AIR2-CT94-1356 Agro-Industrial Applications of Antifungal Proteins from Plant Seeds

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	Proposal No:	AIR2-CT94-1356
	Date Prepared:	September 1999
	Source:	Consolidated progress report summary September 1997

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Consolidated progress report summary September 1997

Introduction

The project aimed to:

• investigate the possibility of improving the antifungal activity of AFPs and synthetic peptides
• study the target sites of AFPs on fungi
• investigate the resistance of AFP-producing transgenic plants
• investigate the possibility of expressing multiple AFPs in a transgenic plant
• test whether the expression of an AFP in an economically-important crop species results in enhanced disease resistance

Scientific objectives

The main goal of the project is to evaluate possible agro-industrial applications of highly-potent antifungal proteins (AFPs) of plant origin. The project will focuss on four parts, two of which are aimed at increasing our basic knowledge of AFPs, while two others are devoted to the exploration of valuable applications.

The first objective is to understand the relationships between the structure and activity of AFPs using state-of-the-art technology for three-dimensional structure determination, active-site modelling and site-directed mutagenesis, as well as a new approach comprising automated chemical synthesis of thousands of variant polypeptides. This information will allow the rational design and selection of novel AFPs with improved activity or altered selectivity.

The second objective is to understand the mode of action of AFPs down to the molecular level. This knowledge will form the rational basis for the use of AFPs in defined combinations in order to increase their antifungal effects or to delay appearance of resistance symptoms in fungal organisms.

The third objective is to express the AFPs in a functional form in at least one economically-important crop, e.g. oilseed rape. The disease resistance of the transgenic crops will be evaluated both experimentally in the glasshouse, as well as by exposure to natural disease pressure in field trials. Special emphasis will be given to the development of strategies aimed at minimising the emergence of fungal resistance to AFPs, such as combined expression to two AFPs with different modes of action.

The fourth objective is to develop methods for producing AFPs or synthetic peptide derivatives of AFPs on a pilot scale, either by chemical synthesis or by fermentation, so that the feasibility of their use as preservatives in agro-industrial applications can be evaluated.

Activities

The work plan for this reporting period included purification of AFPs in large quantities from plant sources in order to supply material for performing NMR and mode-of-action studies, as well as to record H-NMR data of some synthetic peptides and to finalise structure-determination of isolated peptides. Other tasks included the synthesis and testing of linear, cyclic and bicyclic derivatives of various sequences, including those with multiple amino acid permutations; assessment of the activity of synthetic peptides against a broad range of microorganisms; and then the synthesis and testing of the activity of permutation variants of the most active peptide obtained. Further work concerned the characterisation of yeast-expressed AFP variants, as well as an assessment of the possibility of producing AFPs in yeast and cloning the resistance gene of yeast mutants resistant to plant defensin in order to characterise the plant defensin receptor. In addition, tests were carried out on homozygous transgenic oilseed rape plants expressing AFPs for resistance to Phoma lingam via detached leaf assays, while a field trial was performed using AFP-expressing plants. Transgenic plants expressing two different AFPs were analysed.

Achievements

The participants have undertaken a multi-disciplinary collaboration aimed at understanding structure-activity relationships of antifungal proteins, including Rs-AFP1, Ah-AMP1 and Ib-AMP1. These antifungal proteins belong to three different types, namely the morphogenic plant defensins (Rs-AFP2), the non-morphogenic plant defensins (Ah-AMP1) and the four-cysteine type antifungal proteins (Ib-AMP1). Elucidation of the three-dimensional structure of Rs-AFP1 by proton NMR enabled a detailed model to be established, with a RMSD value of 1.03 angstrom for the backbone atoms and 1.20 angstrom for all heavy atoms (based on the best 25 structures). These values are respectively 0.84 angstrom and 1.21 angstrom for the best 25 structures of Ah-AMP1. Ah-AMP1 and Rs-AFP1 have very similar folds with an alpha helix packed against a triple-stranded beta sheet. Three-dimensional structure analysis of Ib-AMP1 was determined and showed a U-shaped structure with three consecutive beta-turns in the core region. In addition, the structure of a synthetic bicyclic decapeptide based on a portion of Rs-AFP2 was determined and found to consist of a very short double-stranded antiparallel beta-sheet. Another synthetic peptide was found to lack a rigid conformation.

In order to define the active site of Rs-AFP2 (a homolog of Rs-AFP2 containing 51 residues) sets of overlapping 6-, 9-, 12-, 15-, 19- and 20-mer synthetic peptides were produced. These investigations led to the identification of bioactive peptides consisting of sequences corresponding to the beta2-beta-turn-beta3 region of Rs-AFP2. A highly-active 0-mer corresponding to this loop was subjected to successive rounds of substitution scans at all possible amino-acid positions. This has resulted in a range of peptides which have up to six alterations relative to the Rs-AFP2 sequence and which have strongly-increased antifungal activity; notably these peptides are more active than native Rs-AFP2 itself in high-salt media.

Synthetic peptides were also synthesised based on the amino acid sequences of other AFPs, including Ah-AMP1, Dm-AMP1 and Ib-AMP1. These peptides, together with the Rs-AFP2-based peptides, were subjected to extensive antimicrobial activity tests on over 20 different microorganisms. A synthetic peptide based on the Ib-AMP1 sequence appeared to be the most potent one and inhibited microorganisms at lower doses than Ib-AMP1 itself. The Ib-AMP1-based peptide was then subjected to substitution scans in order to further optimise its antimicrobial potency.

In the course of this project several antifungal proteins causing rapid ion fluxes (including Ca⁺-influx, K⁺-efflux and H-uptake) upon interaction with fungi were discovered. In order to further understand their mode of action, binding studies were carried out with several radio-labelled antifungal proteins. These studies indicate the presence of high-affinity binding sites for these proteins on plasma membrane preparations of Neurospora crassa and Saccharomyces cerevisiae (yeast). Yeast mutants have been generated which are highly resistant to the plant defensins Dm-AMP1, Ah-AMP1 and Ct-AMP1. These mutants show strongly-reduced binding by radio-labelled Dm-AMP1 compared to wild-type strains, suggesting that they have a receptor with altered affinity. Genetic analysis indicated that the resistance gene is carried on a single, dominant locus. Several attempts to clone this resistance gene by a complementation approach have failed so far, but these efforts will be continued. In addition, alternative biochemical approaches are being deployed.

Genes encoding antimicrobial peptides (either Dm-AMP1, Ace-AMP1 or both) have also been introduced in the economically-important crop species oilseed rape and it has been shown that the proteins are expressed at relatively high levels. A bioassay for disease resistance against Phoma lingam has been worked out. This test can distinguish between a known sensitive cultivar of oilseed rape (Westar) and a field-tested resistant one (Mikado). Extensive testing of transgenic lines has confirmed that some are more resistant than their Westar parent, but the tests did not always give consistent results in the T2 generation plants. A field trial with transgenic canola plants has been performed during the last reporting period but the results are not yet available. Several more field trials will be carried out during the last reporting period.

Conclusions

During this period three-dimensional models were produced for three different antifungal proteins. These models were used for the rational design of peptide mimetics with potent antifungal properties, some of which being clearly more potent than the authentic proteins. These peptides may be useful for initially-unexpected applications such as curing microbial diseases in humans. High-affinity binding sites for antifungal proteins on fungal membranes were identified. In addition, transgenic oilseed rape plants expressing relatively-high levels of antimicrobial proteins were produced, some lines having been found to be resistant to Phoma lingam. Plants have also been produced which express two different types of antimicrobial proteins.