Relationship Between Sphingolipid-metabolizing Enzymes And The Infection And Replication Of Rice Stripe Virus In Small Brown Planthopper(Laodelphax Striatellus Fallén)

Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown.To date,numerous researches have elucidated the interactions between virus infection and sphingolipids in mammals,especially in human cells.However,little has been understood on sphingolipid in insects,especially less in virus infected insects.Rice stripe virus(RSV),which is mainly transmitted by the Laodelphax striatellus(small brown planthopper),is one of the most economically devastating rice diseases that mainly occur in temperate and subtropical areas.In this dissertation,the content of sphingolipid species and the mRNA levels of sphingolipid enzymes,the characteristic of LsSPL and LsSK,the effect of these genes in RSV infection and replication were studied,the results were listed as follows:1)We report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper(Laodelphax striatellus Fallen).According to protein sequence similarity,we identified 12 sphingolipid enzyme genes in L.striatellus.By comparing their mRNA levels in viruliferous versus nonviruliferous L.striatellus at different life stages by qPCR,we found that RSV infection upregulated six genes(LsCGTI,LsNAGA1,LsSGPP,LsSMPD4,LsSMS and LsSPT)in L.striatellus at most stages andfour genes(LsCGTl,LsSMPD2,LsNAGA1 and LsSMS)and three genes(LsNAGA1,LsSGPP and LsSMS)significantly upregulated in third-instar and fourth-instar nymphs,respectively.HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides,such as Cer18:0,Cer20:0 and Cer22:0 species,in third and fourth instar L.striatellus nymphs.Together,these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L.striatellus,indicating thatsphingolipids may be important for RSV infection or replication in L.striatellus.2)To understand the characteristic of sphingosine kinase and the effect of this gene on the response to insecticides in rice stripe virus(RSV)-infected Laodelphax striatellus Fallen(small brown planthopper,SBPH)and virus-free SBPH.We cloned a LsSK sequence from L.striatellus using PCR.qRT-PCR was employed to analyze the expression pattern of LsSK gene in different stages and various organs of the two above mentioned SBPH colonies,as well as the LsSK mRNA level in SBPH after exposure to three insecticides,including deltamethrin,buprofezin and imidacloprid.After knockdown LsSK by using dsRNA injection,SBPH was exposed to 50%lethal concentration of three insecticides and the mortality rate was recorded.We cloned a SK sequence from L.striatellus and its length is 1282 bp(GenBank accession no.KT989975).According to the phylogenetic tree analysis,LsSK of L.striatellus was clustered together with other hemipteran insects and had great distances from insects of other orders,indicating LsSK is very conserved.qRT-PCR results showed SK was highly expressed in the 3 rd,4 th instar nymphs and adults of RSV infected L.striatellus,especially higher in the 4 th instar viruliferous nymphs.Interestingly,LsSK gene showed extremely high expression in the tissues of viruliferous males than viruliferous females and RSV-free adults.After the treatment of exposure to lethal concentrations of three pesticides,LsSK mRNA level of two SBPH colonies was increased,especially significant to buprofezin but without great significance to deltamethrin.The response of LsSK expression to buprofezin was relatively faster than the other insecticides.Injection of L.striatellus with dsRNA against LsSK reduced the mRNA level of LsSK greatly,and silencing of LsSK led to increased susceptibility in virus-infected and virus-free SBPH to these three insecticides.This study firstly identified and cloned the LsSK gene in L.striatellus,and found significantly high expression in the 3th and 4th instar of virus-infected SBPH.Silencing the LsSK resulted to increased susceptibility in SBPH to three insecticides,indicating LsSK plays a role in facilitating SBPH to response to insecticidal stress.3)The full-length cDNA encoding sphingosine-1-phosphate lyase(named as LsSPL)was cloned from L.striatellus.LsSPL consists of 1647 nucleotides that encoded a protein of 549 amino acids.The results of alignment of LsSPL from other insects showed,LsSPL contained a transmembrane region and a conserved pyridoxal-dependent decarboxylase domain.The analysis of qPCR showed,the relative expression levels of LsSPL in RSV-free 1st instar and 4th instar were significantly higher than in other stages.The expression level were significantly higher in 3rd and 4th instar nymphs,especially higher in the 3rd nymphs(18 folds).Furthermore,the mRNA levels of LsSPL were higher in the 3rd,4th and 5th instar viruliferous nymphs than in the same stages of viruliferous L.striatellus.These results suggest that RSV infection may induce the expression of LsSPL in 3rd,4th and 5th instar viruliferous nymphs.According to the expression profiles of LsSPL in different life stages of two colonies,the level was highest in salivary of males and its higher than in females of both two colonies.The expression level was higher in spermary than in ovary.Moreover,the transcript level of RSV-special coat protein was higher in 3rd nymphs,followed by 2nd and 4th nymphs.And the level was higher in male than female.Although the transmission time was not affected after injection of dsRNA,the transmission rate was significantly lower in dsSK group.After feeding dsRNA-injected L.striatellus with viruliferous rice,the time of acquiring virus was not altered while the viruliferous L.striatellus was significantly higher in dsSPL group and significantly lower in dsSK group.Together,all results showed LsSPL and LsSK may play an important role in virus infection and replication.