Identification and evolution of glucosinolate sulfatases in a specialist flea beetle

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.

A specialist flea beetle manipulates and tolerates the activated chemical defense in its host plant

“…Compared to previous studies with P. chrysocephala adults (Beran et al, 2018; Ahn et al, 2019), plant myrosinase activity had less impact on the metabolic fate of ingested glucosinolates in P. armoraciae adults. In quantitative studies, plant myrosinases hydrolyzed approximately 75% of the total ingested glucosinolates in P. chrysocephala , whereas only about 10% of ingested allyl glucosinolate were hydrolyzed in P. armoraciae .…”

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases

“…In previous quantitative feeding studies with P. chrysocephala, plant myrosinases hydrolyzed approximately 75% of the total ingested glucosinolates (Beran et al, 2018;Ahn et al, 2019), whereas only about 10% of ingested allyl glucosinolate were hydrolyzed in our quantitative study with P. armoraciae. (A) Beetles were allowed to feed for 1 min on Arabidopsis leaves with (wild type) or without (tgg) myrosinase activity and were dissected into gut and rest of body 5 min later (n = 3 per plant genotype).…”

Gut microbiota degrades toxic isothiocyanates in a flea beetle pest

“…Both adults and larvae of P. chrysocephala cause crop damage; adult beetles feed on leaves, whereas the larvae mine within the stems and petioles (Vig, 2003; Williams, 2010). Previous research demonstrated that P. chrysocephala uses at least three different strategies to overcome the GLS‐myrosinase defense of their host plants (Ahn et al., 2019; Beran et al., 2018) (Figure 1a). Two distinct mechanisms prevent the hydrolysis of GLS, i.e .…”

“…Herbivorous insects have evolved diverse strategies to minimize their exposure to toxic ITCs (Jeschke et al., 2016a; Winde & Wittstock, 2011). For example, diamondback moth larvae, desert locusts, and cabbage stem flea beetles prevent the hydrolysis of ingested GLS by plant myrosinases through conversion to desulfo‐GLS (Ahn et al., 2019; Falk & Gershenzon, 2007; Heidel‐Fischer et al., 2019; Ratzka et al., 2002), and butterflies in the subfamily Pierinae evolved a biochemical mechanism that promotes the formation of less toxic nitriles instead of ITCs upon GLS hydrolysis (Edger et al., 2015; Wittstock et al., 2004). Other herbivores tolerate ITCs or evolved efficient glutathione‐S‐transferases, which reduce the amounts of free ITCs by conjugating them to glutathione (Gloss et al., 2014; Jeschke et al., 2017).…”