Function Of Sulfatase Modifying Factors In The Development And Host Adaptation Of Plutella Xylostella

Glucosinolates-myrosinase is a widespread substrate-enzyme defense system in Brassicaceae plants.Glucosinolates(GS)and myrosinase exist in separated chambers in healthy plants.When the plant tissue is damaged by herbivorous insects,glucosinolases are rapidly hydrolyzed by myrosinases to produce toxic products.Diamondback moth,Plutella xylostella is one of the most destructive pests of Brassicaceae vegetables.Glucosinolate sulfatases(GSSs)in P.xylostella can desulfurize GS to produce non-toxic metabolites,which is an important strategy for P.xylostella to overcome the defense system of host plants.The activities of the vast majority of sulfesterase need to be activated after being modified by the sulfatase modifying factor(SUMF).GSS as one of the family members of sulfatases,it is still poorly understood on both whether GSS’s activity also needs modification by SUMF and the functions of SUMF in development and host adaptation process of P.xylostella.In our previous studies,gss1 and sumf were identified to be co-expressed and highly expressed in the midgut of P.xylostella.Therefore,in this study,yeast two-hybrid technique was used to further explore the interaction between GSS1 and SUMF in vitro,and CRISPR/Cas9 gene editing technique was used to analyze the in-vivo function of SUMF in the development and host adaptation of P.xylostella.The key findings are listed as below:1.In this study,based on the verified sequences of sumf1 a,sumf1b and gss1 genes of P.xylostella,we constructed the bait plasmid p GBKT7-gss1 and the prey plasmids p GADT7-sumf1 a and p GADT7-sumf1 b and used the yeast two-hybrid system to verify the interaction between GSS and SUMF1A/1B.The results showed that the yeast strains introduced with p GADT7-sumf1a/p GBKT7-gss1 and p GADT7-sumf1b/p GBKT7-gss1 plasmids could produce blue colonies on the resistance selection medium,which indicated that both SUMF1 A and SUMF1 B can interact with GSS1.2.Based on CRISPR/Cas9 gene editing technique,two single guide RNAs(sg RNAs)were designed at exon 1 and exon 2 of sumf1 a and sumf1 b,respectively.These sg RNAs were synthesized by in vitro transcription and co-injected into P.xylostella embryos with the cas9 protein.Homozygous mutants of sumf1a-M1 with deletion of 5 bp fragments and sumf1b-M5 with insertion 25 bp and deletion of 9 bp fragments were screened and established.The results of Western blot detection confirmed that the target proteins were not detected in the corresponding mutant,indicating that the sumf1 a and sumf1 b genes of P.xylostella were knocked out successfully.All mutant strains laid an important foundation for further exploring the function of sumf1 a and sumf1 b genes in P.xylostella.3.Based on the biological parameters of G88 wild type strain and sumf1 a and sumf1 b mutants feeding on artificial diet from egg to adult stage,it was found that the adult eclosion rate of P.xylostella without sumf1 a or sumf1 b was significantly lower than that of wild type,which indicated that sumf1 a and sumf1 b might be involved in the adult emergence stage of P.xylostella.However,there was no significant difference in larval survival between the mutant and the wild type,which indicated that under the feeding condition without host plant defense,the knockout of sumf1 a and sumf1 b genes did not affect the growth and development of P.xylostella larvae.4.Based on the biological parameters of relevant mutants of sumf1 a and sumf1 b feeding on Arabidopsis thaliana from 3rd instar larvae to adults,the daily survival rates of sumf1 a and sumf1 b mutants were significantly different from wild type,and the total larval survival rate of sumf1 a and sumf1 b mutants were significantly lower than wild type,which was different from that feed in artificial diet.It is suggested that SUMF1 A and SUMF1 B play an important role in the adaptation of P.xylostella to host plants.5.Based on extracting A.thaliana leaf protein after short-term fed by wild type of P.xylostella and sumf1a/1b mutants,the GSS1/2 protein was detected by Western blot experiment.It was found that GSS1/2protein was almost not detectable in A.thaliana leaves fed by sumf1 a mutants,while GSS1/2 could still be detected in the treatment of sumf1 b mutants.Combined with previous laboratory studies that the main protein released by P.xylostella on A.thaliana leaves was GSS1 protein,it is inferred that the deletion of SUMF1 A in P.xylostella may affect the release of GSS1 during feeding process,but the specific molecular mechanism needs further study.In this study,the CRISPR/Cas9 system was used to knock out the sulfatase modifying factors genes sumf1 a and sumf1 b of P.xylostella.It was found that knockout of sumf1 a and sumf1 b genes significantly decreased the larval survival rate of P.xylostella after feeding on the host plant of A.thaliana,indicating that the adaptability of P.xylostella to host plants is decreased.It is further revealed that SUMF1 A may affect the important role of GSS1 in plant defense by affecting the release of GSS1 protein from P.xylostella.This study provides a further molecular experimental basis for exploring the co-evolution of P.xylostella and cruciferous plants,and paves the ways for integrated pest management based on biotechnology and promoting the study of plant-insect interaction.