BSPP Presidential Meeting 2000

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

Offered Poster Abstracts - V

The Molecular and Cellular Basis of Spore Adhesion in Colletotrichum lindemuthianum
Sarah L. Rawlings1, H. Bleddyn Hughes1, Richard J. OConnell2, Jonathan R. Green1
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS41 9AF, UK

Fungi of the genus Colletotrichum are successful plant pathogens causing anthracnose diseases in a wide variety of crops. C. lindemuthianum infects Phaseolus vulgaris (French bean). The first feature of successful fungal pathogenesis is the adhesion of spores onto the host surface, without which the infection process could not take place. Within the genus Colletotrichum, very few moleculaes thought to be involved in adhesion have been identified and no adhesive glycoprotein directly involved in adhesion has been cloned. In recent studies, spores of C. lindemuthianum have been shown, by TEM, to possess a pre-formed, carbohydrate-rich, fibrillar spore coat arranged perpendicular to the cell wall that is not found on either germ tubes or appressoria. The spore coat is thought to mediate C. lindemuthianum spore adhesion. A monoclonal antibody (UB20) has been raised which binds to the two major components of the spore coat. We have developed an adhesion assay which has allowed us to investigate the mechanisms of spore adhesion in C. lindemuthianum and the role of the spore coat in this process. Removal of the spore coat by proteases significantly inhibited adhesion onto polystyrene petri dishes. Incubation of spores with purified UB20 IgG also inhibited adhesion. Evidence reported here suggests that the adhesion of C. lindemuthianum spores involves mainly hydrophobic interactions between a pre-formed protein in the spore coat and the hydrophobic surface of polystyrene.

Progress towards cloning an avirulence gene from Arabidopsis thalliana downey mildew (Peronospora parasitica (At))
Anne Rehmany, Laura Grenville, Nick Gunn, Eric Holub and Jim Beynon
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, U.K.

The interaction between Arabidopsis thaliana and the oomycete Peronospora parasitica is mediated by RPP loci (Recognition of P. parasitica) in the plant and corresponding ATR loci (Arabidopsis thaliana-recognised) in Peronospora. Here we describe the map-based strategy in use and progress made towards cloning the ATR1 gene from Peronospora, which interacts with genes at the RPP1 locus in Arabidopsis accessions Nd-1 and Ws-3.

A bulked-segregant analysis using the AFLP technique is in progress and AFLP markers have been identified that span the ATR1 locus. We report the construction of a genomic BAC library from Peronospora and preliminary efforts to construct a BAC contig across the interval.

Histological and cytological expression of the host parasite specificity in Lactuca spp. - Bremia lactucae interaction
Sedlov Michaela & Lebeda Ale
Palack University, Faculty of Science, Department of Botany, lechtitel 11, 78371, Olomouc-Holice, Czech Republic.,

The interaction between lettuce (Lactuca sativa), closely related L. serriola and lettuce downy mildew (Bremia lactucae) follows gene-for-gene relationship. Race-specific resistance is generally expressed as a hypersensitive reaction (1, 3). According to microscopical observations, a great variation in pathogen development, host cells and tissue reaction can be distinguished during initial stages of the host-pathogen interaction based on different resistance mechanisms (2).

Nine genotypes of Lactuca spp. were involved in the study: L. sativa (Cobham Green, UCDM2, Mariska), L. serriola (PIVT 1309, LSE/18), L. saligna (CGN 05147, CGN 05271) a L. virosa (CGN 04683, NVRS 10.001 602) representing different mechanisms of compatibility and incompatibility to B. lactucae (race NL16). Germination, number and size of infection structures (primary and secondary vesicles), formation of infection hyphae of B. lactucae, tissue and cell reaction (HR) of host genotypes were examined on leaf discs 3, 6, 12, 24, 36 and 48 h after inoculation (hai) under light microscope. These features were related to the changes of host cytoskeleton.

Formation of pathogen primary infection structures (PV, SV) in epidermal cells of susceptible genotypes (L. sativa Cobham Green and UCDM 2, L. serriola LSE/18) was faster (6-12 hai) than in resistant genotypes (12-24 hai). The proportion of SV formed from PV was much lower in L. saligna than in other genotypes of Lactuca spp. HR was very rare in compatible interaction and only ever involved one cell per infection site. Great variation was found in the expression of HR among genotypes with different resistance mechanisms. Rapid HR and arrest of the fungus following formation of PV and SV is related to resistance in L. virosa. In CGN 04683 necrosis was detected in about 60% of penetration sites and also included subepidermal necrosis (SEN). In NVRS 10.001 602 both the PN and extent of HR were higher but spread just in attached epidermal cells.

Rearrangement of microtubular cytoskeleton have been investigated in infected epidermal cells by immunofluorescence microscopy (4). Alterations of microtubular array were mostly focused into the infected cells, only in two L. virosa genotypes (where more cells is involved in HR) they occurred also in 1-3 adjacent cells. Timing and extent of all processes taking place during the early stages of infection (pathogen infection structures development, reorganization of cortical microtubules, etc.) were specificaly related to the susceptibility/ resistance mechanism of different Lactuca spp. genotype.

Abbreviations: hai - hours after inoculation; PV, SV primary, secondary vesicle; HR hypersensitive reaction; PN- proportion of infection sites with necrotic epidermal cells; SEN - subepidermal necrosis

This research was supported by grant of Czech Ministry of Education Plant stress and pathological biology, biochemistry and bioenergetics" (MSM 153 100010).

