BSPP Presidential Meeting 2000

Plant-pathogen Interactions:
Understanding Mechanisms of Resistance and Pathogenicity for Disease Control

Offered Poster Abstracts - II

Plate, tube and immuno-chromatographic assays for detection and quantification of Botrytis cinerea in plant tissues and grape juice
Frances M. Dewey (Molly)
1, Ulla Meyer1, Chris Danks2 & Ian Barker2
University of Oxford, Department of Plant Sciences, South Parks Rd, Oxford, OX1 3RB; 2Central Science Laboratories, Sand Hutton, York, YO41 1LZ.

Methods that are relatively rapid and "user-friendly", such as immunoassays, are needed for monitoring the levels of Botrytis in infected plants and juice from wine grapes at harvest time. Previous attempts to develop such methods have not been satisfactory1,2,3,. Using a Botrytis-specific monoclonal antibody, BC-12.CA4, raised at Oxford4, we developed a laboratory-based 3 hour plate-trapped antigen ELISA5, a robust 20 min tube-ELISA, suitable for use on the testing stands at wineries and a semi-quantitative immunochromatographic or Lateral Flow device, that can be completed in 5 min. Additionally, in collaboration with SAPS (Science and Plants in Schools), a mini- plate assay, for the detection of Botrytis in fresh or frozen raspberries, has been developed for use in schools as a teaching exercise that can be completed in 40 mins (now available as a kit from SASA). The Tube and immunochromatographic assay are currently being tested in field trials in wineries in California. The antibody recognizes a water soluble thermostable antigen that is present in the extracellular matrix surrounding the hyphae. Competition assays have shown that the antigen binds to compounds containing rhamnose and molecular sieving indicates that the molecular weight of the antigen is approximately 100 to 30kD.

1. Ricker, R.W., Marois, J.J., Dlott, R.M., and Morrison, J.C. 1991. Immunodetection and quantification of Botrytis cinerea on harvested wine grapes. Phytopathology 81:404-411.

2. Bossi, R. and Dewey, F.M. (1992) Development of a monoclonal antibody-immunodetection assay for Botrytis cinerea (Pers).Plant Pathology 41, 472-482.

3. Dewey, F. M. & Cole, L. (1997) Monoclonal antibody-based assays for the detection and quantification of Botrytis cinerea. In: Diagnosis and Identification of Plant Pathogens, Ed: H.W.Dene; G. Adam; M. Diekmann; J. Frahm

4. Meyer, U and Dewey, F.M.(2000) Efficacv of different immunogens for raising monoclonal antiobides to Botrytis cinerea . Mycological Research, 104, 979-987.

5. Dewey, F.M., Ebeler, S.E., Adams, D.O., Noble, A.C. and U. .M. Meyer (2000). Quantification of Botrytis in grape juice determined by a monoclonal antibody-based immunoassay. American Journal of Viticultre and Enolog in press.

Identification and characterisation of host factors controlling susceptibility to plant viruses
David Edge and Sue Angell
Department of Virus Research, John Innes Centre, Colney Lane, Norwich, NR4 7UH.

Natural variation existing between different ecotype of the model plant species Arabidopsis thaliana has previously been used to elucidate the genetic basis for a number of plant pathogen interactions. In this study screens of Arabidopsis were performed using Potato Virus X tagged with the beta-glucuronidase gene (GUS) (PVX-GUS). A second screen was performed with Tobacco Rattle Virus tagged with the jellyfish green fluorescent protein gene (GFP) (TRV-GFP). The presence of the marker genes allowed the progression of the viral infection to be visualised within the plant. Ecotypes of Arabidopsis showing phenotypic variations in their response to viral infection were identified and the phenotypes observed were further characterised in order to discern the mechanisms underlying the observed phenotypes.The screens have shown that some ecotypes are unable to support long-distance movement of PVX, whereas other ecotypes are fully susceptible to PVX-GUS infection. The TRV screens have revealed ecotypes showing differences in susceptibility to TRV-GFP infection and to a TRV-mediated necrotic response. Genetic analyses on the ecotypes showing phenotypic variation will be performed using a map-based cloning approach in order to clone the loci controlling the observed phenotypes. cleaved amplified polymorphic (CAPS) markers will be used for the mapping and the region containing the locus responsible will be isolated from a bacterial artificial chromosome (BAC) library for further characterisation.

