Identification of the Regulatory Components Mediated by the Cyclic di-GMP Receptor Filp and Its Interactor PilZX3 and Functioning in Virulence of Xanthomonas oryzae pv. oryzae

Abstract

The degenerate GGDEF/EAL domain protein Filp was previously shown to function as a cyclic di-GMP (c-di-GMP) signal receptor through its specific interaction with an atypical PilZ domain protein PilZX3 (formerly PXO_02715) and that this interaction is involved in regulating virulence in Xanthomonas oryzae pv. oryzae. As a step toward understanding the regulatory role of Filp/PilZX3-mediated c-di-GMP signaling in the virulence of X. oryzae pv. oryzae, differentially expressed proteins (DEPs) downstream of Filp/PilZX3 were identified by isobaric tagging for relative and absolute quantitation (iTRAQ). A total of 2,346 proteins were identified, of which 157 displayed significant differential expression in different strains. Western blot and quantitative reverse transcription-PCR analyses showed that the expression of HrrP (histidine kinase-response regulator hybrid protein), PhrP (PhoPQ-regulated protein), ProP (prophage Lp2 protein 6) were increased in the ∆filp, ∆pilZX3, and ∆filp∆pilZX3 mutant strains, while expression of CheW1 (chemotaxis protein CheW1), EdpX2 (the second EAL domain protein identified in X. oryzae pv. oryzae), HGdpX2 (the second HD-GYP domain protein identified in X. oryzae pv. oryzae) was decreased in all mutant strains compared with that in the wild type, which was consistent with the iTRAQ data. Deletion of the hrrP and proP genes resulted in significant increases in virulence, whereas deletion of the cheW1, hGdpX2, or tdrX2 genes resulted in decreased virulence. Enzyme assays indicated that EdpX2 and HGdpX2 were active phosphodiesterases (PDEs). This study provides a proteomic description of putative regulatory pathway of Filp and PilZX3 and characterized novel factors that contributed to the virulence of X. oryzae pv. oryzae regulated by c-di-GMP signaling.