BSPP Presidential Meeting 2003

Plant Pathogen Genomics - From Sequence To Application

Garrett Memorial Lecture

Twenty-five Years of Resistance Genes

Richard W. Michelmore

The Genome Center, University of California, Davis, CA 95616.

There has been remarkable progress over the last twenty-five years in our understanding of plant-pathogen interactions particularly in the area of the molecular bases of specificity. We have advanced from classical genetic and biochemical studies to detailed knowledge of the types of proteins involved and the evolution of the genes that encode them. A significant proportion of genes in plant genomes potentially encode resistance related genes and large numbers of virulence effector proteins are secreted from pathogens into their hosts. This results in at least three types of molecules involved in cycles of selection: the bacterial virulence effector proteins, their plant targets and the plant resistance genes that directly or indirectly detect the virulence effectors. In addition, proteins downstream of the initial recognition events are also subject to cycles of selection and adaptation. Genes encoding virulence effector proteins can move horizontally between bacterial pathogens resulting in the exposure of many plant species to overlapping sets of virulence effectors. We are taking a comparative approach to understanding the evolution of specificity in plant-pathogen interactions using Arabidopsis, lettuce and tomato. A variety of genetic events occur at loci encoding disease resistance in a range of plant species; these include point mutations, insertion/deletions, intragenic and intergenic unequal crossing-over, and gene conversion. The relative frequencies and importance of each of these processes to the evolution of new resistance specificities is now being determined. Clusters of resistance genes exhibit a complex variety of patterns of evolution. Even within a cluster, different groups of genes may exhibit different evolutionary histories. In the major cluster of resistance genes in lettuce, some genes evolve slowly as distinct lineages with little sequence exchange between paralogs. Orthologs of these genes are readily detectable in diverse germplasm. Polymorphism is maintained by balancing selection. Deletion events have led to loss of certain lineages in some haplotypes. Other genes undergo frequent sequence exchange with other paralogs and close orthologs are rare in germplasm. Similar patterns are seen in Arabidopsis and tomato. In vitro evolution using DNA shuffling of the tomato Pto gene is providing additional insights into ligand binding and downstream signaling.

Session 1: Why Use Genomics?

Structural and functional analysis of fungal pathogenesis: the rice blast fungus genome project

Ralph A. Dean and members of the International Rice Blast Consortium

Center for Integrated Fungal Genomics, NCSU, Raleigh NC 27695, USA.

The recent availability of genome sequences for fungi provides unprecedented opportunities to dissect the molecular pathways governing fungal pathogenesis. Rice blast disease caused by Magnaporthe grisea is one of the most devastating threats to food security worldwide. The fungus is amenable to classical and molecular genetic manipulation and is a compelling experimental system for studying host-pathogen interactions. In 1998, an international consortium (IRBGP) was established to sequence the rice blast genome. Initially, a draft sequence (~5X coverage) of chromosome 7 was created using the "BAC by BAC" approach coupled with a comprehensive EST program. In collaboration with the Whitehead Institute Center for Genome Research, a shotgun approach was undertaken to sequence and assemble the entire genome. Sequenced BAC clones, known Magnaporthe genes and ESTs were used to validate the 6X assembly. Based on gene predictions and other evidence, publicly available whole genome oligo-based micro-arrays were developed in partnership with Agilent Technologies. These unique arrays also contain oligos designed on rice genes. These resources in conjunction with a genome-wide gene knock out program are being used to functionally dissect pathogenesis. The current status of the genome project, including annotation, comparative and functional analyses pertaining to pathogenesis, will be presented. Sequence data and other information are publicly available at and

Genome Dynamics in Bacterial Pathogens

Julian Parkhill

The Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, U.K.

Much of the initial emphasis in bacterial genomics has been concentrated on human pathogens. Although plant pathogens are beginning to take their share of this effort, there is still a great deal more primary and comparative data available for human pathogens. It should therefore be instructive to look at some of the results that have come from this analysis in order to consider what kind of data will be available to the plant pathogen community in the near future. One of the strongest of such underlying themes is that of genomic variation and diversity.

