Study On The Mechanism Responsible For The Virulence Variation Of The Rice Brown Planthopper,Nilaparvata Lugens(Stal)

Brown planthopper (BPH), Nilaparvata lugens (Stal)(Homoptera:Delphacidae) is a major pest of rice in China and other countries in Asia. Cultivation of resistant rice varieties is an important method to control BPH. However, a fast change in virulence of BPH makes this method failure in the field. The mechanisms underlying the variation of BPH’s virulence remain unclear. It has been reported that symbionts, saliva and detoxifying enzymes of herbivorous insects played important roles in virulence variation of herbivorous insects. Therefore using BPH populations, with different virulence traits, from two rice varieties Mudgo virulent and TN1avirulent as a research system, comparative transcriptomes of fat bodies, which there were detoxifying enzymes and symbionts, of BPH from the two populations were analyzed. Moreover, the roles of a salivary protein N14777in the virulence change of BPH were investigated. The main results are as follows:1)By de novo transcriptome assembling and massive parallel pyrosequencing of the fat bodies of Mudgo and TN1populations, we obtained32332unigenes for Mudgo population and33775unigenes for TN1population, all of which were combined into42621all-unigenes. For all-unigenes, the best match species was the red flour beetle(Tribolium castaneum). Based on sequence homology, approximately14993and15461of the unigenes of TN1and Mudgo populations, respectively, could be annotated in GO. Both of the two populations had a similar GO distribution. Approximately21142all-unigenes of BPH could be annotated in COG annotation. For KEGG pathway analysis,14656all-unigenes were mapped to241pathways. In the BPH fat-body transcriptome data,2884simple sequence repeats (SSRs) or microsatellites were detected, and (CAG)n were the most frequent motif of SSRs. Totally,7860differentially expressed genes of fat bodies between TNI and Mudgo populations were found. Among these genes,3858genes, which were mainly related to metabolism, signaling, immune responses and transport, were annotated. For the primary metabolism,236genes were found to be differentially expressed. These genes were involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, energy metabolism and xenobiotic metabolism. There were57differentially expressed unigenes that were related to immune responses, of which40genes were upregulated in Mudgo population. These genes were mainly involved in pathogen recognition, immune signaling pathways, genes of AMPs and other immune-related genes. Three hundreds and seventeen differentially-expressed unigenes were related to yeast-like symbiote (YLS). These fungal species belonged to12genera, of which Debaryomyces, Kluyveromyces, Lodderomyces, Saccharomyces, Schizosaccharomyces, Scheffersomyces, Vanderwaltozyma and Zygosaccharomyces was first identified in BPH. Twenty-two genes were related to Wolbachia,20of which were upregulated in the Mudgo population. These findings laid a foundation for the elucidation of mechanisms responsible for virulence variation of BPH.2)Based on the results from comparative analysis of the transcriptomes of the salivary glands of the two BPH populations, we chose a differentially-expressed gene N14777, encoding a putative secretory protein, to study its role in the change of BPH virulence. The protein N14777, including242amino acids, possessed a signal peptide and a trans-membrane domain. The C-terminal sequence of this protein contained two functional EF-hand Ca2+-binding domains. Phylogeny analysis showed N14777was homologous to a multiple coagulation factor deficiency protein. The stability of three candidate reference genes in BPH was detected by QRT-PCR. We found the orders of the stability of the three candidate reference genes in the different tissues and developmental stages of BPH were Flightin>18S rRNA> P-Actin and18S rRNA> β-Actin> Flightin, respectively. According to the QRT-PCR results, level of N14777transcripts was the highest in the salivary glands of BPH, and its level in Mudgo population was18.51times of that in TN1population. Levels of N14777mRNAs could be effectively decreased by injecting double-stranded RNA of N114777. Knockdown of N14777reduced the survival rate of BPH nymphs and the amount of honey dew secreted by female adults but did not affect their fecundity. N14777could be secreted into rice by BPH feeding. Compared to that by normal BPH, infestation by a BPH mutant with knockdown of N14777elicited rice plants lower JA concentrations (at8h after infestation) but higher SA (at24h) and H2O2(at8h) levels. Infestation of the BPH mutant also increased the amount of total volatiles and two single chemicals,2-heptanol and linalool, emitted from rice plants which subsequently enhanced the attractiveness of rice volatiles to Anagrus milaparvatae Pang et Wang, an egg parasitoid of BPH. These results indicated that salivary secretory protein N14777played important roles in the virulence variation of BPH by mediating the rice direct and indirect defense responses.