The Pathogenesis Of Molecular Mimicry Proteins In Bursaphelenchus Xylophilus

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide;however,the underlying mechanism leading to pathogenesis remains unclear.Terpenoid metabolism changes and cavitation of pines are the typical pathological features observed during the development of pine wilt disease.Pathogens may secrete proteins that are structurally or functionally similar to host defense-related proteins,which are called molecular mimicry proteins.The molecular mimicry proteins of B.xylophilus may interfere with the normal defense metabolism and lead to the metabolic disorder in host plants.Therefore,we carried out some experiments to investigate the function of pine wood nematode molecular mimicry proteins in its pathogenic process.The main results are as follows:In this study,we identified molecular mimicry proteins based on comparative B.xylophilus transcriptomics data.The phylogenetic analysis revealed that there was a higher sequence similarity between the Bx-TLP-1,Bx-TLP-2 and Bx-CPI protein sequence and the plant protein sequences,which indicated that they may have similar functions.Meanwhile,the expression of Bx-tlp-1 gene was upregulate after ?-pinene treatment.The results of current study revealed there were two peaks of monoterpene level at days15 and 27,or that of sesquiterpene level at days 18 and 27 in Pinus massoniana seedlings during B.xylophilus infections.Besides,the monoterpene synthase genes increased on days 12 and 24,or the sesquiterpene synthase genes increased on days 15 and 24.The results of the current study revealed high Pm-tlp expression levels at days 3,12 and 27,whereas Bx-tlp-1was highly expressed on days 6,15 and 30.Additionally,the Pm-cpi expression level was relatively high on days 6,15 and 27.In contrast,Bx-cpi was highly expressed on days 9,18 and 24.The defense response of pine was induced by B.xylophilus infections,and the expression of terpene genes was upregulated.Besides,the gene expressions of molecular mimicry proteins were upregulate after terpenes accumulation.It may promote the upregulation of DXS,DXR,MCT,CMK and HDS genes in the terpene synthesis pathway(DXP).Terpenes formed secondary accumulation causing the cavitation of pines.RNA interference and prokaryotic expression were used to investigate the function of Bx-tlp-1 gene in B.xylophilus.A q RT-PCR assay indicated that the effects of Bx-tlp-1 gene RNAi were maintained in the F3 offspring.After RNAi of the Bx-tlp-1 gene,the adaptability of B.xylophilus decreased in the environment,resulting in the reduction of B.xylophilus reproduction.On the other hand,there was ?-1,3-glucanase activity in Bx-TLP-1 protein by the prokaryotic expression.Thaumatin-like proteins exhibit ?-1,3-glucanase activity and may bind to ?-1,3-glucans in plants,which may mediate the plant defense response.It was found that there was a peak of terpenes level at days 18 and that of limonene level at days 12 after pine trees inoculated with B.xylophilus treated with Bx-tlp-1 ds RNA.At the same time,there was no secondary accumulation of terpenes in P.massoniana seedlings during B.xylophilus infections.The secondary accumulation of terpenes in pine trees cannot be promoted within 30 days,thereby reducing the pathogenicity of B.xylophilus and prolonging the disease course of pine trees.The results showed that there was a peak of terpenes level at days 12 and that of limonene level at days 9 after pine trees inoculated with Bx-TLP-1 protein of B.xylophilus.They were upregulated for DXS,DXR,MCT,CMK and HDS genes in the terpene synthesis pathway(DXP)at days 9.At the same time,the Pm-tlp gene was also upregulated at days 6 after Bx-TLP-1protein inoculation.It showed that the Bx-TLP-1 protein of B.xylophilus may induce the upregulated expression of related enzyme genes in the terpene synthesis pathway of P.massoniana,resulting in the accumulation of terpenes.Besides,we designed the PCR primers of Bx-tlp-1,Bx-tlp-2 and Bx-cpi genes to specifically amplify the DNA of B.xylophilus and Monochamus alternatus tissue.It was found that the sizes of fragments were 755 bp,241 bp and 202 bp,respectively.The results showed that the Bx-tlp-1,Bx-tlp-2 and Bx-cpi genes can be used not only to specifically detect B.xylophilus,but also to detect B.xylophilus from M.alternatus in the field.In conclusion,the molecular mimicry protein of B.xylophilus may participate in and regulate the terpene metabolism process of P.massoniana,and the Bx-tlp-1 gene is one of the key pathogenic genes of B.xylophilus.The defense response of pine was induced by B.xylophilus infections,and the expression of terpene genes was upregulated.And the expression of Bx-tlp-1 gene was upregulated after terpenes accumulation.Due to the functional similarity between Bx-TLP-1 of B.xylophilus and Pm-TLP of P.massoniana,Bx-TLP-1 may play a similar role to Pm-TLP in P.massoniana.And it may promote the upregulation of related enzyme genes in the terpene synthesis pathway(DXP).Terpenes formed secondary accumulation causing the cavitation,which led to wilt and death of pines.