Two TAL effectors of Xanthomonas citri promote pustule formation by directly repressing the expression of GRAS transcription factor in citrus.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), poses a significant threat to the citrus industry. Xcc employs the transcription activator-like effector (TALE) PthA4 to target the major susceptibility (S) gene CsLOB1 in citrus, promoting host susceptibility to bacterial canker. However, the contribution of other Xcc TALEs, aside from PthA4, to virulence remains underexplored. In this study, we characterized two PthA1 variants, designated PthA5 and PthA6, which facilitate Xcc infection in susceptible citrus species by promoting the formation of hypertrophy and hyperplasia symptoms. Both PthA5 and PthA6 bind directly to effector-binding elements (EBEs) in the promoter of CsGRAS9, suppressing its expression. CsGRAS9 negatively regulates Xcc growth in citrus and contributes to CBC resistance. Notably, natural variations in the EBEs of the FhGRAS9 promoter, a homolog of CsGRAS9 in Hong Kong kumquat, prevent Xcc from affecting FhGRAS9 expression. Using the PTG/Cas9 system, we generated proCsGRAS9-edited sweet orange lines #18-2 and #23, which contain 86-bp and 62-bp deletions in the EBE regions of the CsGRAS9 promoter. These mutant lines showed enhanced CsGRAS9 expression and increased resistance to CBC during Xcc infection. Several GA-related genes and CsTAC1, regulated by CsGRAS9, were also identified. This is the first report that TALEs act as repressors of a resistance gene to confer host susceptibility.