Caracterização de CsLIEXP1 como um novo gene de susceptibilidade a Xanthomonas citri subsp. citri e edição gênica por CRISPR/Cas9 para tolerância ao cancro cítrico

Author(s)

Reinaldo Rodrigues de Souza Neto

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2022-11-29

Examining board members

Alessandra Alves de Souza; Marcelo Menossi; Henrique Marques Souza; Maxuel de Oliveira Andrade; Marie Anne Van Sluys

• Advisor: Alessandra Alves de Souza

Abstract

Citrus canker is a disease caused by Xanthomonas citri subsp. citri (Xcc). The pathogenicity of this bacterium is dependent on the translocation of effector proteins called Transcription Activator-Like Effectors (TALEs) into the host cell. These proteins work as transcription factors in plants, promoting disease susceptibility genes expression. In Xcc, PthA4 is the main TALE essential for host gene regulation which will facilitate the bacterial colonization. This effector transactivates the CsLOB1 gene, which one is a transcription factor, whose expression is associated with symptoms development. Although CsLOB1 is required to induce canker on different species of Citrus, may other genes could be also involved in canker development. Thus, the goal of this study was to identify new susceptibility genes of sweet orange to Xcc and using CRISPR/Cas9 technology for editing and confirming of functionality in citrus canker development. Therefore, based on transcriptomic data, seven candidate genes to be regulated by PthA4 were identified and validated by relative expression. Among them, an expansin and CsLOB1 genes showed the highest expression levels in all varieties in response to Xcc, but not Xcc-?pthA4 infection. In agroinfiltration assay, the expansin gene was not regulated by PthA4, but it was induced by CsLOB1, which one was induced by PthA4, thus, expansin is indirectly regulated by PthA4. Therefore, this gene was renamed for CsLOB1-INDUCED EXPANSIN 1 (CsLIEXP1). So, the CsLIEXP1 gene was characterized as a susceptibility gene indirectly induced by PthA4. To confirm its role in citrus canker development, a CRISPR-Cas9 vector was developed with 3 sgRNA targeting the editing of first exon of CsLIEXP1 gene. Twnety-two transgenic lines were obtained, and one was selected by High Resolution Melting Analysis (HRMA) as putative edited plant, which was confirmed by genetic sequencing. The. H_CsLIEXP1_1 line presented editing of 6.25% of five nucleotides and 37.5% of one thymine, in total of 43.75% of editing. This plant was challenged with Xcc and the bacterial growth was affected as well as symptoms development. Thus, we demonstrated that editing of CsLIEXP1 gene interfered in citrus canker development, and consequently, affected the bacterial growth. Therefore, in this study, a new susceptibility gene was identified and using genome editing approach citrus canker tolerant plant was obtained (AU)

• FAPESP's process: 17/13885-1 - Use of CRISPR/Cas9 in the editing of susceptibility genes of citrus sinensis to Xanthomonas citri subsp. Citri
• Grantee: Reinaldo Rodrigues de Souza Neto
• Support Opportunities: Scholarships in Brazil - Doctorate