Effect And Molecular Mechanism Of Salivary Protein BtRDP On Bemisia Tabaci Host Adaptation

Whiteflies(Bemisia tabaci),as a type of piercing-sucking insect with a wide range of host plants,have caused a serious impact on agricultural production worldwide.During their feeding process,whiteflies use their stylet-like mouthparts to pierce the phloem of plants,feeding on plant sap and secreting saliva into the plant.The saliva of piercing-sucking insects plays crucial roles in lubricating the stylet,assisting in penetration,and regulating plant defenses during feeding.This saliva is essential for the feeding behavior and adaptation to host plants by insects.Exploring the functions of insect saliva,particularly that of piercing-sucking insects,contributes to a deeper understanding of the interactions between insects and plants.However,research on whitefly saliva remains limited at present.Our study identified the effects of the whitefly salivary protein Bt RDP(Bemisia tabaci RLP-degrading protein,Bt RDP)on its feeding by using bioinformatics,molecular biology and ecological methods,and revealed its molecular mechanism in regulating plant defense.These findings not only enhance our understanding of the interactions between piercing-sucking insects and plants but also provide new strategies and approaches for whitefly control.The main conclusions of this study are as follows:(1)To investigate the effects of whitefly feeding on tobacco defense,changes in gene expression in tobacco before and after whitefly feeding were studied through transcriptome analysis.Functional enrichment analysis of differentially expressed genes revealed that upregulated genes were enriched in pathways related to reactive oxygen species(ROS)response,hormone response,external stimulus response,biotic stimulus response,and innate immune response.Downregulated genes were mainly associated with metabolism and photosynthesis.Furthermore,transcription factors like WRKY TFs and ERFs were significantly upregulated after whitefly feeding.Plant pattern recognition receptors(PRRs),critical components of plant innate immunity,also showed significant changes in gene expression before and after whitefly feeding.(2)In preliminary experiments,the identification of whitefly saliva proteins was conducted through LC-MS/MS and transcriptomic analysis.The results revealed that the saliva protein Bt RDP is highly expressed in the salivary glands of the whitefly.Findings from real-time fluorescent quantitative PCR and immunohistochemical staining further confirmed the specific expression of saliva protein Bt RDP in the salivary glands.Through examinations of different developmental stages of the whitefly,it was observed that saliva protein Bt RDP is highly expressed during the adult stage.The WB detection results showed that the presence of saliva protein Bt RDP was detected in tobacco plants that had been fed upon by whiteflies.In experiments where Bt RDP expression was inhibited using RNAi,a significant reduction in feeding behavior and a notable decrease in egg production by whiteflies were observed.Following transient expression of saliva protein Bt RDP in tobacco plants,a significant increase in egg production by whiteflies was noted,and they displayed a stronger preference for feeding on the tobacco with transiently expressed saliva protein.These results suggest that saliva protein Bt RDP plays a crucial role in the feeding processes of whiteflies on host plants.(3)The receptor-like protein Nt RLP4 was screened from the c DNA yeast library of tobacco by using salivate protein Bt RDP as bait.Nt RLP4 was overexpressed in tobacco,we found that the expression levels of JA hormone pathway related genes FAD7 and PDF1.2 were significantly up-regulated,while those of SA hormone pathway related genes PAL and NPR1 were significantly down-regulated.Moreover,overexpression of Nt RLP4 triggered a noticeable burst of reactive oxygen species.At the same time we alsofound that a significant reduction in fecundity and a preference shift towards control tobacco plants.However,interference with Nt RLP4 expression in tobacco led to contrasting results.WB detection combined with confocal microscopy showed that salivary protein Bt RDP degraded receptor-like protein Nt RLP4 by ubiquitination pathway.After the co-expression of salivary protein Bt RDP and Nt RLP4 in tobacco,the tobacco defense was significantly reduced,the oviposition of whitefly was significantly increased,and the tobacco after co-expression was more attractive to whitefly.(4)Receptor-like proteins need to form receptor complexes with receptor kinases to regulate downstream defenses.Through Bi FC and Co-IP,the interaction between Nt RLP4 and the receptor kinase Nt SOBIR1 was clearly demonstrated.Transient expression of Nt SOBIR1 in tobacco found that the fecundity of whitefly was significantly reduced,and the attractiveness to whitefly was decreased.By silencing Nt SOBIR1,we further clarified the adverse effects of Nt SOBIR1 on whitefly.The results showed that the receptor kinase Nt SOBIR1 plays an important role in tobacco defense against whitefly.(5)A homologous gene of RLP4,Sl RLP4,was identified from tomatoes,another host plant of whiteflies.The interaction between Bt RDP and Sl RLP4 was confirmed through Y2 H and Co-IP experiments.Co-expression of Bt RDP and Sl RLP4 in tobacco demonstrated that salivary protein Bt RDP effectively degrades Sl RLP4 at the protein level,suggesting a conserved function of Bt RDP in whitefly adaptation to different host plants.In addition,through homology alignment,the homologous gene Os RLP4 of Nt RLP4 was identified in the rice genome.By using yeast two-hybrid screening,the brown planthopper saliva protein Nl SP694 that interacts with the rice Os RLP4 was obtained.Co-expression of Nl SP694 and Os RLP4 in tobacco showed that Nl SP694 saliva protein similarly degraded rice Os RLP4 at the protein level.RNAi silencing of brown planthopper saliva protein Nl SP694 significantly reduced honeydew excretion and oviposition,indicating an important role of Nl SP694 in brown planthopper feeding.In conclusion,different insects have evolved saliva effectors targeting plant receptor-like protein RLPs,and this mechanism appears to be conserved across various herbivorous insects.In summary,during the feeding process of whitefly,the salivary protein Bt RDP is secreted into the host plant.The salivary protein Bt RDP regulates plant defense by degrading the receptor-like protein through the ubiquitination pathway,thereby enhancing the host adaptation of whitefly.Additionally,we also discovered that the saliva protein of brown planthopper has a similar function,indicating the possibility of convergent evolution among insect saliva proteins,adopting similar strategies to regulate plant defense.