The gene for gene relationship in plant parasite interactions i r crute, e b holwb, j j burdon (CABI, 1997)

The Gene-for-GeneRelationship

in Plant-Parasite Interactions



The Gene-for-GeneRelationship
in Plant-Parasite Interactions
Editedfor the British Societyfor Plant Pathology by

I.R. Crute and E.B. Holwb
Horticulture Research International
Wellesbourne
UK
and

J.J. Bwrdon
CSIRO Division of Plant Industry
Canberra
Australia

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ISBN 0 85199 164 5

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Contents

Contributors
Preface
Part I: Genetic Analyses and Utilization of Resistance
LR. Crute

ix
xiii
1

1 Organization of Resistance Genes in Arabidopsis
E. B. Holub

5

2 Genetic Fine Structure of Resistance Loci
S. Hulbert, T.Pryor, G. Hu, T.RichterandJ. Drake

27

3 Mutation Analysis for the Dissection of Resistance
P. Schulze-Lefert, C. Peterhaensel and A. Freialdenhoven

45

4 Cultivar Mixtures in Intensive Agriculture

A.C. Newton

65

5 Crop Resistance to Parasitic Plants
J.A. Lane, D. V. Child, G.C. Reiss, V. Entcheva andJ.A. Bailey

81


Contents

vi

Part 11: Population Genetics
J.J. Burdon
6 The UK Cereal Pathogen Virulence Survey
R.A. Bayles, J.D.S. Clarkson and S.E. Slater

99

103

7 Adaptation of Powdery Mildew Populations to Cereal Varieties in
Relation to Durable and Non-durable Resistance
J. K.M. Brown, E.M. Foster and R. B. O’Hara

119

8 Virulence Dynamics and Genetics of Cereal Rust Populations in

North America
J.A. Kolrner

139

9 Interpreting Population Genetic Data with the Help of Genetic
Linkage Maps
U.E. Brandle, U.A. Haemmerli, J.M. McDermott and M.S. W o v e

157

10 Modelling Virulence Dynamics of Airborne Plant Pathogens in
Relation to Selection by Host Resistance in Agricultural Crops
M.S. Hovmaller, H.Ostergdrd and L. Munk

173

11 An Epidemiological Approach to Modelling the Dynamics of
Gene-for-Gene Interactions
M.J. Jeger

191

1 2 Modelling Gene Frequency Dynamics
K.J. Leonard

211

1 3 The Genetic Structure of Natural Pathosystems
D.D. Clarke

231

14 The Evolution of Gene-for-Gene Interactions in Natural
Pathosystems
J.J. Burdon

Part 111: Cell Biology and Molecular Genetics
E.B. Holub
1 5 Phenotypic Expression of Gene-for-GeneInteraction Involving
Fungal and Bacterial Pathogens: Variation Gom Recognition to
Response
J. Mansfield, M . Bennett, C. Bestwick and A. Woods-Tor

245

263

265


Contents

1 6 The Molecular Genetics of SpecificityDeterminants in Plant
Pathogenic Bacteria
A. Vivian, M.]. Gibbon and]. Murillo

vii

293

1 7 Molecular Characterization of Fungal Avirulence
W. Knogge and C. Marie

329

18 The Molecular Genetics of Plant-Virus Interactions
N.]. Spence

347

19 Molecular Genetics of Disease Resistance: a n End to the
'Gene-for-Gene' Concept?
J.L. Beynon
20 Elicitor Generation and Receipt -the Mail Gets Through, But How!
N.T. Keen
2 1 Learning from the Mammalian Immune System in the Wake of
the R-Gene Flood
], L. Dangl

