BSPP Presidential Meeting 2003

Plant Pathogen Genomics - From Sequence To Application


Poster Abstracts

PP1 Identification of race-specific avirulence genes in Phytophthora infestans by transciptional profiling

Rays H.Y. Jiang, Guo Jun, Lars Kamphuis and Francine Govers

Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, The Netherlands.

Phytophthora infestans is a destructive oomycete pathogen causing potato late blight worldwide. Genetic analyses of potato and P. infestans have demonstrated that in this pathosystem, monogenic resistance mediated by resistance (R) genes, is based on a gene-for-gene interaction. We aim at cloning and characterising avirulence genes in P. infestans. We performed cDNA-AFLP analysis on a F1 population carrying different avirulence phenotypes. From the two parental isolates and from 19 F1 progeny mRNA was isolated from the stage that Avr genes are most likely to be expressed, i.e., the germinating cyst stage. Candidate genes were selected by cDNA-AFLP analysis on bulks of samples representing the same phenotype. The association of expression and avirulence phenotype was verified by cDNA-AFLP analysis on individual progeny and by RT-PCR. The transcriptional profiling will provide information on genes differentially expressed in different races and possibly lead to identification of Avr genes of P. infestans.


PP2 A proteomic approach to investigate the infection process of Phytophthora infestans

Pieter van West1, Laura J. Grenville1, Catherine R. Taylor1, Anna Avrova2, Alison Williams1, and Paul R.J. Birch2.

1 University of Aberdeen, Department of Molecular and Cell Biology, Foresterhill, Aberdeen, AB25 2ZD, Scotland UK
2 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland UK
E-mail: p.vanwest@abdn.ac.uk

A thorough understanding of the molecular events taking place during early interactions between P. infestans and host and non-host plants is crucial for developing new control measures. The appressorial stage of the interaction is the first point in which direct contact between the pathogen and the plant occurs via the formation of highly specialised infection structures, such as the appressorium, penetration peg, and the infection vesicle. Moreover, it is during this phase that plant defence responses are initiated. Resistance is based, principally, on recognition of a particular elicitor component from the pathogen. Therefore, we anticipate that secreted and cell wall proteins from the appressorial infection stage of P. infestans are likely to be rich in important signalling molecules involved in disease resistance or establishing a successful infection process. A proteomic approach is employed to accelerate the discovery of novel extra-cellular and appressorial stage-specific proteins. Here we present our latest results.


PP3 Sigatoka Forensics

Skye Thomas-Halla, Susan Porchunb, Juliane Hendersonb, Julie Pattemoreb Elizabeth Aitkena

a Department of Botany, The University of Queensland, St Lucia, QLD, 4072, Australia
b CRC for Tropical Plant Protection, Molecular Diversity and Diagnostics Research Laboratory, Plant Pathology Building, Indooroopilly Research Centre, 80 Meiers Rd, Indooroopilly, QLD, 4068, Australia.

An outbreak of the leaf disease black Sigatoka caused by Mycosphaerella fijiensis, in the Tully banana production area in Australia, April 2001, raised the question as to how this pathogen could have passed through Australias strict quarantine barrier and where did it originate from? The Tully region in north Queensland produces over 65% of Australias bananas , therefore this outbreak was of major concern to the industry, already under pressure from potential imports. A molecular diagnostic based on the ITS region has been crucial for the quick and definitive identification of Mycosphaerella fijiensis, which in its early stages, is indistinguishable from the less aggressive Mycosphaerella musicola (causal agent of yellow Sigatoka). In this study, the ITS region was sequenced from numerous isolates of M. fijiensis and M. musicola from around the world and throughout Australia including those from the recent outbreak. The results were used to validate and improve the molecular diagnostic and to determine the diversity within each species to help trace the origin of certain strains. The data gave clear phylogenetic distinction between the two different fungal species, but only a small level of variation was observed within the M.fijiensis isolates whereas a comparatively large amount of variation was found within the M.musicola isolates, indicating several possible distinct strains of that species. As the ITS region did not vary sufficiently enough to distinguish between isolates of black Sigatoka from different locations, a new region had to be analysed. Sequencing of the 5.5kb IGS (intergenic spacer) region in M.fijiensis revealed a highly variable 2.5kb region. This starts 800bp in, with a mircosatellite region that would often vary in size, followed by a 700bp element (possibly a transposon) that is found in different locations or not at all, then is followed by unique sequence before becoming a conserved 450bp repeat element. The IGS region proved to have enough diversity to distinguish between strains of M.fijiensis from different locations. The ITS and IGS data identified two distinct genotypes of M. fijiensis which had spread throughout the Tully region in roughly equal proportion. One of these genotypes was found on islands in the Torres Straits as well as from the Philippines and Papua New Guinea, the other genotype was different from the entire 220 samples we had in our collection of isolates from all around the world including those from previous incursions from Cape York in Far North Queensland. It therefore seems that the Tully 2001 incursion did not arise from the predicted sources in Cape York or the Torres Straits but possibly from further a field such as PNG, which is one of the proposed centres of diversity.


PP4 Downy mildew genomics: identification and functional analysis of genes encoding secreted proteins

Posthuma, K., Elberse, J., Weisbeek, P. and Van den Ackerveken, G.

Molecular Genetics, Molecular and Cell Biology, Utrecht University, Utrecht, The Netherlands, 3584 CH
Email:
k.i.posthuma@bio.uu.nl

Downy mildews infect many important crops worldwide. To protect crops from downy mildew disease, natural resistance genes have been introduced into cultivars. However, resistance is usually rapidly overcome by the pathogen. The project Downy mildew genomics and plant disease resistance aims to identify new resistance genes that mediate the recognition of important pathogen proteins and may therefore be more durable. A genomics approach is used to identify downy mildew genes that encode secreted proteins and that are specifically expressed during the infection process. Two downy mildew plant interactions are being studied: Peronospora parasitica Arabidopsis thaliana and Bremia lactucae lettuce. Over 3,000 Expressed Sequence Tags (ESTs) have been collected from B. lactucae and P. parasitica conidiospore libraries. These ESTs have been screened for signal peptides and for similarity to genes or proteins in public databases. Microarray technology is being used to study the expression of these genes during infection of the host. In addition, we are collecting as large number of ESTs from a subtracted library of the P. parasitica A. thaliana interaction. Functional studies of selected P. parasitica secretory proteins will be carried out by (transient) expression in A. thaliana and Nicotiana sp. B. lactucae genes encoding secreted proteins will be transiently expressed in lettuce to identify lines reacting with a hypersensitive response. These lines will be tested further for downy mildew resistance and can be used by lettuce breeders to obtain new resistance specificities to downy mildew disease.

This research is funded by the Dutch Technology Foundation (STW).


PP5 Molecular Characterization of Potyvirus infecting sugarcane in India

R.K.Gaur, G.P.Rao and Maneesha Singh

Sugarcane Research Station, Kunraghat, Gorakhpur-273008, UP, India

Sugarcane mosaic virus (SCMV) is a member of the genus potyvirus infecting many of the sugarcane growing belts of India. Comparison of purified virus preparation by western blot analysis revealed a coat protein size of about 39 kDa. Part of the genomic 3 terminal non-coding region was amplified from SCMV-IND isolates by immunocapture reverse transcriptase polymerase chain reaction (IC-RTPCR) using an universal primer and cDNA products was sequenced. BLST search revealed that the C-terminus of SCMV-IND shows 90-95% homology to that of other SCMV isolates reported from other countries. The putative capsid protein (CP) gene of SCMV-IND encoded 365 amino acid and phylogenetic analysis showed that SCMV-IND was closely related to SrMV, MDMV and SCMV. Based on this sequence analysis our result revealed that SCMV-IND could be considered a distinct potyvirus within the SCMV subgroup.


PP6 Secondary metabolism and avirulence in Magnaporthe grisea: is ACE1 part of an avirulence gene cluster?

Heidi U. Bhnert, Anne-Elodie Houlle, Laurent Camborde and Marc-Henri Lebrun.

FRE2579 CNRS / Bayer CropScience, Lyon, FRANCE.

Isolates of the rice blast fungus Magnaporthe grisea that carry the avirulence gene ACE1 are specifically recognized by rice varieties carrying the resistance gene Pi33. ACE1 was isolated by map based cloning and encodes a natural hybrid between a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS). Together with the lovastatin biosynthetic enzyme LNKS from A. terreus, Ace1 defines a novel family of eukaryotic PKS/NRPS that appears to be widespread in ascomycetes but not in basidiomycetes. The M. grisea genome is particularly rich in representatives of this enzyme family. ACE1 is specifically expressed during penetration, and the enzyme is localized in the cytoplasm of the appressorium. Since Ace1 biosynthetic activity is required for avirulence, we believe that the fungal signal recognized by resistant rice plants is the secondary metabolite whose synthesis depends on Ace1. ACE1 is part of a gene cluster. We identified 18 ORFs, 14 of which show homology to genes involved in secondary metabolism. Ten of these were shown to be co-regulated with ACE1 and are therefore potentially involved in the biosynthesis of the avirulence signal. We are currently testing this hypothesis by constructing individual gene deletion mutants. Additionally, we are trying to create a strain that expresses the ACE1 cluster constitutively by overexpressing the putative positive regulator of these genes.


