Genomics and walnut hull proteomics of Xanthomonas arboricola pv. juglandis 417 for the development of new disease control.

Abstract

Xanthomonas arboricola pv. juglandis 417 (Xaj417) is the causal agent of walnut bacterial blight, the most significant above-ground disease of walnuts (Juglans regia L.). Walnut producers have registered losses of up to 40% in local production annually. Disease management uses copper-based pesticides which induce pathogen resistance despite being harmful for the environment. Our aim was to evaluate the genome content of the pathogen, dissect the host-pathogen response to define determinants that regulate the host susceptibility and assess the mutation effect of a conserved secreted protein among plant-associated Xanthomonadaceae. Our study focused on Xaj417 to understand the proteo-genomics attributes to colonize its host. We investigated the genome sequence and proteome of this plant pathogen by performing a comparative analysis with other sequenced Xaj and inoculating walnut fruits with Xaj417. The comparison of 32 Xaj genomes revealed that the adaptive evolution generated by intensive spray application to control bacterial diseases possibly led to selection of resistant bacteria and emergence of pathogenic strains (Chapter I). The results revealed that bacterial virulence and copper resistance emerged by the acquisition of specific sets of pathogenesis-related genes commonly transferred among the members of the Xanthomonas genus on mobile genetic elements. This was evidenced for the reference strain Xaj417, a copper-resistant Californian isolate, that acquired a new copper resistance cassette by HGT associated with a new transposon family in Xanthomonas (TnXaj417). The expansion of mobile genetic elements in the most virulent strains influence the repertoire of virulence effectors and adaptation strategies shaping the evolution of pathogenic strains. On Chapter II, we dissected this pathosystem using tandem mass tag quantitative proteomics. This is the first proteome study of this pathosystem examining the molecular responses during the disease development by comparing the proteomes of infected fruit hulls to healthy tissue. Xaj proteins detected in infected tissues demonstrated its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Finally, on Chapter III the secreted monofunctional chorismate mutase mutant (XajCM) was created in Xaj417 and showed increased virulence in walnut nuts. The bacterial morphology was characterized and IX changes in the protein profile of the mutant in planta were tested. The proteomic results suggested intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Overall, this study offers new insights into the emergence of virulence, adaptation, and tolerance to disease management strategies used in orchard ecosystems. It also provides knowledge into molecular mechanisms highlighting potential molecular tools for early detection and disease control strategies.