359

3 79

389

22 Genetic Disease Control in Plants - Where Now?
S.P. Briggs and R.J. Kemble

40 1

Index

407



Contributors

J.A. Bailey, Institute ofArable Crops Research, Long Ashton Research Station,
Department ofAgricultura1 Sciences, University of Bristol, Long Ashton,
Bristol BSI 8 9AF, UK.
R.A. Bayles, National Institute of Agricultural Botany, Huntingdon Road,
Cambridge CB3 OLE, UK.
M. Bennett, Department of Biological Sciences, W y e College, University of London,
W y e , Ashford, Kent TN25 5AH, UK.
C. Bestwick, Department ofBiologica1 Sciences, W y e College, University of
London, W y e , Ashford, Kent TN25 5AH, UK.
J.L. Beynon, Department of Biological Sciences, W y e College, University of
London, W y e , Ashford, Kent TN25 5AH, UK.
U.E. Brandle, Phytopathology Group, Institute of Plant Sciences, Swiss Federal
Institute of Technology, Universitatstrasse 2, CH-8092 Zurich, Switzerland.
S.P. Briggs, Pioneer Hi-Bred International, Inc., PO Box 1 0 0 4 , Johnston, Iowa
5 0 1 3 1 , USA.
J.K.M. Brown, Cereals Research Department, John Innes Centre, Colney Lane,
Norwich N R 4 7UH, UK.
J.J. Burdon, Centrefor Plant Biodiversity Research, Division of Plant Industry,
CSIRO, PO Box 1 6 0 0 , Canberra, ACT2601, Australia.
D.V. Child, Institute ofArable Crops Research, Long Ashton Research Station,
Department of Agricultural Sciences, University of Bristol, Long Ashton,

Bristol BSI 8 9AF, UK.
D.D. Clarke, Division of Environmental and Evolutionary Biology, Graham Kerr
Building, University of Glasgow, Glasgow G12 8QQ, UK.
ix


X

Contributors

J.D.S. Clarkson, National Institute of Agricultural Botany, Huntingdon Road,
Cambridge CB3 OLE, UK.
J.L. Dangl, Department of Biology and Curriculum in Genetics and Molecular
Biology, Coker Hall 108, University of North Carolina, Chapel Hill, North
Carolina 2 7 5 9 9 , USA.
J. Drake, Department of Plant Pathology, Kansas State University, Manhattan,
Kansas 6 6 5 0 6 - 5 5 0 2 , USA.
V. Entcheva, Institute of Wheat and Sunflower Research, Dobroudja, near General
Toshevo, Bulgaria.
E.M. Foster, Cereals Research Department, John Innes Centre, Colney Lane,
Norwich N R 4 7UH, UK.
A. Freialdenhoven, Rheinisch- Westfaelische Technische Hochschule Aachen,
Department of Biology I, Worringer Weg 1,D-52074 Aachen, Germany.
M.J. Gibbon, Department ofBiologica1 Sciences, University of the West of
England-Bristol, Frenchay Campus, Coldharbour Lane, Bristol BSI 6 1 QY,
UK.
U.A. Haemmerli, Phytopathology Group, Institute of Plant Sciences, Swiss
Federal Institute of Technology, Universitatstrasse 2 , CH-8092 Zurich,
Switzerland.
E.B. Holub, Plant Pathology and Weed Science Department, Horticulture Research

International, Wellesbourne, Warwickshire CV35 9 E F , UK.
M.S. Hovm0ller, Department of Plant Pathology and Pest Management, Danish
Institute of Plant and Soil Science, DK-2800 Lyngby, Denmark.
G. Hu, Department of Plant Pathology, Kansas State University, Manhattan,
Kansas 6 6 5 0 6 - 5 5 0 2 , USA.
S. Hulbert, Department of Plant Pathology, Kansas State University, Manhattan,
Kansas 6 6 5 0 6 - 5 5 0 2 , USA.
M.J. Jeger, Department of Phytopathology, Wageningen Agricultural University,
POB 8025, 6700 EE Wageningen, The Netherlands.
N.T. Keen, Department of Plant Pathology and Genetics Graduate Group,
University of California, Riverside, CA 9 2 5 2 1 , USA.
R.J. Kemble, Pioneer Hi-Bred International, Inc., PO Box 1 0 0 4 , Johnston, Iowa
5 0 1 3 1 , USA.
W. Knogge, Department of Biochemistry, Max-Planck-Institut f u r
Zuchtungsforschung, Caul-von-LinnbWeg lO,D-50829 Koln, Germany.
J.A. Kolmer, Agriculture and Agri-Food Canada, Cereal Research Centre, 1 9 5
Dafoe Road, Winnipeg, Manitoba R3T2A.19, Canada.
J.A. Lane, Institute of Arable Crops Research, Long Ashton Research Station,
Department of Agricultural Sciences, University of Bristol, Long Ashton,
Bristol BSI 8 9AF, UK.
K.J. Leonard, US Department of Agriculture, Agricultural Research Service, Cereal
Rust Laboratory, University of Minnesota, St Paul, M N 55 108, USA.
J. Mansfield, Department of Biological Sciences, W y e College, University of
London, W y e , Ashford, Kent TN25 5AH, UK.


