BSPP Presidential Meeting 2000

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

Offered Poster Abstracts - III

The identification and characterisation of genes involved in appressorium formation and function in the rice blast fungus Magnaporthe grisea
Lucy J. Holcombe and Nicholas J. Talbot
School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG

The rice blast fungus Magnaporthe grisea causes a serious disease of cultivated rice and is widely studied in order to develop an understanding of the mechanisms by which fungi initaite infections of cereal hosts. The life-cycle of M. grisea begins during periods of high humidity with the production of three-celled, asexual conidia. These spores germinate to produce a short germ tube, which differentiates into a specialised infection cell called an appressorium. The formation of a septum at the base of this appressorium leads to the generation of enormous turgor pressure within the cell. Appressorial pressure gives rise to a penetration peg that ruptures the host cuticle, allowing the fungus to ramify within and between plant cells. We are attempting to understand the process of turgor generation by M. grisea in order to determine how appressoria function.. Measurements of the pressure within M. grisea appressoria produce an estimated a value of up to 8 MPa. Biochemical analysis has revealed that the most abundant solute in appressoria is glycerol, and this provides an ideal candidate for a cytoplasmic osmolyte that could generate appressorial turgor. The purpose of this project is to determine the role of glycogen metabolism in the ability of M.grisea to cause plant disease. Previous observations have shown that large numbers of glycogen rosettes are degraded during appressorial turgor generation providing evidence for a role of glycogen degradation in appressorium turgor generation.

Two genes involved in glycogen metabolism have been identified; GPH1, which encodes glycogen phosphorylase, and AGL1, which encodes the glycogen debranching enzyme (amyloglucosidase). Both genes are expressed in appressoria and preliminary characterisation both genes and their products will be presented.

A Molecular Study of the Type III Secretion System in the Potato Pathogen Erwinia carotovora subsp. atroseptica
Maria C. Holeva1, Anna Avrova1, Kenneth Bell1, Glenn Bryan1, Richard Parsons2, Ian Toth1, Paul Birch1
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA Scotland;
Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN; email:

The importance of plant cell wall degrading enzymes in the pathogenicity of the soft rot erwinias has long been established. Recently, however, genes associated with a type III secretion system (hrp, avr, dsp genes) involved in pathogenicity have been identified in E. amylovora, a closely related pathogen. As part of a genomics effort at SCRI to produce a physical map of the soft rot erwinia E. carotovora subsp. atroseptica (Eca), clone 2B8 from a bacterial artificial chromosome (BAC) library of Eca was identified as carrying the entire hrp cluster and part of the dsp gene.

The purpose of this study is to analyse the structure and function of the hrp genes and hrp-associated genes from 2B8 and to compare their structural and functional similarity to other bacterial pathogens. Work has begun on completing the entire sequence of the hrp region by PCR amplifying DNA between sub-clones (produced as part of another project at SCRI) from the 2B8 clone. To investigate the function(s) of this region, the hrp and dsp genes will be mutagenised using an omega-kanamycin cassette. These mutations will then be analysed in planta. The generation of a mutant dspEca is now underway and its comparison with wild type Eca, and with known avirulent dsp mutants in E. amylovora, will shed new light on its role in pathogenicity or host range. This strategy will then be extended to other genes. Furthermore, complementation tests between Eca BAC clones and E. amylovora mutants, together with an investigation into the effect of the hrpEca cluster (clone 2B8) on the non-host response in tobacco, will help to determine the function to this region. Results so far have shown a structural similarity between the hrp/dsp cluster in Eca and E. amylovora, implying a functional similarity. A plant response which resembles a hypersensitive response (HR) has also been shown in a number of Nicotiana species and is now being investigated further.

