Abstract
Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. Several insect herbivores, including the cabbage stem flea beetle (Psylliodes chrysocephala), prevent glucosinolate activation by forming desulfo-glucosinolates. Here we investigated the molecular basis of glucosinolate desulfation in P. chrysocephala, an important pest of oilseed rape. Enzyme activity assays with crude beetle protein extracts revealed that glucosinolate sulfatase (GSS) activity is associated with the gut membrane and has narrow substrate specificity towards the benzenic glucosinolate sinalbin. In agreement with GSS activity localization in vivo, we identified six genes encoding arylsulfatase-like enzymes with a predicted C-terminal transmembrane domain, of which five showed GSS activity upon heterologous expression in insect cells. PcGSS1 and PcGSS2 used sinalbin and indol-3-ylmethyl glucosinolate as substrates, respectively, whereas PcGSS3, PcGSS4, and PcGSS5 showed weak activity in enzyme assays. RNAi-mediated knock-down of PcGSS1 and PcGSS2 expression in adult beetles confirmed their function in vivo. In a phylogenetic analysis of coleopteran and lepidopteran arylsulfatases, the P. chrysocephala GSSs formed a cluster within a coleopteran-specific sulfatase clade distant from the previously identified GSSs of the diamondback moth, Plutella xylostella, suggesting an independent evolution of GSS activity in ermine moths and flea beetles.
Highlights
Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory
Significantly less desulfo-indol-3-ylmethyl glucosinolate was formed in assays with gut homogenates from PcGSS2-double-stranded RNA (dsRNA)-injected adults compared to the other treatments, which shows that PcGSS2 is responsible for glucosinolate sulfatase (GSS) activity towards indol-3-ylmethyl glucosinolate in P. chrysocephala (n = 9–11, One-way analysis of variance (ANOVA); P < 0.001; Fig. 4g)
Glucosinolate desulfation catalyzed by sulfatase enzymes is a well-known detoxification strategy that prevents the activation of ingested glucosinolates to toxic isothiocyanates[16,17,18,19]
Summary
Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. To determine the localization of GSS activity in P. chrysocephala adults, we incubated crude tissue homogenates of dissected guts and the corresponding remaining body tissues with different glucosinolate substrates. Enzyme activity assays performed with gut tissue homogenates of the horseradish flea beetle, Phyllotreta armoraciae, revealed no GSS activity (Supplementary Fig. S1).
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