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The Degenerate EAL-GGDEF Domain Protein Filp Functions as a Cyclic di-GMP Receptor and Specifically Interacts with the PilZ-Domain Protein PXO_02715 to Regulate Virulence in Xanthomonas oryzae pv. oryzae

June 2014 , Volume 27 , Number  6
Pages  578 - 589

Fenghuan Yang,1 Fang Tian,1 Xiaotong Li,1 Susu Fan,1 Huamin Chen,1 Maosen Wu,1 Ching-Hong Yang,2 and Chenyang He1

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193; 2Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee 53211, U.S.A.

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Accepted 8 February 2014.

Degenerate GGDEF and EAL domain proteins represent major types of cyclic diguanylic acid (c-di-GMP) receptors in pathogenic bacteria. Here, we characterized a FimX-like protein (Filp) which possesses both GGDEF and EAL domains in Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Both in silico analysis and enzyme assays indicated that the GGDEF and EAL domains of Filp were degenerate and enzymatically inactive. However, Filp bound to c-di-GMP efficiently within the EAL domain, where Q477, E653, and F654 residues were crucial for the binding. Deletion of the filp gene in X. oryzae pv. oryzae resulted in attenuated virulence in rice and reduced type III secretion system (T3SS) gene expression. Complementation analysis with different truncated proteins indicated that REC, PAS, and EAL domains but not the GGDEF domain were required for the full activity of Filp in vivo. In addition, a PilZ-domain protein (PXO_02715) was identified as a Filp interactor by yeast two-hybrid and glutathione-S-transferase pull-down assays. Deletion of the PXO_02715 gene demonstrated changes in bacterial virulence and T3SS gene expression similar to Δfilp. Moreover, both mutants were impaired in their ability to induce hypersensitive response in nonhost plants. Thus, we concluded that Filp was a novel c-di-GMP receptor of X. oryzae pv. oryzae, and its function to regulate bacterial virulence expression might be via the interaction with PXO_02715.

© 2014 The American Phytopathological Society