Downy mildew of Arabidopsis thaliana caused by Hyaloperonospora parasitica (formerly Peronospora parasitica)

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

Pathogen profile Blackwell Publishing Ltd.

Downy mildew of Arabidopsis thaliana caused by Hyaloperonospora parasitica (formerly Peronospora parasitica) A L A N J. S L U S A R E N KO * A N D N I KO L A U S L . S C H L A I C H Department of Plant Physiology (BioIII), RWTH Aachen, Worringerweg 1, D-52056 Aachen, Germany

SUMMARY Downy mildew of Arabidopsis is not a hugely destructive disease of an important crop plant, neither is it of any economic importance. The most obvious symptom, the aerial conidiophores, might, at a glance to the casual observer, be mistaken for the trichomes normally present on the leaves. However, a huge research effort is being devoted to this humble pathosystem which became established as a laboratory model in the 1990s. Since then, enormous progress has been made in cloning and characterizing major genes for resistance ( RPP genes) and in defining many of their downstream signalling components, some of them RPP-gene specific. Resistance is generally associated with an oxidative burst and a salicylic acid dependent hypersensitive reaction type of programmed cell death. Biological and chemical induction of systemic acquired resistance (SAR) in Arabidopsis protecting against downy mildew were demonstrated early on, and investigations of mutants have contributed fundamentally to our understanding of host–pathogen interactions and the mechanisms of plant defence. This review will attempt to collate the wealth of information which has accrued with this pathosystem in the last decade and will attempt to predict future research directions by drawing attention to some still unanswered questions. Taxonomy: Hyaloperonospora Constant. parasitica (Pers.:Fr) Fr. (formerly Peronospora parasitica), Kingdom Chromista, Phylum Oomycota, Order Peronosporales, Family Peronosporaceae, Genus Hyaloperonospora, of which it is the type species. The taxonomy of the group of organisms causing downy mildew of brassicas has undergone a number of revisions since Corda (1837) originally coined the genus Peronospora. All isolates pathogenic on brassicas were described initially as P. parasitica but Gäumann (1918) classified isolates from different brassicaceous hosts distinctly and thus defined 52 new species

*Correspondence: E-mail: [email protected]

© 2003 BLACKWELL PUBLISHING LTD

based on conidial dimensions and host range. After much debate it was decided to revert to the aggregate species of P. parasitica for all brassica-infecting downy mildews, whilst recognizing that these show some isolate-specific differences (Yerkes and Shaw, 1959). The latest re-examination of P. parasitica by Constantinescu and Fatehi (2002) has placed isolates of P. parasitica and five other downy mildew species in a clear new subgroup on the basis of their hyaline conidiospores, recurved conidiophore branch tips and ITS1, ITS2 and 5.8S rDNA sequence comparisons; meriting the coining of the new genus ‘Hyaloperonospora Constant’. The class Oomycetes in the Kingdom Chromista (Straminipila) comprises fungus-like organisms with heterokont zoospores (i.e. possessing two types of flagellae, whiplash and tinsel). The Oomycetes have non-septate hyphae with cellulose-based walls containing very little or no chitin. The latter is regarded as a major distinction separating the Oomycetes from the true fungi, and reports of the presence of chitin had generally been regarded as due to small amounts of contamination (Gams et al., 1998). However, in view of recent studies by Werner et al. (2002) showing a chitin synthase gene in an Oomycete and demonstrating the presence of the polymer itself by an interaction with wheat germ agglutinin (WGA), it is perhaps safe to say that we have not seen the last taxonomic revision which will affect this group! The families within the Oomycetes show a clear evolutionary trend to a lesser absolute dependence on an aqueous environment and some members of the Peronosporales, e.g. H. parasitica, have no zoosporic stage in the life cycle. Host range: Isolates infecting Arabidopsis thaliana have so far proven to be non-pathogenic on other crucifers tested but exist in a clear gene-for-gene relationship with different host ecotypes. Disease symptoms: Infections are first apparent to the naked eye as a carpet or ‘down’ of conidiophores covering the upper and lower surfaces of leaves and petioles. This symptom is characteristic of this group of diseases and lends it its name. Useful websites: (links to references on Oomycetes), (TAIR, The Arabidopsis Information Resource).

159

160

A. J. SLUSARENKO AND N. L. SCHLAICH

I N T RO D U C T I O N On an evolutionary timescale, downy mildew of Arabidopsis is an ancient pathosystem and it has certainly been known to science for some time. Thus, Gäumann (1918) referred to a herbarium specimen from Schneider dating from 1865 showing H. parasitica on Sisymbrium thaliana (syn. Arabidopsis thaliana). However, the history of Arabidopsis downy mildew as a focus for research in plant pathology is short and well within the memory span of living individuals. Therefore, this review is a somewhat personalized account of developments over the last decade or so. Additionally, this pathogen profile would only be half complete without some reference to the host plant, not least because as an obligate biotroph, Hyaloperonospora parasitica , cannot exist outside of its intimate association with Arabidopsis thaliana. By the late 1980s the pioneering lead of geneticists such as Laibach, Röbbelin and Rédei in using the small brassicaceous weed Arabidopsis as a laboratory genetic model had already grown to see this plant used world-wide to address problems of metabolism and biochemistry (Somerville, 2001). However, the use of Arabidopsis to address plant pathological questions lagged behind for want of a suitable pathosystem. Pioneering work in establishing Arabidopsis as a useful model in plant pathology came first from Keith Davis, then still in Fred Ausubel’s laboratory, which at the time was still devoted largely to Rhizobium. He showed the activation of classical defence responses, such as the accumulation of transcripts for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), and also demonstrated the induction of peroxidase activity in elicitortreated cell suspension cultures (Davis and Ausubel, 1989). In various laboratories, two approaches were used to develop pathosystems with Arabidopsis. One was to take pathogens of related hosts, inoculate them into Arabidopsis and see if they multiplied and caused symptoms (see, e.g. Debener et al., 1991; Dong et al., 1991; Whalen et al., 1991) and the other approach was to search for naturally infected plants in the field. In the late 1980s, Paul Williams (University of Wisconsin) while on a field trip to Europe found Arabidopsis plants which were infected with H. parasitica at several different geographical locations. However, the first detailed description of the pathosystem (Koch and Slusarenko, 1990) came after Eckhard Koch, who had been a visiting post-doctorate in Dr William’s laboratory, returned to Europe and, while a post-doctorate in my lab in Zürich, brought in some naturally infected plants which he had found on a Sunday afternoon walk. The characterization of the interaction phenotypes of susceptible and resistant ecotypes, confirming that host variation in resistance to the pathogen existed and intimating that the advantages of Arabidopsis, could be used to characterize the genetic basis of that variation was an important milestone. The F2 from crosses between resistant and susceptible ecotypes and back-crosses between the F1 and susceptible

