Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs

• Published in: Planta 1998-04, Vol.204 (4), p.472-479
• Main Authors: Urwin, P.E, McPherson, M.J, Atkinson, H.J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Additive effects; Agronomy. Soil science and plant productions; alpha-Amylases - antagonists & inhibitors; Animals; Antibodies; Arabidopsis - immunology; Arabidopsis - parasitology; ARABIDOPSIS THALIANA; Biological and medical sciences; Body Constitution; CELL MEMBRANES; CISTEINA; Cyst nematodes; Cystatins; Cystatins - genetics; Cystatins - metabolism; Cystatins - physiology; CYSTEINE; Cysteine Proteinase Inhibitors - genetics; Cysteine Proteinase Inhibitors - metabolism; Cysteine Proteinase Inhibitors - physiology; ENZIMAS; ENZYME; ENZYMES; ENZYMIC ACTIVITY; Escherichia coli - metabolism; Fertility; Fundamental and applied biological sciences. Psychology; GENE; Gene Expression; GENES; Genetics and breeding of economic plants; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; Memory interference; NEMATODA; Nematoda - metabolism; Nematoda - physiology; NEMATODENRESISTENZ; Nematodes; Pest animals; PEST RESISTANCE; Plant parasitic nematodes; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Proteins - physiology; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants; Plants, Genetically Modified; PLASMAMEMBRAN; PROTEASAS; PROTEASE; Protease inhibitors; PROTEASES; PROTEINASE; RESISTANCE AUX ORGANISMES NUISIBLES; RESISTENCIA A LAS PLAGAS; SERINA; SERINE; TRANSGENIC PLANTS; Trypsin Inhibitors; Tylenchoidea - metabolism; Tylenchoidea - physiology; Varietal selection. Specialized plant breeding, plant breeding aims
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s004250050281

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-IΔD86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.