Study On The Mechanism Of Flagellin In Pine Wilt Disease

Pine wilt disease (PWD), which caused by pine wood nematode (PWN), Bursaphelenchus xylophilus has become a widespread threat to forests. Recently, more and more researches indicate that bacteria associated with PWN play an important role in pine wilt. In order to study the role of flagellin secreted by bacteria carried by PWN in the pine wilt disease, mechanism of cell death of Pinus thunbergii induced by flagellin was studied. The results indicated that cell nuclei were broken into small nuclei, genomic DNA was degraded as analyzed by agarose gel electrophoresis, but no obvious ladder was formed, which suggested that cell death of Japanese black pine induced by flagellin was atypical apoptosis. Then, effects of death of Japanese black pine induced by flagellin on propagation of PWN as well as its carrying Pseudomonas fluorescens GcM5-lA were studied. The results indicated that pre-treatment of Japanese black pine callus cells with flagellin following by inoculation with PWN could promote the propagation of PWN. Within 0-9 days, the longer callus cells were pre-treated, the faster PWN would reproduce. Meanwhile, compared with normal callus of P. thunbergii, callus pre-treated with flagellin could also promote reproduction of P. fluorescens GcM5-1A carried by PWN to some extent. Similar results were got when dead callus caused by boiling was used instead of flagellin pre-treated callus. When PWN was inoculated into flagellin solution and cultured for 5 days, results showed that amount of flagellin decreased, no degradation was found, but number of PWN increased obviously, which suggested that PWN could use flagellin as food to grow and reproduce. The above research illustrated that flagellin could promote propagation of PWN not only by inducing pine cells death, but also acting as nutrition of this pathogen, which probably plays an important role in pine wilt disease.To further investigate the function mechanism of flagellin on pine cells, gene encoding flagellin of P. fluorescens GcM5-lA was cloned and sequenced. The open reading frame (ORF) in this gene was 1659bp in length encoding 552 amino acid residues. The ORF was amplified by PCR and cloned into pET-15b to construct expression plasmid pET-15B-fla, and then this plasmid was introduced into E.coli BL21(DE3) to construct engineering bacterium. Over-expression of recombinant flagellin was achieved with IPTG induction in engineering bacteria. Recombinant flagellin was partially purified by affinity chromatography. Finally, cell immunofluorescence assay indicated that there existed a direct interaction between pine cells and flagellin, which suggested that there was a probably receptor of flagellin on cell membrane involving in atypical apoptosis. The total proteins of cell membrane were extracted from Japanese black pine in order to purify the possible receptor using recombinant flagellin by affinity chromatography.