ecotypes gave the expected 3 : 1 and 1 : 1 segregation ratios and quickly established that resistance was inherited in a Mendelian fashion (Mauch-Mani et al., 1993; Slusarenko and Mauch-Mani, 1991). Thus, we began a map-based cloning project to isolate the major gene in the ecotype RLD conditioning resistance to our isolate of the pathogen. Arabidopsis plants naturally infected with downy mildew had meanwhile turned up in Norwich and East Malling in the UK, and soon a spectrum of different pathogen races showing differential interactions with Arabidopsis ecotypes were characterized (Crute et al., 1993; Holub et al., 1994). Our original isolate of the pathogen became called the ‘WELA’ isolate, using a naming system introduced by Eric Holub (see Dangl et al., 1992). Our isolate had been found in a suburb of Zürich called Weiningen and was virulent on (amongst others) the ecotype Landsberg erecta. The first two letters of the location where the isolate was found (WE), combined with the first two letters of a susceptible ecotype (LA) gave the name of the isolate. Thus, NOCO was found in Norwich and is virulent on Columbia, EMWA at East Malling and is virulent on Wassilewskija, etc. A large degree of host–pathogen variation was soon found in natural populations (Crute et al., 1993; Tör et al., 1994), and to date nearly 30 individual RPP genes have been postulated on the basis of differential interactions. The pathogen and the disease cycle In spring, new infections of Arabidopsis plants occur via oospores which have over-wintered in leaf debris in the soil. Successive rounds of infection on leaves and cotyledons occur via conidia. In contrast to several H. parasitica isolates infecting other brassicas, H. parasitica infecting Arabidopsis is homothallic. Oospores are usually found in the leaves of Arabidopsis a week or so after infection of true- or cotyledon leaves by conidia of a single isolate. The disease cycle is shown in Fig. 1. The disease is typical of cool, moist conditions, but empirical data for optimal field conditions for disease development are, to our knowledge, lacking. Nevertheless, a general rule of thumb for other downy mildews on brassicas is that disease in the field is most problematic between 10 and 15 °C and high humidity. However, it should be borne in mind that different developmental stages of the pathogen might show different optima, as is the case for other crucifera downy mildews. Thus, Felton and Walker (1946) showed that the germination of conidia was most rapid at 8–12 °C, whereas penetration was optimal at 16 °C and haustoria formation at 20–24 °C. For laboratorygrown Arabidopsis, the routine conditions employed for infection and conidiophore production are 16–18 °C and 100% r.h. Sporulation occurs mainly at night and spores are disseminated during the morning as drying conidiophores twist violently and fling conidia into the air. If you want a good infectious preparation of conidia, it is better to be early, otherwise many of the

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD

Downy mildew of A. thaliana

161

Fig. 1 Life cycle of Hyaloperonospora parasitica. (a) infections arise initially from oospores germinating in the soil. (b) Plants are colonized by a coenocytic, intercellularly growing mycelium which swells to fit the intercellular spaces, giving it an irregular appearance. The hyphae put out pear-shaped feeding organs called haustoria into host cells. After a variable period of growth (1–2 weeks) conidiophores, bearing asexual, spherical hyaline conidiospores (c) grow out of stomata. (d) On germination, conidia initiate new rounds of infection. (e–g) Oospores are formed concurrently with asexual spores. (e) The female sexual organs, oogonia, contain an oosphere that is fertilized via a fertilization tube growing through its outer wall from the male antheridium. (f ) The fertilized oosphere develops into a mature oospore. (g) Oospores are very profuse in infected leaves. The components of this diagram are not drawn to scale but the fungal structures are illustrated photographically in Koch and Slusarenko (1990). Reprinted from Mauch-Mani and Slusarenko (1993) with permission from Elsevier Science Publishers.

spores will already have been lost from the conidiophores on the leaves. Infection arises after a conidium germinates to either directly produce an appressorium or after making a short germ tube, generally within 6 h of coming into contact with the leaf. A penetration hypha grows from the underside of the appressorium and this penetrates the leaf at the anticlinal juncture of two epidermal cells (see fig. 1 in Koch and Slusarenko, 1990). Rarely, an appressorium forms over a stomate and the penetration hypha grows into the leaf directly through the stoma. Haustoria are often budded off into the epidermal cells as the penetration hypha grows down between them, and further haustoria are produced into mesphyll cells as the hyphae make intercellular growth. Conidiophores are produced from conidiophore initials which grow out of the stomates (Koch and Slusarenko, 1990; Mauch-Mani and Slusarenko, 1994a). The highest conidiophore density is generally observed where the density of stomata is greatest, i.e. on the underside of the leaves. Incompatible combinations of different Arabidopsis/Hyaloperonospora genotypes can show a range of resistance phenotypes. Thus, leaves of RLD show a rapid and complete cessation of growth associated with the death of one or a few plant cells (i.e. a typical HR) against WELA, whereas in Col-0, WELA makes more

growth and the HR extends well into the mesophyll. In some genotype combinations, for example Columbia with EMOY, necrosis appears behind the advancing hyphal tip (Holub et al., 1994) giving rise to a trailing necrosis phenotype which had previously been noted in some circumstances in susceptible tissue conditioned to systemic acquired resistance (Uknes et al., 1992). In a compatible combination, the degree and rate of colonization and the quantity of conidiophore production also vary in a genotype interaction-specific manner. Thus, in a tour de force, Holub et al. (1994) reported 11 different Arabidopsis/Hyaloperonospora interaction phenotypes in cotyledons and followed their segregation in the F2 after setting up diallel crosses.

C L O N I N G O F M A J O R RE S I S T A N C E G E N E S — T H E RA C E F O R T H E H O L Y G RA I L Ever since Flor (1942; 1955) proposed his gene-for-gene hypothesis to explain the basis of race–cultivar specific interactions between pathogens and their hosts, it became an elusive goal in plant pathology research to isolate and characterize a classical major gene for resistance. These ‘R ’ genes, which were so clearly defined by their phenotype and inheritance, remained for many years beyond reach because the technology to isolate them was lacking.

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

162

A. J. SLUSARENKO AND N. L. SCHLAICH

Fig. 2 Distribution of the mapped RPP genes along the five chromosomes of Arabidopsis thaliana. To the left: a numerical list of the known 27 RPP genes with their chromosomal or MRC location given, where known. RPP3 is not mapped yet, thus the Arabidopsis ecotype and the H. parasitica isolate are given. Underlined RPP genes have been renamed once. RPP genes with an asterisk have been cloned. To the right: graphical representation of the five Arabidopsis chromosomes with chromosome 1 to the left and chromosome five to the right. The centromeres are shown as black boxes. Mapping markers are given to the left of the chromosome. The region of a MRC is indicated with a black bracket and the locus of a specific RPP gene is shown with a black arrowhead to the right of the chromosome.

With the emergence and growth of modern molecular biology and cloning techniques, what had long been fantasy became reality. Thus, map-based or positional cloning strategies to isolate R genes could be begun in the early 1990s (Parker et al., 1993). As more R genes were mapped, it became clear that these were not randomly distributed on the chromosomes but that most fell into larger or smaller clusters, often interspersed with major resistance genes which were active against other pathogens. This led to the coining of the term MRC loci (major recognition gene complexes) with MRC-A, MRC-B, MRC-F, MRC-H and MRC-J on chromosomes 1, 3, 4 and 5, respectively (Holub, 1997). Candidate R genes in Arabidopsis were named RPP genes for recognition of Peronospora parasitica (Crute et al., 1993). Some 19 RPP genes are grouped in the three MRCs and four others are scattered at separate loci on chromosomes 1 and 2 (see Fig. 2 and Table 1). RPP3, which was identified in Oy-0, has not yet been mapped. Of the several RPP loci postulated on the basis of interaction patterns with isolates of H. parasitica, only a minority have been cloned (see Fig. 2 and Table 2). In Zürich we mapped RPP11, which conditions resistance to WELA in RLD, to chromosome 3 at a position 0.4 cM below the marker m249 (Joos et al., 1996). Meanwhile, Jonathan Jones and Jane Parker at Norwich, Jim Beynon and Eric Holub at East Malling and Jeff Dangl across the Atlantic at Chapple Hill were chasing RPP5/RPP1, RPP13 and RPP8, respectively. Despite all the advantages for molecular genetics which Arabidopsis offers, the honour of being the first plant from which an R gene (sensu Flor) was cloned went to tomato, with the isolation of PTO against Pseudomonas syringae pv. tomato (Martin et al., 1993) The first RPP gene whose cloning was reported was RPP5 (Parker et al., 1997), followed closely by RPP1 and RPP8 (Botella et al., 1998; McDowell et al., 1998). Interestingly, while RPP8 conditions resistance against EMCO in Ler-0, alternative alleles condition

