The TALE effector PthA4 of Xanthomonas citri subsp. citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an important citrus disease worldwide. PthA4 is the most important pathogenicity gene of Xcc and encodes a transcription activator like effector (TALE) secreted by the type III secretion system. PthA4 is known to activate the expression of CsLOB1, the canker susceptibility gene and a transcription factor, to cause citrus canker symptoms. Extensive effort was made to identify downstream targets of CsLOB1 to investigate the mechanism underlying canker symptom development. However, none of identified CsLOB1 target genes have been confirmed to be involved in citrus canker development. Here, we first identified the direct targets of CsLOB1 by generating promoter-uidA (GUS) reporter fusion construct for the 13 genes highly induced by both PthA4 and CsLOB1 and monitored the reporter activity in N. benthamiana leaves co-expressing CsLOB1. Agrobacterium tumefaciens-mediated transient expression of CsLOB1 activated seven gene promoters in N. benthamiana including Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410 (CsEXP2), Cs2g27100, Cs2g20750 (CsEG1), and Cs9g17380. Next, we constructed dTALEs to target unique sequences in the promoters of the seven direct target genes of CsLOB1 and transformed them into XccpthA4::Tn5 mutant. Our results indicate that a combination of 5 and 7 dTALEs caused canker-like symptoms in the inoculated citrus leaves. In addition, dTALECsEXP2 and dTALECsEG1 caused water soaking and pustules, which are typical canker symptoms. Taken together, Xcc indirectly activates CsEXP2 and CsEG1 via PthA4-CsLOB1 to cause canker symptoms.