A second resistance gene that confers AVR9 recognition in Lycopersicon pimpinellifolium is distinct from Cf-9 and reveals intragenic recombination between Cf-9 homologues
Renier van der Hoorn, Marco Kruijt,
Pierre de Wit and Matthieu Joosten
Laboratory of Phytopathology, Wageningen University, The Netherlands

The tomato Cf-9 gene confers resistance towards the fungal pathogen Cladosporium fulvum carrying the matching avirulence gene Avr9 and is reported to be introgressed into cultivated tomato from the wild relative Lycopersicon pimpinellifolium. Several accessions of L. pimpinellifolium were identified that show a hypersensitive response upon PVX-based expression of AVR9 (Laug et al. Plant Journal 2000, 25: 735). Here we examined the molecular basis of AVR9 recognition in these accessions. After PCR-based amplification on genomic DNA isolated from AVR9-responsive accessions, fragments of Cf-9 homologues (Hcr9s) were cloned into binary expression vectors and transiently co-expressed with Avr9 through agroinfiltration of tobacco (Van der Hoorn et al. MPMI 2000, 13: 439). Surprisingly, the Cf-9 gene was not identified in this screen. In all 10 AVR9-responsive accessions, a hybrid between Hcr9-9D (a homologue adjacent to Cf-9 at the Cf9 locus) and the Cf-9 gene itself (representing homologue Hcr9-9C), was present (hence called Hcr9-9DC). As a result of the presence of the 5 Hcr9-9D sequence, the encoded 9DC protein carries 61 amino acid substitutions when compared to wild-type Cf-9. Most of these amino acid substitutions are present at putative solvent-exposed positions of the leucine-rich repeats. Despite these differences, the 9DC protein confers AVR9 recognition with the same sensitivity, activity and specificity as Cf-9. Although the L. pimpinellifolium population contains selfing and outcrossing accessions, AVR9 recognition was only present in selfing accessions, collected from the coastal plains in the middle and the southern parts of Peru, the southern part of the L. pimpinellifolium distribution range. The distribution pattern of 9DC genes in the population, and evidence for recombination between 9DC alleles, suggests that the 9DC gene existed in the initial, outcrossing species before the population started to spread over its current distribution range.

Comparison of A group and B group Leptosphaeria maculans ascospores germination and infection on oilseed rape
Y.J. Huang1, J.S. West1, B.D.L. Fitt1, A.M. Hall2
IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK;
Environmental Science, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL 10 9AB, UK

Stem canker (blackleg), caused by Leptosphaeria maculans, is a common disease of oilseed rape worldwide. Previous studies show that the population of L. maculans can be divided into at least two main sub-groups, which are often termed A and B groups. Ascospores released from infected debris are the main source of inoculum. Air-borne ascospores can infect leaves to cause leaf spots, then the fungus can grow systemically down to the leaf petiole to reach the stem and cause stem canker. The poster will report work comparing the germination and infection of ascospores of A group and B group L. maculans, which will provide new evidence whether the two groups are different species.

The A group ascospores were obtained from infected oilseed rape stem from UK debris, while the B group ascospores were obtained from Polish oilseed rape stems. A group and B group ascospore suspensions were inoculated onto water agar slides and detached oilseed rape leaf surfaces and incubated at different temperatures. The percentage germination of ascospores, the lengths of germ tubes, the number of germ tubes per ascospore, the position of germ tubes and the diameter of germ tubes were observed. To study infection, oilseed rape plants were inoculated with A group & B group ascospore suspensions at growth stage 1,3, and ascospore germination and penetration of leaf surfaces were observed.

The results indicated that ascospores of A group and B group L. maculans germinated on water agar and leaf surfaces over a wide range of temperatures (5 - 20C). Nevertheless, germination started later and the percentage of germination was lower at 5C than at 10 - 20C. Compared with germination on water agar, % germination on leaf surface was lower. B group ascospores germinated faster than A group ascospores, but the maximum % germination on leaf surfaces was lower than for A group ascospores. With increasing temperature, the germ tube extension rate of A group ascospores increased more slowly than that for B group ascospores. Thus, the germ tube length of the B group ascospores was longer than that of A group ascospores at 15 - 20C, but the germ tube diameter was smaller than that of A group ascospore.

Under the same conditions, A group ascospores produced more germ tubes than B group ascospores and the positions of germ tubes differed between A group and B group ascospores. B group ascospores generally produced 3.1 germ tubes per ascospore, mainly from terminal cells, whilst the A group ascospores produced 3.8 germ tubes per ascospore, mainly from medial cells. After 24 hours incubation, the hyphae of the B group grew almost in straight lines, whilst the hyphae of the A group grew tortuously. The hyphae of both A group and B group ascospores penetrated the leaf through stomata, but an appresorium-like structure was observed with A group ascospores and not with B group ascospores. The penetration rate of A group ascospores was greater than that of B group ascospores.