resistance to turnip crinkle virus (allele HRT ) and cucumber mosaic virus (allele RCY1) (Cooley et al., 2000; Takahashi et al., 2002). The RPP8 locus is a good example of how recombination slippage and domain shuffling lead to new recognition specificities (Cooley et al., 2000; McDowell et al., 1998). The mapping of RPP13 was published in 1999 (Bittner-Eddy et al., 1999), and was shown to map at the same locus as RPP11 on chromosome 3 (Joos et al., 1996). Following cloning and characterization, it turned out that RPP13 and RPP11 were indeed allelic and recognized different pathogen avirulence determinants (Bittner-Eddy et al., 2000). Thus, RPP13, which was originally cloned from the ecotype Niederzenz, was designated RPP13-Nd, while RPP11, which we had been chasing in the ecotype RLD, was renamed RPP13-RLD (Bittner-Eddy et al., 2000). Cloning of the RPP1 locus from the Ws-0 ecotype revealed that three functional, genetically linked recognition specificities which had been previously designated RPP1, RPP10 and RPP14 were present together. These were re-designated RPP1-WsC, RPP1-WsA and RPP1-WsB, respectively (Botella et al., 1998). Until recently there has been little progress in characterizing the products of, or cloning avirulence genes from, H. parasitica. DNA fingerprinting of the pathogen has been attempted and John Lucas has established a mating system useful for gene mapping with H. parasitica, but this is largely with genetic markers in heterothallic isolates and the situation is compounded by the homothallism observed for the isolates pathogenic on Arabidopsis (Moss et al., 1994; Tham et al., 1994). Nevertheless, the outcrossing of two homothallic H. parasitica isolates revealed subsequent progeny segregation of avirulence loci matching six resistance loci in A. thaliana (Gunn et al. 2002). Additionally, the potential for map-based cloning of genes from H. parasitica was demonstrated in the report by Rehmany et al. (2003), which described the isolation of a BAC contig of four clones spanning

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD

Downy mildew of A. thaliana

Table 1 Recognition specificities of RPP genes. For each RPP gene the MRC or chromosomal location, the formerly assigned number (where applicable), the ecotype and the isolate(s) recognized are given.

Major recognition complexes MRC-A MRC-B

MRC-F

MRC-H

MRC-J

not in MRCs

163

RPP gene

Arabidopsis ecotype

Hyaloperonospora parasitica isolate(s)

no RPP genes RPP6 RPP7 RPP25 RPP27 RPP1-WsA formerly RPP10 RPP1-WsB formerly RPP14 RPP1-WsC formerly RPP1 RPP1-Nd P1-Nd formerly RPP26 RPP13-Nd formerly RPP16 RPP13-Nd formerly RPP17 RPP13-Nd RPP13-RLD formerly RPP11 RPP2 RPP4 RPP5 RPP12 RPP18 RPP8 RPP8 formerly RPP23 RPP21 RPP22 RPP24 RPP3 (not mapped) RPP9 RPP19 RPP20 RPP28

Col-0 Col-0 Nd-0 Ler-0 Ws-0 Ws-0 Ws-0 Nd-1 Nd-1 Nd-1 Nd-1 Nd-1 RLD Col-0 Col-0 Ler-0 Ws-0 Col-0 Ler-0 Ler-0 Ler-0 Ler-0 Ler-0 Oy-0 Wei-0 Col-0 Col-0 Col-0

WELA HIKS AHCO HIKS NOCO; EMOY; MAKS; CALA NOCO; EMOY; MAKS NOCO EMOY; HIKS, WACO ASWA EMCO GOCO; EDCO, MAKS WELA CALA EMOY; EMWA NOCO WELA HIND EMCO GOWA MADI, MAKS ASWA EDCO CALA HIKS HIND4 WAND HIND2

less than 250 kb of DNA over the region containing the ATR1Nd avirulence gene. Using the same strategy employed by Pierre De Wit for Cladosporium and tomato (De Wit and Spikman, 1982) we had some success in demonstrating the race-specific elicitor activity of intercellular washing fluids from infected Arabidopsis leaves (Rethage et al., 2000).

S Y S T E M I C A C Q U I RE D RE S I S T A N C E ( S A R ) Induction of SAR in Arabidopsis effective against downy mildew and other pathogens after chemical (dichloroisonicotinic acid or INA) treatment was shown in 1992 (Uknes et al., 1992) and after biological treatments, such as necrotic infections with Pseudomonas or Fusarium, in 1994 (Cameron et al., 1994; Mauch-Mani and Slusarenko, 1994b). In addition, the chemical inducers benzothiadiazole (BTH) and β-aminobutyric acid (BABA) were later shown to be effective in inducing resistance against downy mildew (Lawton et al., 1996; Zimmerli et al., 2000). The SAR state

is associated with the systemic accumulation of PR-proteins and increased levels of SA, as in several other dicots (Delaney et al., 1994; Uknes et al., 1992). SAR-conditioned plants respond to penetration by an ostensibly virulent genotype of H. parasitica with resistance responses ranging from a phenocopy HR involving one or a few cells at the penetration site, to limited hyphal growth with trailing necrosis eventually catching up with, and halting further colonization (Mauch-Mani and Slusarenko, 1994b; Uknes et al., 1992). The molecular/biochemical basis of SAR is still unclear, however, in order to mount a SAR response against H. parasitica, plants must be able to accumulate SA (Delaney et al., 1994).

R G E N E - M E D I A T E D S I G N A L T RA N S D U C T I O N Signal transduction pathways used by the different RPP genes to mediate resistance are still poorly understood and are complex and diverse. At one extreme there is RPP13-Nd, for which so far no downstream signalling components are known. It functions

