Functional and applied study of the RAP2.2 and RPS5 genes in the Xylella fastidiosa resistance

Author(s)

Willian Eduardo Lino Pereira

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-02-28

Examining board members

Alessandra Alves de Souza; Celso Eduardo Benedetti; Jorge Mauricio Costa Mondego; Ivan de Godoy Maia; Erika de Carvalho Teixeira

• Advisor: Alessandra Alves de Souza

Abstract

The bacterium Xylella fastidiosa causes Citrus Variegated Chlorosis (CVC) in sweet orange (Citrus sinensis), the main specie of economic importance, due to its use for orange juice production, one of the main Brazilian agribusiness commodities. The importance of this bacterium gained more prominence due to its recent occurrence in Europe and the high losses in the olive trees culture. In the citrus case, all varieties of sweet orange are affected by the disease, but mandarins (Citrus reticulata) are resistant. Due to the prior identification of the transcriptome of genes associated with resistance of C. reticulata to X. fastidiosa, it is essential to understand the function of these genes in the plant-pathogen interaction. For this, the use of model plants is the fastest way to get these answers. Although previously described as host for X. fastidiosa, A. thaliana is poorly used because of the lack of a phenotype associated with bacterial infection. Therefore, in chapter 1, A. thaliana was better characterized by factors associated with the pathogenicity of X. fastidiosa, such as migration, colonization and bacterial population during infection. In addition, it was observed and quantified the purple top symptom in infected leaves, due to the anthocyanin accumulation, characterizing a visual symptom for future works involving A. thaliana as a model plant. In the following chapters, functional studies using A. thaliana mutants for genes homologous to those differentially expressed in C. reticulata were performed. Two genes, AtRAP2.2 and AtRPS5, were shown to be involved in resistance to X. fastidiosa, because in their absence the bacterium more efficiently colonized the host. The AtRAP2.2 gene encodes a transcription factor from AP2/ERF superfamily related to an ethylene-mediated signaling pathway that regulates genes associated with the defense against Botrytis cinerea, while the AtRPS5 gene encodes a CC-NBS-LRR type protein, responsible for the recognition of the AvrPphB effector of Pseudomonas syringae and triggering defense response. In C. reticulata there is no information about the role of these genes, so studies with A. thaliana aimed to obtain more information about their functions in resistance to X. fastidiosa. Thus, studies revealed that both the subcellular localization and the 3D structure of CrRAP2.2 and CrRPS5 proteins were conserved relative to their homologs in A. thaliana, suggesting a conservation of functions and consequently functional orthology. A detailed study in A. thaliana showed that both the anthocyanin accumulation symptom and the bacterial population were larger in the rap2.2 mutant and decreased with CrRAP2.2 overexpression at Col-0 or complementation in the mutant. Thus, C. sinensis plants were transformed with CrRAP2.2 and challenged with X. fastidiosa. The event T142 that presented greater expression of CrRAP2.2 was the one that presented greater resistance to CVC, with potential for future field trials. The results of inoculation and confocal microscopy confirmed that increased colonization and migration of X. fastidiosa occurred in the rps5 mutant compared to the wild type, which would classify its probable orthologous CrRPS5 as important in the defense of the plant to X. fastidiosa, being promising for transfer in C. sinensis (AU)

• FAPESP's process: 13/26944-5 - Functional and applied study of the rap2.2 transcriptional factor and rps5 resistance gene in tolerance to phytopathogen Xylella fastidiosa
• Grantee: Willian Eduardo Lino Pereira
• Support Opportunities: Scholarships in Brazil - Doctorate