Contributors

xi

C. Marie, Department of Biochemistry, Max-Planck-Institut fur
Zuchtungsforschung, Carl-von-Linn6 Weg 1 0 , D - 5 0 8 2 9 Koln, Germany.
J.M. McDermott, Phytopathology Group, Institute of Plant Sciences, Swiss Federal
Institute of Technology, Universitatstrasse2, CH-8092 Zurich, Switzerland.
L. Munk, Plant Pathology Section, Department ofplant Biology, The Royal
Veterinary and Agricultural University, DK- 1 8 7 1 Frederiksberg C, Denmark.
J. Murillo, Departamento de Produccion Agraria, Universidad Publica de Navarra,
3 1006 Pamplona, Spain.
A.C. Newton, Department of Fungal and Bacterial Plant Pathology, Scottish Crop
Research Institute, Invergowrie, Dundee DO2 5DA, UK.
R.B. O’Hara, Cereals Research Department, John Innes Centre, Colney Lane,
Norwich N R 4 7UH,UK.
H. OstergArd,Environmental Science and Technology Department, Plant Genetics,
Ris0 National Laboratory, DK-4000 Roskilde, Denmark.
C. Peterhaensel, Rheinisch-Westfaelische Technische Hochschule Aachen,
Department of Biology I , Worringer Weg 1,D-52074 Aachen, Germany.
T . Pryor, Division of Plant Industry, CSIRO, PO Box 1600, Canberra, ACT 2601,
Australia.
G.C. Reiss, Institute of Arable Crops Research, Long Ashton Research Station,
Department of Agricultural Sciences, University of Bristol, Long Ashton,
Bristol BSI 8 9AF, UK.
T. Richter, Department ofplant Pathology, Kansas State University, Manhattan,
Kansas 6 6 5 0 6 - 5 5 0 2 , USA.
P. Schulze-Lefert, The Sainsbury Laboratory, Norwich Research Park, Colney,
Norwich N R 4 7UH, UK.
S.E. Slater, National Institute ofdgricultural Botany, Huntingdon Road,
Cambridge CB3 OLE, UK.
N.J. Spence, Plant Pathology and Weed Science Department, Horticulture
Research International, Wellesbourne, Warwick CV35 9EF, UK.
A. Vivian, Department of Biological Sciences, University of the West of

England-Bristol, Frenchay Campus, Coldharbour Lane, Bristol BSI 6 1 QY,
UK.
M.S. Wolfe, Phytopathology Group, Institute ofplant Sciences, Swiss Federal
Institute of Technology, Universitatstrasse 2, CH-8092 Zurich, Switzerland.
A. Woods-Tor, Department of Biological Sciences, W y e College, University of
London, W y e , Ashford, Kent TN25 5AH, UK.



Preface

This book has its origins back in 1993 when one of us (I.R.C.) accepted the
nomination as Vice-president of the British Society for Plant Pathology. In
the tradition of the Society, the Vice-president becomes President-elect and
President in succeeding years and is accorded the pleasure of choosing the
theme for the main residential meeting of the Society during his presidency.
Consequently, in December 1995, the BSPP Presidential meeting addressed the
theme of: ‘The gene-for-gene relationship: from enigma to exploitation’.
The meeting was planned to explore what was known and unknown
about gene-for-gene specificity in host-parasite interactions at the molecular,
cell, plant and population levels of organization. A further emphasis was the
way in which current knowledge is being exploited for control and how new
insights may lead to new approaches. Recent advances in the isolation and
sequencing of several genes involved in specificpathogen recognition made the
meeting particularly timely and, from the outset, one intention was to provide
a forum for exchange of information and ideas among the diversity of scientists
with an interest in gene-for-gene relationships. For example, the efficient utilization of ‘natural’resistance genes in agriculture currently requires a n understanding of interactions between crop and target pathogen populations; as
resistance genes are moved and utilized, as transgenes, within and between
species, a similar level of understanding will be required to ensure their effective
exploitation. Judged by attendance alone, the meeting was a success comprising a blend of verbal and poster presentations and a delegate list of over 200.

