Γονιδιωματική εξόρυξη και λειτουργική αξιολόγηση βακτηριακών παραγόντων παθογένειας και μελέτη μηχανισμών αλληλεπίδρασης παθογόνου-ξενιστή

Abstract

Intensive research during the last two decades has increasingly focused on the elucidation of the molecular basis of host-pathogen interactions in plant disease. Although many aspects of the genetic, molecular and biochemical basis of microbial and viral pathogenicity and host resistance remain unknown, significant progress has been made in the understanding of basic mechanisms of pathogenicity and host responses during pathogen invasion and host colonization. The availability of complete genome sequences of several pathogenic microorganisms and some their hosts, enables researchers to explore in greater depth the molecular/cellular mechanisms of host-pathogen interactions, focusing on selected model pathogens and model plants. This doctoral dissertation is divided in four chapters. The 1st chapter presents the results of bioinformatic and functional analysis of two putative new bacterial type III effector proteins (T3EPs) from Pseudomonas syringae pv. syringae B728a and other P. syringae pathovars. Furthermore, the in silico analysis and genomic mining for putative type VI secretion systems (T6SSs) coded by genomic islands of three P. syringae pathovars with fully sequenced genomes. These strains carry one or two putative genomic loci with significant degree of homology and synteny to a T6SS locus from P. aeruginosa. The 2nd chapter presents the results of investigations on the role of T3EPs on the RNA mediated gene silencing in plants. These studies revealed that transient expression of genes coding certain bacterial T3SPs, which did not elicit HR in transgenic Nicotiana benthamiana line 16C, enhanced GFP silencing following agro-delivery of GFP and effector gene expressing cassettes. In addition, it was shown that these T3SPs increased GFP-specific siRNA as well as nat-siRNAs and lsiRNAs accumulation levels. These effects were blocked by two viral silencing suppressors that were tested. Further analysis using genetic truncations and site directed mutants showed that the silencing enhancer activity does not involve the receptor-recognition domains of T3EPs. Our results suggest that a subset of P. syringae T3SPs engage the small RNA accumulation pathways in plants, thus providing evidence for a novel function and a basis for a novel functional assay for T3SPs. It appears that phytopathogenic bacteria, like plant viruses, deploy specific protein effectors to manipulate the host RNA silencing machinery in order to cause disease. In the 3rd chapter, the results of manipulating apoplastic oxidation of polyamines (PAs) on plant responses to various plant pathogens are presented. In wild-type tobacco (Nicotiana tabacum ‘Xanthi’) plants, infection by the compatible pathogen Pseudomonas syringae pv. tabaci resulted in increased expression of the endogenous polyamine oxidase (PAO) gene and corresponding PAO enzyme activities. Polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine exodus to the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher accumulation of hydrogen peroxide. Plants overexpressing PAO in the apoplast showed increased tolerance against the biotrophic bacterium P. syringae pv. tabaci and the hemibiotrophic oomycete Phytophthora parasitica var. nicotianae, but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in apoplastlocalized plant-pathogen interactions, and signals parallel defense responses prior to infection, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of biotrophic plant pathogens. Finally, the 4th chapter presents a new approach to the valorization of genomic sequences data for various plant pathogens (bacteria and viruses) and host plants, for the development of a prototype molecular toolkit for disease resistance breeding and functional genome analysis in plants. In this chapter, is presented the “proof of concept” for the use of pathogen effectors as “Functional Markers” for conventional disease resistance breeding, based on Ravr gene interactions to detect, the presence/absence or co-segregation, of functional R gene alleles in a breeding population.