Molecular Study Of GSS And SUMF Of The Diamondback Moth,Plutella Xylostella(L.),in The Detoxification Of Host Plant Glucosinolates

Plants produce phytochemicals in a constitutive or inducible manner to deter herbivory by insects.Successful herbivory of insects relies on their ability to counteract or adapt to the plants' defensive chemicals and this evolutionary arms race is thought to shape the coevolution of plants and herbivores.Cruciferous plants(Cruciferae)possess a strong glucosinolate-myrosinase defense system,known as the "mustard oil bomb",which has been well characterized as a plant chemical defense mechanism.Glucosinolates and myrosinases are separately stored in different compartments in host plant cells.However,insect herbivores,pathogen attacks,and mechanical injuries may trigger interactions between these compounds whereby myrosinase hydrolyzes glucosinolates to generate toxic products,such as isothiocyanate,nitriles and other organic thiocyanates.Insects,in turn,have evolved ingenious detoxification strategies to overcome plant chemical defenses.Corresponding to different glucosinolate profiles,these strategies are also diverse,not only between the groups of generalist and specialist herbivores,but also within each of the groups.As a cruciferae specialist herbivore,the diamondback moth,Plutella xylostella(L.),uses sulfatases(SULF)to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding.Sulfatase activity is regulated by post-translational modification of a cysteine residue by sulfatase modifying factor 1(SUMF1).Therefore,in this paper,the sulfatase and sulfatase modifying factors of different insects were analyzed from the perspective of bioinformatics,and the sulfatase and sulfatase modifying factors of Plutella xylostella were studied from the aspects of in vivo and in vitro system integration and functional verification.Finally,with the hope to provide insights into the evolutionary adaptation of cruciferous plants and insect herbivores.1.Expression and functional charaicteration of sulfatase and sulfatase modifying factor genes in diamondback moth,P.xylostella.We identified 12 SULF genes(PxylSulfs)and two SUMF1 genes(PxylSumf1s)in the P.xylostella genome.Phylogenetic analysis of SULFs and SUMFs from P.xylostella,Bombyx mori,Manduca sexta,Heliconius melpomene,Danaus plexippus,Drosophila melanogaster,Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups,and the SUMFs could be divided into two groups.Profiling of the expression of PxylSulfs and PxylSumfs by RNA-seq and by qRT-PCR showed that two glucosinolate sulfatase genes(GSSs),PxylSulf2 and PxylSulf3,were primarily expressed in the midgut of 3rd-and 4th-instar larvae.Moreover,expression of sulfatases PxylSulf2,PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying factor PxylSumf1a.Small interfering RNAs(siRNAs)were used to down-regulate PxyISulf2,PxylSulf3 and PxylSumf1a expression in the 3rd-instar larvae feeding on radish seedlings.However,resulting in 44-59%mortality after 72 h and 87-90%mortality at adult emergence after feeding radish seedlings,compared with 10.6%and 34%in the control.The core sequence of PxylSulf2 gene was edited by CRISPR/Cas9 technique,and more than 50%of mutant individuals were obtained from GO generation.These mutants were hybridized with wild-type individuals,respectively,and we found not only the same mutant type as GO generation in F1 individuals,but also found two or more different types of mutations that were different from GO generation maternal.This provides diversity for homozygous mutants of the PxylSulf2 gene of P.xylostella.The findings from this study provide new insights into the structure and expression of SUMF1 and PxylSulf genes that are considered to be key factors for the evolutionary success of P.xylostella as a specialist herbivore of cruciferous plants.2.Effects of plant glucosinolates on SULFs and SUMF1 gene expression in generalist and specialist lepidopteransWe compared the feeding preferences of the cruciferae specialist herbivore P.xylostella and the generalist T.ni on different cruciferous plants including artificial diets,broccoli,Arabidopsis thaliana Col-0 wildtype and mutants deficient in myrosinasis(Tgg1/2),aliphatic glucosinolates(Myb28/29),indole glucosinolates(Cyp7982/B3),or both(QKO,quadruple knockout).We compared the gene expression patterns of SULFs and SUMF1 in both insects after feeding on these plants.Each of the two experiments showed that P.xylostella had a larger increase in body weight on the artificial diets than feeding on plants.They were reared on the same food source from 1st to 4th instar and gained more weight than those transferred from artificial