Because of the broadly based interest in the topic of the meeting, it
was decided that a publication would be timely and place on the record the
...

Xlll


xiv

Preface

state of knowledge as the year 2000 approaches and from which progress in
the coming decades can be measured. Although all speakers at the meeting
were invited to contribute to this book, there was never an intention that it
would simply record the proceedings. Additionally, many excellent reviews
have been written about various aspects of the gene-for-gene relationship over
the last 2 5 years or so; no attempt is made in this book to provide a comprehensive restatement of historical findings. Rather, the intention has been, through
multiple authorship of a series of chapters, to attempt a synthesis of the most
exciting recent developments in understanding the gene-for-gene relationship
and the practical utilization of this information.
This book addresses three themes: genetic analyses and utilization of resistance; population genetics: and cell biology and molecular genetics. The
contributions within each theme have been the responsibility of a single editor
whose own perspectives are presented in the form of a preamble to each of the
three sections.
The gene-for-gene relationship has been a compelling and unifying force
in the study of plant-parasite interactions since it was first advanced by Flor
during his classical career-long studies on flax rust in North Dakota starting in
the 1930s. We hope that readers will be both provoked and stimulated by the
contents of this book and will sense the excitement of the authors who are all
active researchers in this rapidly advancing field of enquiry.

Ian Crute
Eric Holub
Jeremy Burdon


Genetic Analyses and
Utilization of Resistance

The elucidation of the gene-for-gene relationship and its acceptance as a framework in which to consider variation for genotype specific interactions between
plants and their parasites results from many painstaking investigations of the
inheritance of resistance and virulence - primarily of course, the pioneering
work of H.H. Flor with flax and flax rust. Additionally, the raw material for
these investigations has come, for the most part, from the practice of plant
breeding for improved resistance to pests and diseases and the frequently observed lack of durability resulting from selection of virulent parasite variants.
The literature on the genetics of interactions between parasites and their
hosts is legion and has been the subject of many useful and comprehensive
reviews of differing flavour and perspectives. It is however possible to make a
few general statements that require further elucidation:
0

0

0

Plants have evolved and maintain a vast genetic repertoire allowing recognition and response to parasitic variation.
Characteristic interaction phenotypes are associated with the operation of
different recognition genes - there are degrees of compatibility.
Genes involved in parasite recognition tend to be organized in distributed
complexes or comprise multiple allelic series.

In recent times, understanding of the above phenomena has been advanced through concentration on some particularly suitable experimental systems: the exploitation of molecular markers and specially constructed mapping
populations to provide high genetic resolution: recognition and elucidation of
non-allelic interactions: and the identification and genetic characterization of
mutants. The first three papers in this section between them provide a clear


2

Part I

statement of advances being made towards an understanding of the fine structure and organization of resistance genes in plant genomes, mechanisms that
are involved in the evolution of specific pathogen recognition capability and
the way genes at different loci interact to bring about the observed phenotypic
variation.
Eric Holub describes how investigations of variation for virulence among
pathogens of Arabidopsis has revealed many specific recognition genes and
several regions of the host genome seemingly of particular importance in
defence. The power of Arabidopsis as a non-crop model for evolutionary and
ecological investigations in addition to its well-established value in plant
molecular genetics is well illustrated.
By reference to several systems but primarily the RPI locus for rust resistance in maize, Scott Hulbert and colleagues describe the fine structure of a
complex resistance locus and the mechanisms of recombination that can result
in the generation of novel recognition capability. Of considerable interest is the
notion of harnessing these mechanisms to produce new genes or gene combinations of particular practical utility and durability for disease control.
Mutation analysis has clearly demonstrated that the expression of resistance requires the concerted action of genes at loci other than those identified among natural variants of a host species and conceptualized as being
involved as primary determinants of gene-for-gene specificity. Paul SchulzeLefert and colleagues describe studies of non-allelic interactions between
specific resistance genes and loci identified by mutation which will surely provide a fuller comprehension of the signal transduction pathways leading to
resistance.
Despite what is frequently written in elementary texts of plant breeding
and pathology, pathotype specific resistance has been and continues to be the

