Cloning And Functional Analysis Of Transient Receptor Potential (TRP) Channels Genes In Nilaparvata Lugens

Nilaparvata lugens Stal,commonly known as the brown planthopper(BPH),is a hemipteran pest of rice that causes severe crop losses throughout Asia.As a typical monophagous phloem feeder,it not only directly damages the rice plants,often leading to plant wilting and subsequent death known as‘hopper burn’,but also acts as a carrier for the grassy stunt and ragged stunt viruses.Screening and breeding of cultivars that harbor planthopper resistance genes(Bph/bph)are considered to be the most desirable and economic strategy for the control of BPH.However,many improved cultivars carrying these genes have lost their resistances to BPH due to the evolution of new biotypes(populations).Bph3,with its dominant gene of plasma-membrane-localized lectin receptor kinases,OsLecRKs,has been widely applied in breeding of rice cultivars.After confined on IR56 rice variety(a BPH-resistant rice variety containing Bph3)over 40 generations,IR56-BPH strain obtained resistance to IR56.However,the molecular mechanism underlying the interaction between IR56-BPH and its host plant has not well been elucidated.In the current work,the transcriptome datasets of two BPH populations(an avirulent TN1-BPH,which is incapable of breaking down the resistance of the rice varieties containing Bph genes,and a virulent IR56-BPH,which has the capacity to break down Bph3-mediated resistance)feeding on IR56 were obtained.The genes of transient receptor potential(TRP)channel family which were differently expressed between the two strains were functionally analyzed by RNA interference(RNAi).The main findings in the study are briefly listed in the following.1.Transcriptome survey of two populations of N.lugensThe transcriptome datasets of two populations TN1-BPH and IR56-BPH which were feeding on TN1 and IR56 rice varieties were obtained by using RNA-seq.After filtering low-quality reads,the average numbers of clean reads were 46.44 and 48.13 million in the sequencing libraries of TN1-BPH and IR56-BPH,respectively.In total,215 genes were differentially expressed between IR56-BPH and TN1-BPH.Out of these genes,135 genes were significantly up-regulated,whereas 77 genes were down-regulated in the IR56-BPH,compared to those in the TN1-BPH.According to the gene ontology(GO)of DEG,twelve GO categories that grouped into three ontologies(biological process,molecular function,and cellular component)were characterized.Among them,peptidase activity,RNA binding,nucleic acid binding,cellular component biogenesis,and cellular component assembly were significantly enriched.The KEGG enrichment analysis showed that chaperones and folding catalysts,protein processing in endoplasmic reticulum,folding and sorting and degradation,human immunodeficiency virus 1 infection,estrogen signaling pathway,immune system were significantly enriched.These findings provided fundamental data for further studying the involved DEGs in virulence BPH.2.Cloning and functional characterization of TRP genesAccording to the transcriptome and DEG analysis,six DEGs belong to TRP family.These six TRP members and the other seven members were cloned by RT-PCR,and designed as NlTrp,NlPain,NlTrpl,NlPyrexia,NlNanchuang,NlTrpa1 NlPkd2,NlNompC,NlTrpy,NlTrpm,NlInactive,NlWtrw,NITRPML,respectively.The phylogenetic tree showed that thirteen groups were formed with N1TRPs and orthologues of insects.The transmembrane domain perdition revealed that 9 members of NITRPs have 2-6 transmembrane domains,respectively,whereas no transmembrane domain was found in four individuals(NlPkd2,NlTrpy,NlInactive,NlTRPML).Beside,ankyrin repeats(1-13)were found in all NlTRPs,except for NlTrpm and NITRPML.The qRT-PCR results demonstrated that NlTrp,NlTrplm and NlPyrexia had higher expression levels in TN1-BPHs,but NlPain had lower expression level,compared with those in IR56-BPHs.RNA interference-aided knockdown by injection of dsRNA derived respectively from several TRP genes(dsPain,dsNanchuang,dsTrpal,dsNompC,dsTrpy,dsWtrw,and dsTRPML)significantly decreased the survival rate of BPH.Further results showed that dsNompC suppressed the expression of NlNompC in both TNl-BPHs and IR56-BPHs.We found that dsNompC injection reduced the survival rates of TNl-BPHs feeding on both TN1 and IR56 rice varieties.The survival rates of BPHs feeding on TN1 rice was decreased more obviously than those of BPHs on IR56.We hypothesize that NlNompC is involved in the adoption of TN1-BPH to resistant rice IR56,to some extent.