| Half of all insects known to date eat plants.Herbivorous insects absorb nutrients from host plants to maintain their survival and reproduction,which has brought huge economic losses to crop production in China.In order to control agricultural diseases and insect pests,pesticide control is the first priority,but the use of chemical pesticides has brought a series of problems to the ecological environment,insecticide resistance and food safety.During the co-evolutionary process of plants and insects,plants will evolve defense strategies against herbivorous insects.At the same time,insects also produce a set of herbivore-associated molecular patterns that are similar to pathogen-associated or microbial-associated molecular patterns in pathogens or microorganisms or secreted effectors to regulate plant defense responses.In recent years,in the study of the relationship between plants and herbivorous insects,insect salivary elicitors and effectors have become the current research hotspots.In-depth study of the molecular mechanisms of plant-insect salivary component interactions can not only provide insight into the synergistic evolutionary process of plants and insects,but also provide new concepts for the breeding of insect-resistant species and the control of pests in the field the molecular mechanisms of plant-insect salivary interactions have become a hot topic of research.Riptortus pedestris is a typical sap-sucking insects,are distributed in all parts of the country,its diets is miscellaneous,the host range is more extensive,and the main harm crop soybeans important agricultural economy in our country,to cluster the pest harm,main sap-sucking legume plant leaves and fruit juices,cause bud and decay,mesophyll of water,fruit malformation,serious when whole plant particles.In recent years,the summer soybean fields in the Yellow and Huaihai Sea region of China have been rampantly affected by the R.pedestris,which is one of the main reasons for the occurrence of the soybean "pod but not real" type "disease green" phenomenon.In addition,the stinging mouthpiece feeding and flying lifestyle also lead to the spread of virus diseases,which can drastically reduce plant yield when infested on a large scale.At present,research on the disease is mainly focused on its biological characteristics,the influence of environmental factors and control techniques,while there is still a gap in the research on the mechanism of interactions between the spotted stink bug and its hosts.Studies have shown that sap-sucking insects feed on plants through their specialized mouthparts,and secrete a large amount of saliva into the host plants,which interferes with the physiological and biochemical status of plants and is conducive to stinging and feeding.Therefore,the salivary components of sap-sucking insects play an important role in the interaction between insects and plants.Previous studies on the salivary proteins of sap-sucking insects mainly focused on aphids.In this paper,the salivary proteins of the sap-sucking insects regulate the plant defense response as the starting point,and the salivary proteins of the R.pedestris are screened and identified.The specific research contents are as follows:1.Screening and identification of salivary elicitor and effector of R.pedestrisIn this paper,we identified genes specifically expressed in the salivary glands by dissecting the salivary glands of the R.pedestris and extracting RNA for transcriptome sequencing analysis.The identified genes were screened with Agrobacterium tumefaciens transient expression system for(1)induction of cell death,(2)inhibition of cell death induced by PAMP INF1,and(3)induction of ROS accumulation.Finally,two genes that can induce cell death and ROS accumulation were identified: Rp302 and Rp309.2.RP309 is a potential salivary elicitor of R.pedestrisIn this study,two salivary proteins,Rp302 and Rp309,which can cause the death of Nicotiana benthamiana and the accumulation of ROS,were identified in the screening of candidate salivary proteins of R.pedestris.These two genes can be expressed in N.benthamiana by Western Blot.The expression levels of RP302 and RP309 in the head,salivary gland and gut of R.pedestris were determined by quantitative PCR,and the results showed that the two genes were up-regulated in salivary gland.The function of RP302 and RP309 signal peptides was confirmed by yeast secretion experiments.In the experiment to explore the effect of RP302 and RP309 on Phytophthora capsici infection,after overexpression of RP302 and RP309 in N.benthamiana,the diameter of the disease spots was measured 24 h and 36 h after inoculation with P.capsici.Compared with GFP,the disease spots showed no significant change,indicating that RP302 and RP309 did not affect the resistance of plants to P.capsici.Plants recognize specific excitons,or HAMPs,in insect saliva through pattern recognition receptors(PRRs)on the surface.BAK1 and SOBIR1 are different PRRs.To investigate whether cell death induced by RP302 and RP309 is dependent on Nb BAK1 and Nb SOBIR1,the VIGS technique was used to silence Nb BAK1 and Nb SOBIR1 in N.benthamiana.After the quantitative PCR validation of silencing efficiency,it was observed that the degree of cell death induced by RP302 was not significantly changed in the silenced plants with GFP,Nb BAK1 and Nb SOBIR1,while the degree of cell death induced by RP309 was significantly decreased in the silenced plants with Nb BAK1 and Nb SOBIR1.Thus,RP309 induced cell death dependent on Nb BAK1 and Nb SOBIR1.Then quantitative PCR was used to detect the expression of GFP and RP309 induced defense genes.The results showed that compared with GFP,RP309 could induce the up-regulation of defense genes related to hormone pathway at 36 h,indicating that RP309 stimulated plant immunity.In summary,this study screened for two salivary proteins,RP302 and RP309,that cause cell death and ROS accumulation in N.benthamiana,and virus-mediated gene silences showed that RP309 induced cell death dependent on Nb BAK1 and Nb SOBIR1,which is a potential salivary elicitor of R.pedestris. |
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