Glucosylation prevents plant defense activation in phloem-feeding insects

Abstract

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.

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Fig. 1: Activation and metabolism of 4-methylsulfinylbutyl glucosinolate (4msob-GSL) in the whitefly Bemisia tabaci.
Fig. 2: 4msob-GSL metabolites in B. tabaci whitefly honeydew.
Fig. 3: Elucidation of the biochemical mechanism for B. tabaci whitefly glucosylation of GSLs.
Fig. 4: Chromatographic analyses of products from BtMEAM1 SUC1–5 enzymes heterologously produced in D. melanogaster S2 cells.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding authors upon reasonable request. Public databases used in the construction of the protein phylogenetic tree are provided in Supplementary Table 7 and are available from the following websites: HGSC (https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project), BIPAA (https://bipaa.genouest.org/sp/acyrthosiphon_pisum/), Whitefly Genome Database (http://www.whiteflygenomics.org/cgi-bin/bta/index.cgi), BIPAA (https://bipaa.genouest.org/sp/myzus_persicae/), NCBI (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Bombyx_mori/101/), HGSC (https://www.hgsc.bcm.edu/arthropods/tobacco-hornworm-genome-project), NCBI (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Pieris_rapae/100/), NCBI (https://www.ncbi.nlm.nih.gov/assembly/GCA_000697945.4), Ensembl (http://metazoa.ensembl.org/Tetranychus_urticae/Info/Index) and dbCAN (http://bcb.unl.edu/dbCAN/). All other data supporting this work, if not already indicated, are available in the Supplementary Information.

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Acknowledgements

We thank A. Douglas (Cornell University) for the SUC1 sequence, K. Falk for assistance with graphics, the MPI-CE, DSMZ and HUJI greenhouse teams for plant and insect maintenance, and other members of the African Cassava Whitefly Project (cassavawhitefly.org) for helpful discussions. This work was supported financially by the Max Planck Society, the Deutsche Forschungsgemeinschaft (DFG Collaborative Research Center 1127 ChemBioSys) and the Natural Resources Institute, University of Greenwich from a grant provided by the Bill and Melinda Gates Foundation (OPP1058938).

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The authors contributed in the following manner: conceptualization (O.M., M.L.A.E.E., J.G., S.M. and D.G.V.); direction and supervision (D.G.V., J.G., O.M. and S.M.); funding acquisition (J.G., J.C., S.M. and D.G.V.); provision of insects and genetic materials (O.M., M.G., B.S., S.W., K.L., A.S., L.L.M., J.C. and S.S.); chemical analysis of honeydew by LC-MS (M.L.A.E.E., D.G.V., M.R., A.S. and O.M.); purification of insect-derived products (M.L.A.E.E. and D.G.V.); execution and interpretation of the NMR analyses of the purified products (C.P.); carrying out labeled artificial diet feedings (M.L.A.E.E., D.G.V., M.G., B.S. and S.W.); performance of phylogenetic analysis (K.J. and O.M.); estimation of gene expression (D.S.-G. and O.M.); cloning of candidate GH13 genes, generation of recombinant proteins and performance of enzyme assays (M.L.A.E.E., with contributions from K.L.); development of methods (M.L.A.E.E., D.G.V., M.R., C.P. and O.M.); drafting of the manuscript (M.L.A.E.E. and D.G.V., with input from J.G., O.M. and S.M.).

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Correspondence to Osnat Malka or Daniel G. Vassão.

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Extended data

Extended Data Fig. 1 Bemisia tabaci glucosylates GSLs with various side chains.

LC-MS traces of allyl-GSL, 4mtb [4-methylthiobutyl]-GSL, 3msop [3-methylsulfinylpropyl]-GSL and 4moi3m [4-methoxyindolyl-3-methyl]-GSL and their glycosides detected in the combined honeydew of 50-100 adult Bemisia tabaci MEAM1 whiteflies feeding on GSL-containing plants (kale or A. thaliana Col-0). The detected parent GSL is indicated with structure and representative color for the mass spectral trace (light gray), and the dectected subsequent glycosides represented as +162 Da and +324 Da (dark gray and black respectively).

Extended Data Fig. 2 GSL glucosylation in the whitefly Bemisia tabaci is catalyzed by a transglucosidase activity.

a, Simplified reaction mechanism of a sucrase-transglucosidase showing the two competing reaction paths: After binding of sucrose to the enzyme (A), hydrolysis of the fructose residue occurs with retention of bound glucose (B). Glucose is released (C) when sucrose concentrations are low, while transglucosidation to an acceptor (C’) occurs when acceptor concentrations are sufficiently high. This product may undergo further transglucosidation (D). b, Depiction of the results from two of the five diets not shown in Fig. 3, those diets with the 13C-labeled monosaccharides glucose and fructose. None gave labeled glycosylated GSLs, unlike feeding with sucrose labeled in the glucose portion. The results are consistent with a transglucosidase activity that initially hydrolyzes sucrose and links the resulting glucose moiety to the plant GSL.

Extended Data Fig. 3 Maximum likelihood circular cladogram showing the relationship of glycoside hydrolase family 13 enzymes from nine chosen herbivore species.

The tree was inferred using a total of 205 sequences. Ultrafast bootstrap45 and Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT)46 validation values lower than 95 are presented close to the corresponding nodes. GH13 members from Bemisia tabaci are highlighted by red nodes. SUC1–5 are indicated by bold text in red. Colors surrounding the cladogram indicate the feeding guild of the corresponding species. Thin colored inner circles specify the subfamily of the corresponding GH13 enzymes. Subfamilies with less than four proteins are marked only by the subfamily number (for more details see Supplementary Table 8). The protein sequences are named according to their GenBank accession numbers, or their names in the released proteome. Species name abbreviations are as indicated: Ld, Leptinotarsa decemlineata; ACYP, Acyrthosiphon pisum; BT, Bemisia tabaci; MPER, Myzus persicae; Bm, Bombyx mori; Msex, Manduca sexta; Prapae, Pieris rapae; FOCC, Frankliniella occidentalis; Tetur, Tetranychus urticae.

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Supplementary Figs. 1 and 2, Tables 1–10 and Note 1.

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Malka, O., Easson, M.L.A.E., Paetz, C. et al. Glucosylation prevents plant defense activation in phloem-feeding insects. Nat Chem Biol 16, 1420–1426 (2020). https://doi.org/10.1038/s41589-020-00658-6

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