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The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection., (© 2019 American Society of Plant Biologists. All rights reserved.)

Many phloem-feeding insects are considered severe pests of agriculture and are controlled mainly by chemical insecticides. Continued extensive use of these inputs is environmentally undesirable, and also leads to the development of insecticide resistance. Here, we used a plant-mediated RNA interference (RNAi) approach, to develop a new control strategy for phloem-feeding insects. The approach aims to silence "key" detoxification genes, involved in the insect's ability to neutralize defensive and toxic plant chemistry. We targeted a glutathione S-transferase (GST) gene, BtGSTs5, in the phloem-feeding whitefly Bemisia tabaci, a devastating global agricultural pest. We report three major findings. First, significant down regulation of the BtGSTs5 gene was obtained in the gut of B. tabaci when the insects were fed on Arabidopsis thaliana transgenic plants expressing dsRNA against BtGSTs5 under a phloem-specific promoter. This brings evidence that phloem-feeding insects can be efficiently targeted by plant-mediated RNAi. Second, in-silico and in-vitro analyses indicated that the BtGSTs5 enzyme can accept as substrates, hydrolyzed aliphatic- and indolic-glucosinolates, and produce their corresponding detoxified conjugates. Third, performance assays suggested that the BtGSTs5 gene silencing prolongs the developmental period of B. tabaci nymphs. Taken together, these findings suggest that BtGSTs5 is likely to play an important role in enabling B. tabaci to successfully feed on glucosinolate-producing plants. Targeting the gene by RNAi in Brassicaceae cropping systems, will likely not eliminate the pest populations from the fields but will significantly reduce their success over the growing season, support prominent activity of natural enemies, eventually allowing the establishment of stable and sustainable agroecosystem., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plants of the Brassicaceae are defended from feeding by generalist insects by constitutively-expressed and herbivory-induced glucosinolates (GS). We induced Arabidopsis plants 1, 16 and 24 h prior to allowing neonate larvae of the generalist Helicoverpa armigera to feed on whole plants for 72 h. These plants were subsequently retested with another group of neonates for a further 72 h. We used wild-type A. thaliana Col-0, and mutant lines lacking indolic GS, aliphatic GS or all GS. We hypothesized that larvae would not grow well on defended plants (WT) compared to those lacking GS, and would not grow well if plants had been primed or fed on for longer, due to the expected induced GS. There was survivorship on all lines suggesting H. armigera is a suitable generalist for these experiments. Larvae performed less well on wild-type and no indolic lines than on no aliphatic and no GS lines. Larvae distributed feeding damage extensively in all lines, more so on wild type and no-indolic lines. Contrary to expectations, larvae grew better on plants that had been induced for 1 to 16 h than on un-induced plants suggesting they moved to and selected less toxic plant parts within a heterogeneously defended plant. Performance declined on all lines if plants had been induced for 24 h, or had been fed upon for a further 72 h. However, contrary to expectation, individual and total GS did not increase after these two treatments. This suggests that Arabidopsis plants induce additional (not GS) defenses after longer induction periods.

