Functional Analysis Of Saliva Proteins Of The Brown Planthopper And The Roles Of The Salivary Gland In The Transmission Of Rice Ragged Stunt Virus

The brown planthopper(BPH,Nilaparvata lugens)is the most destructive piercing-sucking insect and transmitted rice ragged stunt virus(RRSV)through feeding on rice plant.During feeding,the insect secreted two types of saliva(gel and watery saliva),which played important roles in insect and host plant interaction.Moreover,hemipterous pests were important vectors for viral transmission.As the last tissue for viral circulation,the mechanism for virus replication and release from the salivary gland needs further investigation.In this study,the salivary glands and saliva proteins were studied through genomic,transcriptomic,and proteomic approach.The possible role of salivary gland in RRSV profication and transmission was also investigated.The main results were as follow:(1)Using illumina sequencing technology,we generated three salivary gland transcriptomes of BPH,SBPH,and WBPH,respectively.The majority of salivary gland genes participated in energy metabolism,protein synthsis,and post translational modification,which related with secretion property of this tissue.Compared with BPH(a monpohagous insect),the SBPH and WBPH(oligophagous insects)possessed more homology genes in salivary glands,which might explained by different feeding habites.The monpohagous pests have more genes,such as OBP?P450?GST;While the number of genes correlated with CSPs was decresed.(2)A total of 50 watery saliva proteins and 16 gel saliva proteins in BPH were identified through shotgun proteomic detection combined with the genomic and transcriptomic analysis.Most of these saliva proteins were specifically expressed in salivary glands and guts.Using RNA interference,we found annexin-like-5,carbonic anhydrase,salivap-3,salivap-4(NIShp)were essential for BPH survival.The annexin-like was significant different from the annexin in insect species,which might be the results of BPH and rice co-evolution.The annexin-like-5 was indispensable for BPH feeding on phloem or xyloem sap.The salivap-3 and salivap-4 were the main component of salivary sheath.Knocking down the expression of salivap-4 resulted in premature death of insect on rice plant,but not on artificial diet.(3)The saliva protein of NlMul was related with host adaption.Altered gene expression was observed when the insect transferred from TN1 to ASD7.Knocking down the expression of NlMul resulted in decreased salivary sheath and the insect exhibited prolonged development duration.The dsNlMul-treated BPH was lethal on ASD7,but not on TN1,indicating that the NIMul involved in host adaption.(4)RRSV induced apoptosis in the salivary gland cells of its insect vector.The occurance of apoptosis and viral infection was closely related.The RRSV-induced apoptosis was regulated through a caspase-dependent manner.Inhibiting the expression of N.lugens caspase-1 genes signifcantly interfered virus transmission.Our findings established a link between virus-asscociated apoptosis and virus transmission.The induction of viral-asscociated apoptosis might be the mechanism for virus released from salivary glands.(5)The interaction between RRSV Pns10 and NIOSCP was identified through yeast two-hybrid system,GST pull-down assay,confocal miscopy,and TEM.The RRSV directly linked with mitochondria through this ineraction.Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects.Our findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane.