Screening And Functional Analysis Of Effectors From Nilaparvata Lugens

The brown planthopper(BPH),Nilaparvata lugens(St?l),is a major rice pest that has co-evolved with rice for approximately 0.25 million years.During feeding on rice,BPH use their stylets to puncture the plant epidermis,followed by penetration through the plant cell to access the phloem sap.During this process,BPH secretes saliva from their salivary glands into plant cels.The saliva of herbivorous insects plays a crucial role in their interaction with plants,and determines the compatibility between feeding insects and host plants.Broadly,salivary proteins and other molecules that can be secreted by insects into plant tissues and can alter host structures and functions are defined as effectors.However,a limited number of BPH effector proteins have been identified to date.We analyzed the salivary gland transcriptomes of the brown planthopper and subsequently established a N.lugens secretome consisting of 1140 protein-encoding genes.Secretome analysis revealed the presence of both conserved and rapidly evolving salivary proteins.A screen for potential effectors that elicit responses in the plant was performed via the transient expression analysis of 64 BPH salivary proteins in N.benthamiana leaves and rice protoplasts.The salivary proteins Nl12,Nl16,Nl28,and Nl43 induced cell death,whereas Nl40 induced chlorosis and Nl32 induced a dwarf phenotype in N.benthamiana,indicating effector properties of these proteins.Tissue expression analysis showed a higher expression level of the six candidate effectors in salivary glands than in other tissues.Subcellular localization and analysis of domain required for cell death showed a diverse structure and function of the six effectors.Nl28,Nl40 and Nl43 are N.lugens-specific,in contrast,Nl12(protein disulfide-isomerase),Nl16(apolipophorin-III),Nl32(chemosensory protein)are conserved among insects.Our results suggest a higher level of effector conservation exist in BPH compared to that in plant pathogens.Nl40 family has numerous isoforms produced by alternative splicing,exemplifying rapid evolution and expansion of effector proteins in the brown planthopper.We analyzed the protein structure,the subcellular localization and the phenotype of NlCSPs(Nilaparvata lugens chemosensory proteins)in N.benthamiana.Transient transformation experiments in N.benthamiana showed that NlCSPs could induce different degrees of dwarf and cell death phenotypes in N.benthamiana.PVX(PotatoVirus X)is a virus that can infect N.benthamiana.Compared with the control,the expression of PVX-CP gene increased significantly in PVX-NlCSP1 transformed N.benthamiana leaves,indicating that NlCSP1 enhanced the accumulation of PVX RNA and the pathogenicity of PVX.The subcellular localization of NlCSPs in plant cels was analysed.Four NlCSPs(NlCSP1,NlCSP4,NlCSP5 and NlCSP9)were mainly localized in the nucleus of N.benthamiana cells,while the remaining seven proteins were localized in the nucleus and cytoplasm.It was found that the basic amino acids(K,R)at either end or in the middle of OS-D domain of NlCSP1,NlCSP4 and NlCSP5 all affect their nuclear localization,while the nuclear localization signal of NlCSP9 is located in the region of 74-130 aa.The frequency of basic amino acids(K,R)in 11 members of the NlCSP family was counted.It was found that the average frequency of basic amino acids in the whole NlCSP family was more than 15%.Therefore,we thought that NlCSP family is rich in basic amino acids,and is easy to form nuclear localization signals.NlCSPs induced dwarf phenotypes in N.benthamiana through OS-D domain,retaining or removing signal peptides didn't affect the dwarf phenotypes of NlCSPs in N.benthamiana.Amino acids deletion at both ends of the OSD domain of NlCSPs made the protein easier to degrade.Therefore,we believed that the regions at both ends of the OS-D domain of NlCSPs affected the stability of the protein.Transient transformation experiments in N.benthamiana showed that 8 CSPs from 6 different orders of insects could induce dwarf phenotypes in N.benthamiana.Prediction of the three-dimensional structure of proteins revealed that CSPs in six different orders of insects had similar three-dimensional structures,all of which were spherical structures consisting of about six alpha helixes.We selected 14 conserved loci among 8 CSPs and carried out mutation experiments on NlCSP1.The results showed that one or two amino acid mutations were unable to affect the function of NlCSP1.NlCSP1 induced dwarf phenotype and enhanced the pathogenicity of PVX in N.benthamiana,which was similar to the viral RNA silencing suppressors.So we tested the ability of NlCSP1 to inhibit GFP silencing in 16 c GFP-transgenic N.benthamiana and found that NlCSP1 could suppress systemic GFP silencing but not local GFP silencing in N.benthamiana.Therefore,NlCSP1 may be an RNA silencing suppressor in insects.Further analysis showed that NlCSP1 could not induce significant changes of RNA silencing related genes(NbDCL2,NbDCL4,NbRDR1,NbRDR6,NbAGO1 and NbAGO4)in the non-injected leaves of N.benthamiana.35S-NlCSP1 and 35S-GFP were expressed in N.benthamiana for 12 days,through small RNA sequencing,106 and 97 differentially expressed miRNAs,314 and 467 differentially expressed miRNAs target genes were identified in injected N.benthamiana leaves and upper non-injected leaves.GO analysis showed that among the differentially expressed miRNAs target genes,the number of target genes located in the nucleus was the largest.KEGG enrichment analysis showed that the number of miRNAs target genes involved in plant hormone signal transduction was the largest.Nuclear localization of target genes and their involvement in hormone signal transduction are all related to the function of NlCSP1 inducing dwarf phenotype in N.benthamiana.We found that NlCSP1 was highly expressed in the salivary gland of the brown planthopper and could be secreted into rice tissues when the brown planthopper fed on rice,and NlCSP1 transgenic rice could promote the feeding of the brown planthopper.Thus we thought that NlCSP1 function as an effector protein in rice.The function of NlCSP1 as an effector needs to be further studied.