mainstay of crop genetic improvement programmes with many successful applications. However, it is undoubtedly true that intensive agricultural monoculture provides a stern test of the durability for any resistance gene. Among
the several approaches to enhancing the sustainable efficacy of resistance that
have been suggested, the deployment of genotype mixtures is perhaps the most
successful. Such an approach demands a level of knowledge of the pathosystem
that may be available only for host-parasite combinations that have been
intensively researched. Adrian Newton describes the gains to be made from use
of cultivar mixtures, the mechanisms that might bring about these benefits and
the way their use can be successfully integrated with intensive agricultural
practice.
Although it is with fungal and bacterial pathosystems that gene-for-gene
relationships have primarily been established, it is becoming increasingly evident that the outcome of specific interactions between plants and viruses as
well as invertebrates and parasitic higher plants follow the same basic patterns
and are dictated by the status of specific matching gene pairs in either partner.
In addition, a remarkable and unexpected similarity has recently been demon-


Genetic Analyses and Utilization of Resistance

strated among the products of genes from different plant species which are
involved in determining the outcome of specific interactions with a diversity of
microbial parasites. Systems need to be developed to determine if these same
classes of plant genes will prove important in the specific recognition of invertebrate and angiosperm parasites. Athene Lane and colleagues provide an overview of resistance of plants to parasitic higher plants: in relation to
gene-for-gene relationships, a study in its infancy. At the level of available
knowledge, the work forcibly illustrates the need for basic information on
variation for resistance and virulence together with data on genetic control. At
the same time, however, the work discussed shows how it is possible now, as in
the past with other systems, to make practical advances in control without a
highly refined level of knowledge.
Between them, these five chapters on genetic analyses and utilization of
resistance provide a brief but nevertheless embracing appraisal of the state of

current knowledge and its application with optimistic views of how we can
expect understanding to advance.
I.R. Crute

3



Organization of Resistance
Genes in Arabidopsis
Eric B. Holub
Plant Pathology and Weed Science Department, Horticulture
Research International, Wellesbourne, Warwickshire CV35 9EF, UK

We are witnessing a marriage of disciplines between natural history and
molecular biology as a direct consequence of progress being made in the
genetics and molecular biology of plant disease resistance. This is particularly
well illustrated by efforts aimed at mapping genes in the ephemeral crucifer,
Arubidopsis thulianu (mouse-ear cress), that are required for resistance to a wide
spectrum of viruses and both microbial and invertebrate parasites. The theme
of this chapter, therefore, is to examine ways in which the natural history of a
common wild flower, as viewed through molecular investigation of its genome,
may contribute to a greater understanding of how disease resistance has
evolved in plants.

Stamp Collecting Becomes an Empirical Science
From a utilitarian perspective, the activity of mapping genes required for disease resistance in a wild species such as Arubidopsis will provide a genetic
inventory that will aid programmes of crop improvement. Biotechnology will
be advanced by broadening the gene pool from which genes can be transferred
artificially across species barriers, and by unveiling opportunities for genetic

engineering of novel resistance. More importantly, plant breeding will be aided
by the genetic 'road map' of genes and flanking DNA sequence in the wild
species that can be used to develop molecular probes for marker-assisted selection of disease resistance already existing within germ plasm of a crop species
(Michelmore, 1995).
In the scientific quest to understand the molecular nature of disease resistance, gene mapping has been used successfully as a means to an end. For
0199 7 CAB INTERNATIONAL. The Gene-for-Gene Relationship
in Plant-Parasite Interactions (eds I.R. Crute. E.B. Holub and J.J. Burdon)

5


6

E.B. Holub

genes which are known to exist only by virtue of a characteristic phenotype,
the method of positional or map-based cloning has been used routinely by
molecular biologists to pinpoint the location of a gene with flanking markers in
an interval of DNA small enough to be carried by a transformation vector. In
fact, it has been the expectation that ‘anything that can be genetically mapped,
can be cloned’ along with application of advanced molecular techniques that
largely have been responsible for establishing Arabidopsis as a model organism
of plant biologists (Meyerowitz, 1987; Somerville, 1996). Several genes
required for disease resistance have been isolated using variations of the
positional cloning method including two bacterial resistance genes from
Arabidopsis (Bent et al. 1994; Briggs and Johal, 1994; Mindrinos et al., 1994;
Grant etal., 1995; Staskawiczet al., 1995).
The quest to understand how disease resistance has evolved in plants and
how the necessary polymorphism is maintained within a host species has been
an important subject of debate (Bennetzen and Hulbert, 1992; Pryor and Ellis,

