Identification Of Therioaphis Trifolii(Drepanosiphidae) Salivary Protein And Molecular Response Of Laccase-1 To Insect-resistant Substances

Alfalfa spotted aphid is a major pest of alfalfa.In order to explore the molecular mechanism of Alfalfa spotted aphid using salivary protein to adapt to host plant defense response and screen the key effectors of salivary protein.Based on the previous work in the laboratory,we compared the adaptability of Alfalfa spotted aphid on the two hosts with aphid resistant alfalfa(Zhongmu No.1)and aphid susceptible alfalfa(WL343),and obtained two strains of Alfalfa spotted aphid by domestication of the two hosts.Then,the salivary effectors of Alfalfa spotted aphid were identified and screened by salivary gland transcriptome assay and salivary protein mass spectrometry,Finally,the function of a key salivary protein for alfalfa aphid to adapt to host plant defense was verified.We obtained the following test results.The fitness of Alfalfa spotted aphid on aphid resistant alfalfa was significantly lower than that of susceptible alfalfa.The contents of jasmonic acid,hydrogen peroxide,total phenolic compounds,total flavonoids and the activities of cat,SOD and pod in aphid resistant varieties were significantly higher than those in aphid susceptible varieties,and the content of salicylic acid was significantly lower than that in aphid susceptible varieties.It is speculated that aphids fed on insect resistant varieties will produce higher contents of JA and secondary metabolites,reduce the content of SA,increase the activity of related protective enzymes,and then inhibit the growth and development of aphids.Therefore,we screened the r-aphid domesticated by the insect resistant variety alfalfa(Zhongmu No.1)and s-aphid domesticated by the insect susceptible variety alfalfa(wl343),and evaluated the difference of defense response of host plants after damage.The results showed that the survival rate of r-aphid on the host plants of insect resistant varieties was significantly higher than that of s-aphid(P = 0.039),but there was no significant difference on the host plants of insect susceptible varieties(P = 0.139).Therefore,alfalfa,an insect resistant variety,was selected as the host plant for differential evaluation.We extracted the saliva of two aphids(r-saliva and s-saliva)to infiltrate the host plants.It was found that the uninhabited alfalfa spotted aphid was more likely to feed on the host plants infiltrated by r-saliva,and the survival rate of the host plants infiltrated by r-saliva was significantly higher than that infiltrated by s-saliva.Compared with that fed by s-aphid,the content of JA and the expression of related synthetic genes in host plants were significantly decreased after being fed by r-aphid,while the content of JA and the expression of related genes were significantly increased.The change trend of JA and SA in host plants after being infiltrated by r-saliva and s-saliva was the same as that when they were fed.Therefore,we speculate that r-aphid can use r-saliva to activate the host plant salicylic acid pathway and inhibit the jasmonic acid pathway,so as to reduce the host plant defense ability and improve its fitness on the host,while s-aphid is just the opposite.In order to further analyze the mechanism of saliva regulation of host plant defense by alfalfa spotted aphid,we conducted transcriptome analysis on the salivary gland tissues of r-aphid and s-aphid,and protein mass spectrometry analysis on the salivary proteins of r-aphid and s-aphid.Combined with transcriptome data and protein mass spectrometry data,we screened the effectors in their salivary proteins by consulting the literature.Finally,we screened 8 differentially expressed salivary proteins and verified the function of laccase-1 of Alfalfa spotted aphid.We found that the transcriptional level of laccase-1 in r-aphid salivary gland was significantly higher than that in s-aphid,and it existed in the protein mass spectra of r-saliva and s-saliva.The secretory protein ttlac-1 is highly expressed in the salivary gland and midgut of Alfalfa spotted aphid,and the homology between ttlac-1 and Acyrthosiphon pisum is high.And ttlac-1 can help alfalfa aphid resist the defense response of host plants,and can help alfalfa aphid metabolize and degrade plant secondary metabolites tannic acid and flavonoids.This study can enrich the experimental data for revealing the direction of phytophagous insect host plant interaction,and this study is also the first time to screen and explore the salivary protein of Alfalfa spotted aphid,which can provide new green ideas and theoretical support for the control of Alfalfa spotted aphid on alfalfa pasture.