Structure/function analysis of Rx.- Conserved sequences within the ARC domain of Rx are involved in negative regulation of the disease resistance response
Garry Farnham and David C.Baulcombe
The Sainsbury Laboratory, John Innes Centre, Colney Lane Norwich NR4 7UH, UK

The resistance gene Rx from Solanum tuberosum confers extreme resistance to the single-stranded RNA potex virus potato virus X. Rx has the typical modular structure of cytoplasmic plant resistance proteins. It comprises an N-terminal coiled coil or leucine zipper like domain (LZ), a nucleotide binding site (NBS), a domain which shares homology with Apaf1 other plant Resistance (R) genes and CED4 (ARC) and a leucine rich domain (LRR). The similarity between plant R-genes and regulators of apoptosis suggests they may share common mechanistic features.

Here we investigate the contribution of the ARC domain of Rx towards negative regulation of the disease resistance response. A series of C-terminal deletions of Rx were made at conserved motifs in the ARC domain. Deletants were then over-expressed in N. tabacum. The results indicate that successive removal of conserved sequence motifs within the ARC domain cause a more rapid and severe hypersensitive response, when transiently over-expressed in the absence of the elicitor. Protein levels were unaffected by removal of conserved motifs suggesting that the ARC domain is involved in negative regulation of the Rx mediated disease resistance response.

Mycosphaerella fijiensis disease development in leaves on whole plants and in a detached leaf assay
Jill Foundling, Michaela Corsten, Naomi A. Pain
Zeneca Agrochemicals, Jealotts Hill International Research Centre, Bracknell, Berkshire, RG42 6EY

The development of black Sigatoka (Mycosphaerella fijiensis) in detached banana leaves and on intact plants was compared. Disease development on detached leaves was extensive on the external surface of the leaf, with accumulations of fungal hyphae and extracellular material present on or near stomata. On intact plants, far less fungal growth was observed on the surface of the plant. Internal disease development also differed. In detached leaves, plant cells remained relatively intact, inspite of the necrosis which was visible macroscopically. Isolated hyphae could be detected ramifying between intact cells throughout the depth of the plant tissue by freeze fracture SEM. In contrast, in an inoculated area of a leaf on an intact plant, regions of collapsed spongy mesophyll could be observed following freeze fracture and SEM. Significant hyphal development could be observed within the leaf, stretching across the cavities. In addition, plugs of material of unknown origin and composition were observed in stomata on the abaxial surface of the leaf.

HXC2, a new component of the eds-1 pathway leading to resistance in Arabidopsis ?
Godard Franois, Fabienne Persello-Cartieaux, Lummerzheim Marie, Balagu Claudine, and Roby Dominique
Laboratoire de Biologie Molculaire des Relations Plantes-Microorganismes, UMR CNRS/INRA 215, BP 27, 31326 Castanet Tolosan, FRANCE

In plants, one of the most efficient resistance reaction to pathogen attack, is the so-called Hypersensitive Response (HR). By screening an EMS-mutagenized seed library of Col-0 by spray- and/or manual inoculation of leaves with Xanthomonas campestris pv. campestris (strain 147, Xcc 147), we identified novel Arabidopsis mutants displaying alterations in the HR to Xcc 147 (Lummerzheim et al., 1993). An EMS mutant, hxc2 (for hypersensitivity to Xanthomonas campestris) displays a susceptible phenotype in response to Xcc 147, as shown by measurement of in planta bacterial growth, and histochemical detection of GUS-expressing virulent and avirulent strains. The use of common biochemical and molecular markers of disease resistance and susceptibility showed that the mutation causes pleiotropic alterations of defense responses and acts upstream of salicylic acid accumulation in the signalling events leading to the onset of these responses. Genetic analysis showed that the hxc-2 mutation is inherited as a monogenic recessive trait, different from the R gene involved in the recognition of the Xcc 147 strain, and localized on chromosome 3 between mi413 and atpox RFLP markers.

To address the question of the placement of hxc-2 mutation in the signalling pathways leading to resistance, different approaches including testing of virulent and avirulent pathogens and crossing with mutants affected in resistance have been undertaken. The hxc-2 mutation does not affect resistance to avirulent isolates of Pseudomonas syringae DC3000 harboring avrRpm1, avrRpt2 and avrB genes, recognized by the LZ-NBS-LRR class of resistance proteins. In contrast, the mutant is altered in the TIR-NBS-LRR mediated resistance to DC3000/avrRPS4, suggesting that HXC-2 might be a new component of the eds-1 pathway. An additional evidence in favor of this hypothesis comes from the susceptible phenotype exhibited by eds-1 mutant in response to Xcc 147. Experiments involving tests of other pathogens engaging the eds-1 or ndr-1 pathways, and generation of double eds-1/hxc-2 mutant, are underway, and should confirm the role of HXC-2 in the EDS-1 pathway.

Lummerzheim M. et al. Identification of compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris pv. campestris and characterisation of the Hypersensitive Response. Mol. Plant-Microbe Interact. 6 (1993), 532-544.

