Abstract: 【Objective】 Rice foot rot, caused by Dickeya zeae, is one of the important bacterial diseases on rice. Bacterial flagella is an important movement organ, so far, the mechanism of the flagellar system, flhDC and fliA and their regulatory mechanisms are unclear in D. zeae. To clarify the function of these flagellum genes is helpful for further understanding the pathogenicity of integrated control network in D. zeae, developing new drug action targets and making disease prevention and control strategies. The objective of this study is to investigate the function of flagellar system of flhDC and fliA in D. zeae. 【Method】A set of primers were designed based on the genomic DNA of wild strain EC1 of D. zeae. The upstream and downstream fragments of target genes flhDC and fliA to be knocked out were amplified by PCR, respectively. The upstream and downstream fragments were mixed as a template, and then the fusion fragments that lack of flhDC and fliA were obtained by PCR. After dual- enzyme digestion and purification, the fusion fragments were connected to the suicide vector pKNG101, suicide recombinant plasmids pKNG-ΔflhDC and pKNG-ΔfliA with reverse selection marker gene sacB were constructed, then transferred into wild strain EC1, respectively, by tri-parental mating, so the gene deletion mutants ΔflhDC and ΔfliA were constructed after two alleles homologous recombination screening and PCR detection and sequencing verification. The biological characteristics such as extracellular enzyme, toxin, motility, biofilm, virulence to rice and HR on tobacco were compared and analyzed. In addition, bacterial total RNA was extracted, and a real-time quantitative PCR (qRT-PCR) was carried out using 16SrDNA as internal control for normalization. Then the expression of downstream genes flhD, flhC, fliA and fliC in ΔflhDC and ΔfliA was compared.【Result】 Two target gene deletion mutants ΔflhDC and ΔfliA were constructed successfully by genetic manipulation. Phenotypic test results showed that the motility and biofilm formation of wild strain EC1 were very strong, while the motility and biofilm formation of the ΔflhDC and ΔfliA were decreased obviously. The wild strain EC1 had a strong inhibitory effect on rice seed germination, while ΔflhDC and ΔfliA significantly reduced the inhibition of rice seed germination. The rice plants inoculated with the wild strain EC1 showed a brown spot and a large extent of rottenness, while rice plants with ΔflhDC and ΔfliA inoculation only showed water-brown lesions around the inoculated sites. It indicated that ΔflhDC and ΔfliA significantly reduced the virulence to rice plant. Further phenotypic results showed that the activities of extracellular enzymes, toxin and the ability to cause HR on tobacco were not different significantly between the mutants and the wild strain. The results of qRT-PCR showed that in the mutant ΔflhDC, the flhDC and the fliA did not express, while the expressions of the fliA and the fliC decreased obviously compared with the wild strain; In addition, the flhD, flhC and the fliA in the mutant ΔfliA did not express, but the expressions of the fliC decreased obviously. 【Conclusion】The flhDC operon, which regulates the expression of the bacterial flagellum genes, and the fliA, which expresses flagellin specific factor σ²⁸, are important components of the bacterial flagellar system gene cluster. The genes flhDC and fliA significantly affect the motility, biofilm and the germination of rice seeds, and play an important role involving in the virulence in D. zeae.

Key words: Dickeya zeae, flhDC, fliA, motility, virulence