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Phylogenetic analysis of Citrus LRR-RLKs and the involvement of EF-Tu receptor in Xylella fastidiosa recognition

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Author(s):
Diogo Maciel Magalhães
Total Authors: 1
Document type: Doctoral Thesis
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
Cláudia Barros Monteiro Vitorello; Michel Georges Albert Vincentz; Renato Vicentini; Ronaldo Jose Durigan Dalio
Advisor: Alessandra Alves de Souza; Marco Aurélio Takita
Abstract

Pattern recognition receptors (PRRs) are very important in plant defense against microorganisms because they are able to recognize pathogen-associated molecular patterns (PAMPs) in early infection process triggering innate immune responses (PTI). Activation of PTI confers resistance against a wide range of microorganisms and studies involving the identification and functional role of PRRs are of great importance. Citrus crops have great commercial importance worldwide; however, the productivity has decreased due to several diseases that affect this culture, such as citrus variegated chlorosis, caused by Xylella fastidiosa. Since most identified PRRs in plants belong to the leucine-rich repeat receptor-like kinases family (LRR-RLKs), one of the goals of this study was to analyze the evolutionary aspects of LRR-RLKs in economically important citrus species, Citrus clementina and Citrus sinensis. The multiple alignment of conserved kinase sequences and the reconstruction of phylogenetic trees estimated by maximum-likelihood identified 300 and 297 LRR-RLKs in the predicted proteomes of C. clementina and C. sinensis species, respectively. The LRR-RLKs from both species were classified into 16 sub-groups (I to XVI) and further analysis in the subgroup XII, which includes the main PRRs identified in plants, showed a large expansion and clustering in the Citrus genome, which demonstrates the importance of tandem duplication in the evolution of LRR-XII in Citrus. In addition to the in silico analysis, in this study we also evaluated the role of EF-Tu receptor (EFR) in the recognition of X. fastidiosa. Arabidopsis thaliana efr-1 mutants were inoculated with X. fastidiosa, and using quantitative real-time PCR (qRT-PCR) we verified a significant increase of bacterial population in efr-1, during the time-course of infection, compared to the wild type (WT). At 15 days after inoculation (DAI) the bacterial population was almost ten times more in the efr-1 tissues in relation to WT. Fluorescence microscopy analysis confirmed the high bacterial colonization in the tissues of efr-1 plants. The importance of EFR in X. fastidiosa recognition was also evaluated by the overexpression of AtEFR in tobacco host plants, which are susceptible to X. fastidiosa. Twenty transgenic events were identified by conventional PCR and the expression of the EFR transgene was confirmed in six lines by qRT-PCR. Western blot analysis confirmed the presence of the heterologous protein and the increased ROS production observed in the presence of elf18 epitopes from X. fastidiosa, E. coli and Xanthomonas citri subsp. citri indicate PTI activation in the tobacco transgenic plants. The significant reduction in the incidence of symptoms in transgenic plants inoculated with X. fastidiosa showed that EFR increased the tolerance against the bacteria infection. The results obtained from A. thaliana and tobacco indicate that EFR could be a good strategy to transform Citrus plants aiming X. fastidiosa and X. citri resistance (AU)