Identification of CsLOB1 target genes that promote citrus canker disease

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PthA4, a transcription activator-like effector (TALE) from the citrus canker pathogen Xanthomonas citri pv. citri (Xcc), promotes disease via transcriptional activation of the citrus CsLOB1 gene that encodes a lateral organ boundary (LBD) protein. Since LBD proteins are transcription factors its conceivable that not CsLOB1 itself but rather target genes of CsLOB1, herein designated as dtCsLOB1 genes (direct targets of CsLOB1), will induce physiological changes in host cells that promote bacterial disease. We aim to identify dtCsLOB1 genes and intend to clarify how their expression translates into physiological changes that promote bacterial disease. CsLOB1 target promoters will be identified by a combination of transcriptome profiling (RNA-Seq) and chromatin immunoprecipitation (ChIP-Seq). The possible disease contribution of identified dtCsLOB1 genes will be studied by functional complementation of disease-compromised Xcc pthA4 mutant strains (incapable of activating CsLOB1) with dTALEs that specifically induce expression of individual dtCsLOB1 genes. While Xcc effectors act inside host cells, the pathogen multiplies in the apoplast, which implies that TALE-induced CsLOB1 expression should cause changes in apoplastic fluids. Accordingly, we will also study the impact of CsLOB1 expression on the composition of apoplastic fluids from citrus leaves via mass-spectrometry-based metabolite/proteome profiling to identify changes that affect Xcc growth. Metabolite/proteome-profiling in conjunction with the gene/transcript-based approaches should provide indications of how expression of dtCsLOB1 genes translates into physiological responses that promote apoplastic growth of Xcc. We envision that our studies have the potential to uncover novel microbial invasion strategies that are possibly conserved across phylogenetically diverse pathogenic and/or symbiotic plant-associated microbes.