REPOSITORY > RESULTS

Abstract

Plants are constantly exposed to various environmental stressors. Pathogen infections causing disease can affect economically important crops and impact global food production. To counter disease development and spread, it is imperative that we acquire a thorough knowledge of its pathogenesis, as well as of the mechanisms underlying plant immune response. Plant-pathogen interactions mostly play out at the molecular level, encompassing a range of molecular processes and interactions between biological molecules that are unique to each pathosystem. Exploring these complex mechanisms entails using various functional analysis approaches, from high-throughput sequencing analyses to in vitro protein interaction assays. In the scope of this thesis, we adapt and apply different methodologies to study the molecular processes underlying grapevine (Vitis vinifera) infection with phytoplasma and the mechanisms of potato (Solanum tuberosum) immune response against potato virus Y (PVY).
In studying the grapevine-phytoplasma pathosystem, we first optimized an RNA isolation protocol for the efficient isolation of total RNA, including small RNAs, from samples of 'Candidatus Phytoplasma solani' phytoplasma-infected grapevines. Our optimized procedure enabled the isolation of pure RNA of high integrity and concentration, which we used for library preparation and high-throughput sequencing. We further used a protein pull-down assay coupled with mass spectrometry to screen for plant protein targets of a putative 'Candidatus Phytoplasma solani' effector and subsequently confirmed the interactions between the effector and identified targets with in planta co-immunoprecipitation. The results of this study provide important functional information about the poorly known mechanisms of phytoplasma effector-mediated pathogenicity.
To improve our understanding of the potato immune response to PVY infection, we investigated the role of potato TGA transcription factors, regulatory proteins known for their importance in salicylic acid-mediated plant immunity in the model plant Arabidopsis (Arabidopsis thaliana). Our in silico analyses of potato TGA (StTGA) protein sequences revealed the presence of truncated proteins, which we named mini-TGAs. As mini-TGAs are not known in Arabidopsis, we performed different functional analyses to characterize the potato mini-TGA StTGA2.1. The results of this study revealed that StTGA2.1 compensates for salicylic acid deficiency in potato immune response to PVY and associates with other full-length StTGAs to modulate transcription. We showed that StTGA2.1 is involved in the regulation of class III peroxidase gene expression and used advanced computational modeling to gain insight into the structural aspects of mini-TGA function.
Altogether, the results presented in this thesis demonstrate the application of functional analysis methodologies in plants and provide new insights into the mechanisms of plant-pathogen interactions in two economically important crop species: grapevine and potato. The newly acquired knowledge improves our understanding of the molecular biology of plant diseases, which is critical for the development of effective crop protection strategies.