PP7 Microarray data-mining and RNA expression profile of Arabidopsis thaliana glucosyltransferases induced by pathogen challenge

Claire Gachon, Mathilde Langlois-Meurinne, Patrick Saindrenan

Institut de Biotechnologie des Plantes, Btiment 630, Universit Paris XI-CNRS, 91405 ORSAY Cedex

When challenged by a pathogen, plants induce the synthesis of a great number of secondary metabolites. Some of them play important roles in determining the level of resistance to the pathogen, either as signaling molecules or as effectors of the defence responses. Interestingly, most of them do not accumulate in their free form in planta, but rather as a conjugated form, most often to glucose. As a result, glucosyltransferases, the enzymes responsible for transferring the sugar moiety to a secondary metabolite, are prone to play key roles in the synthesis, accumulation and transport of those secondary products. The sequencing of Arabidopsis genome revealed an unexpectedly high number and diversity of glucosyltransferases (121 genes), among which only three are functionnally characterized. As a result, the identification of glucosyltransferases involved in plant defence would be a first step toward the understanding of their physiological role. For this, we took advantage of the increasing number of published microarray results concerning the transcriptome of plants infected by fungal or bacterial pathogens. A bioinformatic analysis focused on glucosyltransferases reveals candidate genes whose expression profiles are strongly affected during the interaction with the pathogens Alternaria brassicicola and Pseudomonas syringae. Their RNA expression profile was examined further by real-time PCR. The phenotypical characterization of the corresponding T-DNA insertion mutants is currently under way. The major results of this work will be presented.

References:

J. Glazebrook et al. The Plant Journal. (2003) 34:217-228

S. van Wees et al. Plant Physiology (2003) 132:607-617

Y. Tao et al. The Plant Cell (2003) 15:317-330


PP8 Identifying expressed sequences within a Peronospora parasitica (At) BAC contig spanning an avirulence gene locus

Anne P. Rehmany and Jim L. Beynon
HRI, Wellesbourne, Warwick. CV35 9EF

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the ATR (A. thaliana-recognised) avirulence genes. We are using a map-based cloning strategy to target ATR1Nd, an avirulence gene recognised by the Arabidopsis resistance gene RPP1Nd, and have constructed and sequenced a BAC contig spanning the ATR1Nd locus. We have used a cDNA enrichment method to specifically identify expressed sequences encoded by the ATR1Nd BAC clones. The identification of expressed sequences from the ATR1Nd interval has assisted in gene characterisation in this "novel" genome.


PP9 Genetic and physiological analysis of mutations in wheat showing enhanced adult plant resistance to yellow rust

James P.E. Melichar, Simon Berry, Mike Field, Lesley. A. Boyd

JIC, Norwich

Puccinia striiformis f.sp. tritici, is a wholly asexual, biotrophic fungal pathogen. It is the causal agent of yellow rust (Yr) in wheat, and is a serious economic foliar disease in most temperate wheat growing areas of the world. It has a distinctive phenotype of intervenal-stripes of yellow pustules (uredia). Control in the UK is based on an integrated approach of resistance breeding with rotation of fungicides of differing modes of action. Mutagenised populations of the wheat cultivars Guardian and Hobbit sib have been screened in field tests, and mutant lines selected that give enhanced Yr adult plant resistance (APR). These include the Guardian mutants M66 and M257, and the Hobbit sib line I3-54. M66 and M257 also show broad-spectrum resistance to powdery mildew, and complete and partial resistance to brown rust respectively, while I3-54 is resistant to powdery mildew but not brown rust. The M66 mutant also shows evidence of necrotic flecking analogous to lesion mimics such as mlo barley lines. To date four double-haploid populations have been created using the maize pollination procedure for I3-54 (30 lines), Guardian (80 lines), M66 (100 lines) and M257 (100 lines), each crossed to the spring cultivar Avocet-S. Screening of SSR markers against parental lines have identified 84 potential polymorphisms. Pathology studies are looking at resistance mechanisms at three distinct growth stages (GS): seedling (GS 12), tillering (GS 22-23), and heading (GS 49-51), in order to elucidate the exact timing and development of APR in these mutants. The synchronous GS will be inoculated with compatible Yr isolates, and pathogen development observed. DAB staining for H2O2 generation in infected mesophyll cells, and NBT staining for superoxide generation will be performed. Fungal structures will be trypan blue stained.


PP10 Curing native plasmids in Pseudomonas syringae pathovar pisi to identify pathogenicity determinants

Butcher1, D., Jackson2, R.W., Roberts, S.J.3, Vivian1, A. and Arnold1, D.L.

1 Centre for Research in Plant Science, UWE-BRISTOL, UK
2 Department of Plant Sciences, University of Oxford, UK
3 Horticulture Research International, Wellesbourne, UK

Pseudomonas syringae pv. pisi (Ppi) is a seedborne pathogen which causes bacterial blight of pea (Pisum sativum). Seven races, comprising two distinct genomic groups (GG), have been identified and these interact with a series of eight differential pea cultivars (cv.). We have cloned plasmid replication genes from strains of Ppi and used them to cure Ppi strains of native plasmids by incompatibility. Race 3A (strain 870A, GG II) was successively cured of the three native plasmids it carries and the resulting cured strains were tested in pea. Pathogenicity to pea appears to be determined primarily by a large plasmid, pAV232 (106 kb). The avirulence gene, avrPpiB1.R3, was confirmed to be located on the smallest plasmid, pAV231 (42 kb). Race 1 (strain 299A, GG I) was successively cured of its two native plasmids. Loss of pAV212 (60 kb) resulted in compatibility to pea cvs. carrying R3, due to the loss of avrPpiB2. Loss of both pAV213 (55 kb) and pAV212 resulted in a novel pathogen phenotype, avirulence on cv. Belinda, that was not due to avrPpiB. Complementation with cosmid library clones positive for pAV213 restored virulence on cv. Belinda. DNA fragments have been cloned from the library clones positive for pAV213 and are being tested on plants to try to identify the novel virulence factor on pAV213.


PP11 Gene identification in the oomycete pathogen Phytophthora parasitica by expressed sequence tag analysis

Franck Panabires, Jo-Yanne Le Berre, Paul Venard and Pierre Abad

INRA, Unit Interactions Plantes-Microorganismes et Sant Vgtale, 62 boulevard du Cap, BP 2078, 06606 Antibes cedex, France.

Phytophthora parasitica Dastur (P. nicotianae Breda de Haan) is a soilborne oomycete pathogen of great importance worldwide. It exhibits an overall broad host range, but some isolates are specialized towards a single host. In order to identify the pathogenic determinants of P. parasitica, we undertook a study to profile gene expression patterns by analysis of expressed sequence tags (ESTs). Two cDNA libraries have been sequenced, the first one constructed from in vitro grown mycelium and the second from 4-day-infected tomato roots. Over 2000 clones from the in vitro library and 4000 clones from the interaction library have been sequenced to date and analyzed for similarity to known sequences, and redundancy within and among libraries. The libraries displayed distinct expression patterns. The analysis revealed ~72-74% unigenes in each library, and 20% of the ESTs are coding for novel genes. In addition, 33% of the sequences from the in vitro-grown mycelium are absent from the compilation of P. infestans and P. sojae sequences maintained at the Phytophthora Genome Consortium Database. Comparison with sequences from other plant pathogens defines hypotheses on the genes required for pathogenicity. The putative roles of some of the identified genes are discussed.


PP12 Quorum Sensing in Bacterial Plant and Animal Pathogens: the Role of luxS

Sarah J. Coulthurst1, C. Lopold Kurz2, Graham Plastow3 and George P.C. Salmond4

1 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK
2 Centre d'Immunologie de Marseille Luminy, Case 906, 13288 Marseille-Cedex 9, France
3 Sygen International Plc, Kingston Business Park, Oxon, OX13 5RF, UK.

Quorum sensing, the mechanism by which bacteria detect and respond to their population cell density, is an important example of bacterial intercellular communication. Autoinducer-2 (AI-2), produced by the enzyme LuxS, has been proposed to be a quorum sensing signal molecule in a variety of bacteria. In this work, the role of luxS has been investigated in the plant pathogen Erwinia carotovora subsp. carotovora (Ecc) and the opportunistic animal pathogen Serratia spp. Production of AI-2 activity was detected, and the luxS locus identified and sequenced, in several strains of Serratia and Ecc. luxS mutants were constructed in Ecc and several strains of Serratia and their phenotypes examined. The phenotypes of the luxS mutants include decreased prodigiosin and haemolysin production in S. marcescens ATCC274 and decreased carbapenem production in Serratia ATCC39006. We have also shown, using a Caenorhabditis elegans model, that the luxS mutant of S. marcescens ATCC274 is attenuated in virulence. In Ecc, a proteomic comparison of the luxS mutant with the wild type identified a limited number of proteins whose expression is altered in the luxS mutant. Finally, we have shown that the culture supernatant of one wild type strain (but not its isogenic luxS- derivative) contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain. We conclude that luxS regulates virulence and antibiotic production in Serratia, in a strain-dependent manner, as well as the expression of a limited set of proteins in Ecc. Also, for at least one phenotype, this regulation is via extracellular signalling.


