Functional Mechanisms Of Rice Plant Volatiles Induced By Rice Dwarf Virus In Rice-virus-leafhopper Interactions

Plant viruses can manipulate their host plants to release odors that are attractive or repellent to their insect vectors,thus may promote the epidemic and dispersal of viruses.However,the volatile organic compounds(VOCs),either individually or as mixtures,which play a key role in the olfactory behavior of insect vectors remains largely unknown.In addition,whether plant viruses influence the expression of the olfactory genes which modulate the hosts selection behavior of insect vectors is not largely explored.Thus,working with the insect vector green rice leafhoppers(GRLHs),Nephotettix cincticeps(Hemiptera: Cicadellidae),rice dwarf virus(RDV)and rice plants(Minghui 63,MH63),the effects of two RDV-induced VOCs((E)-β-caryophyllene and 2-heptanol)on the host selection behavior of non-viruliferous and viruliferous GRLHs were systematically investigated based on insect behavior,chemical ecology,molecular biology and gene-editing methods.In this study,the transcriptome of non-viruliferous GRLHs was also determined to mine the odorant binding proteins(OBPs)and chemosensory proteins(CSPs)genes,and then the effects of RDV infection on the expression of these OBPs and CSPs genes were analyzed,thereafter the target genes of GRLHs olfactory affected by RDV were identified.This thesis will lay a foundation for design of new strategies for disease control and the discovery of potential pest target genes.1.RDV-induced VOCs of rice plant mediate the host selection and odor perception of non-viruliferous and viruliferous GRLHsFirst,the host selection and odor perception of non-viruliferous and viruliferous GRLHs between mock-inoculated and RDV-infected rice plants were tested.Results showed that non-viruliferous GRLHs preferred RDV-infected rice plants compared to mock-inoculated plants,whereas viruliferous GRLHs preferred mock-inoculated plants to RDV-infected plants;RDV-infected rice plants were more attractive to nonviruliferous GRLHs and conversely,they were more repulsive by viruliferous GRLHs.Second,the effects of individual VOCs of(E)-β-caryophyllene and 2-heptanol on the olfactory behavior of non-viruliferous and viruliferous GRLHs were determined.Results suggested that(E)-β-caryophyllene was significantly attractive towards nonviruliferous GRLHs,but exhibited no attraction or repellent effects towards viruliferous GRLHs;2-heptanol neither attracted nor repelled non-viruliferous GRLHs,but was dramatically repellent to viruliferous GRLHs.Thus,we inferred that RDV infection induced the emission of(E)-β-caryophyllene and 2-heptanol from rice plants,and then influenced the host selection of nonviruliferous and viruliferous GRLHs.2.oscas mutant rice plants were constructed by CRISPR/ Cas9 gene editing systemResults found that RDV induced the relative expression levels of(E)-β-caryophyllene synthase OsCAS gene.To further verify the role of(E)-β-caryophyllene as an attractant to non-viruliferous GRLHs,we edited the OsCAS gene using the CRISPR/Cas9 system.A total of 5 types homozygous mutant rice plants were identified.The homozygous mutant of oscas-1 was identified with good editing effects based on the OsCAS gene expression levels in all of the RDV-free or RDV-infected mutants and wild type rice plants.Moreover,the oscas-1 plants showed no significant differences in important agronomic traits compared with MH63 rice plants.Thus,oscas-1 rice plants were used in subsequent experiments.3.Validation of the effects of RDV-induced VOCs of rice plant on GRLHsThe volatiles emitted by MH63,MH63-RDV,oscas-1 and oscas-1-RDV plants were determined.Results suggested that the emissions of(E)-β-caryophyllene from MH63 and oscas-1 plants,as well as oscas-1 and oscas-1-RDV rice plants had no significant difference,but this emission from MH63-RDV were higher than that from oscas-1-RDV plants.The levels of 2-heptanol emitted by the gene edited plants exhibited the same trend as seen for their non-mutant counterparts(MH63 and MH63-RDV)being significantly higher in the oscas-1-RDV plants compared to the oscas-1 plants.The levels of other 13 VOCs emitted from four different rice plants had no significant difference.Bioassay results found that the host selection and odor perception of non-viruliferous GRLHs showed no significant difference between MH63 and oscas-1,as well as oscas-1 and oscas-1-RDV rice plants.But the non-viruliferous GRLHs preferred the MH63-RDV plants compared to oscas-1-RDV plants.In contrast,when oscas-1-RDV rice plants were treated with(E)-β-caryophyllene,the host selection and odor perception of non-viruliferous GRLHs were not significantly different between oscas-1-RDV and MH63-RDV plants.Compared to MH63 rice plants treated with 2-heptanol,the viruliferous GRLHs preferred MH63 rice plants without no treatments.In addition,the viruliferous GRLHs clearly preferred oscas-1 plants to oscas-1-RDV plants.To sum up,all these results showed that the two RDV-induced rice VOCs played key roles in the GRLHs-RDV-rice plants interactions.4.RDV modulates the expression levels of OBPs and CSPs genes in the antennae of GRLHs.The effects of(E)-β-caryophyllene and 2-heptanol on the olfactory of nonviruliferous and viruliferous GRLHs without antenna were determined.Results suggested that(E)-β-caryophyllene had no attractions to non-viruliferous GRLHs without antenna,2-heptanol did not repelled the viruliferous GRLHs without antenna,thereafter indicating that the sensilla of GRLHs sensed the two VOCs were located on its antennae.The head and body transcriptome of non-viruliferous GRLHs was systematically analyzed based on RNA-seq.After assembly of the transcripts into unigenes,46,623 unigenes were obtained.A total of 20 OBPs and 13 CSPs genes were identified from those unigenes.Phylogenetic trees showed that these OBPs and CSPs genes were more closely related to the corresponding genes of Empoasca onukii.The expression profiles of these OBPs and CSPs in different tissues were evaluated by qRT-PCR.There were 9 OBPs and 3 CSPs genes highly expressed in the antenna of GRLHs compared to its other tissues,which may pay key roles in olfactory functions.While the OBPs and CSPs genes highly expressed in other tissues may be involved in other physiological process.Moreover,the expressions of OBP1-9 and CSP2-3 genes were significantly suppressed by RDV.Thus,we speculate that these olfaction genes inhibited by RDV may be involved in the process of perception of these two volatiles in GRLHs.