| Pine wilt disease,caused by pinewood nematode Bursaphelenchus xylophilus(PWN),is an important destructive disease of pine forests in China,threatening forest health and ecological security.cry5Ba3,a toxin-encoding gene cloned from Bacillus thuringiensis zjfc85,was further modified as cry5Ba3Φ according to codon preference in fungi.Cry5Ba3Φ can be successfully expressed in fungus Botrytis cinerea,showing strong nematicidal activity to B.xylophilus.In this study,in order to ascertain the relationship between Cry5Ba3Φ structural integrity and toxicity levels of this protein,six cry5Ba3Φdomain swapping mutants of B.cinerea strains were constructed and their nematicidal activities to PWN were tested;to explore whether toxin adaptability to Cry5Ba3Φ could be enhanced after long-term contact of the protein and to provide potential mechanisms underlying this phenotype,population dynamics of PWN feeding on either wild type or Cry5Ba3Φ transformant strains of B.cinerea were observed across time course and contact frequency and the corresponding community structures of bacterial microbiota associated with PWN were determined.The results are as follows:1.Based on prediction of Cry5Ba3Φ structural domains by bioinformatic analysis,five amino acid loci were selected for truncation,resulting in six different lengths of amino acid sequences of Cry5Ba3Φ,while the putative core toxic fragment of the protein was kept.These fragments were uploaded onto vector p TFCM using Gibson assembly technology,and then were integrated into B.cinerea genome via Agrobacterium tumefaciens-mediated transformation.Nematicidal activities of the six B.cinerea strains to PWN were tested.The biological assay results showed that truncating N-or C-terminal of Cry5Ba3Φ leads to different levels of toxicity to pine wood nematode.2.Compared to the wild-type strain,B.cinerea with cry5Ba3Φ gene greatly controlled the reproduction of B.Xylophilus,however,PWN populations were found to recover gradually.The significant difference in population density between PWN on wild-type and cry5Ba3Φ B.cinerea strains disappeared after 216 h.Likewise,the dead to live nematode ratio for PWN on the two B.cinerea strains was significantly different only during the first contact.Community compositions of bacterial microbiota associated with PWN were determined by DGGE(Denatured Gradient Gel Electrophoresis).Results showed that changes in community structures of bacterial microbiota(either external environment or whole nematode body)couple with PWN adapatability to Cry5Ba3Φ.This study preliminarily explored the effects of Cry5Ba3Φ structural integrity on nematicidal activity and the potential correlation between bacterial community structures and B.xylophilus adaptability to Cry5Ba3Φ protein.These findings may provide guidance for further defining roles of bacterial associates of pinewood nematode in its adaptability to Bt crystal proteins and the potential interacting patterns for bacteria of B.xylophilus with the toxic proteins. |
