The Role Of Salivary Protein NI1860in The Change In Virulence Of The Rice Brown Planthopper,Nilaparvate Lugens,and Its Mechanisms

The brown planthopper (BPH), Nilaparvata lugens (Stal)(Hemiptera: Delphacidae), is a destructive rice pest in Asia. Cultivating resistant rice varieties is the important method to control BPH. However, BPH can quickly overcome rice resistance by evolving new virulent populations, which makes this method failure in the field. Insect herbivore saliva, containing various digestive enzymes as well as other factors, such as elicitors that induce plant defense and effectors that inhibit plant defense, has been reported to play an important role in plant-herbivore interactions, including in plant defense and herbivore virulence. However, thus far little is known about BPH saliva at the molecular level, especially its role in virulence and BPH-rice interaction. Therefore, in this study, we compared the salivary-gland transcriptomes of two BPH populations with different virulence that were maintained on one of two rice varieties:TN1(TN1population) or Mudgo (M population). Moreover, we investigated the role of a salivary protein gene Nll860in BPH feeding and virulence change. The main results were as follows:Using cDNA amplification in combination with Illumina short-read sequencing technology, we sequenced the salivary-gland transcriptomes of TN1and M population of BPH. In total,37,666and38,451unigenes were generated from the salivary glands of these populations, respectively. When combined, a total of43,312unigenes were obtained, about18times more than the number of expressed sequence tags previously identified from these glands. Gene ontology annotations and KEGG orthology classifications indicated that genes related to metabolism, binding and transport were significantly active in the salivary glands. A total of352genes were predicted to encode secretory proteins, and some might play important roles in BPH feeding and BPH-rice interactions. By comparing the salivary-gland transcriptomes of two BPH populations having different virulence traits, we identified5637differentially expressed genes that may be related to this difference. To gain insight into the dominant biological pathways of the differentially expressed genes, a hypergeometric test was performed to explore statistically enriched pathways. This analysis indicated that the difference in’metabolism’and’digestion and absorption’ may contribute to the changes in virulence between the two populations. Moreover,94genes encoding putative secreted proteins were differentially expressed between the two populations, suggesting these genes may be related to the changes in virulence. Our data provide a rich molecular resource for future functional studies on salivary glands and saliva composition of BPH and will be useful for revealing the role of saliva in the changes in virulence of BPH and interactions between BPH and rice.By combining of molecular, RNAi and bioassay approaches, we screened36genes predicted to encode for their potential salivary proteins to influence BPH virulence. We identified a salivary protein Nl1860as such a factor that can regulate BPH virulence. Nl1860shows high homology to endo-β-1,4-glucanase, belonging to glycosyl hydrolase family9. And N11860was highly expressed in the salivary gland and midgut of BPH. Compared to those of TN1population, both mRNA and protein levels of Nl1860were higher in the salivary glands of M population. Using electrical penetration graph, we found that knockdown of Nl1860caused BPH to spend longer time in nonprobing and searching for a nutrient-rich sieve elements but less time in feeding in phloem. Moreover, silencing of Nl1860resulted in fewer BPH individuals finding phloem for feeding during a period of6h, and less amount of honey dew, an indicator of the amount of food intake, secreted by a BPH female adult. Consistent with the results stated as above, knockdown of Nl1860decreased the survival rate of nymphs and the fecundity of female adults of BPH. The corrected survival rates of M population nymphs of BPH feeding on variety Mudgo, TNI and artificial diet were significantly different: the highest is those BPH feeding on artificial diet, followed by feeding on TN1and the lowest is feeding on Mudgo. On the other hand, salivary protein Nl1860induced the production of salicylic acid and H2O2in rice. The results indicate that the protein Nl1860is crucial in the feeding of BPH and plays an important role in the change in virulence of BPH. Moreover, Nl1860is also a elicitor that can activate BPH feeding-induced defense responses in rice.