Form and concentration of nitrogen affects resistance of wheat (Triticum aestivum cv Brigadier) to Septoria nodorum
J. L. M. Greenhouse1, R. Sylvester-Bradley2, J. F. Farrar1
School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW UK
ADAS Boxworth, Boxworth, Cambridge CB3 8NN

The amount of nitrogen supplied to plants can increase or decrease susceptibility to infection by fungal plant pathogens. Both leaf morphology and metabolism are affected by nitrogen supply and these factors may play a role in resistance. The form of nitrogen may also be important in determining disease severity, perhaps influencing either host defence mechanisms or fungal development. The aim of this experiment is to test the hypothesis that plants supplied with low amounts of nitrogen will be more resistant to infection by S. nodorum than those supplied high amounts of nitrogen, and plants supplied with nitrate will be more resistant than those supplied with ammonium.

Wheat seedlings were grown in controlled environment cabinets in hydroponics. Plants were grown in Long Ashton solution minus nitrogen, and supplied either NaNO3 or (NH4)2SO4 at high (2 mmol dm-3) or low (40 mol dm-3) concentrations. When seedlings were 12 d old they were inoculated with S. nodorum. Harvests were carried out 1, 6 and 14 days after inoculation (dai) and at each harvest measurements of disease, chlorophyll contents and leaf areas were made. All plant parts were dried and weighed and the second seedling leaves were analysed for total carbon and nitrogen and soluble carbohydrates. Data was subjected to one way ANOVA (SPSS 9.0).

Plants grown in low amounts of nitrogen were much smaller and had fewer leaves and tillers than those grown in high amounts of nitrogen. Chlorophyll content was lowest in plants supplied low amounts of nitrogen. Chlorophyll content was initially highest in the plants grown in high NH4, but by the final harvest it was 50% lower than plants grown in high NO3. Differences between the two high nitrogen treatments and between the low and high treatments were significant at P <0.01. Specific leaf areas (sla) were significantly different between the treatments although there was no significant difference between the plants grown in low NH4 and those grown in low NO3. Plants grown in high NH4 had the lowest mean sla and plants grown in both low NO3 and low NH4 had the highest mean sla.

Disease severity was assessed at the final harvest. There was no disease on the plants grown in low NO3 and low NH4 and very little on the plants grown in high NO3. The most disease occurred on the plants grown in high NH4 and both lesion number and severity (area of leaf occupied by lesions) were significantly higher than on plants grown in high NO3 (P<0.01). This experiment suggests that both the form and the concentration of nitrogen supplied to a plant can affect its susceptibility to disease. The key questions are whether the fungus is responding to the nutritional status of the host, or whether the hosts defence mechanisms are affected by its nitrogen status? At which stage of infection is resistance expressed? In future work we aim to determine further the mechanisms by which nitrogen affects resistance.

Mapping the Atr1 Locus: An Avirulence Gene in Peronospora parasitica
Laura Grenville1, 2, Anne Rehmany1, Nick Gunn1, Eric Holub1, Chris Caten2 and Jim Beynon1
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

Peronospora parasitica (At) is the causal agent of downy mildew on Arabidopsis. Several Resistance or RPP genes, (Recognition of Peronospora parasitica) have been cloned from the host plant. We are attempting to clone the complementary pathogen ATR genes (Arabidopsis thaliana Recognised), the products of which are recognised by the host resistance genes. Three such ATR loci segregate in the mapping cross and initial AFLP bulk segregant analysis has defined a mapping interval for the ATR1 locus. We have redefined the selective bulks and report new markers linked to the ATR1 locus. These have been used to identify BAC clones linked to ATR1.

To increase the precision of the mapping 60 new F2s have been generated, to add to the 40 F2s used initially. We will also report data that suggest up to nine new ATR genes segregate in the mapping cross.

In planta expressed genes in the interaction between Gaeumannomyces graminis and cereals
Morgane Guilleroux and Anne Osbourn
The Sainsbury Laboratory, John Innes Centre, Colney lane, Norwich, UK

Suppression subtractive hybridization (SSH) has been used to generate a cDNA library enriched for sequences that are differentially expressed during infection of wheat roots by Gaeumannomyces graminis. This library has been assessed to confirm that representative constitutively expressed plant and fungal sequences ((-tubulin and actin, respectively) have been subtracted and XYL1 (a xylanase that is known to be expressed during infection) of GgA is expressed. A pilot study of the subtracted library has been carried out on 215 clones. These clones have been sequenced to check the quality of the library and subjected to a BLASTX search. Of these 215 clones, 150 reliable DNA sequences were obtained, 7 of which showed significant homology with fungal gene sequences available in the databases. However, the small average insert size (200bp) impairs both reliable homology search and hybridizations in Southern and northern blot experiments. A cDNA library has therefore been constructed from mRNA from infected roots, and is being used to isolate full-length cDNAs corresponding to subtracted clones of interest. Larger cDNAs fragments of two of the SSH clones have been used as probes on Northern blots and shown to be upregulated during infection. These clones are both of plant origin. An arrayed genomic DNA library of Gaeumannomyces graminis has been constructed and is being screened with the SSH library and other complex probes to gain a better understanding of the metabolic requirements of this root pathogen during the infection process. Gene function will be tested by gene disruption in related fungus Magnaporthe.