Bacterial genomes are not static; the structure and content of these genomes can vary in a number of ways, and over many different timescales. Such variation can be random or directed, and the rates of change can be affected by environments and evolutionary pressures. These concepts will be discussed with reference to genome sequences from two genera: Bordetella and Bacteroides.

Bordetella pertussis is the causative agent of whooping cough, and is host restricted to humans; Bordetella bronchiseptica is a broad-host range organism, capable of causing chronic and asymptomatic respiratory infections in a number of mammals. Bordetella parapertussis also causes whooping cough and infects humans and sheep. These organisms are extremely closely related at the DNA level, yet have different host tropisms and levels of virulence. Comparative genomics has revealed that Bordetella pertussis and Bordetella parapertussis are independent derivatives of a Bordetella bronchiseptica-like ancestor, and that the recent evolution of the derivative organisms has been almost entirely due to gene loss and degradation.

Bacteroides fragilis is a member of the human gut commensal micro-fauna, and can cause severe opportunistic infections, especially after injury or surgery. Bacteroides is known to exhibit extensive phase-variation of surface polysaccharides, and the genome sequence has revealed an unusual and extensive mechanism for phase-variation involving site-specific DNA inversion. An overview of the system will be presented, along with comparative data against other Bacteroides strains and species.

Using genomics to identify Pseudomonas syringae Type III effectors and determining their activities in plants

James R. Alfano

Plant Science Initiative and the Department of Plant Pathology, University of Nebraska, 1901 Vine St., Lincoln, NE, 68588, USA

The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants and to elicit the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants, is dependent upon the type III secretion system (TTSS). The genome of DC3000 has recently been mined for hop (Hrp outer protein) genes that encode TTSS substrates substantially enlarging the Hop inventory of DC3000. Several of the strategies utilized to identify TTSS substrate genes will be discussed. We developed screens to determine whether any of the Hop proteins could suppress the HR and/or other plant defenses. Cosmid pHIR11 enables nonpathogens to elicit an HR dependent upon the TTSS and effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Another effector, HopPtoD2, possessed protein tyrosine phosphatase activity and was capable of suppressing the HR elicited by an avirulent P. syringae and an HR-like cell death elicited by a constitutively active MAPK kinase, called NtMEK2DD. DC3000 mutants lacking these proteins elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Many of these suppressors inhibited the expression of the tobacco PR1a defense-related gene. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD and/or plant defenses suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.

C. elegans and plant parasitic nematodes

Charlie Opperman

North Carolina State University, Raleigh, USA

During the past ten years, dramatic advances in the study of the free-living nematode Caenorhabditis elegans, have provided insight into the genetics and biochemistry of nematodes. In addition, a genetic system to study plant parasitic nematodes has been established. With these two systems in hand, it has become possible to study the biology of parasitism directly and also in a model system. The remarkable degree of conservation in structure and function thus far observed in Nematoda suggests that data from one system will be transferable to others. The recent completion of the genome sequence of C. briggsae will enable powerful comparative genomic approaches to be undertaken. Other genomics programs that focus upon parasitic nematodes will further empower these comparisons. It is likely that inroads into both the nature and evolution of parasitic abilities will directly result from application of genomic approaches.

Using the tools of genetics, genomics and biochemistry, rapid application of information from one system to the other, and to many other important parasitic nematodes is possible. In addition to specific parasitic abilities, basic mechanisms of nematode development will also be approachable. There are several key things that all parasites must do to successfully complete their life cycles. These include location and penetration of a host, migration to an appropriate feeding site, development to maturity, and reproduction. In addition, all parasites must evade host defense responses. Because all parasites must function in a similar manner, we believe that the basic biological mechanisms used will be conserved between plant and animal parasitic nematodes, although the direct signals and functions may be somewhat different. The conservation of the basic mechanisms makes these parasites vulnerable to the use of their own biology against them in designing new control methods.