Phytophthora functional genomics

Prof Adrienne Hardham

In Australia, plant pathogens in the genus Phytophthora infect and kill thousands of species of plants, including many important crop, horticultural and forest species. Rapid dissemination and the establishment of disease are facilitated by the production of motile spores that swim to potential hosts, attach to the plant surface and then invade the underlying plant tissues1. The aim of our research program is to understand the molecular and cellular basis of infection of host plants by Phytophthora, with a view to being able to apply this knowledge to the development of novel and sustainable ways of controlling Phytophthora diseases.

Complete genomic sequences of two Phytophthora species, P. infestans and P. ramorum, are now available, although annotation is not yet complete and much research is needed before the functions of many of these genes are determined. In our molecular studies, we have made several Phytophthora cDNA and genomic libraries, and have isolated and partially sequenced over 500 Phytophthora genes that are preferentially expressed at certain stages of development, in particular during the formation and growth of the spores that initiate infection2-4. We are currently investigating the function of a selection of these genes, including genes that encode proteins involved in zoospore motility, osmoregulation and signalling. We complement our molecular work with extensive experience in cell biological studies of Phytophthora spores, including ultrastructural analyses and immunocytochemical labelling. Much of our immunocytochemistry uses monoclonal and polyclonal antibodies which we generate and which serve as highly specific probes for selected Phytophthora cell components. The antibodies can be used to localise Phytophthora proteins in fluorescence microscopy or electron microscopy, and to characterise the proteins through western blotting of one- and two-dimensional gels. The molecular, cellular and biochemical approaches are fully integrated so that we can isolate genes encoding proteins identified in the structural studies and we can generate antibodies to localise the encoded proteins of genes identified in our gene-discovery program.

There are a number of potential projects in our research program that will explore the functional analysis of Phytophthora pathogenicity genes and proteins, the structure and function of Phytophthora spores and the cellular and molecular basis of plant-Phytophthora interactions. These projects include:

Characterisation of the Phytophthora spore adhesive
(Supervisors: Adrienne Hardham and Leila Blackman)

We have recently cloned the gene encoding the adhesive protein used by zoospores to attach to host plants. Although adhesion is recognised as being a key aspect of disease development, this is the first spore adhesive gene to be identified and cloned from a filamentous plant pathogen. The Phytophthora adhesin shows intriguing similarities to adhesives produced by animal cells and malarial parasites. This project will contribute to the characterisation of the Phytophthora adhesin, including analysis of gene expression and protein synthesis, the phylogenetic distribution of the adhesin and the role of conserved motifs in the adhesin protein.

Analysis of the role of zoospore motility in plant infection
(Supervisors: Adrienne Hardham and Leila Blackman)

The ability of Phytophthora zoospores to swim chemotactically to the surface of potential host plants is likely to make an important contribution to the establishment of disease. Phytophthora zoospore motility is produced by two flagella which have a structure typical of all eukaryotic flagella. We have recently cloned genes encoding three flagella-associated proteins and this project will aim to characterise the role of these genes and their encoded proteins in flagellar movement and zoospore motility. Antibodies that may inhibit flagella function are available and the study may also include RNAi gene silencing to investigate flagellar protein function and the importance of spore motility for infection.

The role of cell wall degrading enzymes in Phytophthora infection of host plants
(Supervisors: Adrienne Hardham and David Jones)

We have cloned and partially characterised 17 members of a Phytophthora multigene family encoding the pectin-degrading enzyme, polygalacturonase5. A number of the Phytophthora polygalacturonase genes have been transformed into yeast, as have a number of plant genes that encode polygalacturonase inhibiting proteins. This project will use the yeast-expressed proteins to determine the biochemical properties of the Phytophthora polygalacturonases and their potential inhibition by plant polygalacturonase inhibiting proteins. While these plant proteins have been shown to inhibit the wall degrading enzymes produced by true fungi, their activity with Phytophthora polygalacturonases is not currently known.

1. Hardham,A.R. (2001) The cell biology behind Phytophthora pathogenicity. Aust. Plant Pathol. 30, 91-98.
2. Shan,W. & Hardham,A.R. (2004) Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae. Fung. Genet. Biol. 41, 369-380.
3. Shan,W., Marshall,J.S. & Hardham,A.R. (2004) Stage-specific expression of genes in germinated cysts of Phytophthora nicotianae. Mol. Plant Pathol. 5, 317-330.
4. Škalamera,D., Wasson,A.P. & Hardham,A.R. (2004) Genes expressed in zoospores of Phytophthora nicotianae. Mol. Genet. Genom. 270, 549-557.
5. Götesson,A., Marshall,J.S., Jones,D.A. & Hardham,A.R. (2002) Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi. Mol. Plant-Microbe Interact. 15, 907-921.

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