Ian R. Crute

Genetics and Utilization of Pathogen Resistance in Plants.

Until relatively recently, knowledge of plant resistance to patho- gens has resulted primarily from research associated with the selective breeding of crop species. Although resistance is well described at the cellular, whole plant, and population levels in terms of genetics, histology, and associated biochemistry, a full mechanistic understanding of how pathogen resistance is mediated in plants is only now becoming feasible as a re- sult of the isolation and sequencing of severa1 putatively interacting plant and pathogen genes (see Alfano and Colher, 1996; Bent, 1996; Dangl et al., 1996; Hammond-Kosack and Jones, 1996, in this issue). Nevertheless, plant resistance genes have been used beneficially in agriculture for decades, even though their effects have not always been durable. This review provides a short and selective overview of the genetics of pathotype-specific resistance in plants, its past utilization in crop improvement, and some indications of how recent ad- vances may impact the future. Reference to data obtained from investigations with a few well-studied host-pathogen combi- nations (Table

Phenotypic and genotypic variation in the interaction between Arabidopsis thaliana and Albugo candida.

Two biotrophic parasites of the wild crucifer Arabidopsis thaliana (L.) Heynh, are being used to explore the molecular basis and evolution of genotype-spcific recognition and host defense. Genes for recognition of Peronospora parasitica (downy mildew) are numerous in A. thaliana and located on four of the five chromosomes as described previously. Genes for recognition of the closely related parasite Albugo candida (white blister) are described here. In contrast to teh former parasite, less than 15% of the host accessions tested were capable of recognizing either of two isolates of A. candida. The geographic regions represented by these accessions included countries in eastern and western Europe, Asia, North America and Africa. Extensive collections from England and Germany were required to identify examples of incompatible interactions. Phenotypic variation among incompatible interactions included reduced blister formations of complete lack of asexual reproduction by the parasite. Variation in the extent of the host response was also observed. Three host genes for recognition of A. candida (RAC), each associated with different interactions phenotypes, were identified through inheritance studies with three accessions. One of these genes at locus RAC1 appeared to be completely dominant, whereas the other two genes were only partially dominant or recessive under certain conditions, possibly including the effect of genetic background. One of the later two genes defined a second locus RAC2. RAC1 was mapped to the top arm of chromosome 1 in the 1 cM interval between RFLP markers M254 and M253.

Map positions of three loci in Arabidopsis thaliana associated with isolate-specific recognition of Peronospora parasitica (downy mildew)

Our research is aimed al understanding the molecular basis for gene-for-gene interactions between plant parasites and their hosts. A.s a prelude to cloning, the positions in the Arabidopsis thaliana genome were investigated for four of the 10 loci (RPPI, RPP2, RPP4, and RPP7) that have been identified as associated with the genotype-specific recognition of the biotrophic Oomycete Peronospora parasitica (downy mildew). A single cross between accessions Col-5 and Nd-1 was primarily used to map their chromosomal locations. RPPI from Nd-1 was characterized by the absence of asexual sporulation and the occurrence of necrotic pits, visible macroscopically on cotyledons 3 days after inoculation with isolates Emoy2 or Hiksl; this locus was mapped to chromosome 3 in the interval between gll and m 249 (6.6 cM above m249 and 3.9 cM below a RAPD marker OPC121250). RPP2 and RPP7 from accession Col-5(g//) were characterized by the absence of asexual sporulation and the occurrence of necrotic flecks visible 7 days after inoculation with isolates Cala2 and Hiksl, respectively. RPP2 was located between ag and B9 on chromosome 4. RPP7 maps within 13 cM of m422, but linkage with other markers on chromosome 5 was not confirmed. RPP4 from accession Col-5 was characterized by the occurrence of necrotic flecks and delayed, light sporulation 7 days after inoculation with isolates Emoy2 and Emwal; this locus also maps to chromosome 4, 14.8 cM above RPP2.

Genetic and Physical Mapping of the RPP13 Locus, in Arabidopsis, Responsible for Specific Recognition of Several Peronospora parasitica (Downy Mildew) Isolates

Fifteen isolates of the biotrophic oomycete Peronospora parasitica (downy mildew) were obtained from a population of Arabidopsis thaliana plants that established naturally in a garden the previous year. They exhibited phenotypic variation in a set of 12 Arabidopsis accessions that suggested that the parasite population consisted of at least six pathotypes. One isolate, Maks9, elicited an interaction phenotype of flecking necrosis and no sporulation (FN) in the Arabidopsis accession Nd-1, and more extensive pitting necrosis with no sporulation (PN) in the accession Ws-2. RPP13 was designated as the locus for a single dominant resistance gene associated with the resistance in Nd-1 and mapped to an interval of approximately 60 kb on a bacterial artificial chromosome (BAC) contig on the lower arm of chromosome 3. This locus is approximately 6 cM telomeric to RPP1, which was previously described as the locus for the PN interaction with five Peronospora isolates, including resistance to Maks9 in Ws-2. New Peronospora isolates were obtained from four other geographically distinct populations of P. parasitica. Four isolates were characterized that elicited an FN phenotype in Nd-1 and mapped resistance to the RPP13 locus. This suggests that the RPP13 locus contains either a single gene capable of multiple isolate recognition or a group of tightly linked genes. Further analysis suggests that the RPP11 gene in the accession Rld-0 may be allelic to RPP13 but results in a different recognition capability.