Initial events in the colonisation of tomatoes by Oidium lycopersici, a distinct powdery mildew fungus of Lycopersicon species
Hannah Jones1, John Whipps2, Tim Carver3, Barry Thomas3, Sarah Gurr
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK. 2Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK.

Oidium lycopersici is a highly polyphagous pathogen of glasshouse grown tomatoes. The identification of this tomato powdery mildew, in the late 80s, led to a number of hypotheses as to its origin. Our recent work has revealed, from ITS sequence analysis, that O. lycopersici to have a close similarity to the Erysiphe aquilegiae var ranunculi, the buttercup powdery mildew.

The initial events involved in the germination of conidia and subsequent formation of appressoria in the newly-described powdery mildew of tomato, Oidium lycopersici, was studied by light and scanning electron microscopy. The greatest rate of spore germination was determined to be 3 - 5 hours after inoculation and appressoria formed some 6 - 8 hours after inoculation. Scanning electron microscopy revealed the conidial coat to be smooth to slightly rugose and the appressoria to be multi-lobed and attached to the host by a mucilaginous ring of extracellular material.

Further investigations into the early development of O. lycopersici, has revealed timed secretion of specific enzymes which coordinate closely to key stages of development. The results will be presented.

Jones, H.E., Whipps, J.M., Thomas, B.J., Carver, T.L.W., Gurr, S.J. (2000) Initial events in the colonisation of tomatoes by Oidium lycopersici, a distinct powdery mildew fungus of Lycopersicon species. Can. J. Bot. 78: 1 - 6

Purification of oligochitin elicitor-binding protein from plasma membrane of rice cells and survey of its gene
Hanae Kaku, Eiichi Minami and Naoto Shibuya
Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Japan.

N-Acelytchitooligosaccharides (>GlcNAc6) could induce the formation of phytoalexin in suspension-cultured rice cells(1-3). High affinity binding site for this elicitor was detected in the plasma membrane of rice cells (4) and a corresponding binding protein was identified by affinity labelling (5). In the present study, we report the purification of this elicitor-binding protein (EBP) from the plasma membrane (PM) by affinity chromatography using newly designed affinity matrix.

The PM was solubilized with Triton X-100 and the solubilized fraction was applied to a GlcNAc8-APEA-CH-Sepharose column, which was then washed with buffer and several elicitor-inactive sugar solutions. The bound fraction was eluted with Glycine-HCl buffer (pH2.3) and the eluate was immediately neutralised with 1M Tris solution. The purified protein showed the specific binding activity to 125I-labeled GlcNAc8-APEA derivative as proved by the affinity crosslinking with glutaraldehyde. SDS-PAGE followed by silver-staining as well as affinity labelling showed the presence of two protein bands, corresponding to 75 and 55 kDa. The result suggested that EBP was cleaved with protease during purification. The bands detected by the affinity labelling disappeared by the addition of the unlabeled elicitor active sugar. The recovery of EBP obtained by the use of the new affinity matrix was approximately 18 times better than that by GlcNAc7-Lys-Sepharose. The increased recovery of EBP paved the way for the analysis of the N-terminal amino acid sequence. The survey of this EBP gene using a probe corresponding to the N-terminal amino acid sequence of EBP is in progress.

(1) A. Yamada et al., Biosci. Biotech. Biochem., 57, 405 (1993).
(2) T. Yamaguchi et al., Plant Cell, 12, 817 (2000).
(3) D. Y. He et al., MPMI, 11, 1167 (1998).
(4) N. Shibuya et al., Plant Cell Physiol., 37, 894 (1996).
(5) Y. Ito et al., Plant J., 12, 347 (1997).

