Identification of HrpX regulon genes in Xanthomonas oryzae pv. oryzicola using a GFP visualization technique

Xanthomonas oryzae pv. oryzicola is the causal agent of bacterial leaf streak in rice and injects repertoires of T3S effectors (T3SEs), which are normally regulated by a global regulator HrpX, into plant cells to suppress plant innate immunity for disease development. To establish a visualization technique to identify HrpX regulon genes in this pathogen, we chose six known or unknown T3SE genes of X. oryzae pv. oryzicola (strain RS105) as the targets in this report. The promoters of these candidates, whether or not containing a PIP-box, were fused with the gfp (green fluorescent protein) reporter gene, and the gfp reporters were introduced into the wild-type RS105 and the hrpX mutant RΔhrpX. GFP expression in the wild-type strain could be observed under fluorescence microscopy, but fluorescence was not observed in the hrpX mutant. Consequently, transcription and secretion detection demonstrated that these HrpX regulon members were repressed when hrpX was mutated and were not secreted into the hrp-inducing medium XOM3 because of a mutation in hrcV (T3SS deficient). These data suggest that the gfp reporter system is a feasible visualization tool for the identification of HrpX regulon genes and discovery of new T3SEs in the X. oryzae pv. oryzicola-rice pathosystem.