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

164

RPP gene Protein structure Ecotype H. parasitica isolate

RPP1-WsA TIR-NB-LRR Ws-0 CALA, EMOY, HIKS, MAKS, NOCO

RPS5 SGT1b RAR1

NR22 NR22

EDS1

+++3,6,20,21

11

TIR-NB-LRR Ws-0 EMOY, MAKS, NOCO

+++3,6,20,21

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD

NDR1 Oxidative Burst1,20,23 PBS3 PAD1 PAD2 PAD3 PAD4

SA NPR1

RPP2

RPP1-WsB 11

+20,21

+16,18 PHX6+LSD519 PHX11+LSD519

+20,21

+16,18

RPP4 22,24

RPP5 16

TIR-NB-LRR Col-0 CALA

TIR-NB-LRR Col-0 EMOY, EMWA

+9 +++1,22 NR5,22

RPP6 8

RPP8 1,24

TIR-NB-LRR Ler NOCO

Col-0 WELA

++9,14 +++1,22 ++4,5,14,16,22

++22 ++22

+9 ++1 +5

+++3

+++3,16

+++3,21

–/+3,5,7

–/+3,20/ ++5,7,9,14,16

NR3

NR5 NR13 NR13 NR13 +++13 PAD1+PAD213 PAD2+PAD313 PAD1+PAD313 –/+2 –/+2

++5,16 NR13 NR13 NR13 +++13,14,16

CC-NB-LRR Col-0 HIKS

NR9 +++1 NR4,5 RAR1+NDR14 NR2,21 EDS1+NDR12,7

–/+5,9

–/+2,5,7,9

NR5

NR5 NR13 NR13 NR13 NR13,21

+21

PAD2+PAD313 +++1,2,14,15 +++1,2,14,16 DTH916,17 SID116 SID216

RPP7

RPP12 10

CC-NB-LRR Ler EMCO

RPP13-RLD

Ws-0 WELA

CC-NB-LRR RLD WELA

NR22 NR22 –/+2/NR3,21 EDS1+NDR12

RPP13-ND 12

CC-NB-LRR Nd ASWA, EDCO, EMCO, GOCO, MAKS NR14 NR14

+++6

NR3

NR21

++3

NR14 EDS1+NDR1 NR14 NR14

NR14

PAD1+PAD313 +21

++2,15 –/+2

NR2 NR2

++2 –/+2

+++15 +16,18

RPP19

RPP20

RPP21

Col-0 HIND4

Col-0 WAND

Ler MADI, MAKS

+9 +++1 +5

++1 +++5

–/+22 –/+22

12

NR14 NR14

+++3,21

–/+5,9

+5 NR13 +13 +13 +++13 PAD1+PAD213 PAD2+PAD313 PAD1+PAD313 –/+2 ++2

–/+5

+21

++2 –/+2

Quantitative contributions are as follows: NR, not required; –/+, contributes very little to resistance;+ or ++, increasing requirement to resistance; +++, required for resistance. The relative position of the signalling components does not necessarily reflect the correct sequential action of the respective gene product along the signalling pathway. Occurrence of the oxidative burst has been shown only for a few RPPs, however, it is assumed that it is a generally important factor in disease resistance signalling and hence it is shown as if it would occur downstream of all RPPs. References given: 1Tör et al. (2002); 2McDowell et al. (2000); 3Aarts et al. (1998); 4Tornero et al. (2002); 5Warren et al. (1999); 6Parker et al. (1996); 7Century et al. (1995); 8Parker et al. (1997); 9Warren et al. (1998); 10McDowell et al. (1998); 11Botella et al. (1998); 12Bittner-Eddy et al. (2000); 13 Glazebrook et al. (1997); 14Bittner-Eddy and Benyon (2001); 15Delaney et al. (1994); 16van der Biezen et al. 2002; 17Mayda et al. (2000); 18Delaney et al. (1995); 19Morel et al. (1999); 20Rusterucci et al. (2001); 21Feys et al. (2001); 22Austin et al. (2002); 23Aviv et al. (2002); 24Holub (2001). Components modulating resistance reactions to H. parasitica by increasing resistance in compatible interactions are not listed in the table, because they are not attributable to specific RPPs: cpr mutants: Bowling et al. (1994; 1997); Clarke et al. (1998; 2000); Jirage et al. (2001); Yoshioka et al. (2001); lsd mutants: Dietrich et al. (1994); pmr4 mutant: Vogel and Somerville (2000); dnd1 mutant: Yu et al. (1998); ssi2 mutant: Kachroo et al. (2001); Shah et al. (2001); snc1 mutant: Li et al. (2001); hrl1 mutant: Devadas et al. (2002); cim mutants: Maleck et al. (2002); rin4 mutant: Mackey et al. (2002); son1 mutant: Kim and Delaney (2002).

A. J. SLUSARENKO AND N. L. SCHLAICH

Table 2 Contribution of the various signalling components to the resistance reaction mediated by the various RPP resistance proteins (to be read from top to bottom for each RPP column)

Downy mildew of A. thaliana

independently of EDS1, NDR1, SA or NPR1, which in various combinations are required for the function of several other R genes (Bittner-Eddy and Benyon, 2001). At another extreme is RPP4, which seems to depend upon almost all defence signalling components so far known. Thus, RPP4-conditioned resistance against EMOY or EMWA is compromised in Col-0 plants carrying a mutation in EDS1, PAD4; NDR1, PBS3; SGT1b, RAR1; SID1, SID2 or DTH9, respectively (Table 2). Additionally, it is dependent on NPR1 and the accumulation of SA and even requires a functional RPS5 gene, which is the R gene required for the recognition of Pseudomonas syringae pv. tomato isolates carrying the avrPphB gene. Essentially, all RPP genes seem to have individual downstream requirements; being qualitatively or quantitatively dependent on different combinations of, for example, SGT1b, RAR1, EDS1, PAD4 or NDR1 (Table 2). In mutants a complete loss of resistance is often not observed, but rather a reduction of resistance which can be additive if two mutations are combined. Thus, resistance in the ecotype Ler against the EMCO isolate (mediated by RPP8 ) was initially reported to be independent of EDS1 and NDR1 (Aarts et al. 1998) but was later found to be significantly compromised in an eds1/ndr1 double mutant (McDowell et al., 2000). Rather than being a straightforward, a linear path from recognition to resistance expression, the picture which is emerging is of a complicated ‘lattice’ or ‘grid-like’ network of interconnecting signalling circuits (Parker, 2000). What is known in terms of a rather ‘linear’ summary of signalling components required for establishment of full resistance as mediated by the different R genes is given in Table 2. Thus far, signalling through RPP genes appears to be independent of COI1, JAR1 or EIN2; components required for jasmonate and ethylene signalling, respectively. These genes are required for resistance to necrotrophic pathogens such as Alternaria or Botrytis (Thomma et al., 1998).

B I O C H E M I S T R Y O F D I S E A S E RE S I S T A N C E — WHAT DO WE KNOW? We now know many molecular details about how plants recognize pathogens, and the study of mutants has given us much insight into the signal transduction that occurs between recognition and the expression of defence responses. However, we think it is still safe to say that, although there is much correlative data, we still do not really know how resistance is manifested at the biochemical level. Plants can employ chemical defence either as pre-formed antimicrobial substances (phytoanticipins) or as induced antimicrobial substances which accumulate after contact with the pathogen (phytoalexins, see below) (VanEtten et al., 1994). In Arabidopsis, as in other brassicas, glucosinolates come into question as potential phytoanticipins. However, phytoanticipins are generally correlated with non-host resistance (Mansfield, 2000),

165

Fig. 3 The oxidative burst as a response of Arabidopsis to attempted infection by H. parasitica. Leaves of Col-0 plants have been infected with conidiospores (C) of the WELA isolate and stained with diaminobenzidine for the production of H2O2 in the presence of peroxidases. The brown insoluble precipitate can be seen around the penetration hyphae (PH) at the end of a germ tube (Gt). The penetration attempt through a stomate is causing an oxidative burst in a guard cell (GC). (Photo by M. Hermanns.)

