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Use of CRISPR/Cas9 in the editing of susceptibility genes of citrus sinensis to Xanthomonas citri subsp. Citri

Grant number: 17/13885-1
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): December 01, 2017
Status:Discontinued
Field of knowledge:Agronomical Sciences - Agronomy
Principal Investigator:Alessandra Alves de Souza
Grantee:Reinaldo Rodrigues de Souza Neto
Home Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil
Associated research grant:13/10957-0 - Xylella fastidiosa-vector-host plant interaction and approaches for citrus variegated chlorosis and citrus canker control, AP.TEM
Associated scholarship(s):18/21990-2 - Use of CRISPR-Cas9 to edit citrus canker susceptibility genes to obtain commercial sweet orange varieties resistant to Xanthomonas citri subsp. citri, BE.EP.DR

Abstract

Citrus Canker is a disease caused by the bacterium Xanthomonas citri subsp. citri (X. citri) and it has been associated to important economic losses in citrus orchards at worldwide. The symptoms of Citrus Canker are characterized by eruptive lesions that form on the surface of leaves, branches and fruits. With the development of the disease, there are the abscission of leaves end premature fall of fruits, reducing the plant production. It is known that the pathogenicity of X. citri is depend on translocation of effector proteins known as Transcription Activator-Like Effectors (TALEs) to the inside of plant cell. TALEs working as transcription factor in host cell and binding to Effector Binding Elements (EBE) on promoters of specific plant genes activating its expression. These genes are denominated of "susceptibility genes" because their expression promote to development of disease and bacterial colonization. PthA4 is a TAL effector of X. citri essential to induction of hypertrophy and hyperplasia in citrus. It is known that PthA4 trans-activate LOB1 gene in sweet orange, whose expression is correlated with Citrus Canker development. However, the only expression of LOB1 is not enough to generate Citrus Canker symptoms, suggesting that other activated genes by PthA4 will be necessary to development of disease. Thus, such genes became good targets to genome edition aiming to obtain resistant plants to X. citri. Therefore, the goal of this study is identifying new susceptibility genes to X. citri aiming to edit their EBE regions using the CRISPR/Cas9 system. Based on previously transcription data and bioinformatic analysis, eight genes of sweet orange were selected, which are showing the target sequence of PthA4 in their promoter regions. The relative expression of these genes will be evaluated in different varieties of sweet orange. The most induced genes, whose expression be depends on PthA4, will be selected to edition by CRISPR/Cas9 by transient expression and genetic transformation. This project intends to identify new direct targets of PthA4in commercial varieties of sweet orange and obtaining edited resistant plants by CRISPR/Cas9 to X. citri. (AU)