Cloning And Function Analysis Of Three Autophagy-related Genes In The Brown Planthopper

The brown planthopper,Nilaparvata lugens（St（?）l）,is one of the most serious rice pests in Asia countries.As the brown planthopper has strong adaptability to traditional chemical insecticides and resistant rice,we urgently need to find new control targets.Autophagy plays an important role in the growth and development of insects.The study of the functions of autophagy-related genes in the brown planthopper is of great significance for the prevention and control of the brown planthopper.In the early stage,by analyzing the transcriptome data,we found that the expression level of the autophagy-related gene ATG1 in brown planthoppers on resistant rice was significantly higher than that in brown planthoppers on susceptible rice,suggesting that autophagy may be involved in the adaptation process of brown planthoppers to resistant rice.In this study,three autophagy-related genes,ATG5,ATG12 and ATG3,were selected for research to evaluate their potential as targets for brown planthopper control.ATG5 and ATG12 are key proteins involved in the elongation of phagophores and are necessary for the formation of autophagosomes.As an E3 enzyme,the ATG12-ATG5 complex determines the location of the autophagy marker protein ATG8 on the autophagy bilayer membrane in Saccharomyces cerevisiae.In addition to playing a role in autophagy,Atg5 and Atg12 proteins also participate in the regulation of apoptosis.In the process of autophagosome formation,Atg3 acts as an E2 enzyme to catalyze the binding of Atg8 and PE.In this study,the full-length cDNAs of NlATG5,NlATG12 and NlATG3 genes of the brown planthopper were cloned,and the spatiotemporal expression patterns were analyzed by RT-qPCR.Using the method of RNAi combined with transcriptome analysis,the target specificity and off-target effects of RNAi of NlATG5 gene were studied.The effects of RNAi of NlATG5,NlATG12 and NlATG3gene on the survival rate of brown planthopper were compared.On this basis,NlATG3gene with better lethal effect was selected to further study the effect of RNAi on the fecundity of brown planthopper,and the potential of NlATG3 in the control of brown planthopper was comprehensively evaluated.The specific research results are as follows:1.Cloning and expression pattern analysis of autophagy-related genes NlATG5,NlATG12 and NlATG3 in the brown planthopperThe full-length cDNA of NlATG5 is 1334 bp,with an open reading frame of 819bp,encoding 272 amino acids.The full-length cDNA of NlATG12 is 837 bp,with an open reading frame of 420 bp,encoding 139 amino acids.The full-length cDNA of NlATG3 is 1431 bp,with an open reading frame of 1002 bp,encoding 333 amino acids.RT-qPCR results showed that the expression levels of NlATG5,NlATG12 and NlATG3 were stable in the nymph stage,but there were differences in the adult stage.NlATG5 and NlATG3 were highly expressed in females and males,respectively,while there was no significant difference in the expression of NlATG12 between females and males.Both NlATG5 and NlATG12 were highly expressed in the head of the brown planthopper,while NlATG3 was highly expressed in the fat body.2.Transcriptome analysis of brown planthopper after RNAi of NlATG5Comparing the differences in the transcriptome of the brown planthopper between the RNAi treatment group and the control group,27 differentially expressed genes were screened,including 14 up-regulated genes and 13 down-regulated genes,indicating that the RNAi of NlATG5 had little effect on the transcriptome of the brown planthopper.The differentially expressed genes contained 4 known genes,including the target gene ATG5 for RNA interference and the other 3 genes TXN2,resilin,and Unc5c.Sequence alignment showed that among the 27 differentially expressed genes,except NlATG5,there was no homologous sequence（>20bp）that may cause interference effect with interference fragments（ds NlATG5）,suggesting that the differential expression of these genes may not be caused by off-target effect,but may be functionally related to NlATG5.Therefore,the target specificity of RNAi of NlATG5 is relatively ideal and suitable for subsequent functional studies of specific genes.3.Functional studies of autophagy-related genes NlATG5,NlATG12 and NlATG3in the brown planthopperRT-qPCR results showed that RNAi can effectively inhibit the expression level of target genes.The relative expression levels of NlATG5,NlATG12 and NlATG3 were30.0%,27.6%,4.1%of the control group 4 days after injection.RNAi of NlATG3 had the greatest effect on the survival of nymphs,followed by NlATG5 and NlATG12.The survival rate of the 4^th-instar nymphs decreased to 45.8%（P<0.01）2 days after injected with ds NlATG3,and to zero 3 days after injection.The 5^th-instar nymphs injected with ds NlATG3 also showed the same high lethal effect,and all the nymphs of the treatment group died 5 days after injection.In addition,after RNAi of NlATG3,all nymphs died at the end of instars without molting,while the control group of brown planthopper had successfully completed molting and entered the next instars.After RNAi of NlATG5and NlATG12,the survival rate of 4^th-instar nymphs decreased to 46.7%and 67.5%（P<0.01）,respectively,while the survival rate of the control group was above 90.0%at this time.Since none of the ds NlATG3-treated brown planthopper could successfully molt,the effects of interference with NlATG3 on epidermis and related structures were examined by transmission electron microscopy.The results showed that the autophagy of the brown planthopper was significantly inhibited after RNAi of NlATG3.After ds NlATG3 injection,the new cuticle showed a loose filamentous structure,while the new and old cuticle in the control group showed a dense and well-organized structure.In addition,the nymphs injected with ds NlATG3 were unable to molt due to abnormal epidermal development,so they stayed at the 4th instar for a long time and could not enter the 5^th-instar after 3 days.The nymphs in the control group have entered the early5^th-instar at this time and have begun to develop new epidermis to prepare for the next molting（eclosion）.These results indicated that RNAi of NlATG3 inhibited autophagy and thus affects the cuticle formation and development of the nymphs.Therefore,high mortality of brown planthopper after ds NlATG3 injection is associated with autophagy dysfunction and failure in molting.In addition,RNAi of NlATG3 also led to slow movement and decreased ATP content of the nymphs.In order to comprehensively evaluate the potential of NlATG3 gene which has the most significant effect on the survival rate of brown planthopper after RNAi as a control target,the effect of RNAi of NlATG3 gene on adult fecundity was further studied.The results showed that RNAi of NlATG3 significantly inhibited the reproductive capacity of the adults,especially the reproductive capacity of females.When ds NlATG3-treated females mated with ds GFP-treated males（ds NlATG3♀×ds GFP♂）,the average number of eggs laid by females decreased by 80.4%from 163 to 32,and the hatching rate of eggs decreased from 95.7%to zero.When the ds NlATG3-treated males mated with the ds GFP-treated females（ds NlATG3♂×ds GFP♀）,there was no significant difference in the average amount of eggs,but the hatching rate was reduced by 46.7%（P<0.01）.In order to further study how RNAi of NlATG3 affects the reproductive capacity of brown planthopper,the morphology of internal reproductive organs of adults were observed under microscope.The results showed that there was no significant change in the morphology of the internal reproductive organs of ds NlATG3-treatment males.However,RNAi of NlATG3 significantly changed the morphology of females’internal reproductive organs.There were many typical banana-shaped eggs in the ovaries of the control females injected with ds GFP,but no such typical eggs were found in the ovaries of the females injected with ds NlATG3,with only milky white,swollen structures.In addition,females injected with ds NlATG3 also showed significant swelling of the lateral ovary fallopian tubes.In this study,the functions of three autophagy-related genes in brown planthopper were studied,and their potential as targets for brown planthopper control was evaluated,which is expected to provide new clues for the control of brown planthopper.