and there is no evidence that glucosinolates play a role in the interaction of Arabidopsis with H. parasitica. The accumulation of defence gene transcripts such as PAL and CHS, and the increase in peroxidase activity in cell suspension cultures treated with elicitor was reported by Davis and Ansubel (1989) and indicates that stimulation of phenylpropanoid metabolism in Arabidopsis could be expected to be associated with resistance, as observed in several other species (Dixon and Paiva, 1995). In the Arabidopsis/H. parasitica interaction an early oxidative burst of H2O2 production is observed on penetration of the epidermis by an avirulent race of the pathogen (Fig. 3). Following the oxidative burst is the genetically programmed hypersensitive cell death response (HR). In an HR against an avirulent Pseudomonad a shift from house-keeping to defence metabolism which affected an estimated 10% of the transcriptome was demonstrated (Scheideler et al., 2002), and this is presumably similar in the HR against H. parasitica. Since H. parasitica is an obligate biotroph, the rapid hypersensitive response (HR) which occurs in the epidermal cells adjacent to the penetration hyphae, and sometimes an additional few cells in the mesophyll (Koch and Slusarenko, 1990), would be expected to be sufficient to effectively condition resistance by preventing the establishment of the highly co-evolved nutritional relationship between host cell and pathogen, which depends on haustoria. Nevertheless, the HR to H. parasitica is associated with the accumulation of at least one antimicrobial phytoalexin, camalexin (Slusarenko and Mauch-Mani, 1991; Tsuji et al., 1992) and it was reported that the phytoalexin deficient mutants (pad1-1, pad2-1 and pad3-1 and the double mutants pad1-1/pad2-1, pad1-1/pad3-1

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

166

A. J. SLUSARENKO AND N. L. SCHLAICH

and pad2-1/pad3-1) showed increased susceptibility in incompatible combinations with five races of H. parasitica, namely EMOY, EMWA, CALA, HIKS and HIND (Glazebrook et al., 1997 and Table 2). Camalexin, the only Arabidopsis phytoalexin so far described, is an indole thiazole derivative, as are other phytoalexins so far reported from the Brassicaceae. Thus, camalexin is synthesized from tryptophan and not from phenylalanine, which is a precursor for phenolics in plants. This metabolic distinction was made use of to assess the relative contributions of phenolics and phenolic polymers to the resistance of Arabidopsis to H. parasitica, separately from phytoalexins (Mauch-Mani and Slusarenko, 1996). Thus, in Arabidopsis it is possible to inhibit phenolic metabolism without directly interfering with phytoalexin synthesis (Fig. 4). Using specific inhibitors of PAL (2-aminoindan2-phosphonic acid, AIP) and cinnamyl alcohol dehydrogenase (2-hydroxyphenyl-aminosulphinyl acetic acid 1,1-dimethyl ester, OH-PAS) to inhibit phenolic metabolism in general or lignification in particular, it was shown that loss of lignification caused a mild shift towards susceptibility, but that a more general inhibition of the phenolic metabolism resulted in complete susceptibility (Mauch-Mani and Slusarenko, 1996). Feeding salicylic acid (SA) back into the system restored resistance in the presence of AIP and was interpreted as showing the dependence of resistanceexpression on SA. These results conform with the observations of

Fig. 4 Simplified scheme of the biosynthesis of the defence related compounds camalexin, salicylic acid and lignin in Arabidopsis. Chorismate is the first branch point, since camalexin arises via tryptophan, while salicylic acid is synthesized via isochorismate and phenylalanine and lignins arise via phenylalanine. Chorismate is converted to isochorismate by isochorismate synthase (ICS). Phenylalanine ammonia-lyase (PAL) converts phenylalanine to cinnamic acid and is specifically inhibited by aminoindan phosphonic acid (AIP). From cinnamic acid the pathways branch to produce salicylic acid or, via cinnamaldehydes and monolignols, lignin. The conversion from cinnamaldehydes to monolignols by cinnamyl alcohol dehydrogenase (CAD) is inhibited by hydroxyphenylaminosulphinyl acetic acid dimethyl ester (OH-PAS).

Delaney et al. (1994) for transgenic Arabidopsis carrying the bacterial nahG gene and are therefore unable to accumulate SA. However, despite the overwhelming effect of SA, the contribution of lignification to resistance against H. parasitica was shown, and this highlights the multifactorial nature of the resistant response. It has been suggested that the SA that is required for PR1 gene expression and SAR might be predominantly synthesized via isochorismate whereas the SA which modulates cell death in the HR might arise predominantly from phenylalanine (Fig. 4) (Wildermuth et al., 2001). The idea that plants might lignify not only their own walls to strengthen them as barriers to pathogen spread (Hijwegen, 1963), but that the pathogens themselves might be inactivated by polymerization of monilignols to lignin in their walls as part of the peroxidase-catalysed intercellular free radical condensation reaction, was proposed by Hammerschmidt and Kuc (1982) and Ride (1983), and in the course of our investigations we also found evidence for the active lignification of hyphae in intercellular spaces (Mauch-Mani and Slusarenko, 1996). Arabidopsis is not an easy subject for biochemical study, and comparative studies are complicated by leaf-age- and ecotypespecific differences in basal enzyme activity levels (Mauch-Mani et al., 1993). After the inoculation of plants with virulent or avirulent isolates of H. parasitica, we did not observe a significant pattern of change in the activities of superoxide dismutase, catalase, ascorbate peroxidase, lipolytic acyl hydrolase, lipoxygenase or linolenic acid 13-hydroperoxide decomposing activity. All these enzymes have been reported to be important in one or more pathosystems. It is possible that in Arabidopsis these enzymes have no role in resistance against H. parasitica or, because only relatively few cells show an HR, any changes are diluted-out in comparison with the bulk of non-stimulated cells in the leaf. In contrast, transcripts for lipoxygenase were reported to be induced by treatment with Pseudomonas (Melan et al., 1993), which leads to a quantitatively greater leaf response than in the H. parasitica pathosystem where we found no increased transcript levels using the same probes (Mauch-Mani et al., 1993; Slusarenko, 1996). Thus, SA seems to be important for defence against most H. parasitica races, presumably because of its role as a signal amplifier, and it seems that lignification and camalexin might be needed for full resistance, since some resistance is lost when lignification is blocked or in pad4 or pad1+2, 1+3 or 2+3 mutants.

P RO S P E C T S The Arabidopsis/H. parasitica pathosystem has proven useful for identifying genes involved in gene-for-gene resistance. In addition to direct mutant screens on wild-type plants to identify genes affecting resistance/susceptibility, H. parasitica can also be used to identify suppressors of already known mutations. Moreover,

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD

Downy mildew of A. thaliana

this highly co-evolved pathosystem might be useful for studying newly emerging topics in plant disease resistance, e.g. agerelated resistance. Reduced susceptibility in older Arabidopsis plants has thus far been demonstrated with bacterial pathogens (Kus et al., 2002). This neatly connects to another so far neglected area: the study of the molecular basis of organ specificity. Why is a certain host organ with the same genetic background as the rest of the plant not ‘palatable’ to a pathogen? Thus, different responses to H. parasitica between cotyledons and leaves of ndr1 plants have been documented (Aarts et al., 1998). Another challenge will be to bring the Arabidopsis/H. parasitica pathosystem up to date in the era of genomics. This means that attempts should be made to obtain insights into the genome of H. parasitica. Being an obligate biotroph does not make that particularly easy, but since the host genome is known, it can be ‘subtracted’ from the DNA sequence of H. parasitica infected leaves. Thus, an initial attempt has been made using cDNA-AFLP fragments from infected and non-infected leaves; most differential gene fragments obtained were from H. parasitica (van der Biezen et al. 2000). This, together with the exciting news that a contig of H. peronospora BAC clones has been produced which spans the ATR1Nd avirulence allele (Rehmany et al., 2003), hold out the promise that we shall soon know very much more about the pathogen side of the interaction of H. parasitica with its host. Looking back at some 10 years of research with this pathosystem, we feel that it is still quite young and, while having contributed to answering several profound questions in plant pathology, it has unveiled even more intricate problems still to be addressed.