PP13 Non-host resistance in Arabidopsis against wheat powdery mildew

Yun B-W.1, Atkinson H.1, Gaborit C.2, Greenland A.2, Read N.1, Pallas J.A.2 and Loake G.J.1

1ICMB, University of Edinburgh, Edinburgh, EH9 3JH, UK. 2Syngenta, Berkshire, RG42 6EY, UK.

We are investigating the genetic basis of non-host resistance (NHR) in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Attempted infection occasionally resulted in successful penetrations that lead to the development of abnormal unilateral haustoria. Routinely, however, Bgt triggered reactive oxygen intermediate (ROI) accumulation and the rapid hypersensitive cell death of directly challenged epidermal cells. The expression of luciferase reporter genes responsive to ROIs and salicylic acid but not methyl-jasmonate, were also engaged. These defence responses were attenuated in eds1, pad4 and nahG plants but not other defence-related Arabidopsis mutants. Furthermore, Bgt often developed bilateral haustoria in eds1, pad4 and nahG plants that closely resembled those formed in wheat. A similar decrease in NHR was observed in Arabidopsis plants with a defective actin cytoskeleton. In eds1 mutants, loss of actin cytoskeletal function severely compromised NHR in Arabidopsis against Bgt. These plants permitted the infection, growth and asexual reproduction of Bgt. However, colonisation was not as extensive as the compatible interaction on wheat. Genetic screens are currently in progress to uncover the nature of non-host Bgt recognition and additional components required for the expression of NHR against this pathogen and other non-host fungi.


PP14 Studies on cultural characteristics and nitrogen assimilation in Macrophomina phaseolina infecting cluster bean (Cyamopsis tetragonoloba

Sharmishtha Purkayastha1, Bhavneet Kaur1, Neeraj Dilbaghi2, J. Brian Power3, Michael R. Davey3 and Ashok Chaudhury1, 3 

1 Department of Biotechnology, Guru Jambheshwar University, Hisar 125 001, Haryana, India
2 Department of Food Technology, Guru Jambheshwar University, Hisar 125 001, Haryana, India
3 Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. 
E-mail: ashokchaudhury@hotmail.com

Thirty four isolates of Macrophomina phaseolina, collected from four major cluster bean growing states (Haryana, Rajasthan, Punjab and Gujarat) of northern India were grown on four different culture media and subjected to chlorate resistance tests. The pathogen showed maximum growth (abundant mycelia as well as sclerotia) on semi-synthetic media such as potato dextrose agar (PDA) followed by corn meal agar (CMA). Sclerotia formation was rare on synthetic media such as Czapeks Dox agar (CZDA) and Richards agar (RA). There was a distinct variation in the colour of the mycelium from black grey (PDA), dark brown (CMA) to olive green (RA and CZDA). Mycelial texture was fine on semi-synthetic media, but cottony and fluffy on synthetic media. The chlorate phenotype of each isolate was determined following growth on a defined medium containing 120 mM potassium chlorate; chlorate is a nitrate analog. Among the isolates, 70.6% were chlorate-resistant and grew normally, producing numerous dark microsclerotia on medium containing potassium chlorate. The remaining isolates (29.4%) were chlorate sensitive and could be divided into two classes. One class of sensitive isolates showed sparse growth with a feather-like microsclerotial pattern, whereas radial growth of the other sensitive class was almost completely restricted. All the thirty-four isolates from cluster bean were found to be differentially sensitive to chlorate.  This information can be useful in analyzing and exploring genetic variation among these important fungal isolates infecting not only cluster bean but also a wide range of crop plants from cotton to soybean. Comparison of DNA polymorphism of fifty-six isolates from cluster bean, soybean, cotton and okra is in progress using RAPD and PCR-RFLP analyses. This is the first report of the cultural characteristics and chlorate phenotypes of M. phaseolina which infects industrially and economically important cluster bean crops worldwide.


PP15 Tissue culture of Lilium and its applications in fungal resistance

Gracia C. Ribas-Vargas, Michael R. Davey and J. Brian Power

Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK
E-mail: sbxgcr@nottingham.ac.uk 

Many members of the genus Lilium are self-incompatible and tissue culture, aided by embryo rescue, has been used to generate interspecific hybrids as part of conventional breeding strategies. Lilium is susceptible to several fungal pathogens, including Fusarium oxysporum, Botrytis elliptica and Rhizoctonia solani, which infect different parts of the plant leading, ultimately, to a reduction in the yield of cut flowers. In order to produce transgenic plants expressing novel characteristics, such as fungal resistance, reproducible in vitro regeneration protocols are essential as a baseline for transformation procedures. In vitro propagation and regeneration of L. longiflorum White Heaven and the oriental hybrid Star Gazer have been achieved by the culture of pedicel and stem explants on semi-solid Murashige and Skoog (MS) based medium supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (BAP), both at 0, 0.5, 1.0, 2.0 or 5.0 mg l-1. Explants (1 per well) were placed in 25-well plates (Bibby-Sterilin) with 2 ml of medium per well. Dishes were maintained at 23 1C under a 16 h photoperiod (80 *mol m-2 sec-1), or with continuous darkness. Somatic embryos developed, in both the light and dark, after 12 weeks of culture of pedicel and stem explants on MS medium containing 0.5 mg l-1 2,4-D and 1.0 mg l-1 BAP.  Adventitous bulblets developed on explants incubated on medium with 1.0 mg l-1 of both 2,4-D and BAP with a 16 h photoperiod. This micropropagation protocol will form a basis for the induction, selection and regeneration of transgenic Lilium plants carrying a rice chitinase gene (RCH10). It has been demonstrated previously that the action of chitinase enzymes can inhibit the growth of many fungal species as a result of lysis of their hyphal tips. This, in turn, could provide a basis for the introduction of disease resistance into Lilium species.


PP16 Integrative and conjugative elements: A possible role in transmission of pathogenicity factors in Pseudomonads 

Pitman, A. R.1, Jackson, R. W.2, Mansfield, J. W.3, Vivian, A1, and Arnold, D. L.1

1 Centre for Research in Plant Science, University of the West of England, Bristol, UK
2
Department of Plant Sciences, University of Oxford, Oxford, UK
3 Agricultural Sciences Department, Imperial College, Ashford, Kent, UK
Email
Andrew.Pitman@uwe.ac.uk

Plant pathogenic bacteria possess a myriad of factors that enable them to infect and colonise a variety of hosts. Many of these proteins, known as effectors, are secreted directly into plant cells through the type III secretion system. These effectors are described as avirulence factors if they interact with a host resistance protein, triggering a reaction in the plant that leads to a resistance response called the hypersensitive reaction (HR). Although the mechanisms by which Type III effectors elicit plant responses, including the HR, are now being elucidated, little is known about their evolution or the means by which these effectors have been transferred amongst pathogens. Several genes encoding type III effectors have been found to be located on mobile elements including reversibly integrating plasmids and genomic islands. Pseudomonas syringae pathovar phaseolicola (Pph) is the causal agent of halo blight disease in bean (Phaseolus). However, the type III effector, AvrPphB, from Pph race 4, was found to be responsible for the initiation of a hypersensitive response in cultivars of bean carrying the resistance gene R3. Inoculation of resistant cultivars with Pph race 4 usually resulted in a HR, but occasionally variants proliferated to cause disease. Further investigation established that race change was due to the loss of in excess of 40 kb of chromosomally located DNA carrying the avrPphB gene. We present evidence that the deleted region of DNA responsible for race change in Pph race 4 is actually 100 kb in size and is a member of a subclass of integrating and conjugative genomic islands termed ICElands. Comparative genomics suggests the ICEland has a modular organisation, consisting of a conserved backbone structure interrupted by several variable regions thought to be mobile gene cassettes. Components of this family of ICElands were found in a variety of other strains of Pseudomonas syringae suggesting that this family of islands could be widespread amongst pseudomonads and that they could be responsible for horizontal transfer in these plant pathogens. We identified another gene known to encode a type III effector, avrRpt2, which appears to be located on an ICEland suggesting these mobile elements could be especially important in the transmission of pathogenicity factors in Pseudomonas.


PP17 Transcription profiling of the Mycosphaerella graminicola (Septoria tritici) - Wheat interaction 

John Keon, John Hargreaves, Kim E. Hammond-Kosack, and John Antoniw 

Wheat Pathogenesis Programme, Rothamsted Research, Plant-Pathogen Interactions Division, Harpenden, Herts, AL5 2JQ, UK 

The Mycosphaerella graminicola (Septoria tritici) - Wheat interaction project is part of the new Wheat Pathogenesis programme at Rothamsted Research which aims to identify the common molecular themes underlying disease formation on cereal hosts caused by non-biotrophic fungi.  The two other pathogens investigated are the ear blight pathogens Fusarium graminearum and F. culmorum and the eyespot pathogen Tapesia yallundae. In this project we are undertaking micro-array based experiments to identify genes from  M.graminicola up or down regulated during invasion of wheat leaves and growth under in vitro conditions that simulate this environment. 

