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Abstract

Two-component activated chemical defenses are a major part of many plants’ strategies
to disrupt herbivory. The activation step is often the b-glucosidase-catalyzed removal of
a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While
some b-glucosidases have been well studied, several aspects of their roles in vivo, such
as their precise sites of enzymatic activity during and after ingestion, and the importance
of particular isoforms in plant defense are still not fully understood. Here, plant defensive
b-glucosidases from maize, white mustard and almonds were shown to resist digestion
by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the
ingested activities toward both general and plant pro-toxic substrates was recovered
in the frass. Among other proteins potentially involved in defense, we identified specific
plant b-glucosidases and a maize b-glucosidase aggregating factor in frass from plantfed
insects using proteomic methods. We therefore found that, while S. littoralis larvae
efficiently degraded bulk food protein during digestion, b-glucosidases were among a
small number of plant defensive proteins that resist insect digestive proteolysis. These
enzymes remain intact in the gut lumen and frass and can therefore further catalyze
the activation of plant defenses after ingestion, especially in pH-neutral regions of the
digestive system. As most of the ingested enzymatic activity persists in the frass, and
only particular b-glucosidases were detected via proteomic analyses, our data support
the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in
defense in vivo.