diets to plants at the beginning of the 4th instar.The generalist T.ni had the highest weight gain on broccoli and similar to P.xylostella.They were reared on the same food source from 1st instar to 5th instar and gained more weight than those transferred from artificial diets to plants at the beginning of the 5th instar.However,unlike P.xylostella,the weight gain on the two plants was significantly greater than those on the artificial diet after 8 h feeding on plants.After 24 h,the difference was less.The expression of PxyIGSSl was related to plant glueosinolates,whether P.xylostella were reared on the same food source from 1st instar to 4th instar,or transferred from artificial diets at the 4th instar to plants.Especially with broccoli,there was a significant induction response,and the gene expression of PxylGSS1 was consistent in two independent experiments.In contrast to the PxylGSS1 expression pattern,PxylGSS2 expression responded mainly to A.thaliana plants,whereas there was no significant response to broccoli with higher glucosinolate content.Unlike the expression patterns of the two PxylGSSs genes,PxylSumf1 gene expression was significantly induced going from artificial diets at the 4th instar,and the expression of PxylSumf1 in P.xylostella that were reared on the same food source from 1st instar to 4th instar was not affected by a change in plant glucosinolates.The two independent experiments on T.ni showed that,whether its larvae were constantly reared on the same food source or transferred at the early stage of the 5th instar,the expression of TniSulf1 was significantly increased after feeding on broccoli.There may exist a certain correlation between the larger weight gain on broccoli and the gene expression induction.The expression of TniSulf2 was significantly increased on several plants at the beginning of the 5th instar,mainly in broccoli and wild-type A.thaliana,but the expression of TniSulf2 in T.ni that were reared on the same food source from 1st instar to 5th instar did not change significantly in either of the experiments.Being more significantly than TniSulf1 expression pattern changes,TniSulf1 expression not only induced response to broccoli,but also significantly increased on wild-type A.thaliana.In general,the results of the first experiment showed that the expression of TniSumf1 was not significantly increased regardless of the larvae reared on the same food source or transferred at the beginning of the 5th instar.The expression pattern of TniSumf1 was increased after 8 h feeding on the indole glucosinolates mutant at the beginning of the 5th instar.In this study,we compared the gene expression patterns of SULFs and SUMF1 in both generalist and specialist lepidopterans in response to the host plant glucosinolates in order to better understand the parallel evolution of insect-plant interaction from the perspective of comparative genome.Our findings provide new insights into the relationship between plant glicosinolate contents and composition and their involvements with the expression of glucosinolate detoxication genes in P.xylostella.3.The P.xylostella cell lines expressing midgut PxylGSSs and PxylSumf1genesSix new cell lines were established from embryonic tissue of the diamondback moth,P.xylostella.The primary cultures were seeded in Grace's medium supplemented with 10%fetal bovine serum and incubated at 27?.Currently,the five cell lines have been passaged more than 100 times.The cell lines were selected for differential characteristics,including growth in attachment or in suspension,susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxication pathway in P.xylostella larvae.The cell lines have three cell types:spindle-shaped,epithelial-like and round cells;Five of them grew as an attached monolayer,and FAFU&CU-Px-2 grew as suspended cells.The results of the growth curve test showed that the population doubling times for all cell lines ranged from 17.74 to 23.27 hrs.The mitochondrial cytochrome c oxidase subunit I gene sequence analysis showed that the diamondback moth cell lines(FAFU&CU-Px-1-6)were derived from the DBM larvae in the laboratory.The FAFU&CU-Px-1-6 were also used to detect the gene expression of P.xylostella glucosinolate sulfatases(PxylGSSs)and sulfatase modifying factor(SUMF1),which were highly eo-expressed in the midgut of 3rd-and 4th-instar larvae.The PxylGSSs have been confirmed as playing important roles in DBM detoxification of host plant glucosinolates.Therefore,the P.xylostella cell lines developed in this study could be useful in vitro research systems for studying complex molecular mechanisms in glucosinolate detoxication and insect-plant interactions.