Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (13): 2563-2570.doi: 10.3864/j.issn.0578-1752.2014.13.008

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Characterization of the Negative Regulator PXO_02944 in Virulence, Extracellular Polysaccharide Production and Biofilm Formation in Xanthomonas oryzae pv. oryzae

 LI  Xiao-Tong, YANG  Feng-Huan, LIANG  Shi-Min, TIAN  Fang, CHEN  Hua-Min, HE  Chen-Yang   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2013-12-30 Online:2014-07-01 Published:2014-01-24

Abstract: 【Objective】Xanthomonas oryzae pv. oryzae (Xoo) is the causing pathogen of bacterial blight of rice. Xoo can use extracellular polysaccharide (EPS) and enzymes, T3SS and its effectors for sucessful infection of rice. It has been shown that cyclic di-GMP signaling plays an important role in regulation of virulence in Xoo. PXO_02944, the GGDEF, EAL and REC domain containing protein, belongs to c-di-GMP signaling protein family. The objective of this study is to demonstrate the structure and function of the PXO_02944 in regulation of virulence expression of Xoo. 【Method】 Based on the sequences of PXO_02944, the specific primers were designed to amplify the full length of gene using the genomic DNA of the wildtype strain PXO99A as the template. In silico analysis of the GGDEF and EAL domains of PXO_02944 with the conserved ones from other bacteria were done. Then the upstream and downstream fragments of PXO_02944 were amplified, and ligated to vector pK18mobsacB to get the plasmid pK2944. Then a Gm resistance gene (GmR) was inserted into pK2944 resulting in plasmid pK2944-GmR to construct the gene deletion mutation by marker exchange. The full length of PXO_02944 was cloned and ligated into the vector pHM1, and the plasmid was electroporated into ΔPXO_02944 to get the complementory strain. The virulence, EPS production, enzymatic activities, biofilm formation and motility of ΔPXO_02944 were tested compared with PXO99A, ΔPXO_02944 and complemented strain. And the expression of EPS production and virulence related genes were analyzed.【Result】Bioinformatic analysis indicated that PXO_02944 was a response regulator of two-component regulatory system (TCS), which contained the REC input domain and the GGDEF and EAL output domains. In the mutant, PXO_02944 was deleted by replacing of the GmR. The mutation in PXO_02944 led to significant increases in bacterial virulence on rice, EPS production, biofilm formation, hrpG and gumG gene transcripts compared to PXO99A. Interestingly, no changes in activities of cellulase and xylanase and flagellar motility were observed in ΔPXO_02944. The deficiency above in the mutant could be restored to the wild-type level by in trans expression of the PXO_02944. 【Conclusion】 PXO_02944 might function as a negative regulator in bacterial virulence, EPS production and biofilm formation in Xoo.

Key words: Xanthomonas oryzae pv. oryzae , c-di-GMP signaling , virulence expression , response regulator

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