1993) together with the role of symbiosis or ’evolution by association’ as a
major driving force of speciation and biodiversity (Sapp, 1994; Margulis and
Sagan, 1995). Empirical examination of the theories has only recently begun
to be possible in plant biology from fine-scale molecular genetics and the
molecular isolation of individual genes (see Beynon, Chapter 19; Hulbert et al.,
Chapter 2; Keen, Chapter 20; and Knogge and Marie, Chapter 1 7 this volume).
Further mutational dissection of the signal transduction pathways responsible
for disease resistance will certainly continue this trend (see Dangl, Chapter 2 1;
and Schulze-Lefert et al., Chapter 3 this volume). However, to develop fully the
evolution of disease resistance in plants as an empirical science, investigations
must be advanced with respect to understanding the kinds of genes and biochemical pathways involved in plant defence, the numbers of genes in each
functional class that exist within a genome, the organization of those genes
throughout the genome, and how these genes work in concert physiologically
and genetically (e.g. suppression or enhancement of recombination).
Ideally, it will be most instructive to investigate all four aspects of disease
resistance (kind, number, organization, and how the genes work) in the context of a single plant species. Parallel studies in different species are certainly
essential for purposes of comparison such as examining the collinearity of DNA
sequence between species in those regions of each genome that have been
associated with disease resistance. In any case, a systematic approach to gene
mapping and DNA sequencing will provide the basic framework to assemble a
more complete knowledge of the evolution of disease resistance in plants.
Arabidopsis provides one suitable biological system for empirical investigations. This wild flower is among the easiest of organisms in which to map the
location of a gene on a fine scale. Detailed genetic maps based on phenotypic
and several types ofmolecular markers have been created (reviewed by Koornneef, 1994) with the density of markers on these maps enabling researchers to
position a new gene within an average distance between loci of 1.5 cM. AS


Organization of Resistance Genes in Arabidopsis

7

described below, yet another detailed genetic map is emerging from efforts to
map parasite recognition and defence-related genes. DNA sequence of the entire Arabidopsis genome is expected within the decade as a primary objective of
an internationally coordinated programme (Somerville, 1996). A physical
map of the genome will provide the necessary skeleton for the sequence information. This is being constructed from a contiguous sequence of overlapping
yeast artificial chromosomes (YACs); with a given YAC carrying an insert of
100-800 kb of Arabidopsis DNA. The first of the five Arabidopsis chromosomes
has already been reconstructed as a single YAC contig (Schmidt et al., 1995).
One approach to building up a database of DNA sequence has been via the EST
(expressed sequence tags) sequencing project in which partial sequence is obtained from random cDNA clones (Hofte et al., 1993; Newman et al., 1994;
Somerville, 1996). Partial sequences of over 20,000 expressed genes have
already been produced and made available to the research community.
There are certainly limitations to what can be learned from Arabidopsis,
but the technical power and research opportunities of this wild flower are
impressive. One can imagine from the activities described above that the task of
cloning a gene will be as routine as mapping its location, searching the
database of Arabidopsis sequence to identify candidate genes in the vicinity, and
testing those genes via transformation to determine which candidate is the
targeted gene. Even the procedure of Agrobacteriurn-mediated transformation
by vacuum infiltration has greatly enhanced the prospects of cloning a gene by
overcoming the need for tissue culture (Bechtold et al., 1993; Chang et al.,
1994). Researchers can now justify shot-gun transformation experiments involving a hundred or more candidate clones.
Ultimately, genetic and physical maps of recognition and defence-related
genes in Arabidopsis and functional analyses of these genes will serve as a
chronicle of the ways in which a wild host species has evolved in part from past
encounters with parasites. Biologists in this field of research are therefore embarking, intentionally or not, on an exploration of the natural history of disease
resistance in plants.

Plant Parasites as Physiological Probes
Less than a decade ago, Homo sapiens was widely regarded as the only organism

capable of benefiting from Arabidopsis. Since then, researchers have described
Arabidopsis as a host for a growing list of pathogenic opportunists that include
numerous examples of prokaryotic (bacteria and mollicute) and eukaryotic
(plasmodiophoromycete, oomycete, ascomycete and basidomycete) microorganisms, viruses and invertebrates (nematode).This topic has been reviewed
by several authors in recent years (Dangl, 1993; Crute et al., 1994; Sijmons
et al., 1994; Simon, 1994; Kunkel, 1996).