Glucosinolates are activated plant defenses common in the order Brassicales that release isothiocyanates (ITCs) and other hydrolysis products upon tissue damage. The reactive ITCs are toxic to insects resulting in reduced growth, delayed development and occasionally mortality. Generalist lepidopteran larvae often detoxify ingested ITCs via conjugation to glutathione (GSH) and survive on low glucosinolate diets, but it is not known how this process influences other aspects of metabolism. We investigated the impact of the aliphatic 4-methylsulfinylbutyl-ITC (4msob-ITC, sulforaphane) on the metabolism of Spodoptera littoralis larvae, which suffer a significant growth decline on 4msob-ITC-containing diets while excreting ITC-glutathione conjugates and their derivatives in the frass. The most striking effects were a decrease of GSH in midgut tissue and hemolymph due to losses by conjugation to ITC during detoxification, and a decline of the GSH biosynthetic precursor cysteine. Protein content was likewise reduced by ITC treatment suggesting that protein is actively catabolized in an attempt to supply cysteine for GSH biosynthesis. The negative growth and protein effects were relieved by dietary supplementation with cystine. Other consequences of protein breakdown included deamination of amino acids with increased excretion of uric acid and elevated lipid content. Thus metabolic detoxification of ITCs provokes a cascade of negative effects on insects that result in reduced fitness., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glucosinolates are plant secondary defense metabolites confined nearly exclusively to the order Brassicales. Upon tissue rupture, glucosinolates are hydrolyzed to various bioactive breakdown products by the endogenous plant enzyme myrosinase. As the feeding of chewing insect herbivores is associated with plant tissue damage, these insects have developed several independent strategies for coping with the glucosinolate-myrosinase defense system. On the other hand, our knowledge of how phloem-feeding insects interact with the glucosinolate-myrosinase system is much more limited. In fact, phloem feeders might avoid contact with myrosinase altogether so their susceptibility to intoxication by glucosinolate hydrolysis products is unclear. Previous studies utilizing Arabidopsis thaliana plants accumulating high levels of aliphatic- or indolic-glucosinolates indicated that both glucosinolate groups have moderate negative effects on the reproductive performance of Bemisia tabaci, a generalist phloem-feeding insect. To get a deeper understanding of the interaction between B. tabaci and glucosinolate-defended plants, adults were allowed to feed on artificial diet containing intact glucosinolates or on Brussels sprout and A. thaliana plants, and their honeydew was analyzed for the presence of possible metabolites. We found that B. tabaci is capable of cleaving off the sulfate group of intact glucosinolates, producing desulfoglucosinolates that cannot be activated by myrosinases, a mechanism described to date only in several chewing insect herbivores. The presence of desulfated glucosinolates in the honeydew of a generalist phloem-feeder may indicate the necessity to detoxify glucosinolates, likely due to some level of cellular damage during feeding, which results in glucosinolate activation, or as a mechanism to circumvent the non-enzymatic breakdown of indolic glucosinolates.

Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant immunity to molluscs is limited. We found slugs to be natural herbivores of Arabidopsis thaliana and therefore investigated possible resistance mechanisms of this species against several molluscan herbivores. Treating wounded leaves with the mucus residue ('slime trail') of the Spanish slug Arion lusitanicus increased wound-induced jasmonate levels, suggesting the presence of defence elicitors in the mucus. Plants deficient in jasmonate biosynthesis and signalling suffered more damage by molluscan herbivores in the laboratory and in the field, demonstrating that JA-mediated defences protect A. thaliana against slugs and snails. Furthermore, experiments using A. thaliana mutants with altered levels of specific glucosinolate classes revealed the importance of aliphatic glucosinolates in defending leaves and reproductive structures against molluscs. The presence in mollusc faeces of known and novel metabolites arising from glutathione conjugation with glucosinolate hydrolysis products suggests that molluscan herbivores actively detoxify glucosinolates. Higher levels of aliphatic glucosinolates were found in plants during the night compared to the day, which correlated well with the nocturnal activity rhythms of slugs and snails. Our data highlight the function of well-known antiherbivore defence pathways in resistance against slugs and snails and suggest an important role for the diurnal regulation of defence metabolites against nocturnal molluscan herbivores., (© 2013 John Wiley & Sons Ltd.)

The defensive properties of the glucosinolate-myrosinase system in plants of the order Brassicales have been attributed to the formation of toxic isothiocyanates generated upon tissue damage. Lepidopteran herbivores specialised on brassicaceous plants have been shown to possess biochemical mechanisms preventing the formation of isothiocyanates. Yet, no such mechanisms are known for generalist lepidopterans which also occasionally but successfully feed on plants of the Brassicales. After feeding on Arabidopsis thaliana plants, faeces of Spodoptera littoralis larvae contained glutathione conjugate derivatives (cysteinylglycine- and cysteinyl-isothiocyanate-conjugates) of the plant's major glucosinolate hydrolysis product, 4-methylsulfinylbutyl isothiocyanate. When caterpillars fed on leaves of A. thaliana containing [¹⁴C]₄-methylsulfinylbutyl glucosinolate, more than half of the ingested radioactivity was excreted as the unmetabolised corresponding isothiocyanate, and only 11% as glutathione conjugate derivatives. However, these conjugates were demonstrated to be the major metabolites of isothiocyanates in S. littoralis, and their abundance was shown to correlate with the amount of isothiocyanates ingested. Analysis of larval faeces from several species of generalist lepidopterans (Spodoptera exigua, S. littoralis, Mamestra brassicae, Trichoplusia ni and Helicoverpa armigera) fed on different Brassicaceae revealed that glutathione conjugates arise from a variety of aliphatic and aromatic isothiocyanates derived from dietary glucosinolates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)