The project has involved four phases:- 

Phase 1 Accumulation of gene sequence data

5,000 clones derived from three cDNA libraries were sequenced and a gene inventory containing 2,926 unisequences was obtained 1. The sequences of these are available in the COGEME (Consortium for the Functional Genomics of Microbial Eukaryotes) database (http://cogeme.ex.ac.uk/) 2.

1. Keon, J., Bailey, A., and Hargreaves, J. (2000).  A group of expressed cDNA sequences from the wheat fungal leaf blotch pathogen, Mycosphaerella graminicola (Septoria tritici) . Fungal Genetics and Biology 29:118-133.

2. Soanes DM, Skinner  W, Keon J, Hargreaves J, Talbot NJ. (2002) Genomics of phytopathogenic fungi and the development of bioinformatic resources.  Mol. Plant Microbe Interact. 15:421-427. 

Phase 2 Fabrication of micro-arrays

2,568 unisequences were used for micro-array assembly. Inserts from the cDNA clones were initially amplified by PCR.  Micro-arrays were printed on glass slides at Manchester University in the laboratory of Prof. Stephen Oliver as part of the BBSRC COGEME initiative. 

Phase 3 Hybridisation of labelled M. graminicola cDNA  to micro-arrays

Direct labelling of cDNA was done using Superscript Reverse Transcriptase

and Cy-Dye derivatised UTP. Initial experiments have focussed on investigating mycelial growth under high nutrient and low nutrient conditions in vitro.  Present experiments are investigating gene expression during sporulation of the pathogen on wheat leaves.

Current results will be presented. 

Phase 4 Bioinformatic analysis

The microarray results were analysed statistically using Informax Vector Xpression software.  Gene expression profiling is being used to identify the key metabolic pathways that are affected under specific growth conditions3.  The Genome Information Management System (GIMS database and client software) developed for yeast is also being used to analyse the M. graminicola gene expression data, sequences and associated functional information.  This latter activity is undertaken in collaboration with the University of Manchester, COGEME II 4.

3. Keon, J., Curtis, R., Cabrera, H. and Hargreaves, J. (2003)

A genomics approach to crop pest and disease research. Pest Manag. Sci. 59:143-148

4. Cornell, M. et al. (2001)., GIMS - A Data Warehouse for Storage and Analysis of Genome Sequence and Functional Data, to be published in Proc. 2nd IEEE International Symposium on Bioinformatics and Bioengineering (BIBE), IEEE Press, 15-22, 2001. 

Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. This work is supported in part by a grant from the BBSRC to JK & JH as part of the COGEME initiative.


PP18 The powdery mildew fungus From genomics to proteomics

Carsten Pedersen, Ziguo Zhang, Gerhard Saalbach and Hans Thordal-Christensen

Plant Research Department, Ris National Laboratory, Denmark 

Powdery mildew caused by Blumeria graminis is one of the most severe diseases in cereals, especially wheat and barley. The plant-pathogen interaction is controlled by specific resistance genes in the plant and matching fungal avirulence genes according to the gene-for-gene hypothesis. B. graminis is an obligate fungus that develops haustoria inside the epidermal cells and it is important for the fungus to keep the cells alive in order to obtain nutrients from the plant. We are studying the fungus and the interaction at the genome level, the transcriptome level and the proteome level. A detailed study at the genome level has been carried out by sequencing and analysing 74 kb of genomic DNA. It showed a complex mixture of genes, various kinds of retrotransposable elements and other types of repetitive DNA elements. The study gives a glimpse of what to expect of a whole-genome sequencing project, which has now been prioritised by the Fungal Genome Initiative. At the transcriptome level we are looking at expression profiles of a unigene-set of about 1520 genes from an EST-project using high-density arrays on filters. We have compared 5 isolates at three different stages and are now undertaking a more detailed study of expression during the infection process. Our proteome analysis is focusing on the intimate interaction. We have developed a method to isolate haustoria from infected leaves and are now doing proteome analysis on this material aiming at identifying proteins involved in uptake and transport of nutrients as well as proteins involved in signalling and communication with the plant. Results from our studies on the three levels will be presented.


PP19 Unravelling the genetics of durable resistance to Puccinia striiformis f.sp tritici 

C M Lewis, R M D Koebner and L A Boyd

Disease and Stress Biology Department, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH. UK

Yellow rust (caused by the fungal biotroph Puccinia striiformis Westend. f. sp. tritici) is a serious fungal pathogen of wheat in the UK.  It is widely known that there are many single genes (race-specific) that confer yellow rust resistance but such resistance is liable to rapidly decline as the pathogen population mutates to virulence.  This leads to outbreaks of yellow rust under field conditions and consequent yield reductions. Breeders are now sourcing multi-genic resistance (non-race specific) for its potential to improve the adult plant resistance (APR) of wheat cultivars.  Such resistance is commonly termed durable and is characteristically observed when a cultivar has maintained its resistance for several years under widespread cultivation in an environment favourable to the pathogen. Claire (Nickerson Seeds) is a wheat variety that has the durable cultivar Carstens V in its pedigree.  This cultivar has long been believed to be the donor of non-race specific resistance in many durable varieties. The main objective of this project is to determine the genetics of the resistance observed in Claire and see if it contains the durable APR from Carstens V.  The techniques employed (SSR, AFLP, ESTs) are well suited as potential markers to update and improve efficiency when breeding for durability. The specific nature of durable resistance will also be investigated microscopically to determine the wheat growth stage at which it is initiated and the defence mechanism by which the resistance manifests. 


PP20 Endochitinase - a potential gene for phylogenetic studies in Trichoderma

H.P.Narra, A.Culham and R.T.V.Fox

School of Plant Sciences, The University of Reading, Whiteknights, Reading, RG6 6AS, U.K.

Species of Trichoderma (Ascomycetes, Hypocreales) are known for their biocontrol potential against a wide range of fungal pathogens. They are able to produce a large number of extracellular enzymes involved in the degradation of polysaccharides. The 42 kDa endochitinase enzyme plays a major role in mycoparasitism through its control of the ability to break down cell walls.  The nuclear single copy endochitinase gene encoding this protein was used for reconstructing the phylogenetic relationships in the genus Trichoderma. A primer set was used to partially amplify the gene in 40 isolates. The amplified fragment was sequenced for all the isolates to determine their relationship.  Alignment and subsequent cladistic analysis were conducted on this 580bp sequence. Results obtained from sequencing the endochitinase gene clearly differentiated the Trichoderma species under study indicating the potential of this region as a molecular marker. 


PP21 Metal transporters in Blumeria graminis f.sp. hordei, the barley powdery mildew

Alice Smith, J.L. Hall and Lorraine E. Williams

School of Biological Sciences, University of Southampton, UK

Powdery mildew fungi infect a wide range of plant species causing significant losses in a number of economically important crops such as barley and wheat. When the biotrophic fungus invades a plant epidermal cell, a complex interface known as the haustorial complex is formed between the host plant and the pathogen. It is believed that all nutrient transfer from host to pathogen occurs via the haustorium. Previous research has suggested that infection with powdery mildew fungi can result in considerable modification of host photoassimilate production and partitioning. The fungus relies on the host not only for sugars but also for essential mineral nutrients. We are studying the molecular basis of micronutrient uptake (eg. iron, copper, manganese and zinc) by the barley powdery mildew fungus, Blumeria graminis f.sp. hordei. Here we describe the cloning of several putative metal transporter cDNAs from Blumeria graminis which may play an important role in heavy metal transport and homeostasis. Based on sequence infromation of metal transporters in other organisms, RT-PCR was used o amplify cDNAs from the CPx-ATPase and NRAMP families. Analysis of the expression and mechanism of these transporters should provide a basis for further characterisation of nutrient transfer in this plant pathogen interaction. Efficient nutrient uptake from the host plant is essential for fungal survival; therefore understanding the basis of this process may enable the development of fungal control strategies.


PP22 Coordinated expression of Type III secretion system genes and effector proteins in Pseudomonas syringae pv. phaseolicaola

R Thwaites1, P Spanu2, C Stevens3 and JW Mansfield1

1 Department of Agricultural Sciences, Wye Campus, Imperial College London, Wye, Ashford, Kent TN25 5AH
2 Department of Biology, Imperial College London, Exhibition Road, London SW7 2AZ
3 Present address: International Rice Research Institute, Dhaka Office, GPO Box 64, Ramna, Dhaka 1000, Bangladesh 

Pseudomonas syringae pv. phaseolicola, like many other Gram-negative bacterial pathogens, delivers proteins directly into host cells through a type III secretion system (TTSS).  The regulation of TTSS and effector proteins in other bacteria, notably human pathogens such as Yersinia and Salmonella, has been studied in some depth.  These studies, as well as molecular and microscopic observations of the complexity of the interaction between P. syringae pv. phaseolicola and its host, suggest that genes encoding components of the TTSS and effector proteins are likely to be regulated in a deliberate and coordinated manner. We have used  Taqman quantitative reverse-transcriptase polymerase chain reaction amplification to examine the transcriptional regulation of genes encoding components of the P. syringae pv. phaseolicola TTSS, as well as those coding for effector proteins.  TTSS and effector genes are known to be activated when the bacterium enters its plant host or when cultured in a minimal medium which mimics the plant apoplast environment. In phytopathogenic Pseudomonas spp. this induction is mediated by hrpL, an alternative sigma factor encoded within the hrp cluster. Primers targeted to genes representing all operons within the hrp cluster have revealed that there is a hierarchy of expression amongst TTSS genes in both the host (bean) and minimal medium.  Expression in planta was, however, consistently higher than that in vitro. Furthermore, we have been able to separate bacteria attached to plant cells from those unattached in the apoplasm, and have revealed that expression of TTSS and effector genes is higher in cells attached to the plant host.  These results suggest that an ability to sense contact with the plant as well as responsiveness to the apoplastic environment or minimal medium is important in regulating TTSS and effector genes in P. syringae pv. phaseolicola. In addition to TTSS genes associated with pathogenicity we have also found another gene which is induced in a hrpL-dependent manner.  The fliC gene, which in P. syringae pv. phaseolicola encodes the flagellin subunit, is itself exported via a TTSS previously thought to be unconnected with the hrp secretion system.  However, we have found that, although fliC is transcribed constitutively in rich medium, its expression is greatly increased on culture in minimal medium or following infiltration into bean leaves.  There may, therefore, be a complex regulatory network linking different TTSS systems within the same bacterium. 


