Use of CRISPR-Cas9 to edit citrus canker susceptibility genes to obtain commercial sweet orange varieties resistant to Xanthomonas citri subsp. citri

Abstract

Citrus canker is a disease caused by Xanthomonas citri subsp. citri. 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 function as transcription factors in plants. They recognize specific regions in promoters of target genes known as Effector Binding Elements (EBEs). The binding of TALEs to corresponding EBEs promotes transcription of disease susceptibility genes. In X. citri, PthA4 is the main TALE essential to elicit canker on citrus. This effector transactivates the LOB1 gene in sweet oranges, whose expression is associated with symptoms development. Although LOB1 is required to induce canker on citrus, microarray and RNA-seq data suggest that other genes are also involved in canker development and could be also direct targets of PthA4. Thus, the EBE sequence of such genes could be good targets for gene editing by CRISPR-Cas9 aiming resistance to X. citri. Based on previous results of transcriptome and bioinformatic analyzes, we selected nine genes as potential targets of PthA4. The expression profile of these genes was analyzed by qPCR in three sweet orange commercial varieties used in Brazil. Among the nine genes tested, expansin, pectate lyase C and LOB1 showed high expression levels in all varieties in response to X. citri, but not X. citri pthA4 mutant infection. Thus, the promoters of these genes were cloned and sequenced to design sgRNAs for CRISPR-Cas9 vector construction and to verify the possible direct regulation of these genes by PthA4. Therefore, the goal of this proposal is to confirm that expasin and pectate lyase C are directly regulated by PthA4 and using LOB1 as concept proof. After this, to use CRISPR-Cas9 vector to obtain edited plants resistant to X. citri.