BSPP Presidential Meeting 2003

Plant Pathogen Genomics - From Sequence To Application


Session 3: Tools - Functional Genomics/ Bioinformatics

Functional genomics of plant infection by the rice blast fungus Magnaporthe grisea

Nicholas J. Talbot, Martin J. Gilbert, Michael J. Kershaw, Darren M. Soanes, Zheng Yi Wang, Joanna M. Jenkinson, Lucy J. Holcombe, and Gurpreet Bhambra.

School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, UK.
E mail: N.J.Talbot@exeter.ac.uk

The rice blast fungus Magnaporthe grisea causes one of the most serious diseases of cultivated rice, and understanding the early events of the infection is of paramount importance if durable control measures are to be developed. M. grisea forms a specialised infection structure called an appressorium which is used to penetrate the tough outer cuticle of rice leaves, allowing the fungus entry to the underlying tissues (Talbot, 2003). Appressoria are melanin-pigmented, dome shaped cells, which form in response to the hydrophobic leaf surface and generate massive turgor pressure. Turgor is translated into mechanical force and a narrow penetration hypha is formed at the base of the appressorium, puncturing the cuticle We are using a multi-disciplinary approach, involving gene functional analysis, cell biology and analytical biochemistry, to investigate the biology of appressorium-mediated plant infection.

Insertional mutagenesis previously identified a P-type ATPase-encoding gene, PDE1, which plays a role in elaboration of the penetration peg during cuticle penetration (Balhadre and Talbot, 2001). This protein is a plasma membrane-localised aminophospholipid translocase and related to the Saccharomyces cerevisiae DNF3 gene. We will present evidence that a second P-type ATPAse, encoded by DRS2, also plays a significant role in appressorium function, associated with Golgi function and exocytosis, implicating this class of protein in microbial virulence. We will also present the results of experiments aimed at understanding the process of appressorium turgor generation, based on utilisation of the M. grisea genome to guide the large-scale analysis of genes encoding the likely biosynthetic enzymes for glycerol production in the appressorium during turgor generation. We have investigated glycogen, trehalose, and lipid metabolism, fatty acid beta-oxidation and the glyoxylate cycle and have evidence that each of these processes contributes to virulence, albeit at different stages of pathogenesis-related development. The results have begun to provide an insight into the processes that occur within a M. grisea spore during germination, germ tube elongation, appressorium formation and subsequent plant infection.

References:

Balhadre, P.V., and Talbot, N.J. (2001) PDE1 encodes a P-type ATPase involved in appressorium mediated plant infection by Magnaporthe grisea. The Plant Cell 13: 1987-2004

Talbot, N.J. (2003) On the trail of a cereal killer: investigating the biology of Magnaporthe grisea. Annual Review of Microbiology 57: 177-202


ColiBASE, an online resource for E. coli, Salmonella and Shigella comparative genomics

Roy R. Chaudhuri and Mark J. Pallen

Bacterial Pathogenesis and Genomics Unit, University of Birmingham.

We have developed coliBASE, an online resource for E. coli comparative genomics. At the heart of coliBASE is a relational database containing data from all completely sequenced E. coli, Salmonella and Shigella strains, together with preliminary sequence data from a number of genome projects currently in progress. Genes of interest can be retrieved based on their annotation text using a flexible and intuitive Google-like search interface. Alternatively a gene can be selected by chromosomal location or by sequence homology using a customised BLAST search.

Once a gene is retrieved a clickable image of the surrounding chromosomal region is generated, allowing easy navigation of the genome. Annotation from GenBank and Swissprot is displayed, including a list of relevant articles. Numerous analytical tools are also provided, including sequence retrieval, BLAST, the applet version of Artemis, a primer design facility based on Primer3 and links to other relevant online resources (including PubMed, CDD, ViruloGenome and RegulonDB). Graphical comparisons of the chromosomal region with homologous regions of other strains, as determined using MUMmer, can be displayed using a novel genome alignment viewer.

coliBASE is available online at http://colibase.bham.ac.uk/

More recently, we have adapted the coliBASE schema for other pathogens, including clostridia (http://clostri.bham.ac.uk) and campylobacters (http://campy.bham.ac.uk). We hope to extend the family of databases to include selected plant pathogens and welcome collaboration with the plant pathogen research community.


Exploring the potential of plant virus sequences

Mike Adams and John Antoniw

Plant Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK

The public nucleotide databases contain over 9000 accessions from plant viruses, viroids and satellites, totalling well over 13 million nucleotides. Accessing and comparing the data, except for relatively small sets of related viruses, can be very time-consuming. As part of the electronic version of the Association of Applied Biologists Descriptions of Plant Viruses, we developed and provided software (DPVMap) to display selected virus sequences interactively. A separate enhanced feature table (EFT) file was written for each sequence containing the start and end nucleotide positions of the features (e.g. open reading frames, untranslated regions) within the sequence. In DPVMap any of the features of the sequence could be dragged into a sequence editor to display its nucleotide sequence (as RNA or DNA), or the predicted amino acid sequence of an open reading frame. The number of sequences provided has steadily increased and now includes all complete sequences of plant viruses, viroids and satellites and all sequences that contain at least one complete gene (>5700 sequences; 31 October 2003). The information contained in the individual EFT files is especially valuable because it is checked for accuracy and is often more detailed than that provided in the original sequence file. These data were therefore transferred into a database so that multiple data sets could be selected and extracted easily and then used for further analysis.

