Studies On The Interaction Mechanism Between Nilaparvata Lugens And Metarhizium Anisopliae Based On Dual RNA-Sequencing

The brown planthopper（Nilaparvata lugens,BPH）is one of serious insect pests of rice in China.Metarhizium anisopliae can kill the BPH by contact with the body wall.However,its control efficiency is largely restricted by the host defense system.To study the interaction between BPH and M.anisopliae and analyze the molecular mechanism of host defense and fungal pathogenicity in this process are great theoretical and practical significance for the development of effective fungal insecticides against BPH.In this study,dual RNA-seq technology was used to analyze differentially expressed genes in mixed samples of adult brown planthopper infected with M.anisopliae at different time points.By using the RNAi technology to study the biological functions of the peroxidase gene Nl POD1 from the Nilaparvata lugens whose expression level was significantly up-regulated during the process of fungal-insect interactions.The main results are as follows:1.Transcriptomic analysis of BPH in response to M.anisopliae infection by dual RNA-seq:An Illumina high-throughput sequencing platform was used to analyze the transcriptomes of infected and uninfected BPH 2d（CK 2d、Ma 2d）and3d（CK 3d、Ma 3d）after M.anisopliae treatment.The differentially expressed genes（DEGs）of BPH and M.anisopliae were analyzed by bioinformatics method.The results showed as follows:Compared with CK 2d,7208 DEGs from BPH were found in Ma 2d samples,of which 3633 were up-regulated and 3575 were down-regulated.Compared with CK 3d,there were 2769 DEGs from BPH in Ma 3d samples,including1361 up-regulated genes and 1408 down-regulated genes.The Ma 2d and Ma 3d comparison revealed that 1722 host DEGs were co-expressed at two post-infection time points,including 942 co-upregulated and 780 co-downregulated genes.Compared with Ma 2d,105 DEGs were noted in Ma 3d samples.Of those,96 were upregulated and the rest nine were downregulated.GO and KEGG analyses based on DEGs of BPH showed that 28 GO terms and 11 KEGG Pathways were enriched in the Ma 2d sample,23 GO terms and 16 KEGG Pathways were enriched in the Ma 3d sample,respectively,compared with the control group.GO and KEGG analyses were performed on the differentially expressed genes of M.anisopliae.The results showed that compared with Ma 2d,40 GO terms and 1 KEGG Pathway were annotated in the Ma3d samples.DEGs analysis showed that when BPH was infected by M.anisopliae,many genes involved in host epidermal formation（e.g.,cuticle protein）,immune regulation（e.g.,serine protease,C-lectin）,cell detoxification（e.g.,peroxidase,cytochrome oxidase P450）,and biological macromolecular metabolism（e.g.,fatty acid synthesis,amino acid degradation）were upregulated.Accordingly,the expression levels of many genes related to fungal stress resistance and virulence in M.anisopliae were significantly up-regulated,such as heat shock protein,chitinase and small molecule channel protein genes.2.Cloning,identification and functional verification of the peroxide gene Nl POD1 in N.lugens:Based on the results of the comparative transcriptome analysis,a host antioxidant defense-related gene Nl POD1,encoding a peroxidase,which was significantly up-regulated（Log₂ Fold change=4.99）during the interaction between BPH and M.anisopliae was selected to verify its function.The full-length c DNA sequence of Nl POD1 gene was cloned.The temporal and spatial expression characteristics and microbe induced expression pattern of Nl POD1 were determined by q RT-PCR,and the expression level was down-regulated by RNAi technology to verify the role of Nl POD1 gene in the resistance of BPH to M.anisopliae infection.The results showed that the c DNA sequence of Nl POD1 gene（Gen Bank Accession Number:MZ682107）is 2 049 bp in length,encoding 682 amino acids with a typical animal heme peroxidase domain（An peroxidase domain）and a predicted signal peptide consisting of 29 amino acid residues at the N-terminus.The phylogenetic analysis showed that Nl POD1 is closely related to the PODs of other hemipteran insects,and has the highest homology with Halyomorpha halys POD.The q RT-PCR results showed that Nl POD1 was expressed in all tested developmental stages and tissuses.The lowest and highest expression levels were observed in the eggs and the5th nymphs,respectively.The transcript levels of Nl POD1 in the haemolymph and gut were significantly higher than those in the head and fat body of the 5th instar nymphs.The expression of Nl POD1 in the 5th instar nymphs was significantly up-regulated within 48 h post injection with E.coli.However,the expression level of Nl POD1increased firstly and then stabilized when the BPH was challenged by S.aureus and M.anisopliae.RNAi results showed that the expression levels of Nl POD1 could be significantly inhibited by microinjection of ds Nl POD1.Inhibition of Nl POD1expression caused no changes in the survival rate of the 5th instar nymphs of BPH,but significantly decreased their resistance to the infection with M.anisopliae.In conclusion,our study presented a genome-wide view of the expression characteristics of host defense and fungal pathogenicity related genes in the interaction between BPH and M.anisopliae by dual RNA-seq,providing scientific basis and gene resources for the development of BPH biocontrol technology based on M.anisopliae.