Study On The Molecular Role Of Bxtlp1,the Key Pathogenic Factor Of Bursaphelenchus Xylophilus

As a worldwide quarantine pest,the pine wood nematode(Bursaphelenchus xylophilus)seriously damages Pinaceae plants.It was first found in Nanjing’s Zhongshan Mausoleum in China in 1982 and has had a huge impact on China’s forest ecosystem,causing immeasurable economic losses.According to the latest announcement from the National Forestry and Grassland Administration,the disease has now occurred in 19 provinces,municipalities,and autonomous regions in China.With the continuous evolution of the pine wood nematode,the trend of "spreading northward and westward" is becoming more serious.Therefore,research on the pathogenic mechanism of pine wood nematode disease is urgent.Considering the complex disease system of pine wood nematode disease and the involvement of human factors,the pathogenic mechanism of pine wood nematode has been controversial for many years.According to the previous experimental results of the laboratory,a sweet like protein "Bxtlp1" closely related to the pathogenesis of B.xylophilus will be secreted during the pathogenesis of B.xylophilus.The interacting proteins with the host Pinus massoniana will be screened by immunohistochemistry and yeast two hybrid,in order to analyze the molecular mechanism of bxtlp1 in the pathogenesis of B.xylophilus from the molecular and cellular levels;The changes of microbial community,the screening of key metabolites and the transcription of the whole genome of P.massoniana after the invasion of B.xylophilus were studied by microbiomics,metabolomics and transcriptomics.The main results are as follows:(1)Based on the analysis of the secreted protein components of B.xylophilus and preliminary research,important secreted proteins TLPs in the process of pine wood nematodeinduced disease in pine trees were screened.q RT-PCR quantification results showed that the expression levels of the Bxtlp1,Bxtlp7,and Bxtlp8 genes were significantly different in different developmental stages of the nematode.The expression level of Bxtlp1 peaked in second instar larvae(L2),while the expression levels of Bxtlp7 and Bxtlp8 peaked in third instar larvae(L3).The pine wood nematode BxTLP1 protein expressed through prokaryotic expression system did not have β-1,3-glucanase activity,while BxTLP7 and BxTLP8 proteins did have β-1,3-glucanase activity.By adjusting and optimizing the nematode interference system,the double-stranded RNA interference method was used to silence the Bxtlp1,Bxtlp7,and Bxtlp8 genes in B.xylophilus.The results showed that the best ds RNA interference primers were ds TLP1-2,ds TLP7-3,and ds TLP8-1;the optimal interference time was 36 h;the developmental stage of the nematode did not significantly affect the interference efficiency;silencing of the Bxtlp1 and Bxtlp7 genes reduced the feeding and reproductive rates of the B.xylophilus,while silencing of the Bxtlp8 gene did not affect the reproduction and feeding of the nematode.Transient expression of Bxtlp1,Bxtlp7,and Bxtlp8 in Nicotiana benthamiana showed that Bxtlp1 was located in the cell membrane and nucleus,while Bxtlp7 and Bxtlp8 were only observed in the nucleus.In the infection process of B.xylophilus,the localization suggested that Bxtlp1 had more potential to interact with P.massoniana.Immunohistochemical experiments showed that the B.xylophilus Bxtlp1 was localized in the esophageal gland and intestine of the nematode,indicating that the BxTLP1 protein can be secreted outside the B.xylophilus.(2)Through analysis of transcriptomic data from P.massoniana,we identified the most central gene module "MEmagenta" that responds to pine wood nematode in early-stage Masson pine.The genes in this module were highly enriched in oxidative phosphorylation,amino sugar and nucleotide sugar metabolism,and plant MAPK signaling pathway.(3)Using a yeast two-hybrid membrane protein system,BxTLP1 target interacting proteins were screened from a B.xylophilus yeast library that infects P.massoniana.Selfactivation assays confirmed that BxTLP1 has no self-activation phenomenon.Eight positive clones were selected from 30 candidate target genes for one-on-one validation in yeast,and the interaction results were confirmed in yeast.Co-immunoprecipitation(Co-IP)experiments revealed that only four target genes,p MS-3(containing RPN7/PCI domain of 26 S proteasome),p MS-6(containing Ribosomal L40 e superfamily protein),p MS-7(Ubiquitin-like ubiquitin protease),and p MS-26(superoxide dismutase),could interact with BxTLP1 among the eight positive clones.(4)LEf Se(Linear discriminant analysis Effect Size)was used to compare and analyze healthy and wilted pine trees to determine the major differences in microbial communities between the two.The results showed that Actinobacteria and Firmicutes were the differential marker communities in the bacterial community of wilted pine trees,while Rhizobiales in the Proteobacteria phylum was the main marker in the bacterial community of healthy pine trees(Fig.2-3B).At the same time,the differential markers in the fungal community of healthy pine trees were Malasseziales,Tremellales,Sordariales,and Fusarium,while Pleosporaceae was the key marker in the fungal community of wilted pine trees.(5)LC-MS/MS-based metabolomics was used to investigate the differences in host metabolites during the pathogenesis of pine wood nematode.A total of 307 different metabolites were identified,including 246 significantly up-regulated metabolites and 61 significantly down-regulated metabolites.KEGG enrichment analysis and differential metabolite annotation analysis showed that polyethylene metabolites accumulated significantly in withered pine trees.After analyzing the relative contents,a total of 12 core metabolites were identified,including 10 flavonoid metabolites and 2 aromatic polyketone compound metabolites.In conclusion,four target proteins were screened by establishing a yeast two hybrid library of the interaction between P.massoniana and B.xylophilus.It is speculated that Bxtlp1,the key pathogenic factor of B.xylophilus,may affect the host immune defense process by mediating ubiquitin regulation,oxidative stress,MAPK molecular signal transduction pathway,thereby aggravating the disease;The effect of B.xylophilus invasion on the changes of microbial community may lead to the enrichment of the community secreting toxins,which may cause damage to the host cells;The invasion of B.xylophilus caused a significant accumulation of polyethylene metabolites in the host,mainly flavonoids metabolites.The excessive accumulation of these substances may be toxic to the host cells,thus affecting the host defense response.These works help to deepen the research on the pathogenesis of B.xylophilus,and provide a theoretical basis for the production of prevention and control technology of B.xylophilus.