Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

Hosoyama, Hiroshi; Irie, Kentaro; Abe, Keiko; Arai, Soichi

doi:10.1007/bf00193714

Cited by 11 publications

(3 citation statements)

References 19 publications

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“…In practice, the addition of any fusion partner to the primary structure of a proteinase inhibitor to increase its biological activity may be useful as long as its inhibitory efficiency is not altered negatively. Previous studies suggested a functional stability of the model cystatin, OCI, after fusion partners such as GST or β-glucuronidase were fused at its C or N termini (Michaud et al, 1994;Hosoyama et al, 1995;Tudyka and Skerra, 1997). In line with these observations, the data presented here, using tomato CDI as a fusion partner, strongly suggest that plant cystatins such as OCI or CCII may retain full inhibitory activity against insect cysteine proteinases when a fusion partner is attached at the N terminus.…”

Section: September 2005supporting

confidence: 88%

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Brunelle

Girard

Cloutier

et al. 2005

Arch. Insect Biochem. Physiol.

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Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.

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Section: September 2005supporting

confidence: 88%

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Brunelle

Girard

Cloutier

et al. 2005

Arch. Insect Biochem. Physiol.

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“…1987b). However, we need further experimentation to confum in a final manner the safety of the transgenic rice whose cystatin content has been enriched artificially (Hosoyama et al 1994;Irie et al 1996).…”

Section: Insect Resistancementioning

confidence: 99%

Phytocystatins and Their Target Enzymes: From Molecular Biology to Practical Application: A Review

Arai

Matsumoto

Abe

1998

J Food Biochemistry

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Cystatins are protein inhibitors of cysteine proteinases. Oryzacystatin I (OC‐I) has been cloned and is apparently the first well‐defined cystatin of plant origin (phytocystatin). Besides OC‐I, various other phytocystatins occur in rice, corn, wheat and soybean. Phytocystatins have endogenous target enzymes whose expression is induced by gibberellin, and also exogenous target enzymes from viruses and insects. It is thus likely that phytocystatins are involved in the regulation of intracellular protein catabolism as well as in the defense from infection. Better understanding of this phenomenon and cloning of appropriate genes will facilitate the creation of transgenic crops with enhanced resistance to viruses and insects.

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“…Plant transformation is an effective method for obtaining crop plants protected from their pests and diseases, Therefore, we have attempted the introduction of the OC gene and its overexpression in the resultant transgenic rice plants [12,13]. However, the level of expression of the introduced OC gene was still insufficient for the effective protection against insect pests.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1996

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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.

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Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

Cited by 11 publications

References 19 publications

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Phytocystatins and Their Target Enzymes: From Molecular Biology to Practical Application: A Review

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

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