Glucose Oxidase of a Crucifer-Specialized Insect: A Potential Role in Suppressing Plant Defense via Modulating Antagonistic Plant Hormones.

Glucose oxidase (GOX) is a representative compound found in most insect saliva that can suppress plant-defensive responses. However, little is known about the origin and role of GOX in the crucifer-specialized pest Plutella xylostella. In this study, we showed obvious regurgitation from the larval gut of P. xylostella and identified abundant peptides highly similar to known GOX. Three PxGOX genes were verified with PxGOX2 preferentially expressed in the gut. The heterologously expressed PxGOX2 confirmed its function to be a GOX, and it was detected in plant wounds together with the gut regurgitant. Further experiments revealed that PxGOX2 functioned as an effector and may suppress defensive responses in plant through the production of H2O2, which modulates levels of antagonistic salicylic acid and jasmonic acid. However, excessive H2O2 in the host plant may be neutralized by peroxidase, thus forming defensive feedback. Our findings provided new insights into understanding the GOX-mediated insect-plant interactions.

An inducible gene from glycoside hydrolase one family of Plutella xylostella decreases larval survival when feeding on host plant.

Glycoside hydrolase family 1 (GH1) members exhibit a broad substrate spectrum and play important roles in insect-plant interactions, such as the defensive ß-glucosidase and ß-thioglucosidase (so-called myrosinase). However, knowledge about the expression profiling and function of glycoside hydrolase family 1 members in a specialist pest of crucifers Plutella xylostella is still limited. In this study, 13 putative glycoside hydrolase family 1 members of P. xylostella were identified based on the sequence characteristics, while no myrosinase activity was detectable in P. xylostella using gas chromatography-mass spectrometry (GC-MS). Expression profiling of these glycoside hydrolase family 1 members identified the midgut-specific gene Px008848 that is induced by host plant. Further experiments revealed that the in vitro expressed Px008848 protein had ß-glucosidase activity and the survival rate of the larvae feeding on wounded Arabidopsis thaliana leaves declined when leaves were treated with purified Px008848 protein. When CRISPR/Cas9-based homozygous mutant larvae of Px008848 and wild-type larvae were respectively transferred onto the A. thaliana, the larval survival rate of the mutant larvae was significantly higher than that of the wild-type individuals. Our work showed that certain insect glycoside hydrolase family 1 gene may have negative effect on the development of larvae feeding on the host plant, which broadened our understandings on the evolutionary function of this gene family in the insect-plant interaction.

Functional Characterization of Two RNA Methyltransferase Genes METTL3 and METTL14 Uncovers the Roles of m6A in Mediating Adaptation of Plutella xylostella to Host Plants.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

Glucosinolate Sulfatases-Sulfatase-Modifying Factors System Enables a Crucifer-Specialized Moth To Pre-detoxify Defensive Glucosinolate of the Host Plant.

Numerous herbivores orally secrete defense compounds to detoxify plant toxins. However, little is known about the role of orally secreted enzymes by a specialized pest, Plutella xylostella, in the detoxification of plant defense compounds. Three glucosinolate sulfatases (GSSs) or two sulfatase-modifying factors (SUMF1s) mutant strains were established on the basis of CRISPR/Cas9 technology to validate the existence of a species-specific GSSs-SUMF1s system. In comparison to the bioassay data from mutant strains of GSS1/GSS2 or SUMF1a/SUMF1b, GSS3 had a minimal role because no significant change was found in GSS3-/- under different feeding contexts. Antibody-based technologies were used to examine GSSs-related deficient strains, and the results showed that the GSS1 protein was primarily released through larval oral secretion. On the basis of high-performance liquid chromatography, we found that GSS1 was secreted to pre-desulfate the typical plant defensive glucosinolates known as 4-(methylsulfinyl)butyl glucosinolate (4MSOB-GL) to suppress the production of the toxic substance, which is referred to as pre-detoxification strategy. These findings highlighted that the GSSs-SUMF1s system is the key factor for counteradaptation of P. xylostella to cruciferous plants, which strengthens the concept that herbivores deploy pre-detoxification strategies to disrupt the plant chemical defenses to facilitate the colonization process.

Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella.

Evolutionary adaptations of herbivorous insects are often dictated by the necessity to withstand a corresponding evolutionary innovation in host plant defense. Glucosinolate sulfatase (GSS) enzyme activity is considered a central adaptation strategy in Plutella xylostella against glucosinolates (GS)-myrosinase defense system in the Brassicales. The high functional versatility of sulfatases suggests that they may perform other vital roles in the process of growth and development. Here, we used a CRISPR/Cas9 system to generate stable homozygous single/double mutant lines of gss1 or/and gss2 with no predicted off-target effects, to analyze the functions of the pair of duplicated genes in the development and host adaptation of P. xylostella. The bioassays showed that, when fed on their usual artificial diet, significant reduction in egg hatching rate and final larval survival rate of the single mutant line of gss2 compared with the original strain or mutant lines of gss1, revealing unexpected functions of GSS2 in embryonic and larval development. When larvae of homozygous mutant lines were transferred onto a new food, Arabidopsis thaliana, no induced effect at protein level of GSS1/2 or gene expression level of gss1/gss2 was detected. The absence of GSS1 or GSS2 reduced the survival rate of larvae and prolonged the duration of the larval stage, indicating that both GSS1 and GSS2 played an important role in adaptation to host plants. The versatile functions of duplicated GSSs in this study provide a foundation for further research to understand potential functions of other sulfatase members and support evidence of adaptation in herbivorous insects.