Functional Characterization Of Key Genes For Terpene Biosynthesis Involved In Defense To Pine Wood Nematode In Pinus Massoniana

Pinus massoniana,a pioneer tree species with drought and barren tolerance in afforestation of barren mountains in southern China,grows fast and has strong adaptability to environment,which is widely used in industries such as building materials,pulp and paper,and oleoresin extraction,occupying an extremely vital position in forest base construction.Pine wood nematode disease(PWD)is a kind of extremely devastating forest disaster caused by PWN,propagated by Monochamus alternatus.It seriously jeopardized the forest ecological security with huge economic losses to forestry,which is difficult in prevention due to the PWN spreading rapidly.P.massoniana is one of the main damage targets of pine wood nematodes(PWN,Bursaphelenchus xylophilus),which has seriously hindered the development of the masson pine industry nowadays.However,PWN-resistance genotypes exist in P.massoniana population,meaning that replacing susceptible varieties with disease-resistant varieties is one of the most economical and effective ways to control the forest diseases.In the early stage,our research team had bred a batch of resistant P.massoniana families and clones in Anhui,Zhejiang and other places,finding that yield and some chemical component content of resin were significantly related to the resistance of P.massoniana.Transcriptome high-throughput sequencing was performed on the genotypes of resistant and susceptible masson pine,and a batch of candidate genes for resistance to PWN were selected.Based on the previous study,the resistant and susceptible masson pine clones were used as test materials,focusing on the gene cloning,expression pattern detection and functional characterization of key four genes in terpene biosynthesis(resistant terpene genes),including PmGPPS1,PmTPS4,PmTPS21 and PmCYP720B11v2 among candidate genes against PWN,which might lay a theoretical foundation for revealing the resistance mechanism and early selection of resistant varieties from P.massoniana.The main results are as follows:(1)PmGPPS1,PmTPS1,PmTPS4,PmTPS21 and PmCYP720B11v2 are differentially expressed in the transcriptomes of resistant and susceptible P.massoniana,the gene families of which related to terpenoid synthesis among the candidate genes for resistance to PWN were comprehensively identified based on the full-length transcriptome of masson pine.11 members of Pm IPS gene family,54 members of PmTPS gene family and 14 PmCYP720 B members of gene family were identified in the full-length transcriptome of P.massoniana.Pm IPS gene family members were classified into 3 clades including IPS-a,IPS-b and IPS-d and had 20 motifs by constructing a phylogenetic tree,among which PmGPPS and Pm GGPPS family members shared more conserved motifs.PmGPPS1,the disease-resistance candidate gene,was located in the IPS-b subfamily and may participate in GPP synthesis.In the PmTPS gene family,mono-TPS,sesqui-TPS and di-TPS members were located in the 3 subfamilies TPS-d1,TPS-d2 and TPS-d3,respectively.PmTPS1 and PmTPS4 in disease-resistance candidate genes belonged to TPS-d1 and may be involved in monoterpene synthesis.PmTPS21 were classified in TPS-d2 and may participate in the sesquiterpenes synthesis.PmCYP720 B gene family members were divided into 4 clades,in which clades ? and ? covered more PmCYP720 B members.Candidate PmCYP720B11v2 was located in clade ? and may participate in multiple DRAs synthesis.The conserved motifs of different subfamily members in the IPS,TPS and CYP720 B gene families of P.massoniana were highly conserved,and each subfamily also contained its own specific conserved motifs.Members with close evolutionary relationships had similarly conserved motifs.(2)The key PWN-resistance genes for terpenoid synthesis was highly expressed specifically in the resin duct epithelial cells in resistant P.massoniana.Based on the analysis of the expression patterns of the candidate PWN-resistance genes under the stress of PWN invasion,it was found that the expression levels of PmGPPS1,Pm Pin S,PmTPS4,PmTPS21 and PmCYP720B11v2 from resistant P.massoniana were significantly higher than the susceptible ones at 1 d,15 d and 30 d after inoculation.The analysis of the expression patterns of the above genes in different tissues showed that PmGPPS1,PmTPS4,PmTPS21 and PmCYP720B11v2 were significantly upregulated in the stem tissues of resistant P.massoniana after PWN inoculation.Specific antibodies of PmGPPS1,PmTPS4,PmTPS21 and PmCYP720B11v2 were hybridized with paraffin sections of stem tissues from resistant P.massoniana with the method of immunohistochemistry,so as to analyze the tissue location of candidate genes in resistant P.massoniana at the protein level.The results showed that the specific antibodies of candidate genes had specific-expression signals in the xylem,cambium and phloem of resistant P.massoniana,especially in the resin ducts epithelial cells of the xylem.