Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Abstract

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.

Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins

“…Inclusion of PMC in the diet of such insects at levels found in tubers significantly inhibits their growth (Orr et al, 1994). Hence, protease inhibitors in general are valued as potential insecticides (Ryan, 1990;Jongsma and Bolter, 1997;Schuler et al, 1998;Zavala et al, 2004), and transformation of plants with genes encoding Cys protease inhibitors imparts resistance against select insect pests (Irie et al, 1996).…”

Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job's Tears (Coix lacryma-jobiL. var.Ma-yuenStapf)

“…12) Transgenic rice into which a corn cystatin gene has been introduced inhibits insect-gut proteinases. 13) After the identiˆcation and characterization of other phytocystatins, it may be possible to use some to confer insect pest resistance on crop plants.…”

Molecular analysis of maize cystatin expression as fusion product in Escherichia coli

“…Various physiological functions have been attributed to phytocystatins that are mostly described in regulation of endogenous proteinases during seed maturation and germination (Arai et al 2002;Martinez et al 2005b;Hong et al 2007), participating in defense mechanism against attack by insects and nematodes that generally contain cysteine proteinases in their guts (Irie et al 1996;Urwin et al 1997;Pernas et al 1998;Rahbé et al 2003), inhibition of viral infections (Gholizadeh et al 2005;Campos et al 1999;Cipriani et al 2000;Shah and Bano 2009) and modulating programmed cell death (PCD) during plant development, senescence and HR responses (Belenghi et al 2003;Solomon et al 1999). Moreover, the most recent findings signify the involvement of abiotic environmental stresses such as drought and salt in phytocystatins induction (Gaddour et al 2001;Corre-Menguy et al 2002;Diop et al 2004;Petya et al 2007;Girard et al 2007).…”