Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (4): 675-684.doi: 10.3864/j.issn.0578-1752.2014.04.007

• PLANT PROTECTION • Previous Articles     Next Articles

Functional Analysis of HrpX/HrpY in Dickeya zeae Virulence

 CHEN  Xue-Feng, WEI  Chu-Dan, ZHANG  Qing, LIU  Qiong-Guang   

  1. College of Natural Resources and Environment/Key Laboratory of Microbial Signals and Disease Control, Guangdong Province, South China Agricultural University, Guangzhou 510642
  • Received:2013-07-19 Online:2014-02-15 Published:2013-09-21

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Abstract: 【Objective】Rice bacterial foot rot caused by Dickeya zeae is one of the important bacterial diseases on rice. The virulence and regulation mechanism of D. zeae are imperfect at present. This paper aims to analyze the sequence characteristics of HrpX/HrpY, a two-component regulatory system (TCS) in D. zeae, and study on the roles of this system in D. zeae virulence and the relationships between HrpX/HrpY and its downstream genes.【Method】The sequences of HrpX/HrpY were analyzed by bioinformatics, a suicide recombinant plasmids pKNG-ΔhrpX and pKNG-ΔhrpY with reverse selection marker gene scaB were constructed, then transferred into wild strain EC1, respectively, by triparental mating, so the gene deletion mutants ΔhrpX and ΔhrpY were constructed after two alleles homologous recombination screening. The biological characteristics such as growth rate, extracellular enzyme, toxin, motility, biofilm, virulence on rice and HR on tobacco were compared and analyzed. In addition, bacterial total RNA was extracted, and real-time quantitative PCR was carried out so as to assess the expression of downstream genes of HrpX/HrpY.【Result】In the genome of D. zeae strain EC1, hrpX and hrpY were single copy. The coding region of hrpX was 1 473 bp, which encoding 490 amino acids, while hrpY with the size of 642 bp encodes 213 amino acids. There were only 32 base pairs between hrpX and hrpY. They were in the same transcriptional direction, and had functional relationship. HrpX was a histidine protein kinase combined with the bacterial membrane and HrpY was a response regulator in the cytoplasm. They constitute a two-component system and regulate the downstream genes expression of hrp. Further by genetic manipulation, two gene deletion mutants ΔhrpX and ΔhrpY were constructed successfully. The results of phenotypic determination showed that ΔhrpX and ΔhrpY lost motility, could not form pellicle (biofilm), weakened the inhibitory effect on rice and seed germination. Furthermore, mutants virulence tests showed that the symptoms alleviated than the wild strain EC1 did when the bacterial suspensions were poured into the root of rice plant. There were no significant differences between mutants and EC1 in growth rate, activities of extracellular enzyme and toxin production, they could also induce HR on tobacco. Real-time fluorescent quantitative PCR results showed that HrpX/HrpY regulated positively some downstream hrp genes, and the expression quantity of hrpA, hrpF and hrpN decreased obviously. Further analysis found that the regulation effect of HrpY was more significant than HrpX. 【Conclusion】HrpX/HrpY is a two-component regulatory system in D. zeae. It regulates bacterial motility, biofilm, and virulence, and regulates the expression of downstream hrp genes positively.

Key words: Dickeya zeae , HrpX , HrpY , motility , virulence

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