Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes

Abstract

Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

Digestive Duet: Midgut Digestive Proteinases of Manduca sexta Ingesting Nicotiana attenuata with Manipulated Trypsin Proteinase Inhibitor Expression

“…It is well established that insects respond to high dietary PI levels either by producing proteinases of similar substrate specificity that are sterically insensitive to the inhibitor or by degrading the inhibitors [24], [25], [27], [63]. For example, H. armigera larvae that ingest high levels of serine PI proteins increase the accumulation of transcripts and proteins of not only midgut serine proteinases (trypsin/chymotrypsin) [21], but also proteinases that are insensitive to the inhibitors that digest the ingested PI proteins [26].…”

“…The most widespread strategies insects use to counter PIs is to produce proteases that are insensitive to the inhibitor [23]–[25] and/or to proteolytically inactivate PIs with midgut proteases [26], [27]. Evidence for the effects of PIs on gut proteinases comes from experiments with insects that fed on plants heterologously expressing pi genes or artificial diets containing PIs; no study to date has altered the expression of an endogenous pi gene in a host plant to examine its effect on lepidopteran digestive enzymes.…”

Mechanism of Resistance in Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae)

“…In C. maculatus , the expression of scN-insensitive CmCatB occurs through the regulation of positive HNF-4 and negative CmSvp factors (Zhu-Salzman et al, 2003). Bruchids have developed adaptation to toxic compounds by the over-production of various enzymes including glutathione S -transferases, cytochrome P450 monooxygenases (P450s) and esterases (Zhu-Salzman et al, 2003; Ahn et al, 2004, 2007; Chi et al, 2009). Some molecular understanding of bruchid adaptations to plant secondary metabolites has been carried out but the upstream regulatory mechanism(s) that induce the transcriptional and post-transcriptional regulation in bruchids when fed on legumes including mungbean is limited.…”

Antagonistic Regulation, Yet Synergistic Defense: Effect of Bergapten and Protease Inhibitor on Development of Cowpea Bruchid Callosobruchus maculatus

“…Attempts to use protease inhibitors in transgenic crops, however, have been largely unsuccessful because insects rapidly adapted to the presence of inhibitors in their diet. Strategies utilized by insects include (i) overproduction of digestive proteases to out-titer the inhibitors in insects [21]–[23]; (ii) increased expression of inhibitor-insensitive protease isoforms [24]–[26]; and (iii) activation of proteases that hydrolyze and thus detoxify plant inhibitors [27]–[29].…”