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. When transiently expressed in Drosophila S2 cells, CmHNF-4 substantially increased CmCatB expression through COUP binding. CmSvp inhibited CmHNF-4-mediated transcriptional activation even in the absence of its DNA-binding domain. Thus antagonism resulted, at least in part, from protein-protein interactions between CmSvp and CmHNF-4. Association of the two transcription factors was subsequently confirmed by glutathione S-transferase pulldown assays. Interestingly, anti-CmHNF-4 serum caused a supershift not only with nuclear extracts of scN-adapted insect midgut but with that of unadapted control insects as well. The presence of CmHNF-4 in unadapted insects further supported the idea that interplay between CmSvp and CmHNF-4 controls CmCatB transcription activation. Together, these results suggest that coordination between CmHNF-4 and CmSvp is important in counter-defense gene regulation in insects.
Highlights
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)
We demonstrated that specific association of a chicken ovalbumin upstream promoter (COUP)-element and cis-element for Seven-up (CmSvp), a homolog of the COUP-interacting transcription factor (COUP-TF), resulted in repression of CmCatB (12)
Because a DNA-protein complex was formed in soybean cysteine protease inhibitor (scN)-challenged insects where CmCatB was highly induced, it seemed reasonable to hypothesize that this nuclear protein could serve as a trans-activator that enhanced CmCatB expression through binding to target promoter elements
Summary
Insect Treatment and Midgut Nuclear Extract—Recombinant scN was expressed in Escherichia coli BL21 (DE3) and purified via Ni2ϩ-chelating Sepharose affinity chromatography (Amersham Biosciences) as previously described (7). Freshly dissected midguts free from gut contents were placed in a pre-chilled, sterile Dounce homogenizer, and homogenized in cold hypotonic buffer with 1 mM DTT and 0.1% (v/v) detergent supplied by the manufacturer. The cell pellet was resuspended in 50 l of hypotonic buffer, incubated on ice for 15 min, and lysed by adding detergent to 1% (v/v) followed by vigorous vortexing for 10 s and centrifugation for 30 s at 14,000 ϫ g. Nuclear proteins were extracted by gently shaking on ice for 30 min. After centrifugation for 10 min at 14,000 ϫ g, the supernatant (containing nuclear proteins) was transferred to a new tube in aliquots and stored at Ϫ80 °C till use. Probes P1 to P5 were randomly labeled with 1.5 M [␣-32P]dCTP by PCR amplification (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s for 25 cycles)
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