Seven‐up facilitates insect counter‐defense by suppressing cathepsin B expression

Abstract

Herbivorous insects are constantly challenged by a broad spectrum of toxins and antinutritional factors produced by their host plants. The insect alimentary tract thus becomes the front line of insect counter-defense. It actively responds to dietary challenges by readjusting expression of its transcriptome and changing the repertoire of proteins in cells that line the digestive tract. Insect digestive enzymes, broadly classified into serine, cysteine, aspartate and metallo-proteases [1], play an important role in protecting the vulnerable cells and tissues of the insect body, in addition to functioning in food breakdown. The cowpea bruchid Callosobruchus maculatus dramatically remodels its profile of midgut digestive enzymes in response to the When challenged by the dietary soybean cysteine protease inhibitor scN, the cowpea bruchid (Callosobruchus maculatus) adapts to the inhibitory effects by readjusting the transcriptome of its digestive system, including the specific activation of a cathepsin B-like cysteine protease CmCatB. To understand the transcriptional regulation of CmCatB, we cloned a portion of its promoter and demonstrated its activity in Drosophila cells using a chloramphenicol acetyltransferase reporter system. EMSAs detected differential DNA-binding activity between nuclear extracts of scN-adapted and -unadapted midguts. Two tandem chicken ovalbumin upstream promoter (COUP) elements were identified in the CmCatB promoter that specifically interacted with a protein factor unique to nuclear extracts of unadapted insect guts, where CmCatB expression was repressed. Seven-up (Svp) is a COUP-TF-related transcription factor that interacted with the COUP responsive element. Polyclonal anti-(mosquito Svp) serum abolished the specific DNA-binding activity in cowpea bruchid midgut extracts, suggesting that the protein factor is an Svp homolog. Subsequent cloning of a cowpea bruchid Svp (CmSvp) indicated that it shares a high degree of amino acid sequence similarity with COUP-TF ⁄ Svp orphan nuclear receptor family members from varied species. The protein was more abundant in scN-unadapted insect guts than scN-adapted guts, consistent with the observed DNA-binding activity. Furthermore, CmCatB expression was repressed when CmSvp was transiently expressed in Drosophila cells, most likely through COUP binding. These findings indicate that CmSvp may contribute to insect counter-defense, in part by inhibiting CmCatB expression under normal growth conditions, but releasing the inhibition when insects are challenged by dietary protease inhibitors.Abbreviations CAT, chloramphenicol acetyltransferase; CmCatB, Callosobruchus maculatus cathepsin B-like cysteine protease; COUP, chicken ovalbumin upstream promoter element; COUP-TF, COUP-transcription factor; DBD, DNA-binding domain; 20-E, 20-hydroxyecdysone; LBD, ligandbinding domain; scN, soybean cysteine protease inhibitor; Svp, Seven-up.

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

“…Probes P6 to P8 resulted from annealing two complementary oligonucleotides. These oligonucleotides were first end-labeled separately with 0.73 M [␥-32 P]ATP using T4 DNA polynucleotide kinase, then mixed in complementary pairs as previously described (12). After annealing, the double-stranded oligonucleotide probes were purified with QIAquick Nucleotide Removal kit (Qiagen).…”

“…We detected retarded bands of different mobility in control (scN-unadapted) and scN-fed (-adapted) insects, which implies that different binding proteins might be interacting with the CmCatB promoter in the two insect groups. In unchallenged control insects, we demonstrated that specific association of a COUP-element and CmSvp, a homolog of the COUP-interacting transcription factor (COUP-TF), resulted in repression of CmCatB (12). Its high abundance in unadapted insects and reduced expression in adapted insects supported the repressor function.…”

“…Expression Plasmid Construction-All the chloramphenicol acetyltransferase (CAT) reporter plasmids, pAc-CatB/CAT, pAc-CatB⌬COUP/CAT, pAc3075, and pAc-IE1/CAT, as well as the expression plasmid pAc5-CmSvp, have been previously described (12). To make the pAc5-CmHNF-4 construct, the entire coding region of CmHNF-4 was amplified by PCR (95°C for 30 s, 68°C for 2 min for 35 cycles) using the following primers: sense, 5Ј-AAGCTGAATTCCAAAAGATGCCTTC-CTCGT-3Ј; and antisense, 5Ј-GCTCACTCGAGAAAAC-TATGCTGCTCCTCCTT-3Ј.…”

“…Transient Transfection and CAT Assays-Transfection experiments were performed as previously described (12). Drosophila Schneider 2 (S2) cells were maintained in Shields and Sang M3 insect medium (Sigma) with supplements at 27°C.…”

“…In an effort to understand CmCatB transcriptional regulation, we cloned a CmCatB promoter and adopted electrophoretic mobility shift assays (EMSAs) to identify potential regulatory elements using nuclear extracts from bruchid midgut tissue (12). We detected retarded bands of different mobility in control (scN-unadapted) and scN-fed (-adapted) insects, which implies that different binding proteins might be interacting with the CmCatB promoter in the two insect groups.…”