Characterisation and cloning of a wide spectrum nematode resistance gene (Hero) of tomato (Lycopersicon esculentum L.)
Kumar, A., Ernst, K., Sobczak, M., Phillips, M., Ganal, M.
Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland and IPK, Corrensstr. 3, D-06466 Gatersleben, Germany

Potato cyst nematodes (PCN; Globodera rostochiensis and G. pallida) are major pests worldwide. However, major resistance genes to both species are lacking in potato cultivars and their related wild Solanum species. In tomato, we have shown that the major nematode resistance gene (Hero) confers complete resistance to G. rostochiensis and 80% to G. pallida pathotypes. Thus, the Hero gene is a wide spectrum nematode resistance gene. This gene has been introgressed from the wild tomato species L. pimpinellifolium into the cultivated tomato in early 1970. To-date, such a resistance gene has not been identified within Solanum species and thus the Hero gene could be valuable for incorporating PCN resistance in potato cultivars.

Comparative histological studies of the infected in vitro roots of susceptible Money Maker and Hero tomato lines with G. rostochiensis Ro1 have revealed that the functional syncytia were developed in Money Maker roots whereas the syncytia induced in Hero roots were mostly found to degenerate a few days after their induction. Some syncytia developed and supported the development of males rather than females. Thus, the ratio between males and females development was biased towards males on Hero roots whereas it was approximately equal on MM roots. Furthermore, microscopic analysis has revealed that the resistant response conferred by the Hero gene is activated after establishment of a functional syncytium. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Thus, a combination of events, involving reduction in the number of functional syncytia developing together with biasing the sex ratio towards males, is responsible for drastically lowering the rate of nematode multiplication in the resistant plants. This is in contrast to the tomato Mi gene-induced resistance, which is based on a rapid hypersensitive response.

Previously, the Hero gene has been mapped onto the short arm of tomato chromosome 4, which is not equivalent to any of the previously mapped G. rostochiensis and G. pallida resistance genes in potato1. A number of cosmid clones ranging from 10-20 kb spanning the Hero locus have been identified and sequencing analyses have revealed that the Hero gene belongs to a multigene family, which comprises 13 copies of the Hero gene homologue. Recently, a number of cosmid-based transformation constructs in Agrobacterium have been used to transform the PCN susceptible tomato line Money Maker. PCN tests on these transgenic tomato plants have revealed that one of the cosmid clones contains the functional copy of the Hero gene.

1. GANAL, W.M., SIMON, R., BROMMONSCHENKEL, S., ARNDT, A., TANKSLEY, S.D., PHILLIPS, M. KUMAR, A. 1995. Genetic mapping of a wide spectrum nematode resistance gene, Hero, against Globodera rostochiensis in tomato. MOLECULAR PLANT-MICROBE INTERACTIONS. 8: 886-891.

Analysis of Bax-induced cell death and N-mediated hypersensitive response to TMV
Christophe Lacomme, Simon Santa Cruz*
Unit of Cell Biology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland
(*) Horticulture Research International, East Malling, West Malling, ME 19 BJ, Kent

A well-studied plant response to invading pathogens involves localized cell death at the infection sites. This programmed cell death (PCD), referred to as the hypersensitive response (HR), is often associated with pathogen resistance. Similarly animal cells can also undergo PCD in the face of invading pathogens. Although some similarities exist between the ultrastructural and physiological hallmarks of PCD in animals and plants evidence for common pathways leading to cell suicide are limited. In animal systems, studies of PCD have identified many regulators of death-inducing stimuli including the prodeath protein Bax. We previously showed (Lacomme and Santa Cruz, PNAS, 96:7956-7961) that murineBax protein induces HR-like cell death in tobacco. Some similarities are observed between Bax-induced cell death in plants and the HR, as they require both an active host response mediated via transient activation of protein phosphatases and lead to PR1 accumulation. Structure-function analysis indicates that the cell-death function requires domains of the Bax-protein involved in homodimerization and mitochondrial localization to respectively potentiate or trigger cell death. This supports the hypothesis that mitochondria may play an active role in Bax-induced cell death in plants as described in other systems. We address the question to what extent cellular events preceding the TMV-induced HR-cell death can be compared to PCD mechanisms in other eukaryotes. Data concerning ultrastructural observations and molecular approaches used to study the TMV-HR pre- and post-necrotic stage will be presented.