PP23 Cytokinins as Modulators of Plant Defence Responses 

Thea K. Davies and Paul Kenton

Institute of Biological Sciences, University of Wales, Aberystwyth, SY23 3DA, UK 

A number of chemical signals, including jasmonates, salicylate and ethylene, have been shown to play key roles in the regulation of plant responses to pathogens. There has been some interest, over several decades, in the possible role(s) of cytokinins in plant defence. Some pathogens, chiefly biotrophs, synthesise cytokinins probably in an attempt to maintain tissue viability and establish a nutrient sink. Exogenous application of cytokinins has shown inhibition of defence responses (especially the hypersensitive response; HR) in some cases, whilst in others treatment with cytokinins appears to enhance defence. We have shown that, whilst cytokinins have a phenotypic effect on HR development, suggestive of compromised resistance, this effect was correlated with neither a delay or reduction in cell death, nor a decrease in the pathogen-induced oxidative burst. Rather, alterations in the extent of HR lesion development may reflect changes in the movement of the pathogen itself. Surprisingly, avirulent bacterial pathogens appear to escape from HR tissue (even in fully resistant wild-type plants), and the extent of the movement of these bacteria is affected by cytokinin treatment. We have shown that, whilst cytokinins have a phenotypic effect on HR development, suggestive of compromised resistance, this effect was correlated with neither a delay or reduction in cell death, nor a decrease in the pathogen-induced oxidative burst. Rather, alterations in the extent of HR lesion development may reflect changes in the movement of the pathogen itself. Surprisingly, avirulent bacterial pathogens appear to escape from HR tissue (even in fully resistant wild-type plants), and the extent of the movement of these bacteria is affected by cytokinin treatment.


PP24 Transmembrane Transport of Salicylate 

Andrew Clarke, Robert M. Darby  and Paul Kenton

Institute of Biological Sciences, University of Wales, Aberystwyth, SY23 3DA, UK

Salicylate (SA) is a key regulator of plant defence responses. Although much progress has been made in several key areas of SA biology, surprisingly little is known about the mechanisms of transport across the plasma membrane. We have been using an Arabidopsis suspension cell model to study transmembrane SA transport and these studies have shown that: SA is rapidly taken up into cells and then almost completely exported. There is little evidence for any conjugation or storage in the vacuole; SA uptake is subject to prolonged inhibition following the first uptake phase; SA is pH-dependent and is associated with an inward flux of H+; SA uptake is associated with transient acidification of the cytosol but relatively prolonged alkalinisation of the apoplast; SA uptake is via a non-specific route and is subject to competitive inhibition by SA analogues.


PP25 Characterisation and Marker-aided Selection of eyespot Resistance in wheat

Natalie Chapman, P. Nicholson and R.M.D. Koebner

Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, U.K

Eyespot is a fungal disease of the stem base of cereal crops.  It causes lodging and premature ripening of grain in the host plants and reduces crop yield.  The causal agents of eyespot are Tapesia yallundae and Tapesia acuformis.  A potent resistance gene Pch1, found in a wild related grass species Aegilops ventricosa, was introduced into wheat to produce the line VPM (Maia, 1967) and is located on chromosome 7D. Unfortunately there appears to be a linkage drag between Pch1 and genes deleterious to yield.  A single chromosome substitution line in which chromosome 7D from VPM was substituted into a Hobbit-sib background (Hobbit-sib (VPM7D)) was crossed with Hobbit-sib to produce a recombinant population (RVPM). The RVPM population differing in number and sizes of Ae. ventricosa segments on chromosome 7D was mapped with  simple sequence repeat markers (SSR).  An endopeptidase isozyme marker Ep-D1b, derived from Ae.ventricosa has been shown to be closely linked to the resistance gene Pch1 (McMillin, et al. 1986).  One SSR (wmc14) mapped to the same location as isozyme marker Ep-D1b and can be used for the selection of eyespot resistance.  RVPM line 25, containing a reduced Ae. ventricosa segment, was identified and crossed with Hobbit-sib to produce a recombinant population with smaller Ae. ventricosa segments to refine the mapping of Pch1 and the link to deleterious effects.  Due to a limited amount of SSR markers that map to the distal long arm of chromosome 7D, the cDNA-AFLP technique is being used to develop markers for Pch1. A second eyespot resistance gene (Pch2) is located on Chromosome 7A and is present in Cappelle Desprez (CD) and absent in Chinese Spring (CS). As above a single chromosome substitution line was available in which Cappelle Desprez 7A is substituted into Chinese spring.  A CS: CS (CD 7A) recombinant Pch2 mapping population is being developed and will be used to screen for eyespot resistance and identify suitable DNA markers for the fine mapping of Pch2.  The cDNA-AFLP technique is also being used to identify genes differentially expressed between CS and chromosome substitution CS (CD 7A), in the search for Pch2.


PP26 Functional genomics of Pasteuria penetrans, a bacterial parasite of plant-parasitic nematodes

Keith G. Davies1,2, Andrew Warry1, Jenora Waterman2, David McK. Bird1,2 and Charlie H. Opperman1,2 

1 Nematode Interaction Unit, Rothamsted Research, Harpenden, Hertfordshire UK
2
Centre for the Biology of Nematode Parasitism, North Carolina State University, Raleigh, North Carolina, USA
Email:
Keith.Davies@bbsrc.ac.uk

Pasteuria penetrans is part of a group of Gram-positive endospore forming bacteria that are reported to infect nematodes and Cladocera (Water fleas).  Research into this bacterium has been focused on them being hyperparasites with potential for development into biological control agents of plant parasitic nematodes.  However the inability to culture large quantities of the bacterium in vitro for large field scale applications, and the fact that one population of the bacterium will attach to and infect one population of nematodes but not another has prohibited its commercial development. We have constructed four genomic libraries using a whole shot gun sequencing approach and have analysed 9,074 sequencing reads, resulting in 2,840,162 bp of primary sequence.  This initial sequence assembled into approaximately 1,500 contigs and covers more than 1.5 Mb of the Pasteuria penetrans genome.  Approximately 50% of the sequences have yielded significant (e-value < 1.0e-10) similarities to known genes in the NCBI database. Mapping our sequences onto the completed genomes of other Bacilli, using ARTEMIS, we have observed significant co-linearity between P. penetrans and other closely related Bacilli such as B. subtilis, B. halodurans and B. anthracis.  Key stages in the life cycle of Pasteuria include endospore adhesion, germination and sporulation and knowing the complete genome sequences of these other closely related bacteria enables us, by comparing genomes, to generate hypothesis that will help us to understand the unique biology, ecology and evolution of this important hyper-parasite. Comparative genomics could also provide an invaluable tool to help solve the problems prohibiting this organism from being developed into a biological control agent.  


PP27 DiGE Analysis of the Regulation and Secretion of Virulence Factors in Erwinia carotovora subspecies atroseptica 

Mark Corbett, Sam Virtue, Kathryn Lilley and George Salmond 

Department of Biochemistry, University of Cambridge, UK 

The Gram-negative bacterium Erwinia carotovora subspecies atroseptica (Eca) is a member of the Enterobacteriacae, and causes blackleg disease in the stems and soft rot in the tubers of potato plants. Several factors have been shown to contribute to the virulence of Erwinia infections, including motility, and some strains produce the antibiotic carbapenem. Particularly important in the pathogenicity of Eca is the production of extracellular enzymes, including proteases, cellulases and pectate lyases. The production of these exoenzymes is regulated by an N-acyl homoserine lactone quorum sensing system, which delays full synthesis until population density has reached a threshold. The secretion of the exoenzymes out of the bacterial cell occurs by various routes. Cellulase and Pectate Lyase are exported to the periplasm before being secreted by the Out apparatus, a Type II secretion system. We have used 2D PAGE to investigate the array of proteins secreted by Eca in liquid culture including, although not limited, to the exoenzymes above. This is allowing the discovery of previously unidentified secreted putative virulence factors. In addition the technique is allowing further investigation of the known virulence factors, including conditions inducing their production and post translational modifications. Difference gel electrophoresis (DiGE) involves fluorescent labelling of protein and allows multiple samples to be compared on 2D gels. This technique has been used to compare the secreted proteins of various mutants, including strains deficient in secretion. An Out- mutant has revealed new proteins that use this virulence associated secretion pathway. In addition a quorum sensing mutant has shown that the production of many of the secreted proteins, including "novel" proteins, is subject to this regulation, suggesting they may contribute to virulence. This technique offers a new route to identifying factors in Eca virulence, and a greater understanding of its pathogenicity. In collaboration with the Scottish Crop Research Institute, Dundee, genes encoding novel putative virulence factors (targeted via the Type II pathway) are being mutated in a functional genomics programme.


