Functional Analysis Of Clpxoo Binding To C-di-GMP And Regulating The Transcription Of Down-stream Genes

The bacterial leaf blight of rice, caused by Xanthomonas oryzae pv. oryzae （Xoo） is one of themost important bacterial diseases worldwide. Rice-Xoo interaction has a series of signaling transductionwhich facilitates its infection to the rice plant. Cyclic-diguanylate （c-di-GMP） is a universal bacterialsecond messenger, involved in the regulation of many physiological processes. Metabolism ofc-di-GMP is mainly affected by two types enzymes containing opposite activities: guanosine cyclase（DGC） and phosphodiesterase （PDE）. The important part of c-di-GMP regulation network is a receptorthat response to signal level changes of c-di-GMP in the bacteria. In Xoo, transcriptional regulationfactor Clpxoo （PXO₀4006） may act as a receptor protein of c-di-GMP. Binding to small moleculeswith its cNMP domain might affect its conformational structure, thus to regulate various physiologicalfunctions in bacteria. In this study, we intended to elucidate the functional mechanism of Clpxoo byfocusing on its two domains. We have made the following progress：（1） To check with Clpxoo interacted with cAMP or c-di-GMP, we conductedsurface plasmon resonance （SPR） experiment. The result shows Clpxoo interacted with c-di-GMPspecifically, with a capacity of1.64μM. Isothermal titration calorimetry （ITC） assay showed theratio of binding between Clpxoo and c-di-GMP is1:1. In conclusion, the Clpxoo is a new type ofc-di-GMP receptor protein in Xoo.（2） We constructed a strain Δclp（pClp-HA） for chromatin immunoprecipitation （ChIP） experiment. Thecomplex of Clpxoo-HA-DNA was pulled down by protein G and anti-HA antibody. The DNA wassequenced by high-throughput sequencing technology. Finally, the sequencing data was analyzed bybioinformatics tools and identified78peaks that directly regulated by Clpxoo.（3） We chose41gene promoter for electrophoretic mobility shift assay （EMSA） to validate ChIP-seqresults. The results show that most of the fluorescent probes specific bound with Clpxoo, and thebinding ability will be changed at the presence of c-di-GMP. Besides, the binding capacity betweenClpxoo and promoter is1e-7M by SPR. We speculate that binding of c-di-GMP to Clpxoo willfacilitate the formation of dimer before binding to its target DNA sequence.（4） We detected the transcription level of the18target genes in the Δclp by RT-qPCR. When there is noc-di-GMP treatment, six genes （PXO₀0043, PXO₀0877, PXO₀2019, PXO₀2190, PXO₀3438,PXO₀4151） were up-regulated by Clpxoo; nine （PXO₀0486, PXO₀0778, PXO₀1741,PXO₀2166, PXO₀2464, PXO₀2485, PXO₀3636, PXO₀0043, PXO₀3877, PXO₀4151）were down-regulated; and three （PXO₀0376, PXO₀2535, PXO₀2675） were unaffected.In conclusion, this study identified that78peaks were directly regulated by Clpxoo. We confirmedthat Clpxoo is a receptor of c-di-GMP in Xoo. These results are helpful to reveal the molecular basis ofc-di-GMP specific recognition by receptors and the downstream signaling mechanism.