ACKNOWLEDGEMENTS Eric Holub, Jane Parker, John McDowell, and Jeff Dangl are thanked for critical comments on the MS and for providing material prior to publication. Research in the authors’ laboratories was supported by the Deutsche Forschungsgemeinschaft (SPP1005 S1 30/1-3).

R E F E RE N C E S Aarts, N., Metz, M., Holub, E., Staskawicz, B.J., Daniels, M.J. and Parker, J.E. (1998) Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R. gene-mediated signaling pathways in Arabidopsis. Proc. Natl Acad. Sci. USA, 95, 10306 –10311. Austin, M.J., Muskett, P., Kahn, K., Feys, B.J., Jones, J.D. and Parker, J.E. (2002) Regulatory role of SGT1 in early R. gene-mediated plant defenses. Science, 295, 2077–2080. Aviv, D.H., Rusterucci, C., Iii, B.F., Dietrich, R.A., Parker, J.E. and Dangl, J.L. (2002) Runaway cell death, but not basal disease resistance, in lsd1 is SA- and NIM1/NPR1-dependent. Plant J. 29, 381–391. van der Biezen, E.A., Freddie, C.T., Kahn, K., Parker, J.E. and Jones, J.D. (2002) Arabidopsis RPP4 is a member of the RPP5 multigene family of

167

TIR-NB-LRR genes and confers downy mildew resistance through multiple signalling components. Plant J. 29, 439 – 451. van der Biezen, E.A., Juwana, H., Parker, J.E. and Jones, J.D.G. (2000) cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana. Mol. Plant-Microbe Interact. 13, 895– 898. Bittner-Eddy, P.D. and Beynon, J.L. (2001) The Arabidopsis downy mildew resistance gene, RPP13-Nd, functions independently of NDR1 and EDS1 and does not require the accumulation of salicylic acid. Mol. Plant Microbe Interact. 14, 416 – 421. Bittner-Eddy, P., Can, C., Gunn, N., Pinel, M., Tör, M., Crute, I., Holub, E.B. and Beynon, J. (1999) Genetic and physical mapping of the RPP13 Locus, in Arabidopsis, responsible for specific recognition of several Peronospora parasitica (downy mildew) isolates. Mol. Plant-Microbe Interact. 12, 792– 802. Bittner-Eddy, P., Crute, I., Holub, E.B. and Beynon, J. (2000) RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica. Plant J. 21, 177–188. Botella, M.A., Parker, J.E., Frost, L.N., Bittner-Eddy, P.D., Beynon, J.L., Daniels, M.J., Holub, E.B. and Jones, J.D.G. (1998) Three genes of the Arabidopsis RPP1 complex resistance locus recognize distinct Peronospora parasitica avirulence determinants. Plant Cell, 10, 1847–1860. Bowling, S.A., Clarke, J.D., Liu, Y., Klessig, D.F. and Dong, X. (1997) The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1independent resistance. Plant Cell, 9, 1573–1584. Bowling, S.A., Guo, A., Gordon, A.S., Klessig, D.F. and Dong, X. (1994) A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell, 6, 1845–1857. Cameron, R.K., Dixon, R.A. and Lamb, C. (1994) Biologically induced systemic acquired resistance in Arabidopsis thaliana. Plant J. 5, 715–725. Century, K.S., Holub, E.B. and Staskawicz, B.J. (1995) NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both bacterial and fungal pathogen. Proc. Natl Acad. Sci. USA, 92, 6597– 6601. Clarke, J.D., Liu, Y., Klessig, D.F. and Dong, X. (1998) Uncoupling PR gene expression from NPR1 and bacterial resistance. Characterization of the dominant Arabidopsis cpr 6-1 mutant. Plant Cell, 10, 557–569. Clarke, J.D., Volko, S.M., Ledford, H., Ausubel, F.M. and Dong, X. (2000) Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. Plant Cell, 12, 2175–2190. Constantinescu, O. and Fatehi, J. (2002) Peronospora-like fungi (Chromista, Peronosporales) parasitic on Brassicaceae and related hosts. Nova Hedwigia, 74, 291–338. Cooley, M.B., Pathirana, S., Wu, H.-J., Kachroo, P. and Klessig, D.F. (2000) Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. Plant Cell, 12, 663–676. Corda, A.C.J. (1837) Icones Fungorum Hucusque Cogitorum, 1, 20. Crute, I.R., Holub, E.B., Tör, M., Brose, E. and Beynon, J.L. (1993) The identification and mapping of loci in Arabidopsis thaliana for recognition of the fungal pathogens Peronospora parasitica (downy mildew) and Albugo candida (white blister). In Advances in the Molecular Genetics of Plant–Microbe Interactions (Nester, E.W. and Verma, D.P.S., eds), Vol. 2, pp. 437–444. Kluwer Academic Press. Dangl, J.L., Holub, E.H., Debener, T., Lehnackers, H., Ritter, C. and Crute, I. (1992) Genetic definition of loci involved in Arabidopisis– pathogen interactions. In Methods in Arabidopsis Research (Koncz, C., Chua, N-H. and Schell, J., eds.). Singapore: World Scientific.

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

168

A. J. SLUSARENKO AND N. L. SCHLAICH

Davis, K.R. and Ausubel, F. (1989) Characterization of elicitor-induced defense responses in suspension-cultured cells of Arabidopsis. Mol. Plant-Microbe Interact. 2, 363–368. De Wit, P.J.G.M. and Spikman, G. (1982) Evidence for the occurrence of race and cultivar-specific elicitors in intercellular fluids of compatible interactions of Cladosporium fulvum and tomato. Physiol. Plant Pathol. 21, 1–11. Debener, T., Lehnackers, H., Arnold, M. and Dangl, J.L. (1991) Identification and molecular mapping of a single Arabidopsis thaliana locus determining resistance to a phytopathogenic Pseudomonas syringae isolate. Plant J. 1, 289 –302. Delaney, T.P., Friedrich, L. and Ryals, J.A. (1995) Arabidopsis signal transduction mutant defective in chemically and biologically induced resistance. Proc. Natl Acad. Sci. USA, 92, 6602–6606. Delaney, T.P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessmann, H., Ward, E. and Ryals, J. (1994) A central role for salicylic acid in plant disease resistance. Science, 266, 1247–1250. Devadas, S.K., Enyedi, A. and Raina, R. (2002) The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid and ethylene signalling in cell death and defence against pathogens. Plant J. 30, 467–480. Dietrich, R.A., Delaney, T.P., Uknes, S.J., Ward, E.R., Ryals, J.A. and Dangl, J.L. (1994) Arabidopsis mutants simulating disease resistance response. Cell, 77, 565–577. Dixon, R.A. and Paiva, N.L. (1995) Stress induced phenylpropanoid metabolism. Plant Cell, 7, 1085 –1097. Dong, X., Mindrinos, M., Davis, K.R. and Ausubel, F.M. (1991) Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell, 3, 61–72. Felton, M.W. and Walker, J.C. (1946) J. Agric. Res. 72, 69– 81. Feys, B.J., Moisan, L.J., Newman, M.A. and Parker, J.E. (2001) Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J. 20, 5400–5411. Flor, H.H. (1942) Inheritance of pathogenicity in cross between physiologic races 22 and 24 of Melampsora lini. Phytopathology, 32, 5. Flor, H.H. (1955) Host–parasite interaction in flax rust—its genetics and other implications. Phytopathology, 45, 680–685. Gams, W., Hoekstra, E.S. and Aptroot, A. (1998) CBS Course of Mycology. Baarn, The Netherlands: Centraalbureau voor Schimmelcultures. Gäumann, E. (1918) Über die Formen der Peronospora parasitica (Pers) Fries. Ein Beitrag zur Speziesfrage bei den parasitischen Pilzen. Beih. Bot. Zbl. 35, 395–533. Glazebrook, J., Zook, M., Mert, F., Kagan, I., Rogers, E.R., Crute, I.R., Holub, E.B., Hammerschmidt, R. and Ausubel, F.M. (1997) Phytoalexindeficient mutants of arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics, 146, 381–392. Gunn, N., Byrne, J. and Holub, E.B. (2002) Outcrossing of two homothallic isolates of Peronospora parasitica and segregation of avirulence matching six resistance loci in Arabidopsis thaliana. In Advances in Downy Mildew Research (Spencer-Phillips, P.T.N., Gisi, U. and Lebeda, A., eds), pp. 185–188. Dordrecht, Netherlands: Kluwer Academic Publishers. Hammerschmidt, R. and Kuc, J. (1982) Lignification as a mechanism for systemic acquired resistance in cucumber. Physiol. Plant Pathol. 20, 61– 71. Hijwegen, T. (1963) Lignification, a possible mechanism of active response against pathogens. Neth. J. Plant Pathol. 69, 314–317.