Artificial induction of a Cf9/Avr9 mediated HR induces enhanced resistance to Leptosphaeria maculans in Brassica napus L
Caroline Hennin, Monica Hfte and Elke Diederichsen
Faculty of Agricultural and Applied Biological Sciences, Laboratory for Phytopathology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium
Aventis CropScience NV Belgium, J. Plateaustraat 22, B-9000 Gent, Belgium

The hypersensitive response (HR) is a rapid and strictly localised cell death at the infection site in the host plant, limiting the spread of the pathogen and preventing its propagation through the plant. Based on the gene-for-gene concept, it is accepted that a dominant plant resistance (R) gene and the corresponding dominant pathogen avirulence (Avr) gene are the basic components required for a HR.

The tomato Cf9 resistance gene confers resistance to particular races of Cladosporium fulvum that express the corresponding avirulence gene Avr9. Injection of the Avr9 peptide into leaves of Cf9 tomato plants induces an oxidative burst, electrolyte leakage, production of ethylene, salicylic acid (SA), pathogenesis related (PR) proteins and hypersensitive cell death at the injection site. The Cf9/Avr9 system has been successfully transferred to other Solanaceous species such as tobacco and potato.

In our work, we investigated if the specificity of the Cf9/Avr9 interaction could be demonstrated in an unrelated plant species and transformed the Cf9 and Avr9 genes into oilseed rape, Brassica napus spp. oleifera L. We have studied whether the Cf9 and Avr9 genes can be functionally expressed in oilseed rape and whether the presence of their gene products induces defence responses that are effective to control diseases. We successfully expressed Cf9 and Avr9 genes in oilseed rape. We demonstrated that transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as has been reported for Cladosporium fulvum. Cf9 oilseed rape exhibited necrotic symptoms upon injection of intercellular fluid containing the Avr9 peptide. Phytopathological analyses revealed that induction of a HR by Avr9 injection on the pathogen inoculation site delayed the development of Leptosphaeria maculans. Reciprocal crosses of Cf9 oilseed rape to Avr9 oilseed rape did not result in seedling death of the F1 progeny. However, the F1 (Cf9 X Avr9) plants were initially slightly more resistant to L. maculans. This is the first report of the functional expression of a stable integrated disease resistance gene and its corresponding Avr gene in a plant species taxonomically not related to the original host plant species.

Effect of plant age on resistance to snow mould in perennial rye-grass and expression of PR genes
Ingerd Hofgaard, Leslie A. Wanner and Anne Marte Tronsmo
Planteforsk, Plantevernet avd plantesjukdommer, Hgskoleveien 7, 1432 s, Norway

Microdochium nivale causes pink snow mould on cereals and grasses. This fungus is widely distributed in the temperate and cooler zones and is the most common snow mould on over-wintering grasses and cereals worldwide. There is great variation in resistance to snow moulds between species of grasses, and perennial rye-grass (Lolium perenne) is among the most susceptible grass species in Northern Europe. Earlier studies have shown increased resistance to snow mould with increasing plant age and after hardening. The variation in resistance could be due to differences in size, morphology, or carbohydrate content. PR gene expression is induced in response to snow moulds and other pathogens, and has also shown to be enhanced during hardening. We are studying the effect of age and hardening on resistance to M. nivale in perennial rye-grass. The objective of this work was to determine whether plants of different ages or hardiness differ in their ability to express PR genes, which could be a possible explanation for their different snow mould resistance.

The age of plants at inoculation was 4, 5, or 6 weeks, or 4 weeks plus 2 weeks hardening at 2C. Resistance to M. nivale increased with plant age and after hardening. Hardened plants were smaller in size and had less dry weight than non-hardened plants sown at the same date, but had the same degree of snow mould resistance, measured as the relative dry weight (inoculated divided by control plants) after re-growth. Resistance increased with increasing age of non-hardened plants. Expression of chitinase and PR1a mRNA was stronger in inoculated compared to control plants after 6 days, but there was no clear difference in expression of these PR genes between plants of different ages, or in hardened versus non-hardened plants. Therefore age- and hardening-related differences in resistance to snow mould in rye-grass do not seem to be explained by differences in capacity to express these PR genes.