Systemic resistance to anthracnose disease in cowpea seedlings treated with acibenzolar-S-methyl
Olu Latunde-Dada and John Lucas
IACR-Long Ashton Research Station, Long Ashton, Bristol, BS41 9AF, UK.

Plant defence activators are non-fungicidal compounds which alter the susceptibility of plants to microbial pathogens. The mechanisms of induction and expression of resistance following treatment with defence activators are not fully understood. Cowpea (Vigna unguiculata (L.) Walp.) seedlings, raised from seeds of a susceptible cultivar treated with acibenzolar-S-methyl (= benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester; BTH), were inoculated with the fungal pathogen Colletotrichum destructivum. The penetration of treated tissues was reduced markedly with intracellular infection vesicles of the fungus restricted to the initially infected epidermal cells. The destructive secondary phase of disease development, in which spreading lesions are formed, was effectively blocked, thereby protecting seedlings against damping-off. This enhanced resistance of BTH-treated tissues was associated with rapid, transient increases in the activities of two key enzymes of the phenylpropanoid/flavonoid pathway, phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI). Subsequently, there was an early, accelerated accumulation of the isoflavonoid phytoalexins kievitone and phaseollidin in treated hypocotyls. In addition, a number of PR protein bands were observed exclusively in the electrophoresed extracts of inoculated, BTH-treated tissues. These responses were not observed in induced, uninoculated tissues. The results suggest that the defence activator protects cowpea seedlings by potentiating an early defence response rather than by altering the constitutive resistance of tissues.

Inhibition of phenylpropanoid metabolism breaks non-host resistance in wheat
Loades C.J. and Barber M.S.
University of Southampton, Division of Cell Sciences, Bassett Cresent East, Southampton S016 7PX.

Recently, several new enzyme inhibitors of general phenylpropanoid and lignin specific pathways have been identified that are likely to be of value in the investigation of lignin dependant processes in plants. The inhibitors that are likely to be most useful must demonstrate highly specific inhibition of the target enzyme, be water soluble and capable of reducing lignification in planta at non-toxic concentrations. The current work assesses the ability of a range of previously known and recently identified inhibitors for their ability to reduce defensive lignification and break non-host resistance in wheat. The inhibitors were screened for their ability to reduce lignification induced by Botrytis cinerea, their ability to break non-host resistance and for their fungal and phytoxicity. Lignification was quantified by Fast GG staining combined with a scanning densitrometic procedure. Breaking resistance was defined as hyphal growth in the tissues beyond the point where normal defensive lignification would have occurred and was assessed by alcoholic lactophenol cotton blue staining and light microscopy. Fungal toxicity was monitored by light microscopy to directly observe inhibition of conidia germination. Phytoxicity was assessed spectrophotometrically by measuring the reduction in extractable chlorophyll. Most of the compounds tested reduced lignification, but many were phytoxic or had adverse effects on fungal germination. Unfortunately, all of the lignin specific pathway enzyme inhibitors were ruled out due to lack of potency or toxicity problems. The possible exception was the cinnamyl alcohol dehydrogenase (CAD) inhibitor coniferal thiol (ML19) that broke resistance, albeit at levels approaching toxicity. In contrast, the most promising compounds identified in this study were the inhibitors of the general phenylpropanoid pathway enzymes phenylalanine ammonia-lyase (PAL), cinamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Two of the widely used PAL inhibitors, 2-aminoindan-2-phosphonic acid (AIP) and a -aminooxi-b -phenylproponic acid (AOPP) substantially reduced lignification and broke resistance. Similarly, the recently identified C4H inhibitors 1-aminobenzotriazole (ABT), and the two hydroxynapthoic acids (1-OH-2-NA and 2-OH-1-NA) dramatically reduced lignification, although only 1-OH-2-NA broke resistance. The 4CL inhibitor methylenedioxycinnamic acid (MDCA) was capable of reducing lignification, although its ability to alter resistance has yet to be assessed. The work provides some insight into the causal relationship between non-host resistance and lignification in wheat, but more importantly identifies several new tools to enable lignin dependent processes to be investigated in plants.