Holub, E.B. (1997) Organization of Resistance Genes in Arabidopsis. In The Gene-for-Gene Relationship in Plant–Parasite Interactions (Crute, I.R., Holub, E.B. and Burdon, J.J., eds), pp. 5–26. Wallingford, UK: CAB International. Holub, E.B. (2001) The arms race is ancient history in Arabidopsis the wildflower. Nature Rev. Genet. 2, 516 –527. Holub, E.B., Beynon, J.L. and Crute, I.R. (1994) Phenotypic and genotypic characterization of interactions between isolates of Peronospora parasitica and accessions of Arabidopsis thaliana. Mol. Plant-Microbe Interact. 7, 223–239. Jirage, D., Zhou, N., Cooper, B., Clarke, J.D., Dong, X. and Glazebrook, J. (2001) Constitutive salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4. Plant J. 26, 395– 407. Joos, H.-J., Mauch-Mani, B. and Slusarenko, A.J. (1996) Molecular mapping of the Arabidopsis locus RPP11 which conditions isolate-specific hypersensitive resistance against downy mildew in ecotype RLD. Theor. Appl. Genet. 92, 281–284. Kachroo, P., Shanklin, J., Shah, J., Whittle, E.J. and Klessig, D.F. (2001) A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc. Natl Acad. Sci. USA, 98, 9448–9453. Kim, H.S. and Delaney, T.P. (2002) Arabidopsis SON1 is an F-box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance. Plant Cell, 14, 1469–1482. Koch, E. and Slusarenko, A.J. (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell, 2, 437– 445. Kus, J.V., Zaton, K., Sarkar, R. and Cameron, R.K. (2002) Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae. Plant Cell, 14, 479–490. Lawton, K.A., Friedrich, L., Hunt, M., Weymann, K., Delaney, T., Kessmann, H., Staub, T. and Ryals, J. (1996) Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. Plant J. 10, 71–82. Li, X., Clarke, J.D., Zhang, Y. and Dong, X. (2001) Activation of an EDS1mediated R.-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance Mol. Plant-Microbe Interact. 14, 1131–1139. Mackey, D., Holt, B.F., Wiig, A. and Dangl, J.L. (2002) RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell, 108, 743–754. Maleck, K., Neuenschwander, U., Cade, R.M., Dietrich, R.A., Dangl, J.L. and Ryals, J.A. (2002) Isolation and characterization of broad-spectrum disease-resistant Arabidopsis mutants. Genetics, 160, 1661–1671. Mansfield, J.W. (2000) Antimicrobial compounds and resistance. The role of phytoalexins and phytoanticipins. In Mechanisms of Resistance to Plant Diseases (Slusarenko, A.J., Fraser, R.S.S. and van Loon, L.C., eds), pp. 325–370. Dordrecht, Netherlands: Kluwer Academic Publishers. Martin, G.B., Brommonschenkel, S.H., Chunwongse, J., Frary, A., Ganal, M.W., Spivy, R., Wu, T., Earle, E.D. and Tanksley, S.D. (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science, 262, 1432–1436. Mauch-Mani, B., Croft, K.P.C. and Slusarenko, A.J. (1993) The genetic basis of resistance of Arabidopsis thaliana L. Heyhn. to Peronospora parasitica. In Arabidopsis Thaliana as a Model for Plant–Pathogen Interactions (Davis, K.R. and Hammerschmidt, R., eds), pp. 5–20. St Paul, MN: APS Press. Mauch-Mani, B. and Slusarenko, A.J. (1993) Arabidopsis as a model host for studying plant–pathogen interactions. Trends Microbiol. 1, 265– 270.

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD

Downy mildew of A. thaliana

Mauch-Mani, B. and Slusarenko, A.J. (1994a) Downy mildew of Arabidopsis thaliana. In Arabidopsis—an Atlas of Morphology and Development (Bowman, J. ed.), pp. 414– 417. New York: Springer. Mauch-Mani, B. and Slusarenko, A.J. (1994b) Systemic Acquired Resistance in Arabidopsis thaliana Induced by a Predisposing Infection with a Pathogenic Isolate of Fusarium oxysporum. Mol. Plant-Microbe Interact. 7, 378–383. Mauch-Mani, B. and Slusarenko, A.J. (1996) Production of salicylic acid precursors is a major function of phenylalanine ammonia lyase in the resistance of Arabidopsis to Peronospora porasitica. Plant Cell, 8, 203 – 212. Mayda, E., Mauch-Mani, B. and Vera, P. (2000) Arabidopsis dth9 mutation identifies a gene involved in regulating disease susceptibility without affecting salicylic acid-dependent responses. Plant Cell, 12, 2119–2128. McDowell, J.M., Cuzick, A., Can, C., Beynon, J., Dangl, J.L. and Holub, E.B. (2000) Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation. Plant J. 22, 523–529. McDowell, J.M., Dhandaydham, M., Long, T.A., Aarts, M.G.M., Goff, S., Holub, E.B. and Dangl, J.E. (1998) Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis. Plant Cell, 10, 1861–1874. Melan, M.A., Dong, X., Endara, M.E., Davis, K.R., Ausubel, F.M. and Peterman, T.K. (1993) An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant Physiol. 101, 441–450. Morel, J.B. and Dangl, J.L. (1999) Suppressors of the Arabidopsis lsd5 cell death mutation identify genes involved in regulating disease resistance responses. Genetics, 151, 305–319. Moss, N.A., Crute, I.R. and Lucas, J.A. (1994) Laboratory production of oospores of Peronospora parasitica (crucifer downy mildew) and the recovery and characterization of sexual progeny from crosses between isolates with different host specificity. Plant Pathol. 43, 713– 725. Parker, J.E. (2000) Signalling in plant disease resistance. In: Molecular Plant Pathology. Annual Plant Reviews. (Dickinson, M. and Beynon, J., eds), 4, 144–174. Sheffield, UK: Sheffield Academic Press. Parker, J.E., Coleman, M.J., Szabo, V., Frost, L.N., Schmidt, R., van der Biezen, E.A., Moores, T., Dean, C., Daniels, M.J. and Jones, J.D.G. (1997) The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the Toll and Interleukin-1-Receptors with N and L6. Plant Cell, 9, 879–894. Parker, J.E., Holub, E.B., Frost, L.N., Falk, A., Gunn, N.D. and Daniels, M.J. (1996) Characterisation of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell, 8, 2033–2046. Parker, J.E., Szabo, V., Staskawicz, B.J., Lister, C., Dean, C., Daniels, M.J. and Jones, J.D.G. (1993) Phenotypic characterization and molecular mapping of the Arabidopsis thaliana locus RPP5, determining disease resistance to Peronospora parasitica. Plant J. 4, 821–831. Rehmany, A.P., Grenville, L.J., Gunn, N.D., Allen, R.L., Paniwnyk, Z., Byrne, J., Whisson, S.C., Birch, P.R. and Beynon, J.L. (2003) A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd. Fungal Genet. Biol. 38, 33– 42. Rethage, J., Ward, P.I. and Slusarenko, A.J. (2000) Race-specific elicitors from the Peronospora parasitica/Arabidopsis thaliana pathosystem. Physiol. Mol. Plant Pathol. 56, 179–184.

