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.

简并显示，简并的GGDEF / EAL域蛋白Filp通过与非典型PilZ域蛋白PilZX3（以前称为PXO_02715）的特异性相互作用而起环状di-GMP（c-di-GMP）信号受体的作用，并且这种相互作用与调节黄单胞菌PV中的毒力。菌。作为了解Filp / PilZX3介导的c-di-GMP信号在米曲霉pv毒力中的调节作用的一步。水稻，通过等压标记对Filp / PilZX3下游的差异表达蛋白（DEP）进行了相对和绝对定量（iTRAQ）鉴定。总共鉴定出2346种蛋白质，其中157种在不同菌株中显示出明显的差异表达。Western印迹和定量逆转录PCR分析显示，距离像（组氨酸激酶反应调节杂交体的表达p rotein），PhrP（PhoPQ调节蛋白），丙（原噬菌体Lp2的蛋白6）Δ分别增加的filp，Δ pilZX3和Δ的filp Δ pilZX3突变株，而CheW1的表达（趋化蛋白CheW1），EdpX2（在标识的第二EAL域蛋白质X.菌PV。米曲霉），HGdpX2（在标识的第二HD-GYP域蛋白质X.菌PV。米曲霉）的所有突变体菌株与在野生型，这与所述的iTRAQ数据一致相比降低。hrrP和proP基因的删除导致毒力的显着增加，而cheW1，hGdpX2或tdrX2基因的删除导致毒力降低。酶分析表明，EdpX2和HGdpX2是活性磷酸二酯酶（PDE）。这项研究提供了蛋白质组学描述的公认的Filp和PilZX3调控途径，并表征了导致米曲霉毒力的新因子。pv。米曲霉由c二GMP信令调节。