8

E.B. Holub

In many cases, the pathogen isolates used by researchers were collected
originally from other hosts such as brassica or tomato, and were assessed for
their ability to infect and colonize accessions of Arabidopsis. Notable exceptions
include Xanthornonas carnpestris pv. carnpestris (black rot)(Tsuji and Somerville,
1992) and two obligate biotrophs common in Europe, Peronospora parasitica
(downy mildew) and Albugo candida (white blister) (Koch and Slusarenko,
1990; Holub and Beynon, 1996; Holub eta]., 1996), which have been obtained from field collections of Arabidopsis. Several pathogens can be observed
to affectplants grown in protected conditions under glass or in growth chambers. Common examples, particularly in plants that have reached the bolting
stage, include Erysiphe cruciferarurn (powdery mildew) and Botrytis cinerea
(blossom, silique and stem rot). However, there are as yet no published reports
in which strains of these fungi, that were originally collected from Arubidopsis,
have been utilized in genetic analyses of disease resistance.
In keeping with the contemporary use of Arabidopsis as a favoured subject
for laboratory investigation, there is little debate amongst practitioners about
the relative merits of investigating naturally-adapted compared with nonadapted (i.e. without known history) pathogens in this model host. The pathogen isolates are in effect regarded as physiological probes for genetic
polymorphism in the host, much the same as molecular probes (e.g. restriction
fragment length polymorphism, RFLP) are useful tools to identify interesting or
unique DNA in the genome. Standard isolates are used to screen Arubidopsis

germ plasm in a search for clear phenotypic difference (or functional dimorphism) between a pair of host accessions. If a difference is found which can be
distinguished reliably, and if the trait is simply inherited in a cross between the
two accessions, then a suitable target for gene cloning has been identified. In
the current mindset of researchers, the natural history of the hostlparasite
combination is superfluous. Nevertheless, the procedure is in theory very
simple, and one which in practice could be optimized with a plant species such
as Arabidopsis to document systematically the relative position of a large number of functionally, and perhaps evolutionarily, related genes.
Isolate collections of different parasites and pathogens are a n invaluable
resource for further analyses as described below. Several examples are proposed including the use of standard isolates as a bioassay for determining the
specificity of a naturally polymorphic gene or mutant allele in response to
infection, and for purposes of comparative biology.

Differences in Kind: Classifying the Genes Required for
Disease Resistance
Natural host and parasite variation has been the fountainhead for pathology in
Arabidopsis. Most of the host genes are expected to be somehow involved in


Organization of Resistance Genes in Arabidopsis

9

genotype-specific recognition of the parasite, either in producing a receptor
molecule that will interact with a gene product from the parasite, or as some
other naturally polymorphic component of signalling events that serve as a
trigger for plant defence. Indeed, all three of the genes isolated thus far encode
what appear to be receptor molecules that are similarly characterized by a
nucleotide binding site and sequence domain of leucine-rich repeats (see
Beynon, Chapter 19 this volume: Bent et al., 1994; Mindrinos et al., 1994:
Grant et al., 199 5). Examples of parasites and locus names for the corresponding recognition genes include: Peronosporaparasitica, RPP; Albugo candida, RAC;