(3)PmGPPS1,a candidate gene in P.massoniana for resistance to PWN,promoted the accumulation of monoterpenoids and provided synthetic precursors for oleoresin to participate in stress of PWN invasion.Compared with the wild type,the expression level of PmGPPS1 in tobacco transgenic lines was significantly up-regulated.The overexpression of PmGPPS1 could effectively promote the expression of genes in the MVA and MEP pathways.Combined with the key genes of DXS and DXR in MEP pathway and the key genes of HMGS and HMGR in MVA pathway,the expression level results showed that expression levels of Nb HMGS,Nb HMGR,Nb DXS,Nb DXR and Nb GGPPS3 increased by 2.4,2.8,8.4,2.4 and 4.8 times,respectively in the PmGPPS-overexpression tobaccos,which revealed the overexpression of PmGPPS1 could activate the metabolic flux of MEP and MVA pathways through a feedback regulation mechanism,thereby significantly increased the production of D-limonene and eucalyptol in transgenic tobacco plants.D-limonene and eucalyptol could be used as bacteriostatic agents,so it was speculated that PmGPPS1 played a positive regulatory role in the resistance process to PWN.In addition,there were significant phenotypical differences between the transgenic lines and the wild type.At the same time,overexpressing PmGPPS1 gene into tobacco could promote the accumulation of GA3 and ZA content in tobacco plants,enhancing tobacco growth and development,early flowering,leaf enlargement,flower numbers,and promoting vegetative growth and reproductive growth at the same time.(4)The PWNs were cultured in vitro using the enzymic products of resistant terpene synthase,which confirmed that the enzymic products of PmTPS4 and PmTPS21 could inhibit the activity of PWN.The results of enzyme activity tests in vitro showed that PmTPS4 was a multi-product ?-pinene synthase,which could simultaneously synthesize 4 monoterpenes of?-pinene,?-pinene,?-myrcene and D-limonene.PmTPS21 could synthesize sesquiterpene(longifolene)and monoterpene(?-pinene)at the same time with FPP as the sole substrate,and synthesize monoterpene(?-pinene)with GPP,which was a longifolene synthase with dual functions.The results of tobacco transient transformation were nearly consistent with the results of their enzyme activity.Based on the enzymic products of PmTPS4 and PmTPS21,as well as the composition and content of terpenoids of in P.massoniana oleoresin,we set up the serial dilutions of ?-pinene,?-pinene,?-myrcene,D-limonene,longifolene,as well as their mixture treating on the cultured PWN in vitro respectively so as to detect the effect of terpenes on the PWN.The results showed that in the pure solution of ?-pinene,D-limonene,longifolene and ?-myrcene,the average survival rate of PWN was significantly reduced as the compound concentration and treatment time increased,among which,D-limonene had the most obvious inhibitory effect on PWN.In low-concentration mixed solution,the survival rate of PWN was approximately down to zero after 6 h of treatment.Therefore,it was preliminarily concluded that ?-pinene,?-pinene,?-myrcene,D-limonene and longifolene played a significant role in the defense process against PWN,as the main enzymic products of PmTPS4 and PmTPS21 and the main components of oleoresin.(5)PmCYP720B11v2 could promote the accumulation of diterpene resin acid(DRAs)compounds to improve the resistance of P.massoniana.The prokaryotic expression vector,p ET28a-PmCYP720B11v2,was constructed by homologous recombination,and the recombinant protein obtained in E.coli was expressed in a large amount,with13-hydroxy-8(14)-abietene used as the substrate for enzymatic reaction.The results showed that PmCYP720B11v2 could catalyze the formation of 13-hydroxy-8(14)-abietene to produce four diterpene resin acids: levopimaric acid,abietic acid,and neoabietic acid.According to the result,it was concluded that PmCYP720B11v2 exhibited catalytic activity with biochemical function,which could produce levopimaric acid,abietic acid,and neoabietic acid,belonging to DRAs,which played a vital role in the defense system of conifer species.Therefore,PmCYP720B11v2 may be involved in the defense process of resistant P.massoniana against PWN.