Molecular Characterization And Functional Analysis Of A Serine Protease From The Nematophagous Fungus Esteya Vermicola

Esteya vermicola was the first reported nematophagous fungus with highly virulence against Bursaphelenchus xylophilus and has the potential as a biocontrol agent.In this study,a serine protease gene Evsp(GenBank accession No.KP698922)from E.vermicola strain NKF 13222 was firstly cloned using homologous cloning and rapid amplification of cDNA ends(RACE).The full-length cDNA of Evsp contains 2280 nucleotides with a 1656 bp ORF encoding a protein with 551 amino acids.The deduced amino acids sequences of Evsp gene showed highly homology with the catalytic domains in subtilisin serine proteases.The genomic Evsp includes two exons(396 bp and 1260 bp)divided by an intron(207 bp),which began with GT and ended with AG.Southern blot analysis revealed that only a copy of Evsp gene in the fungal genome.Phylogenetic analyses based on the protein sequences revealed that E.vermicola is closed to endoparasitic fungi,opportunistic fungi,toxin-producing fungi and entomopathogenic fungi,but separated from nematode-trapping fungi.The serine protease Evsp was expressed using a prokaryotic expression system.The pET28a(+)expression vector was constructed on basis of cDNA sequences of Evsp gene.The vector was transformed into cells of Escherichia coli BL21(DE3)and the expression of Evsp was induced by IPTG.The SDS-PAGE analysis showed that the recombinant protein expressed in E.coli was in the form of insoluble inclusion body.The actively refolded rEvsp was obtained after the inclusion bodies were washed,solubilised and purified.The enzyme activity of rEvsp was detected by Folin phenol method which indicated the casein was enzymatic hydrolysized.The nematicidal activities of rEvsp against Bursaphelenchus xylophilus revealed that the cuticle of nematode was degraded,which suggested it may play an important role in infection process of Esteya vermicola on nematodes.The homologous recombination fragment of 3.6 kb for Evsp gene was constructed by overlapping extension PCR and successfully transferred into cells of E.vermicola by PEG-mediated protoplast transformation.A mutant strain ES84 was confirmed with the deletion of Evsp gene by PCR detection and Southern blot.The attachment and infection to Bursaphelenchus xylophilus by the lunate conidia of mutant strain ES84 and wild strain NKF 13222 of E.vermicola were compared.There were no significant differences in attachment and infection rates between two strains.The study provided not only the molecular evidences for supporting the infection of E.vermicola against B.xylophilus,but also the basis for further functional study of serine protease Evsp.