The Identification and Mapping of Loci in Arabidopsis thaliana For Recognition in the Fungal Pathogens: Peronospora parasitica (Downy mildew) and Albugo candida (White blister)

Forms of the specific crucifer pathogens: Peronospora parasitica (Pp) (downy mildew) and Albugo candida (Ac) (white blister) occur naturally on Arabidopsis thaliana (At) in the UK. Accessions of At have been shown to vary for isolate specific response to both pathogens. The existence of at least eight specific recognition (resistance) alleles are required to explain the pattern of isolate x accession responses observed. All field-collected isolates of Pp characterised so far have proved to express different specific virulence phenotypes. A fourteen parent diallel cross has been completed and, for a sub-set of eight parental accessions, the segregation of response to two isolates of Pp at F2 has been studied. In two crosses involving three parents (Col-gl, NO and OyO) segregation at F3 and cosegregation studies with RFLPs has enabled the identity of five RPp alleles to be confirmed and the chromosome location of three to be established. Each RPp allele conditions a different and characteristic response phenotype. RPpl (from NdO) is located on chromosome 3 between the locus identified by probe M249 and the morphological marker glabrous-1; this allele conditions a response characterised by lack of asexual sporulation and the occurrence of large, spreading, necrotic “pits”. RPp2 and RPp4 (from Col-gl) are located together on chromosome 4 between the loci identified by probes M326 and M600 and probably on either side of the locus identified by probe M557. RPp2 conditions a response characterised by lack of asexual sporulation and necrotic “flecking” while RPp4 also conditions a response characterised by “flecking” but this is accompanied by light asexual sporulation that is delayed in its appearance in comparison with plant lines lacking an active RPp allele. The chromosome location of RPp3 (from Oy0) and RPp7 (from Col-g1) have yet to be determined. An allele for specific recognition of Ac from the field accession Kes37 (RAc1) conditioning a response phenotype characterised by lack of asexual sporulation has been mapped to chromosome 1 below the locus identified by probe M215. Several different response phenotypes to Ac have been observed among At accessions and on this basis the existence of further RAc alleles is suspected; genetic analyses are in progress. These studies are the prelude to efforts to clone one of more specific recognition alleles employing a map-based strategy.

Phenotypic Variation and Non-Allelic Interaction in the Gene-for-Gene Relationship Between Arabidopsis Thaliana and Peronospora Parasitica (Downy Mildew).

Considerable variation is evident among interaction phenotypes when a range of Arabidopsis thaliana (At) accessions are inoculated with different isolates of Peronospora parasitica (Pp). Considering only those phenotypes that can be discriminated readily among segregating individuals, alleles controlling this genotype specific variation have been identified at nineteen loci (RPP) in the At genome and most have now been mapped with varying degrees of resolution. RPP loci occur in several clusters and at least two clusters also contain loci identified by their involvement in the recognition of other pathogens. A cluster on chromosome III is of particular interest because it consists of RPP loci whose expression is associated with a range of phenotypes. Non-allelic additive genetic variation is not usually considered to be a feature of a gene-for-gene relationship (the most obvious demonstration of this being the phenotypic expression of an allele conditioning resistance even though there may be alleles for susceptibility present at many other loci). In contrast, epistatic genetic variation is considered to be the rule, with the observed phenotype being that associated with the allele conditioning the highest manifestation of incompatibility. However, there are examples where this intuitive assumption has not been substantiated. Additive variation is evident in the altered phenotype associated with the expression of some RPP alleles in combination with mutant alleles of other loci involved in plant defence.

27 Microbial Pathogenesis of Arabidopsis

A PRIMER ON PLANT PATHOGENESIS In common with all other terrestrial angiosperms, Arabidopsis provides ecological niches for an array of microorganisms. They may inhabit the aerial parts of the plant or be confined to the roots; they may live within the plant or on its outer surfaces; and they may have detrimental, beneficial, or neutral effects. In this chapter we consider the relationships between Arabidopsis and its pathogens; that is, microorganisms causing overt symptoms of disease. Plant pathogens are in no sense a biologically homogeneous assemblage of organisms, and their diversity is well illustrated by the fungi and bacteria, listed in Tables 1 and 2, respectively, capable of parasitic growth on Arabidopsis. Despite this biological diversity, there is well-founded optimism that studies of Arabidopsis as a host to a variety of pathogens will facilitate a deeper understanding of common processes in microbial pathogenesis of plants. Modes of Parasitism Microbial plant parasites obtain their nutrients either “biotrophically” from living cells or “necrotrophically” from cells which they have killed (Lewis 1973). Examples of the former include fungi, such as powdery and downy mildews, and mollicutes, such as mycoplasma-like organisms which are obligately biotrophic and have not thus far been cultured on any synthetic medium. These obligate biotrophs invade and extensively colonize susceptible plants in such a way that host cells either remain alive or die only after the pathogen has grown on to exploit other living cells. Many of the most destructive plant pathogenic fungi (e.g., Botrytis spp. and Pythium spp.) and...