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NOVEL ANTIFUNGAL PROTEINS : APPLICATIONS IN CROP PROTECTION

Objective

The project is aimed at the development of new molecular strategies for the control of pathogenic plant fungi. Genetic engineering will be used as a tool to confer antifungal protein production on crop plants. The basis of the approach is the discovery of a lectin-like protein (UDA) with a broad antifungal activity spectrum in the wild plant Urtica dioica (stinging nettle), along with the indication from screening experiments that other plant species may also contain unique antifungal proteins.
The project aims to widen the field of application of deoxyribonucleic aid (DNA) recombinant technology to include development of transgenic plants that are resistant to phytopathogenic fungi. The basis of this work is an extensive screening of wild and cultivated plants for the presence of proteins with antifungal activity.

After isolation and characterization of the active proteins and their corresponding genes, the genes can be transferred in a purposeful way to cultivars in order to increase their resistance to fungi. At present' about 680 plant species have been processed through the screening programme. 12 promising plant species have been retained and their antifungal proteins have been intensively studied at the molecular level. The purified proteins are inhibitory to the growth of a broad spectrum of plant pathogenic fungi, but in contrast, they have no adverse effects on cultured mammalian cells. Genes encoding antifungal proteins were cloned from 3 different plant sources and transgenic tobacco plants are now being constructed that overexpress these proteins. This will allow the evaluation of the level of resistance against challenge inoculation with tobacco pathogens.

The project is aimed at the further development of new molecular strategies for the control of plant pathogenic fungi. Purified proteins (Mj-AFP and Rs-2S) were tested for in vivo antifungal activity on 5 plant-pathogen combinations. Wheat-Septoria nodorum and grapevine-Plasmopara viticola were successfully controlled by the Mj-AFPs, whereas the Rs-2S proteins were not active in these tests.
The proteins did not cause phytotoxic effects throughout the in vivo tests.
The Mj-AFPs were tested on 10 different plant pathogenic fungi in vitro. All of them were successfully inhibited.
The Mj-AFP's were tested on 3 different plant pathogenic bacteria but failed to show antibacterial activity, suggesting a high degree of specificity for fungi.
The Mj-Afp's were tested for insect paralysing activities and possible effects on nerve transmission. Injection into 3 different insects at high doses did not result in adverse effect on the insects. Moreover, electrophysiological test performed with isolates from insect nerves showed that the Mj-AFP's do not have neurotoxic properties.

The project is aimed at the further development of new molecular strategies for the control of plant pathogenic fungi.
Vegetative tissues from indoor and outdoor plant supplies have been collected and stored by -80 C freezing.
Seeds have been collected from outdoor collections.
Large scale growth of Bifora testiculata for antifungal protein isolation.

The project is aimed at the further development of new molecular strategies for the control of plant pathogenic fungi. 462 different plant species were screened by testing extracts for antifungal activity against Fusarium culmorum, Botrytis cinerea, Rhizoctonia solani and Pyrenophora tritici-repentis. 17 % of the seed extracts and 3% of the vegetative tissue extracts inhibited 2 or more fungi completely after 3 days of incubation. The pufified antifungal proteins fall into one of six distinct protein families. The genes encodiing these proteins have been cloned and transferred to transgenic plants. These plants are being evaluated for enhanced resistance against fungal diseases.
The tasks of the research work are as follows:

Screening for antifungal proteins. To extend the knowledge on antifungal plant proteins, a systematic screening will be performed on about 500 plant species. Four different phytopathogenic fungi will be used in initial tests to detect antifungal activity. The screening procedure will allow the elimination of ubiquitous antifungal proteins, such as chitinases and glucanases.

Isolation and evaluation of antifungal proteins. After selection of the most active plant extracts, antifungal proteins will be isolated on a preparative scale by FPLC and/or affinity chromatography. Physical and chemical characterization of the purified proteins will be carried out. Their antifungal activity spectrum will be determined by in vitro and in vivo tests, and their possible toxic effects on animals will be investigated.

Cloning of antifungal protein genes. cDNA clone banks will be constructed. Genes encoding antifungal proteins will be selected by oligonucleotide probes and/or immunoprobes.

Plant transformation and evaluation. Coding sequences of the genes will be coupled to adequate regulatory sequences and the constructs transferred to crop plants. Transformation experiments will be directed towards tomato as a model system, but could be extended to other crop plant systems. Transgenic plants will be tested for resistance against pathogens and for genomic stability.

Benefits which are expected from the proposed disease control strategy, based on the use of engineered resistant lines possibly in combination with a more rational and refined chemical protection, include reduction of the concerns for effects on man and environment from agrochemicals; reduction of the crop production costs entailed by field spraying; reduction of selection pressure on fungal pathogens; because of a better confinement of the active substances, new possibilities for the control of vascular and soil pathogens (by regulation of the expression of fungitoxic proteins in vascular and root tissue, respectively); and new possibilities for the cheaper and safer control of postharvest fungal diseases (by regulation of the expression of antifungal protein during crop maturation).

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

KATHOLIEKE UNIVERSITEIT LEUVEN
Address
Oude Markt 13
3000 Louvain / Leuven
Belgium

Participants (1)

IMPERIAL CHEMICAL INDUSTRIES PLC
United Kingdom
Address
Millbank Imperial Chemical House
SW1P 3JF London