Anabolism Of Homoterpenes Involved In Indirect Defense Of Gossipium Hirsutum

Cotton is a worldwide economic crop.Its production is significantly affected by herbivore infestation.Chemical communication among plant-herbivore-carnivore plays a key role in pest control,and semiochemicals involved in tritrophhic interactions can be employed to make appropriate plant protection strategies.In this study,we are focusing on(E)-4,8-dimethylnona-l,3,7-nonatriene(DMNT)and(E,E)-4,8,12-trimethyltrideca-l,3,7,11-tetraene(TMTT)which confer indirect defence to plants.Two CYP genes regulated DMNT and TMTT biosynthesis were identified.And then,combined with GhTPSs which are responsible to(E)-nerolidol and(E,E)-geranyllinalool formation in vitro,co-expression of GhTPSs and GhCYP involved in homoterpenes biosynthesis were investigated,which laid a foundation to study homoterpenes over-emitted cotton cultivar and its possible application in agricultural pest control.1.Influence of DMNT and TMTT on behavioral preference of parasitic wasps,natural enemies of cotton major pests,in the laboratory.Firstly,chewing caterpillars Helicoverpa armigera-infested or sucking bugs Apolygus lucor?m-infested cotton plant volatiles were collected and analysed,both DMNT and TMTT were induced by herbivore damage.Secondly,electroantennogram recordings showed that electroantennal responses of Microplitis mediator and Peristenus spretus to DMNT and TMTT were both dose dependent.Laboratory behavioural bioassays suggested that females of M.mediator responded positively to DMNT and TMTT,and females of P.spretus showed preferrence to TMTT.These results indicated that DMNT and TMTT play an important role in cotton indirect defense.2.Analyzation of all P450 gene families in Gossipium hirsutum.According to the genome data of G.hirsutum,G.raimonddi,G.arboreum as well as P450 s of G.raimondii and Arabidopsis thaliana published on P450 Homepage,all the P450 s identified from G.hirsutum genome data were Blast,and then designated according to nomenclature of P450.In total,565 P450 s were identified,and classified to 9 CYP clans,42 CYP families,except two sequences with identity lower than 40%.P450 s in G.hirsutum and A.thaliana were both belong to nine CYP clans,and CYP702,CYP705,CYP708,CYP709,CYP720 and CYP721 families are excusively in A.thaliana,while CYP736 and CYP749 are only belongs to Gossipium.CYP71 clan which responds to stress has 298 family members,more than half of total GhCYPs.Secondary structure was analyzed to CYP82 family belonged to CYP71 clan.Phylogenetic tree was constructed between GhCYP82 members and CYP82 with reported functions from other plants,which provide supports to the functional analysis of GhCYP82 members.3.Identification of P450 s involved in homoterpenes biosynthesis.According to genome data,A.lucor?m-infested transcriptome data of G.hirsutum and AtCYP82G1 which are reported to regulate TMTT synthesis in A.thaliana,eight candidate genes were filtered.Yeast recombinant expression and enzyme assays indicated that the two GhCYP82 Ls are both responsible for the conversion of(E)-nerolidol to DMNT and(E,E)-geranyllinalool to TMTT.The two heterologously expressed proteins without cytochrome P450 reductase fail to convert the substrates to homoterpenes.Quantitative real-time PCR(qPCR)analysis suggested that expression of the two GhCYP82 L genes were significantly up-regulated in leaves and stems of G.hirsutum after herbivore attack.Transgenetic tobacco plants containing GhCYP82L1 or GhCYP82L2 were generated by genetically modification.However,there was no DMNT or TMTT emission neither from plants containing GhCYP82L1 nor GhCYP82L2.When the tobacco plants were exposed to the environment with Nerolidol,DMNT was detected.But TMTT was not collected from plants in the Geranyllinalool environment.We presumed that transgenetic plants cannot produce homoterpenes due to lack of substrates of GhCYP82 Ls,and further investigations need to be performed.When GhCYP82L1 or GhCYP82L2 gene was scilenced by virus induced gene scilencing,the gene expression level of the gene scilenced palnt was decreased more than half of the TRV2-treated control,and the peak area of DMNT in chromatogram was also less than the control,which suggested that the emission amount of DMNT was influnced significantly after scliencing the target CYP genes.4.Co-expression of GhTPS and GhCYP involved in homoterpenes biosynthesis.Firstly,gene expression profiles of GhTPS22,GhTPS23 and GhTPS24,which are responsible for(E)-nerolidol and(E,E)-geranyllinalool formation,as well as GhCYP82L1 and GhCYP82L2 were analyzed by qRT-PCR.These genes were both upregulated in acrial parts of the herbivore-damaged plants,but not in roots.Secondly,genes of GhTPS22 and GhCYP82L1,GhTPS22 and GhCYP82L2,GhTPS23 and GhCYP82L1,GhTPS23 and GhCYP82L2,GhTPS24 and GhCYP82L1,GhTPS24 and GhCYP82L2 were co-expressed in yeast cells,and enzymatic assays showed that GhTPS22 + GhCYP82L1,GhTPS22 + GhCYP82L2,GhTPS23 + GhCYP82L1,GhTPS23 + GhCYP82L2,GhTPS24 + GhCYP82L1 and GhTPS24 + GhCYP82L2 were able to catalyze FPP or GGPP to produce DMNT.Thirdly,genes of GhTPS22,GhTPS23,GhTPS24,GhCYP82L1 and GhCYP82L2 were constructed to pYLTAC380 H vector,and then transformed to tobacco plants.Plants with gene GhCYPL1,GhCYPL2,GhTPS22,GhTPS23,GhTPS24,GhCYPL1 + GhTPS22,GhCYPL2 + GhTPS22,GhCYPL1 + GhTPS23,GhTPS23 + GhCYPL2,GhCYPL1 + GhTPS24 and GhCYPL2 + GhTPS24 were generated,and plants expressing pYLTAC380 H were taken as control.qPCR analysis suggested that target genes were highly expressed in tobacco plants.GC-MS analysis showed that tobacco plants containing GhTPS22 could emit(E)-nerolidol,tobacco plants containing GhTPS22 + GhCYP82L1 as well as GhTPS22 + GhCYP82L2 were able to produce DMNT,tobacco plants containing GhTPS24 could synthesize linalool.Volatiles from tobacco plants with GhTPS23,GhCYPL1 + GhTPS23 or GhTPS23 + GhCYPL2 needed to be analyzed further.These results revealed that DMNT emitted in tobacco plants was regulated by GhTPS22 and GhCYP82 Ls,which lay the foundation for development cotton cultivar with high DMNT emission.