Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs |
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Authors: | Peter E Urwin Michael J McPherson Howard J Atkinson |
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Institution: | (1) Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds, LS2 9JT, UK, GB |
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Abstract: | 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.
Received: 3 July 1997 / Accepted: 8 August 1997 |
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Keywords: | :Arabidopsis Galactose oxidase Metallothionein Metallothionein-like protein (PsMTa) Nematode Proteinase inhibitor |
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