Inhibition of plant pathogen adhesion and virulence by single chain recombinant antibodies

A project undertaken at the Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra and supervised by Adrienne Hardham

Plant pathogens in the genus Phytophthora cause major diseases of a wide variety of plants including important agricultural and horticultural species as well as thousands of Australian native plants (Figs 1 & 2; Hardham, 2005). One of the critical steps in the establishment of disease in plants, as it is in animals, is the attachment of pathogen cells to the host surface (Fig. 3). In previous studies, we identified the adhesive protein responsible for attachment of Phytophthora spores to the surface of host plants. We were able to identify the adhesin protein and ultimately clone the gene that encoded it by using monoclonal antibodies we generated that specifically recognised the adhesin protein (Fig. 4; Hardham and Gubler, 1990; Robold and Hardham, 2004; Robold and Hardham, 2005).

It has been shown that antibodies directed towards adhesive proteins produced by some animal pathogens, can inhibit their adhesive function and decrease the pathogen’s ability to infect its host. This observation suggested to us that the resistance of plants to Phytophthora pathogens might be increased if they could make and secrete antibodies directed towards the Phytophthora adhesin. As the first step in this approach, we developed single-chain antibodies that reacted with the Phytophthora adhesin. In the project supported by the Hermon Slade Foundation, we have evaluated the use of single-chain and conventional antibodies for their ability to inhibit pathogen spore adhesion and virulence.

In this project, we found that instability of the single-chain antibodies interfered with their ability to recognise the Phytophthora adhesive, thus detracting from their use to inhibit spore adhesion. We developed in vitro and in vivo assays to quantitatively measure pathogen spore adhesion. The in vitro assay revealed that the spores apparently release a variety of adhesive molecules during their attachment to the plant. The ability of the spores to adhere to hydrophobic surfaces, for example, appears not to involve the adhesin we have cloned and characterised. Nevertheless, using the in vivo assay we were able to show that monoclonal antibodies we had generated can indeed inhibit the pathogen’s ability to infect normally-susceptible host plants, presumably through an inhibition of spore adhesion (Fig. 5). This exciting outcome, achieved through the support of the Hermon Slade Foundation, will form the basis of further studies of the role of the Phytophthora spore adhesin in plant infection. The bioassay will also be of great value in future studies in which we will assess the effects of silencing putative Phytophthora pathogenicity genes.

References

Hardham AR (2005) Pathogen profile: Phytophthora cinnamomi. Molecular Plant Pathology 6, 589-604.

Hardham AR, Gubler F (1990) Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube. Cell Biology International Reports 14, 947-956.

Robold AV, Hardham AR (2004) Production of monoclonal antibodies against peripheral vesicle proteins in zoospores of Phytophthora nicotianae. Protoplasma 223, 121-132.

Robold AV, Hardham AR (2005) During attachment Phytophthora spores secrete proteins containing thrombospondin type 1 repeats. Current Genetics 47, 307-15.

Figure 1. Healthy bushland in the Brisbane Ranges National Park, Victoria

Figure 2. Brisbane Ranges National Park infected with P. cinnamomi.

Figure 3. Adhesive material secreted by a P. cinnamomi spore attaches the spore to the surface of a host plant.

Figure 4. The adhesive material secreted by a Phytophthora spore is fluorescently labelled by an antibody that specifically reacts with the adhesin protein.

Figure 5. A. Lupin roots pre-treated with water or a control protein are infected by P. cinnamomi. B. Lupin roots pre-treated with the antibody directed towards the spore adhesin are protected from infection by P. cinnamomi.