Study On Gut Regurgitant And Effector Proteins In The Diamondback Moth,plutella Xylostella(L.)

The diamondback moth(DBM),Plutella xylostella,is an important insect pest of crucifers,which can quickly develop resistance to various insecticides making it difficult to control.Examining the co-evolutionary relationshipw between DBM and cruciferous vegetables can help understand host plant resistance mechanisms and DBM’s sensitivity to host defense,two aspects important for DBM management.The oral secretion of phytophagous insects which plays crucial roles in the adaptation to their host plants,mainly comes from salivary and gut regurgitant.However,the origin and composition of oral secretions in DBM remain unclear.By feeding the DBM with fluorescent dye-containing artificial diet,the study revealed that DBM gut regurgitant could be secreted into plant wound during feeding.Furthermore,the study confirmed that DBM feeding on Arabidopsis thaliana induced the up-regulation of jasmonic acid(JA)synthase genes AOS and OPR3 and salicylic acid(SA)upstream signaling genes EDS5,PAD4 and SID2.However,SA-responding genes PR1,PR5 and BGL2,the key enzyme genes of indole glucosinolate biosynthesis CYP79B2 and CYP79B3,and myrosinase gene TGG1 were all down-regulated in response to DBM feeding.In addition,simulation of DBM feeding with gut grinding fluid resulted in the down-regulation of JA synthase genes AOC and AOS and JA response gene JAR1,SA upstream signal genes EDS5,PAD4 and SID2,the key enzyme genes of indole glucosinolate biosynthesis CYP79B2 and CYP79B3,and myrosinase gene TGG1.The results showed that the biosynthetic pathways of JA and SA were induced by DBM feeding,but their downstream response genes were inhibited,in which the DBM gut regurgitant might play an important role in the process of the inhibition.To further explore the protein composition and abundance of DBM gut regurgitant,the study determined the DBM gut proteome using LC-MS/MS approach.The DBM gut proteome consists of a large class of digestion-related proteins,some detoxification-related proteins and a group of ubiquitin-proteasome degradation system-related proteins.The study in silico predicted some ubiquuitination-related proteins which may be critical components of the gut regurgitant and function as effectors to impact on plant defense system and associated signaling pathways.Moreover,the glucose oxidase(GOX)that plays important roles in feeding success and adaptation to host plants in insects was identified,with high abundance in the DBM gut content.It is widely accepted that GOXs are mainly distributed in the salivary glands of insects,and get the plant wound via oral secretions during the feeding.However,the expression level of Pxgox2 in midgut was much higher than that in salivary glands.The GOX activity in intact DBM gut is 2419.5 nmol·min-1·mg-1,which is very close to the GOX activity,2680.6 nmol·min-1·mg-1,in salivary glands of Helicoverpa zea,the highest GOX activity recorded so far.On the basis of DBM genomics data,the study cloned three DBM GOX genes.The open reading frame(ORF)of Pxgoxl is 1827 bp,encoding a predicted protein of 608 amino acids(aa),the molecular weight(MW)and isoelectric point(IP)of wichi are 70.0 kDa and 6.0,respectively.The ORF of Pxgox2 is 1821 bp,encoding a predicted protein of 606 aa,the MW and IP of which are 66.7 kDa and 4.7,respectively.The ORF of Pxgox3 is 1812 bp,encoding a predicted protein of 603 aa,the MW and IP of which are 66.6 kDa and 6.5,respectively.Pxgox1 is expressed at a very low level throughout the life cycle,and Pxgox3 is expressed mostly in adults.Pxgox2 is larva-preferentially expressed and showed higher expression levels especially in midgut and salivary glands than the other GOX homologs.Therefore,Pxgox2 was considered to be the GOX-encoding gene in the DBM.However,prokaryotic expression of Pxgox2 in Escherichia coli revealed that the protein product was mainly in a form of inclusion body,and the concentration of soluble GOX proteins in the cell supernatant was very low,making it difficult to meet the needs of experiment of GOX effects on plant defense response.The study is significant for the elucidation of the co-evolutionary relationship between DBM and cruciferous vegetables,and can also provide a theoretical basis for understanding the feeding and detoxification mechanisms of the insect.