PP28 Metabolic fingerprinting and profiling discriminates between resistant and susceptible interactions involving the rice blast pathogen Magnaporthe grisea and Brachypodium distachyon

Allwood, J. W.*; Ellis. I. D. , Heald, J. K.*, Draper, J.*; Goodacre, R. and Mur, L. A. J.*

*Institute of Biological Science, University of Wales Aberystwyth, Ceredigion, Wales, SY23 3DA. UK, Department of Chemistry, UMIST, PO Box 88, Sackville Street, Manchester, M60 1QD, U.K.

Currently, functional genomic approaches to biological phenomena are mostly focused at the levels of the transcriptome and proteome. Nevertheless, the accurate discrimination of the metabolome, although technologically more demanding, has a broader range of application and is much more information rich. We have applied two metabolomic approaches to elucidate and identify the biochemical changes occurring within the pathogen and host during the interaction of Magnaporthe grisea the cause of rise blast disease1 and the emerging Pooid model species Brachypodium distachyon2. Two scenarios were investigated; the establishment of disease symptoms and events associated with elicitation of a hypersensitive response (HR). Fourier-transform Infrared spectroscopy (FT-IR) was employed for high-through put metabolomic fingerprinting3, 4 (the generation of a metabolome signature with no regard for individual metabolites) the interactions of M. grisea with the B. distachyon ecotypes ABR1 (susceptible) and ABR5 (resistant) at 0, 3 and 5days post challenge (n=3 biological replicates).  Multi-variant statistical approaches, particularly discriminant function analysis (DFA), allowed the discrimination ("clustering") between developing disease symptoms and the HR at each time point. To validate the observations, the results for two replicates were combined and the third replicate ("test-set") was projected on to these data.  DFA again showed tight clustering indicating a high degree of agreement between data and demonstrating that the experimental approach produced highly reproducible data. Metabolomic profiling allows the detection, quantification, and identification of pre-defined target compounds within the metabolome, for which we employed electrospray ionisation mass spectrometry (ESi:MS). Focusing on the interaction of M. grisea and B. distachyon ABR1 and ABR5 at the 3 day stage of infection DFA analysis of four replicates again revealed tight clustering of challenged from healthy material.  Subtraction spectra revealed those ions that separated healthy from infected material, those which were also present in the most significant DFA loadings, were attributed as substances involved in the fungal reprogramming of the host metabolome or the plants resistance to the fungus.  These substances were identified, the identity of which are presently being confirmed via Tandem-MS.  Ten replicates of each experimental class were used for data validation.  The ten replicates showed tight clustering in DFA ran to 12 principle components, again indicating a highly reproducible system for metabolomic analyses. Our data will undoubtedly reveal novel insights, particularly into in planta fungal development and illustrate the potential of a metabolomic approach to investigate plant-pathogen interactions.

References: 

1Talbot et al., (1995).Trends Microbiol. 3:9-16; 2Draper J,* Mur LA*, et al., (2001). Plant Physiol.127:1539-55. * joint first authors 3Goodacre et al., (1998) Microbiology 144: 1157-1170; 4Vaidyanathan et al., (2001) Anal. Chem. 73:4134-4144.


PP29 Development of phoma canker (Leptosphaeria maculans) on stems of winter oilseed rape in England in relation to thermal time

Bruce D. L. Fitt, Ping Sun, Julie M. Steed, Claudia T. Underwood and Jonathan S. West 

Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

In winter oilseed rape experiments at Rothamsted in 1997/98 (cvs Lipton and Capitol), 1998/99 (cv. Apex) and 1999/2000 (cvs Apex, Lipton and Capitol), development of crown canker and phoma stem lesions in spring was related to development of phoma leaf spot in the previous autumn/winter.  There were differences in thermal time (degree-days) from the first appearance of phoma leaf spot (autumn) to the first appearance of crown canker (spring) between cultivars (cvs Lipton and Capitol, 1220-1240; cv. Apex, 1120-1140 degree-days) but not between growing seasons.  In 1998/99 and 1999/2000, fungicide (November) treatment delayed the start of crown canker development in the spring but did not affect the rate of increase in severity.  In 1997/98, fungicide treatments did not delay the appearance of crown canker but decreased the rate of increase in crown canker severity.  In all three seasons, fungicide treatments generally decreased the proportions of plants at harvest with crown canker severity scores 3 or 4 and increased the proportions with scores 0 or 1.  There were differences between seasons in the distributions of crown canker severity scores at harvest.  The severity of both crown canker and phoma stem lesions increased linearly with accumulated degree-days in plots with or without fungicide treatment in 1997/98 (cv. Lipton), 1998/99 (cv. Apex) and 1999/2000 (cv. Apex).  Regressions showed that severity of crown canker at harvest in July was related to severity in the spring in 1997/98 (early June, cv. Lipton), 1998/99 and 1999/2000 (April, cv. Apex).


PP30 Relations between light leaf spot (Pyrenopeziza brassicae), pod canopy size and winter oilseed rape yield in England

Bruce D. L. Fitt1, Gavin D. Lunn2, Julie M. Steed1, Andreas Baierl1, Neal Evans1 and Peter Gladders3 

1 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
2 University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK
3 ADAS Boxworth, Cambridgeshire CB3 8NN, UK

Mechanisms of yield loss from light leaf spot (Pyrenopeziza brassicae) and effects of tebuconazole fungicide regimes were examined in winter oilseed rape at Rothamsted in 1997/98.  There was no evidence that light leaf spot decreased plant populations, although disease-related winter kill can decrease yield.  The best light leaf spot control was with routine or autumn/spring applications of tebuconazole, with responses greater for cv. Bristol (susceptible) than cv. Capitol (resistant).  Cv. Capitol had a greater pod green area index (GAI, estimated from light interception measurements) than cv.  Bristol and routine tebuconazole treatments produced largest GAIs for both cultivars.  Cv. Capitol yielded more (mean of all treatments 3.70 t ha-1) than cv. Bristol (3.23 t ha-1).  Routine fungicide treatments most increased yield over the untreated (by 1.29 t ha-1 for cv. Capitol and 1.93 t ha-1 for cv. Bristol).  Other treatments also increased yield, with best responses for treatments including a spring fungicide application.  Yield was positively related to pod GAI in June, indicating that the main mechanisms of yield loss from light leaf spot were reductions in numbers of pods and canopy GAI (which reduced capacity to capture light).


PP31 Appressorium and germinating cyst-specific gene discovery in Phytophthora infestans  

Anna Avrova1, Steve Whisson1, Laura Grenville2, Pieter van West2 and Paul Birch1  

1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA
2 University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD. 

Phytophthora infestans, the causal agent of potato and tomato late blight, produces several different cell types prior to penetration of the host plant and during the early stages of infection. Cell types including sporangia, zoospores, germinating cysts, and appressoria can be generated in the absence of the host plant and so form the basis for stage-specific gene discovery. Amplified fragment length polymorphism based mRNA fingerprinting (cDNA-AFLP) and suppression subtractive hybridisation (SSH) are being used to target transcripts specifically up-regulated during cyst germination and appressoria formation; structures formed just prior to infection of potato. These structures are likely to contain many transcripts involved in successful penetration of the host, and establishment of a compatible interaction. Transcripts identified encode proteins potentially involved in adhesion, cell wall degradation, signalling, virulence, amino acid and protein biosynthesis, stress response, and detoxification. Expression of genes identified using SSH and cDNA-AFLP is being quantified by real-time RT-PCR, relative to the constitutively expressed actB gene, in vegetative mycelium, sporangia, zoospores, germinating cysts, germinating cysts with appressoria and at several time points post-inoculation of susceptible potato cultivar Bintje. Based on the expression profile of the identified transcripts, they will be prioritised for functional analysis to determine their role in the pathogen lifecycle and interaction with the host plant, potato.


