduced pesticide use. In conclusion, this comprehensive study delves into the intricate network of interactions involving SBT-PME, and PME-RALF-FERONIA in the plant's immune response against microbial threats. The findings provide a best understanding of the mechanisms governing PME activity in plant defence, paving the way for targeted approaches to enhance plant resistance to pathogens.