Cloning And Function Analysis Of A FlhA Gene In Pseudomonas Fluorescens Strain 7-14

Fluorescent Pseudomonas is an important group of plant growth-promoting rhizobacteria. They can produce a series of antimicrobial factors, including antibiotics, protease, siderophore and HCN, and are described as useful agents for biological control of plant diseases. In this study, a flagella biosynthesis gene flhA was cloned and its function was analyzed by bioinformatics and molecular technology.First of all, a BLASTP search was performed in the genome of Pseudomonas fluorescens strain 5 (Pf-5) (NC₀04129) and P. fluorescens strain 0-1 (Pf0-1)(NC₀04792) using FlhA protein from P. aeruginosa UCBPP-PA14 (NC₀08463.1) as a bait, and the FlhA homolog, named as F1hAPf-5 (YP₂58790) and F1hApPf0-1(YP₃47292), was obtained from Pf-5 and PfO-1 genome, respectively. According to the nucleic acid sequence of flhAPf-5 and flhAPf0-1,a pair of degenerate primers, termed as flhA-F/flhA-R, was designed and used to amplify the flhA gene from P. fluorescens strain 7-14 (Pf7-14). By PCR amplification, a 2037-bp DNA fragment, named as flhPf7-14 was obtained and sequenced from Pf7-14. The sequence analysis of flhA-Pf7-14 gene or FlhAPf7-14 protein was performed by using software, including CLUSTALW and MEGA3. The result of sequence analysis revealed that FlhAPf7-14 shared 93% and 94% amino acid similarity with that from Pf-5 and Pf0-1, respectively. Interestingly, the FlhAPf7-14 also shared relative high amino acid similarity with HrcV protein, which is a conserved component of typeâ…¢secretion system (T3SS) in Xanthomonas oryzae pv. oryzae (Xoo). The result of Southern blot suggested that flhA had a single copy in Pf7-14 genome.In the following steps, based on the result (single copy) of Southern blot of flhA, the flhA gene was disrupted by single-cross recombinant technology. Meanwhile, the corresponding flhA complementation strain was constructed by expression of a broad-host vector pBCLPFLHA in the flhA mutant. The flhA mutant abolished flagella and was unable to mobile in semi-solid medium, while the corresponding flhA complementation strain had the wild-type flagella and the ability to mobile. Furthermore, the flhA mutation in Pf7-14 reduced the colonization ability in rice leaves and significantly reduced biological control efficiency (20.2%) for rice sheath blight, caused by Rhizoctonia solaini, whereas the flhA complementation strain restored the wild-type ability to colonize in rice leaves and biocontrol efficiency (biocontrol efficiency for flhA complementation strain and wile type was 47.8% and 52.4%, respectively) for rice sheath blight. Interestingly, the flhA mutation in strain Pf7-14, on one hand, did not affect its ability to form biofilm and produce protease and siderophore, on the other hand, did not affect its antifungal activity against R. solani and Sclerotinia sclerotiorum.