PP32 A candidate Avr3 gene from Phytophthora infestans  

Steve Whisson1, Miles Armstrong1, Sophien Kamoun2, Anne Rehmany3, Jim Beynon3, Anna Avrova1, Paul Birch1 

1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA
2 Dept. Plant Pathology, Ohio State University, OARDC, Wooster OH 44691, USA
3 Horticulture Research International, Wellesbourne, CV35 9EF

Late blight of potato and tomato, caused by the oomycete Phytophthora infestans, is a devastating disease of these important crop plants worldwide. P. infestans has a narrow host range and exhibits race/cultivar specificity matching the gene-for-gene model.  As a first step in studying the molecular basis of host/oomycete recognition and specificity, we have set out to clone avirulence genes from P. infestans.  Data mining of P. infestans ESTs yielded several genes with avirulence gene-like features, such as small size, cysteine-rich, and a predicted signal peptide.  An association genetic screen of SNPs in these potential avirulence genes has led to the identification of a strong candidate for the Avr3 gene.  Real-time RT-PCR and proteomics have revealed the gene expression profile and secretion of the gene product, respectively.  The genomic organisation of the candidate gene has shown that four copies exist in the P. infestans genome, all clustered within an approximately 250 kb region.  The association with avirulence has been pinpointed to one of these copies and the observed association breaks down rapidly with physical distance from the candidate gene.  The genomic region surrounding the candidate gene shows conserved micro-synteny with the ATR1 avirulence locus from a second oomycete pathogen, Peronospora parasitica (Arabidopsis downy mildew).  The similarity in pathogenicity and physiology shared by these diverse oomycetes suggests that the observed synteny is biologically relevant.  Recombinant P. infestans lines carrying the candidate gene are currently being evaluated to determine if the candidate gene conditions avirulence towards the cognate potato resistance gene R3.


PP33 Regulation of infection plaque formation in Tapesia spp. the cereal eyespot fungi

Kerry Maguire1, Matthew Dickinson 2, Paul Bowyer1, John Lucas1 and Kim Hammond-Kosack1

1 Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ
2 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough

Cereal eyespot, caused by the closely-related fungi Tapesia yallundae and T. acuformis, is one of the most important diseases of wheat, barley, and rye in temperate regions of the world. These pathogens infect the stem base, predisposing the plant to lodging; if vascular tissues are penetrated the ears become white and fail to mature. This results in poor grain quality and lower yields. Tapesia infects the plant by two different mechanisms. Initially, conidia germinate and form simple infection hyphae that penetrate the coleoptile. A mechano-stimulus, created by contact between the coleoptile and the first leaf sheath, is then sensed by Tapesia and triggers the development of an infection structure or plaque. Plaques are complex multicellular mats with multiple infection hyphae required for penetration of the next leaf sheath. Mechano-sensing is therefore key to the infection process. The object of this project is to examine the mechanisms involved in mechano-sensing. An in vitro technique has been developed to test mechano-sensing and plaque formation. Both T. yallundae and T. acuformis respond to the in vitro mechano-stimulus but the architecture of the plaque is different in the two species. T. yallundae forms dense plaques whereas T. acuformis forms digitate plaques. The second part of the project involves screening for mutants, of T. yallundae, that are unable to sense pressure or to form functional plaques. Mutants have been created using N-methyl-N-nitro N-nitrosoguanidine (NTG), a chemical mutagen. Colonies have been identified that form more plaques than the wild type but to date mutants unable to form plaques have not been found. A range of chemicals that are known to alter signal transduction pathways have illustrated that there are two G proteins involved in plaque formation. Site directed mutagenesis and gene knockouts are being used to dissect the role these G proteins have on plaque formation. Work has also begun to look at the initial receptor for this mechano-stimulus. This research will provide information on how the mechano-stimulus is sensed and on the signal transduction pathway that leads to infection structure formation.


PP34 Expression of a WKRY-like transcription factor in potato roots in response to infection by potato cyst nematodes (PCN)

Blok, V.C., Armstrong, M., Phillips, M.S., Paterson, A., Young, V., Avrova, A. and Birch, P.

Plant Pathogen Interaction Program, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA
Email vblok@scri.sari.ac.uk

Expression of the WRKY-like transcription factor, StWRKY-1, is upregulated after inoculation by pathogens Erwinia carotovora subsp. atroseptica and Phytophthora infestans in leaves and is also induced by potato cyst nematodes, Globodera pallida in roots. This suggests activation of common host defence responses either directly by these pathogens or indirectly by plant degradation products generated following infection of the host. Potato cv Desiree was transformed with a construct encoding the St-WRKY1 transcript and expression of the transcript in roots and leaves determined by TaqMan. The levels of expression differ between the transgenic lines. In a preliminary experiment levels of reproduction of PCN have been assessed and differences were found relating to different expression levels of the StWRKY1 transgene. The authors thank Dr Erika Schulte-Kappert, Planta, Einbeck, Germany for production of the transgenic lines and acknowledge funding from EU PL98-4235 (No Nematode) and the Scottish Office Agriculture, Environment and Fisheries Department.


PP35 Mode of Action Studies of Quinoxyfen, a highly efficacious barley powdery mildewicide

Susannah Lee

University of Oxford

Quinoxyfen is a new, low-risk, protectant fungicide which controls powdery mildew by interfering with normal appressorium formation. The exact mode of action remains unknown. Quinoxyfen-resistant isolates of Blumeria graminis f. sp. hordei produce fewer conidia, which germinate and form appressoria more promiscuously than wild type conidia. This suggests Quinoxyfen is perturbing signal perception and / or transduction in B. graminis.. Molecular data support this hypothesis, since Quinoxyfen alters the accumulation of Protein Kinase C (pkc), pkc-like and catalytic subunit of Protein Kinase A (cpka) transcripts, revealed by RT-PCR profiles. Differential display RT-PCR indicates the involvement of a GTPase activating protein (GAP), which is absent or much less abundant in Quinoxyfen-resistant isolates compared to wild type, although transcript sequences were identical so this GAP is not the target of Quinoxyfen. These data suggest Quinoxyfen inhibits mildew infection by disrupting early cell signalling events. Scanning electron microscopy (Cryo-SEM) reveals that Quinoxyfen-resistant isolates form colonies with unusual morphology, characterised by looping, aerial hyphae. This is reminiscent of filamentous fungi in which polarity is disrupted and signal transduction is perturbed. This result has prompted a comprehensive series of "scoring" experiments, where the morphology of wild type and Quinoxyfen-resistant conidia with and without Quinoxyfen are examined by light microscopy. Wild type conidia allowed to germinate on barley leaves pre-treated with Quinoxyfen show pleiotropic unusual phenotypes, such as multiple germ tubes and long, swollen but undifferentiated germ tubes. I have exploited our PCR-based "walking" technique to define the sequence for the B. graminis cdc42 gene, a key gene linking polarity to signal transduction, and plan to search for other important genes linking these processes. I will use confocal microscopy to explore the skeletal architecture of the developing appressoria, and Real Time RT-PCR to profile key gene expression over time. It is hoped that these studies will lead to the elucidation of the mode of action of Quinoxyfen.


PP36 Stem canker of oilseed rape: molecular tools and mathematical modelling to deploy durable resistance

Neal Evans1, Bruce Fitt1, Frank van den Bosch1, Maria Eckert1, Yong-Ju Huang1, Stephane Pietravalle1, Zbigniew Karolewski1,10, Thierry Rouxel2. Marie-Helene Balesdent2, Simon Ross2, Lilian Gout2, Hortense Brun3, Didier Andrivon3, Lydia Bousset3, Peter Gladders4, Xavier Pinochet5, Annette Penaud5, Malgorzata Jedryczka6, Piotr Kachlicki6, Anna Stachowiak6, Julia Olechnowicz6, Anna Podlesna8, Ingrid Happstadius7, Jan Meyer7 and Michel Renard9

1 Rothamsted Research, Harpenden, Herts., AL5 2JQ, U.K. E-mail:neal.evans@bbsrc.ac.uk
2
INRA, Unit PMDV, Route de St Cyr, Versailles 78026, France. E-mail: rouxel@versailles.inra.fr
3 INRA UMR BiO3P and 9UMR APBV, Domaine de la Motte BP 35327, Le Rheu Cedex 35653, France. E-mail: brun@rennes.inra.fr
4
ADAS Boxworth, Cambridge, CB3 8NN, UK. E-mail: peter.gladders@adas.co.uk
5 CETIOM, B.P. no. 4, Thiverval-Grignon 78850, France. E-mail: pinochet@cetiom.fr
6 IGR PAN, Strzeszynska 34, Poznan, 60-479, Poland. E-mail: mjed@igrnov.igr.poznan.pl
7 Svalf Weibull AB, Svalv, SE-268 81, Sweden. E-mail: ingrid.happstadius@swseed.se
8 IUNG-Pulawy, Czartoryskich 8, 24-100 Pulawy, Poland.
10
Dept. of Phytopathology, August Cieszkowski Agricultural University, Dabrowskiego 159, 60-594 Poznan, Poland.
E-mail: karolew@au.poznan.pl

SECURE (Stem canker of oilseed rape: molecular tools and mathematical modelling to deploy durable resistance; QLK5-CT-2002-01813) aims to deliver a model for deployment of cultivars with resistance to Leptosphaeria maculans (phoma stem canker/blackleg) to improve durability of resistance and minimise risk that the resistance will break down. The objectives are: 1. To construct a model of the life cycle of L. maculans and validate it with existing data; 2. To compare the fitness of virulent/avirulent races of the pathogen and develop genomic analysis of avirulence and virulence loci; 3. To analyse effects of plant genetic background and environmental factors on durability of resistance, both in field and controlled conditions; 4. To model effects of resistance deployment strategies on durability of resistance and recommend a sustainable strategy. Results will be disseminated and discussed during the course of the project using a website (www.secure.rothamsted.ac.uk), scientific and popular publications and workshops. The SECURE project is supported by the European Commission under the Fifth Framework Programme.