169

Ride, J.P. (1983) Cell walls and other structural barriers in defence. In Biochemical Plant Pathology (Callow, J.A., ed.), pp. 215–236. Chichester, UK: Wiley. Rusterucci, C., Aviv, D.H., Holt, B.F., Dangl, J.L. and Parker, J.E. (2001) The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis. Plant Cell, 13, 2211–2224. Scheideler, M., Schlaich, N.L., Fellenberg, K., Beissbarth, T., Hauser, N.C., Vingron, M., Slusarenko, A.J. and Hoheisel, J.D. (2002) Monitoring the switch from housekeeping to pathogen defense metabolism in Arabidopsis thaliana using cDNA arrays. J. Biol. Chem. 277, 10555–10561. Shah, J., Kachroo, P., Nandi, A. and Klessig, D.F. (2001) A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens. Plant J. 25, 563–574. Slusarenko, A.J. (1996) The role of lipoxygenase in plant resistance to infection. In Lipoxygenase and Lipoxygenase Pathway Enzymes (Piazza, G.J. ed.), pp. 176–197. Champaign, IL: American Oil Chemists Society Press. Slusarenko, A.J. and Mauch-Mani, B. (1991) Downy mildew of Arabidopsis thaliana caused by Peronospora parastica: a model system for the investigation of the molecular biology of host_pathogen interactions. In Advances in the Molecular Genetics of Plant–Microbe Interactions (Hennecke, H., ed.), Vol. 1, pp. 280–283. Dordrecht, Netherlands: Kluwer Academic Press. Somerville, C.R. (2001) An early Arabidopsis demonstration. Resolving a few issues concerning photorespiration. Plant Physiol. 125, 19–23. Takahashi, H., Miller, J., Nozaki, Y., Sukamto, Takeda, M., Shah, J., Hase, S., Ikegami, M., Ehara, Y. and Dinesh-Kumar, S.P. (2002) RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene, conferring resistance to cucumber mosaic virus requires salicylic acid, ethylene and a novel signal transduction mechanism. Plant J. 32, 655–667. Tham, F.Y., Lucas, J.A. and Wilson, Z.A. (1994) DNA fingerprinting of Peronospora parasitica, a biotrophic fungal pathogen of crucifers. Theor. Appl. Genet. 88, 490–496. Thomma, B.P., Eggermont, K., Penninckx, I.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A. and Broekaert, W.F. (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl Acad. Sci. USA, 95, 15107–15111. Tör, M., Gordon, P., Cuzick, A., Eulgem, T., Sinapidou, E., Mert-Turk, F., Can, C., Dangl, J.L. and Holub, E.B. (2002) Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes. Plant Cell, 14, 993–1003. Tör, M., Holub, E.B., Brose, E., Muskar, R., Gunn, N., Can, C., Crute, I.R. and Beynon, J.L. (1994) Map positions of three loci in Arabidopsis thaliana associated with isolate-specific recognition of Peronospora parasitica (downy mildew). Mol. Plant-Microbe Interact. 7, 214–222. Tornero, P., Merritt, P., Sadanandom, A., Shirasu, K., Innes, R.W. and Dangl, J.L. (2002) RAR1 and NDR1 contribute quantitatively to disease resistance in Arabidopsis and their relative contributions are dependent on the R gene assayed. Plant Cell, 14, 1005 –1015. Tsuji, J., Jackson, E., Gage, R., Hammerschmidt, R. and Somerville, S.C. (1992) Phytoalexin accumulation in Arabidopsis thaliana during the hypersensitive reaction to Pseudomonas syringae pv. Syringae. Plant Physiol. 98, 1304–1309. Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E. and Ryals, J. (1992) Acquired resistance in Arabidopsis. Plant Cell, 4, 645–656.

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170

170

A. J. SLUSARENKO AND N. L. SCHLAICH

VanEtten, H.D., Mansfield, J.W., Bailey, J.A. and Farmer, E.E. (1994) Letter to the editor: Two classes of plant antibiotics: phytoalexins versus ‘phytoanticipins’. Plant Cell, 6, 1191–1192. Vogel, J. and Somerville, S. (2000) Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. Proc. Natl Acad. Sci. USA, 97, 1897–1902. Warren, R.F., Henk, A., Mowery, P., Holub, E. and Innes, R.W. (1998) A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell, 10, 1439–1452. Warren, R.F., Merritt, P.M., Holub, E. and Innes, R.W. (1999) Identification of three putative signal transduction genes involved in R. genespecified disease resistance in Arabidopsis. Genetics, 152, 401– 412. Werner, S., Steiner, U., Becher, R., Kortekamp, A., Zyprian, E. and Deising, H.B. (2002) Chitin synthesis during in planta growth and asexual propagation of the cellulosic oomycete and obligate biotrophic grapevine pathogen Plasmopara viticola. FEMS Microbiol. Lett. 208, 169–173.

Whalen, M.C., Innes, R.W., Bent, A.F. and Staskawicz, B.J. (1991) Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell, 2, 49 –59. Wildermuth, M.C., Dewdney, J., Wu, G. and Ausubel, F.M. (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 414, 562–571. Yerkes, W.D. and Shaw, C.B. (1959) Taxonomy of the Peronospora species on Cruciferae and Chenopodiaceae. Phytopathol. 49, 499 –507. Yoshioka, K., Kachroo, P., Tsui, F., Sharma, S.B., Shah, J. and Klessig, D.F. (2001) Environmentally sensitive, SA-dependent defense responses in the cpr22 mutant of Arabidopsis. Plant J. 26, 447– 459. Yu, I.C., Parker, J.E. and Bent, A.F. (1998) Gene-for-gene disease resistance without the hypersensitive response in the Arabidopsis dnd1 mutant. Proc. Natl Acad. Sci. USA, 95, 7819 –7824. Zimmerli, L., Jakab, G., Métraux, J.-P. and Mauch-Mani, B. (2000) Potentiation of pathogen-specific defense mechanisms in Arabidopsis by β-aminobutyric acid. Proc. Nat. Acad. Sci. USA, 97, 12920 –12925.

MOLECULAR PLANT PATHOLOGY (2003) 4(3), 159–170 © 2003 BLACKWELL PUBLISHING LTD