Three examples are used to demonstrate the usefulness of this information:

The data have been used to generate text files that run batch jobs in GCG for pairwise comparisons of large numbers of related sequences. Results are presented showing the distribution of nucleotide and amino acid identity amongst all coat protein genes of the genus Carlavirus and the family Potyviridae. Such data help provide a robust basis for species demarcation criteria.

A web-enabled Windows client application (VirusCodon) was written in Delphi for IBM-compatible PCs to access the database tables and calculate various codon statistics. Codon usage was analysed for each gene of one example of each fully-sequenced plant virus. Selected results are presented to show that mutational bias rather than translational selection appears to account for most of the variation detected.

A second client application (TMpredict) works in a similar way but each complete ORF was translated into its amino acid sequence and transmembrane (TM) regions predicted using the TMPRED algorithm.

The database and tools should provide a powerful resource for many types of comparative genome analysis. They have been placed on a public internet site, DPVweb (http://www.dpvweb.net/analysis/index.php) from where the client applications can be freely downloaded. Further applications are planned.


RNAi in Plants

Jasmina Dedic, Varsha Wesley, Chris Helliwell, Ming-Bo Wang, Neil Smith, John Watson, and Peter Waterhouse

CSIRO Plant Industry, PO Box 1600, Canberra, ACT, Australia

As early as the 1920s it was known that plants could be protected against a severe virus by prior infection with a related but mild strain of the virus. The mechanism providing this "vaccination-like" protection has remained largely unknown until recently. However, over the last 6 or so years we, and others, have elucidated the mechanism and shown it to be involved in defence against plant viruses. It operates by the sequence-specific degradation of single-stranded RNA and is targeted by small fragments of double-stranded (ds) RNA. This discovery has led to an extremely powerful technology for the destruction of, not only viruses, but also of any specific mRNA, and hence the silencing of any gene, within a cell. The specificity of the destruction is simply governed by the sequence of the dsRNA introduced into the cell. In this talk, I will describe how dsRNA is being expressed in plants as a tool for gene discovery, gene validation and to engineer desirable traits in plants. During the last year, it has become clear that this dsRNA-induced mechanism also plays a key role in regulating the growth and development of almost all eukaryotes ranging from fungi to humans. I will discuss the widening perspective of how this regulatory pathway, that was not contemplated fifteen years ago, plays a central role in multi-cellular life.


Survival, surfaces and susceptibility the sensory biology of pathogens

John Lucas

Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK.
Email: john.lucas@bbsrc.ac.uk

Ever since validation of the germ theory of disease in the late 19th century, the basis of microbial pathogenicity has fascinated and frustrated generations of scientists. As new and more powerful tools and technologies have become available, from electron microscopes to molecular genetics and now comparative genomics, the special properties of pathogens have been continually revisited, usually without definitive answers. Pathogenicity has proved to be, in the large majority of cases, a complex and elusive trait. The advent of genomics, and especially the capacity to compare gene and genome sequences across contrasting pathogen types, has for the first time opened up the prospect of identifying conserved and divergent genetic blueprints required for the disease process in plant and animal hosts. Is there a common core of genes necessary for pathogenesis, and if so, what are they? One thread already emerging from functional genomic analyses is the involvement of conserved signal networks in disease initiation by a variety of fungal pathogens with contrasting infection strategies and different hosts. This is not surprising, given the need for the successful pathogen to locate, penetrate and colonise a living host, a developmental programme involving precise regulation of "when to go", "where to go" and how to survive a harsh environment which includes both basal and innate host defence responses.

The hyphal habit of filamentous fungi is uniquely adapted for exploratory growth on surfaces, within the pore space system of soils, organic substrates, or plant and animal hosts. Optimum deployment of energy and resources relies on appropriate responses to environmental cues. In rust fungi, environmental sensing starts in the hydrated spore, which is photosensitive. This adaptation synchronises germination with periods when the risk of death by dessication or UV radiation is least. Contact sensing then aids orientation of the germ tube to stomatal penetration sites. Topographical signals trigger appressorium formation. The cereal eyespot fungus, Tapesia, forms specialised infection plaques in the tight spaces between leaf sheaths at the base of the plant. The signal for plaque induction appears to be mechanical pressure. This mechano-sensing response partly explains the location of eyespot lesions and can be used to extend the range of plant organs and hosts attacked. In contrast, the leaf blotch pathogen Mycosphaerella graminicola (Septoria tritici) enters through stomata without forming highly differentiated infection structures, and then grows for an extended period within intercellular spaces. The signal-response pathways controlling this endophytic phase and the transition to necrotic lesion formation are not known, but analysis of the transcriptome at specific stages should provide clues. A unigene set representing almost one third of the M. graminicola genome has been arrayed at Manchester University and is now being interrogated with cDNA probes prepared from the pathogen growing under a range of conditions, including within host tissues. Metabolic regulation in these different circumstances will, we hope, reveal pathways necessary for pathogenesis, and novel targets for intervention in the disease process.

To date, less than fifteen fungal genomes have been sequenced. More are arriving in an accelerating pipeline. But the scale of the task should not be underestimated. Recent calculations based on biodiversity indices suggest that there may be 1.5 million fungal species, the second largest group after the insects. Of these a high proportion form associations with plants and animals, in a myriad different relationships. We are sitting on the tip of a vast and unexplored iceberg awaiting discovery.