A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

Abstract

SummaryA defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed.Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

“…Together, disease resistance assays demonstrated that expression of the mpi‐pci fusion gene in rice, either the mpi‐2A‐pci or the mpi‐C‐pci gene, confers enhanced resistance against the rice blast fungus M. oryzae . Furthermore, these results indicated that the MPI‐C‐PCI fusion protein might be effective for inhibition of fungal proteinases (as it was previously demonstrated for PCI; Quilis et al ., ). Knowing that the fusion protein is partially processed in rice leaves of mpi‐2A‐pci (see Figure a), the contribution to the phenotype of disease resistance that is observed in these plants might be due to the activity of the MPI‐2A‐PCI protein and the PCI protein originating from the fusion protein.…”

“…(b) Affinity chromatography on immobilized CPA of pure PCI (Sigma) and protein extracts from rice plants constitutively expressing pci (line 7.34) , mpi‐C‐pci plants (line 7) and mpi‐2A‐pci (line 7) plants. Eluted fractions were analysed for inhibition of bovine CPA activity using AAFP as the substrate (Quilis et al ., ). Lanes E1a to E1c, fractions eluted using buffer at pH 9.0.…”

“…On the other hand, results previously reported by our group indicated that production of the PCI inhibitor alone in the rice plant might be counterproductive in terms of insect resistance, as the weight gain of C. suppressalis larvae fed on rice plants constitutively expressing the pci gene was significantly larger than that of larvae fed on wild‐type plants (Quilis et al ., ). It can then be hypothesized that the production of the two plant inhibitors, MPI and PCI, compensates the negative effects that occur in C. suppressalis larvae fed on rice plants expressing the pci gene alone.…”

“…PCI inhibits digestive carboxypeptidase activities from the lepidopteran insect Helicoverpa armigera (Bay es et al, 2003). However, the constitutive expression of pci in rice has an apparent stimulatory effect on the growth of C. suppressalis larvae fed on the pci transgenic plants, suggesting an adaptive mechanism in the larval gut to compensate for the effect of PCI ingestion (Quilis et al, 2007).…”

Cloning, characterization and expression analysis of a novel gene encoding Kunitz-type protease inhibitor from Dolichos biflorus

“…The gene size and coding regions of these inhibitors are small, devoid of introns (Wang et al 2008) and comprise readily identifiable core region covering the invariant cysteine residues. The potential of PI has been demonstrated by the transfer of these genes from different sources to several plants of economic interest, resulting in transgenic plants more resistant to predation (Lingling et al 2005; Pujol et al 2005) and pathogens (Qu et al 2003; Quilis et al 2007). …”