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Phosphohexose mutase of Xanthomonas oryzae pv. oryzicola is negatively regulated by HrpG and HrpX, and required for the full virulence in rice

Abstract

The genome of Xanthomonas oryzae pv. oryzicola annotates one uncharacterized gene, XOC_3841, only one ORF in this strain is annotated to encode Phosphohexose mutase (XanA), which reversibly converts glucose 1-phosphate to glucose 6-phosphate that implicates in the carbon metabolism pathways. However, it is unclear whether the XanA-coding gene is involved in the full virulence of X. oryzae pv. oryzicola. In this report, we showed that the mutagenesis in unique xanA, led the pathogen effectively to unable to utilize glucose and galactose for growth. The expression of xanA was strongly induced by glucose, sucrose, fructose, mannose or galactose at least 3 times higher than that by non-sugar NY medium. Intriguingly, xanA promoter region contains an imperfect PIP-box (plant-inducible promoter) (TTCGC-N16-TTCGA), and the expression of xanA was inducible in rice suspension cells rather than in a nutrient-rich (NB) medium and negatively regulated by a key hrp regulatory HrpG and HrpX cascade. More importantly, mutation in xanA resulted in impairment of bacterial growth and virulence in planta, and reduced bacterial cell motility and extracellular polysaccharides (EPS) production in media. In addition, the lost properties mentioned above in RΔxanA were completely restored to the wild-type level by the presence of xanA in trans. All these results suggest that xanA is required for EPS production, cell motility and the full virulence of X. oryzae pv. oryzicola.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31301633), and the research project of Department of Education of Zhejiang Province (Y201328481).

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Correspondence to Gong-You Chen.

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Fig. S1

Schematic map and molecular analysis of RΔxanA in X. oryzae pv. oryzicola. The positions and orientations of XOC_3841, encoding Phosphohexose mutase, and other adjacent ORFs are shown by referring to the genome sequence of X. oryzae pv. oryzicola BLS256 (http://www.ncbi.nlm.nih.gov/nuccore/CP003057.1). Arrows indicate locations and orientations of the ORFs, and lines indicate the intergenic sequences. ▼above ORF XOC_3841 presents the insertion site of a Tn5 derivative in mutant Mxoc1594. Arrow above the triangle indicates the insertion orientation of the Tn5. A non-polar construction of a xanA deletion mutant was sketched (see MATERIALS AND METHODS for details). The brown and gray box indicated where the left and right flanks targeted xanA, the white box stands for a 960 bp deletion of xanA. xanA was knocked out after two homologous crossover events occurred, and then was validated by PCR with the primer pairs 1F/2R and 3F/3R (Table S1). λ-EcoT14 or DL2000 DNA marker (TaKaRa, Dalian, China). (DOC 36 kb)

Table S1

Primers used in this study (DOC 166 kb)

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Guo, W., Chu, C., Yang, XX. et al. Phosphohexose mutase of Xanthomonas oryzae pv. oryzicola is negatively regulated by HrpG and HrpX, and required for the full virulence in rice. Eur J Plant Pathol 140, 353–364 (2014). https://doi.org/10.1007/s10658-014-0469-7

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Keywords

  • Xanthomonas oryzae pv. oryzicola
  • Phosphohexose mutase
  • HrpG and HrpX
  • Extracellular polysaccharide
  • Virulence