Coordination of Hepatocyte Nuclear Factor 4 and Seven-up Controls Insect Counter-defense Cathepsin B Expression

Abstract

CmCatB, a cathepsin B-type cysteine protease, is insensitive to inhibition by the soybean cysteine protease inhibitor (scN). Cowpea bruchids dramatically induce CmCatB expression when major digestive proteases are inactivated by dietary scN, which is presumably an adaptive strategy that insects use to minimize effects of nutrient deficiency. In this study, we cloned the cowpea bruchid hepatocyte nuclear factor 4 (CmHNF-4) and demonstrated its involvement in transcriptional activation of CmCatB in the digestive tract of scN-adapted bruchids. Electrophoretic mobility shift assays demonstrated that CmHNF-4 binds to a CmCatB promoter region containing two tandem chicken ovalbumin upstream promoter (COUP) sites, which is also the cis-element for Seven-up (CmSvp), a previously identified transcriptional repressor of CmCatB. Although CmSvp is predominantly expressed in unadapted insect midgut, CmHNF-4 is more abundant in adapted bruchids. The battle between plants and herbivorous insects employs a variety of defense and counter-defense strategies. Insect feeding elicits synthesis of a wide array of plant-defensive compounds, which in turn induce insect counteractive tactics to minimize the impact of plant defense. Analysis of these plantpest interactions has become a research hotspot for understanding insect counter-defense mechanisms. Many insect survival mechanisms involve remodeling of the insect digestive system. Adjustments in cells lining the digestive tract allow pests to deal with a variety of plant toxins and anti-nutritional metabolites and are crucial for insect survival.A number of studies on insect response to plant protease inhibitors indicate that one of the tactics used by insects is activation of inhibitor-insensitive digestive enzymes (1-7). Under regular growth conditions, these inhibitor-insensitive enzymes are often in low abundance, accounting for only a minor portion of the entire proteolytic activity. They can be strongly induced, however, and play an important role in protein degradation once the major digestive enzyme function is blocked. The cowpea bruchid (Callosobruchus maculatus) cathepsin B-like cysteine protease (CmCatB) 2 has been shown to be such an enzyme. The cowpea bruchid is a grain storage insect pest; its larvae feed and develop inside seeds. Adults no longer feed after emergence from infested seeds but mate and lay eggs on the surface of cowpea seeds. Multiple generations of the insect per year lead to severe grain damage.CmCatB, which is solely expressed in larvae (6), was identified as one of the midgut genes that was most highly induced by dietary scN in a cDNA microarray, although it was undetectable in insects feeding on regular diet (8). Expression of CmCatB protein revealed that it possesses cathepsin B-like cysteine protease activity. Interestingly, this proteolytic activity was unaffected by scN, in marked contrast to the major cathepsin L-like digestive enzymes that share similar tertiary structures with CmCatB (6, 9). Most likely, the occluding loop in CmCatB blocked ...

Discrimination of Differentially Inhibited Cysteine Proteases by Activity-Based Profiling Using Cystatin Variants with Tailored Specificities

“…Conclusive cases of insect resistance have been reported for a number of plants expressing recombinant protease inhibitors, but the pesticidal effects of these proteins proved negligible in many other instances despite protease inhibitory effects observed in vivo . Insect herbivores have evolved a range of strategies to cope with dietary protease inhibitors, notably involving complex digestive systems with proteases from diverse functional classes and families, − target protease overexpression to outnumber the inhibitory proteins, , and the production of protease isoforms weakly sensitive to the inhibitors ingested. ,− Other adaptations to protease inhibitors include their degradation by nontarget midgut proteases, ,− the up-regulation of proteases from alternative functional classes, ,− and a reallocation of metabolic resources toward inhibitor-induced compensatory responses. , It is now well established that protease–inhibitor interactions in plant–insect systems are the result of a long, coevolutive process triggering the continuous diversification of insect protease and plant protease inhibitor functions. − In most, if not all, cases the ectopic expression of a protease inhibitor in planta not only alters the activity of constitutively expressed target proteases in the midgut of naive herbivores but also induces a significant remodelling of both the midgut transcriptome and digestive protease complement. ,,, …”