Cloning And Function Analysis Of The Salivary Gland Protein Genes In Brown Planthopper,Nilaparavata Lugens

The brown planthopper(BPH),Nilaparavata lugens(St?l)is the most dreadful rice pest in China.It was categorized into class-I crop pests by the ministry of agriculture and rural affairs state council of the people’s republic of China.The effective management of brown planthopper plays a pivotal role in rice production.Screening and breeding of cultivars that harbor planthopper resistance genes is considered to be the most desirable and economic strategy for the control and management of BPH,avoiding the issues and challenges of 3R activities(resistance,resurgence,and residue)by the abuse of chemical insecticides.However,there were lines of evidents supported that BPH resistance in those cultivars carrying the Bph genes was broken down from the emergence of new BPH biotypes or populations.To reveal the underlying mechanisms for this shift in virulence,it will be make great contributions to delay the resistance breaking-down in rice resistant cultivars.BPH secretes components including gelling and watery saliva from their salivary glands into plant cells during feeding on rice,and potentially direct or indirect interaction with rice protein to regulate rice immune system,conversely counteract the virulence of the brown planthopper.However,the mechanism of BPH and rice interaction are unclear.In current work,two different BPH populations i.e.TN1 population(TN1-BPH,reared and maintained on TN1 rice)and IR56 population(IR56-BPH,reared and maintained on IR56)were used to study the role of salivary proteins in virulence shift.Three genes(Nl MLP,Nl30 and Nl15)encoding salivary protein were cloned,and their temporal and spatial expression patterns were determined.In addition,the functional roles of those proteins were characterized in IR56 population.The main findings are listed as follows:1.Three brown planthopper c DNAs(Nl MLP,N130 and Nl15)were cloned by RT-PCR,with the lengths of 2271 bp,819 bp and 1431 bp,respectively.These three genes contain complete open reading frames in length.Nl MLP,Nl30 and Nl15 encode 569-,221-,and 335-amino-acid protein respectively.Homologous searches revealed that Nl MLP protein showed high sequences similarity with those,mucin-like protein from insects,and Nl30 and Nl15 proteins were predicted to be hypothetical proteins.The typical signal peptide with 19,or 23-residues can be found at the N-terminal region,respectively.These results suggests the three proteins could be secreted into rice.2.Real-time qPCR was used to determine the temporal and spatial expression patterns,and population expression patterns of these three genes in TN1-BPH and IR56-BPH.The results showed that the three genes were expressed throughout the development of brown planthopper.However,there were significant differences among developmental states.The first instar nymph had highest transcriptional levels of Nl MLP and N130 than those in examined nymphal instars.Nl15 gene was highly expressed in the third instar.The spatial expression patterns showed that there were significant difference of m RNA.We found that these three genes were enriched in the head,followed by the other examined tissues.It can be found that IR56 population had higher m RNA levels of the three genes than those in the TN1 population.3.The RNA interference was used to characterize the functional roles of Nl MLP,Nl30,and Nl15 in IR56-BPH.q PCR results showed that the targeted transcript significantly downregulated at 3 days after injection.The amounts of honeydew and weight gain of BPH fed on IR56 rice were decreased significantly(except for ds Nl15 decreased the weight gain of BPH,although there was not statistical significance);knocking-down of Nl MLP,N130 and Nl15,the survival rates of IR56 population were decreased significantly on 2-,4-,and 6-days after ds RNA injections.The expression of Os Lec RK4 in rice was increased significantly,when RNAi-treated planthoppers fed on IR56 rice.In addition,the expressions of Os MPK10,Os NPR1,Os Lox and Os GNS5 were up-regulated in IR56 rice which was fed by ds Nl15-treated planthoppers.The results described above preliminarily reveal roles of salivary gland protein in BPH related to virulence shift,provide ideas for further revealing the mechanism of virulence shift in brown planthopper,and propose the sustainable utilization strategy of rice resistant varieties.It can provide foundation for using green and sustainable control methods.