PP37 Genetic analysis of disease susceptibility in the Arabidopsis thaliana-Peronospora parasitica interaction

Van Damme M, Andel A, Huibers R, Weisbeek P, and Van den Ackerveken G.

Molecular Genetics Group, Dept. Molecular and Cellular Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Plants are susceptible to a limited number of pathogens. They resist most infections by early pathogen recognition and the subsequent activation of plant defense responses. To grow and reproduce on plants and to avoid recognition, pathogens have evolved advanced mechanisms to attack host cells. The oomycete pathogen Peronospora parasitica interacts with the plant through haustoria, feeding structures that invaginate host cells but remain surrounded by a host membrane. To gain insight into the genetic basis of disease susceptibility and haustorium function, Arabidopsis EMS-mutants were created in the eds1-2 background. Fifteen downy mildew resistant (dmr) mutants were isolated so far, of which 8 are characterized in more detail. These represent 6 different dmr loci, that we have mapped. Microscopic analysis showed that in many cases Peronospora haustoria are surrounded by callose and/or have an aberrant form, indicating a distortion of the plant-pathogen interaction. dmr3, 4 and 5 show induced activation of defense responses. In addition dmr3 and dmr4, but not dmr5, show resistance to Pseudomonas syringae. We hypothesize that in dmr1, 2,and 6 important cellular targets for Peronospora infection are disrupted. We will report on the fine mapping of the six dmr mutations and our effort to clone the corresponding DMR genes. Their functional analysis will provide new insights into disease susceptibility and the molecular processes that occur at the host-pathogen interface.


PP38 Molecular aspects of pathogenicity of Clavibacter michiganensis ssp. sepedonicus

I. Holtsmark1, G. D. Mantzilas3, V. Eijsink2, M.B. Brurberg1

1The Norwegian Crop Research Institute, s, Norway 2Dept. of Chemistry and Biotechnology, Agricultural University of Norway, s, Norway 3Dept. of Biochemistry, University of Oslo, Norway

Potato ring rot is caused by the Gram-positive coryneform bacterium Clavibacter michiganensis ssp. sepedonicus and is an economically important disease in potatoes. There are currently no strategies to combat this disease once it has established itself in a crop. We are studying molecular aspects of Clavibacter pathogenicity, the ultimate goal being identification of new targets and strategies for disease control. We have initially been focusing on antimicrobial compounds and secreted enzymes. An antimicrobial protein produced by strains of Clavibacter michiganensis ssp. michiganiensis has been identified. This protein has a narrow activity spectrum comprising its close relative C. m. ssp. sepedonicus, which makes it a potential control agent against the latter. The protein was purified from spent culture supernatant, by ammonium sulphate precipitation, followed by chromatography on SP-sepharose and Resource reverse phase columns.


PP39 Mutagenesis and phenotypic analysis of all potential glycosylation sites in the Cf-9 resistance protein

Brande B.H. Wulff*1, Susana Rivas*1, Renier A.L. Van der Hoorn2, Pierre J. G. M. de Wit2 and Jonathan D.G. Jones1

1 The Sainsbury Laboratory, Norwich, United Kingdom. 2Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands.

The tomato Cf-9 gene confers resistance to the fungal pathogen Cladosporium fulvum through recognition of the avirulence determinant Avr9. The Cf-9 sequence predicts an extracytoplasmic membrane anchored leucine-rich repeat glycoprotein with 22 putative glycosylation sites distributed throughout its sequence. Cf-9 induces an Avr9-dependent hypersensitive response (HR) in tobacco species, that has been widely used as an experimental tool to study Cf-9 function. In this study, all 22 putative glycosylation sites in Cf-9 were mutagenized and the effect of the engineered mutations on the Cf-9/Avr9-dependent HR was analysed.

S.R. and B.B.H.W. contributed equally to this work


PP40 Saponin Glycosyl Hydrolases as Virulence Determinants in Septoria lycopersici

Diaz-P. N., Bouarab K., Melton R., Carter J. and Osbourn A.

The Sainsbury Laboratory. Norwich, England
E-mail: annie.osbourn@sainsbury-laboratory.ac.uk

A number of plant pathogens are known to degrade the antimicrobial secondary metabolites of their hosts and in some cases this phenomenon has been shown to be associated with virulence. The fungus Septoria lycopersici (a foliar pathogen of various Solanaceous species) produces the extracellular enzyme tomatinase, which hydrolyses the tomato steroidal glycoalkaloid saponin alfa-tomatine to the less toxic product, beta-2-tomatine. Tomatinase-minus mutants of S. lycopersici are unable to degrade alfa-tomatine and display enhanced sensitivity to this compound. These mutants are still able to cause disease on tomato leaves, but trigger enhanced cell death and elevated expression of plant defence genes during the early stages of infection. A more striking effect of loss of tomatinase is seen on an alternative Solanaceous host, Nicotiana benthamiana. Tomatinase-minus mutants of S. lycopersici are unable to cause disease on this host and elicit a hypersensitive-like response. Our results indicate that tomatinase is involved in a novel two-component process in which host saponins are hydrolysed and the resulting hydrolysis products then suppress induced defence responses via interference with fundamental disease resistance signal transduction processes. Further characterisation of the role of tomatinase in interactions between S. lycopersici and Solanaceous hosts is in progress.


PP41 Novel developmental processes associated with infection of roots by the rice blast fungus

Ane Sesma and Anne E. Osbourn

The Sainsbury Laboratory, John Innes Center, Colney Lane, Norwich NR4 7UH, U.K
Email: ane.sesma-galarraga@sainsbury-laboratory.ac.uk, annie.osbourn@sainsbury-laboratory.ac.uk

Magnaporthe grisea is responsible for the devastating blast disease of rice and other economically important grasses such as wheat, barley and maize. M. grisea is closely related to other pathogenic (M. poae, M. rhizophila and Gaeumannomyces graminis) and non-pathogenic (Phialophora spp.) root-infecting fungi. Although the rice blast fungus M. grisea is traditionally regarded as a foliar pathogen, this fungus can also cause disease symptoms on cereal roots. We have used cytological methods to investigate this root-infection process in more detail. We generated GFP-tagged transformants of different M. grisea wild-type strains. Chlorazol Black E was also used to visualise the infection process by standard light microscopy. Remarkably, M. grisea is capable of undergoing a range of developmental processes that are typical of root pathogens and forms microsclerotia, runner hyphae and hyphopodia. Bulbous swollen hyphae can be observed within the root cortex, and the fungus progresses through the cortex and invade the stele. Genetic determinants of foliar pathogenicity have been extensively characterised in M. grisea, and a number of mutants with impaired ability to penetrate and invade leaf tissues are available. We have assessed a collection of these mutants for the ability to infect cereal roots and have identified fungal genes that are specifically required for infection of either leaves or roots, and also those that were general determinants of pathogenicity to both tissues. Infection studies with GFP-expressing transformants indicate that M. grisea can spread from the roots to the leaves and produce lesions, suggesting that the soil may be a source of inoculum for the establishment of rice blast disease in the field.


PP42 Genetic diversity in the perennial ryegrass crown rust pathogen Puccinia coronata Corda f. sp. lolii

J.W. Forster1,3, J.L. Dumsday1,3, N.M McFarlane2,3, R.S. Olle2,3, J. Batley1,3, N.O.I. Cogan1,3, K.F. Smith2,3

1 Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, Australia
2
Pastoral and Veterinary Institute, Primary Industries Research Victoria, Department of Primary Industries, Mount Napier Road, Hamilton, Victoria 3300, Australia

3
Molecular Plant Breeding Cooperative Research Centre, Australia

Puccinia coronata Corda f.sp. lolii is the casuative organism of crown rust, the most important foliar disease of perennial ryegrass (Lolium perenne L.). Both qualitative and quantitative disease resistance mechanisms have been observed, and genetic variability in the sexually reproducing pathogen may lead to differing interpretations of the mode of genetic control. Evidence has been obtained for different physiological races between and within single pathogen populations, indicating heterogeneity with respect to virulence. In order to investigate genetic diversity in this obligate biotrophic species, we have developed a suite of molecular markers for in planta genotyping of single pustule-derived uredospore isolates. A number of publicly available genomic DNA-derived simple sequence repeat (SSR) loci from the yellow rust pathogen of wheat (Puccinia striiformis f.sp. tritici) have been evaluated for cross-amplification and polymorphism detection in P. coronata. In parallel, specific marker development has been performed based on EST-SSR discovery. RNA obtained from isolated uredospores has been used to construct cDNA libraries, allowing the identification of 55 unique EST-SSR loci following sequence analysis. The EST-SSR clones have been analysed for annotation by known sequences, and derived primer pairs have been evaluated for efficient amplification of samples from crown rust and other Puccinia taxa. A method for genomic DNA extraction from single pustules based on multiple displacement amplification (MDA) has been optimised. Genetic variation has been detected within and between geographical populations from New Zealand, and a strategy for isolate sampling on an international basis is being implemented to determine global levels of genetic diversity.