Pseudomonas syringae, RPS and R P M (pv. maculicola); Xanthomonas campestris,
R X C and Erysiphe spp., R P W (powderymildew).
The importance of examining natural genetic variation of the host may be
obvious to plant pathologists, but it contrasts markedly with most other topics
of Arabidopsis biology in which researchers have concentrated their efforts
entirely on genetic variability created by artificial mutagenesis of a few standard accessions (Landsberg erecta, Ler-0; Columbia, Col-0; and Wassilewskija,
Ws-0). Arabidopsis responds well to treatments of ionizing radiation and chemical mutagens for the purpose of selecting artificial mutants: a feature which
attracted many plant geneticists to Arabidopsis research before the burgeoning
of molecular biologists in the recent decade (RCdei and Koncz, 1992). In the
past two years, Arabidopsis pathology researchers have also been employing
mutagenesis for dissecting biochemical pathways such as systemic acquired
resistance (Ryals et al., 1994) and programmed cell death (Jones and Dangl,
1996), which are thought in some cases to be linked functionally with the
natural polymorphic genes.
Researchers have used various approaches to select artificially-induced
mutations of Arabidopsis in a search for alterations in parasite recognition and
defence-related responses. The simplest approach has been to screen populations of mutagen-treated plants with an incompatible parasite isolate and to
select individuals that exhibit a shift towards susceptibility. A majority of mutations selected in this way have resulted from a change in the specific recognition gene being investigated. This has typically been verified genetically by
mapping the location of the mutated gene to the same interval of close flanking
molecular markers as that previously determined for a wild-type recognition
gene. Such a mutant is invaluable in efforts to demonstrate that the wild-type
gene has been cloned by using the mutant as the recipient for a transformation
vector containing the putative gene (for example, see cloning of the R P S 2 and
RPMZ genes, Bent et al., 1994; Mindrinos et al., 1994; Grant et al., 1995).
Comparison of DNA sequence between the wild-type and mutant alleles provides further confirmation that the same gene is being investigated as well as
determining the actual structural nature of the mutation (base pair change,
deletion or rearrangement).
Mutant screening with incompatible isolates also provides a powerful
means for determining whether a cluster of parasite-specific recognition genes

10

E.B. Holub

exists at the same locus. When more than one parasite isolate is thought to be
recognized by the same host gene, a mutant screen using one isolate can be
used to determine whether mutants can be selected which exhibit a shift in
compatibility that is specific to that isolate. This approach has been used to
distinguish between RPPI, RPPZO and RPP26 specificities on chromosome 3
in the accessions Wassilewskija and Niederzenz that otherwise have not been
separated by genetic recombination (Bittner-Eddy and Holub, unpublished:
Redmond, M. et al., unpublished: Holub and Beynon, 1996). Alternatively,
artificial mutation can reveal the dual specificity of a single host gene capable
of recognizing different pathogen gene products (Grant et al., 1995).
In several cases, screening with an incompatible isolate has yielded mutations in genes other than ones that are specific to the corresponding parasite
genotype (Table 1.1).For example, Col-ndrl was selected as a shift in macroscopic symptoms towards susceptibility following inoculation with an incompatible isolate of Pseudornonas syringae in a search for mutants of RPS2, and
Ws-eds1 was selected as a shift towards profuse reproduction by an incompatible isolate of Peronospora parasitica in a search for mutants of RPPZ. These
mutations are to a large degree parasite non-specific: the former mutant confers susceptibility to a prokaryotic pathogen, and also exhibits a partial shift
towards susceptibility to several (but not all) incompatible isolates of the
eukaryote Peronosporaparasitica (Century et al., 1995); and the later mutation
appears to negate the resistance conferred by known RPP genes from chromosomes 3 and 4 in Wassilewskija (Parker et al., 1996). Interestingly, Ws-edsl
also supports low to moderate sporulation by P. parasitica and A. candida isolates from Brassica oleracea and Capsella bursa-pastoris. Isolates of P. parasitica
from B. oleracea represent the largest group tested; six isolates have now been
tested, and all appear to reproduce in the same manner.
From this evidence, it would appear that wild-type EDSZ is a parasite
non-specific gene required for function of all RPP genes. However, several
exceptions have been observed. Low sporulation of isolates from other crucifers
suggests that residual downy mildew resistance can still exist in the presence of
edsl , Most experiments have been conducted in cotyledon tissue: however,

residual resistance has been observed in true Ws-edsl leaves with at least one
P. parasitica isolate (Ernoy2) from Arabidopsis (Parker et al., 1996). Most
interestingly, exceptions have been suggested from a cross between Ws-eds 1
and Ler-0. Ler-0 carries at least five RPP genes in the MRC-J region of chromosome 5 (RPP8, RPP21-24), each identified by recognition of a different Wscompatible isolate (see below: Holub and Beynon, 1996).From F2 segregation,
at least two of these genes (RPP8 and RPPZI) appear to confer downy mildew
resistance with apparently no attenuation by the edsz mutation. Using a gI3-yi
double mutant of Ler (flanking phenotypic markers), the MRC-J region from
Ler-0 currently is being backcrossed into the Ws-edsZ background. A new
homozygous combination of edsl from Ws-0 with the Ler-0 RPP genes from