High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

CsBZIP40 confers resistance against citrus bacterial canker by repressing CsWRKY43-CsPrx53/CsSOD13 cascade mediated ROS scavenging

As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.