Lebeda, A., Pink, D.A.C.: Histological aspects of the response of wild Lactuca spp. and their hybrids, with L. sativa to lettuce downy mildew (Bremia lactucae). Plant Pathol. 47, 723-736, 1998.
Lebeda, A., Pink, D.A.C., Mieslerov, B.: Host-parasite specificity and defense variability in the Lactuca spp. Bremia lactucae pathosystem. J. Plant Pathol., 2000 (in press)
Lebeda, A., Reinink, K.: Histological characterization of resistance in Lactuca saligna to lettuce downy mildew (Bremia lactucae). Physiol. Mol. Plant Pathol. 44, 125-139, 1994.
Sedlov, M., Binarov, P., Lebeda, A.: Changes in microtubular alignment in Lactuca spp. epidermal cells during early stages of infection by Bremia lactucae. Phyton, 2000 (in press).

Molecular analysis of Avra12 in the barley powdery mildew pathogen, Erysiphe graminis fsp.hordei
Paraskevi Skamnioti, Christopher J. Ridout, and James K.M. Brown
Cereals Research Department and Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH

The avirulence gene Avra12 segregates in a cross between virulent (CC52) and avirulent (DH14) isolates of Erysiphe graminis f.sp. hordei (barley powdery mildew). Avra12 is recognised by the corresponding resistance gene in barley, Mla12, an allele of the Mla gene which was cloned recently in the Sainsbury Laboratory. Molecular characterization of Avra12 will help in determining the basis of gene-for-gene interactions and the origin of genetic variation in the fungus. It may also be useful in genetic manipulation of resistance, by recognition-based induction of plant defences.

First, Avra12 was mapped as a pre-requisite to map-based cloning. In an attempt to map the gene more precisely, bulk segregant analysis was conducted. 951 AFLP primer combinations, scanning over 65,000 loci, were used, but Avra12 was consistently located at the end of linkage group III. This suggests that the gene is in a region of high recombination, possibly in a subtelomeric region. Therefore, to find markers distal to Avra12, we are investigating polymorphism in PCR products with homology to the consensus ascomycete telomere sequence, so enabling telomere-associated sequences to be mapped in relation to Avra12.

To complement the mapping approaches, we are investigating differences in expressed genes which may be associated with the Avra12 phenotype, using suppression subtractive hybridisation. Lastly, a new cross (CC148 x DH14) is being set up, in order to map Avra12 in relation to other avirulence genes that segregate in this cross.

Light and electron microscopy of the compatible interaction between Arabidopsis and downy mildew pathogen Peronospora parasitica
E. Mine Soylu1, Soner Soylu1 and John Mansfield2
University of Mustafa Kemal, Faculty of Agriculture, Department of Plant Protection. 31034 Antakya/Hatay. Turkey
University of London, Imperial College at Wye, Department of Biology, Wye, Ashford, KENT. TN25 5AH. U.K

Peronospora parasitica causes downy mildew disease in a number of crucifers including Arabidopsis thaliana. In this study we focused on compatible interactions between Peronospora and Arabidopsis using light and electron microscopy (E.M). Light microscopy of compatible interactions revealed that sporangia germinated and penetrated along the junction of line of the anticlinal cell walls of two epidermal cells. Penetration of a mesophyll cell and formation of first haustorium occurred within 12 hr after inoculation. Rapid spreading of the fungal hyphae with formation of numerous haustoria was subsequently followed by profuse sporulation 5 days after inoculation, in the absence of host cell necrosis. During the time course, examination of infected cells under UV radiation did not reveal any autofluorescence indicating the absence of the hypersensitive reaction. E.M observations revealed that coenocytic hyphae ramified and spread intercellularly throughout the host tissue. The cytoplasm of intercellular hyphae was bounded by the fungal membrane and contained typical organelle. Further growth of hyphae within the intercellular spaces and penetration of individual host mesophyll cells led to the formation of haustoria. Intracellular haustoria were lobed with the diameter of 6-7 m. Each haustorium was connected to intercellular hyphae by a wide and very short neck. The cytoplasm of the haustorium included the organelles characteristic of the fungus, vacuoles were seldom formed. The haustorial body contained numerous mitochondria which were much more frequent than elsewhere in the fungus. Callose like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. No obvious response was observed in host cells following formation of haustoria. By 24 hr, one or two haustoria were often observed in single mesophyll cells but as many as 4-5 haustorial profiles were found within a single cell at 5 dai. Apart from a few callose ensheatments, most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell suggest that the fungus exerts considerable control over basic cellular processes and in this respect, response to this biotroph fungus differs considerably from responses to other pathogens such as necrotrophs.

Histochemical localisation of hydrogen peroxide during compatible and incompatible interaction between Arabidopsis and Peronospora parasitica
E. Mine Soylu1, Soner Soylu1 and John Mansfield2
University of Mustafa Kemal, Faculty of Agriculture, Department of Plant Protection, 31034 Antakya, Hatay. Turkey
University of London, Imperial College at Wye, Department of Biology, Wye, Ashford, KENT. TN25 5AH

Diaminobenzidine (DAB) was used to determine the localisation and timing of the accumulation of a component of active oxygen species, hydrogen peroxide (H2O2) in planta. H2O2 was visualised histochemically by its reaction with DAB in the presence of peroxidase to produce brown-red colour staining. Treatment of tissue with catalase either abolished or reduced the level of staining suggesting that the staining was dependent on the presence of H2O2. In uninoculated Arabidopsis cotyledon and the infected cotyledons from compatible interaction, (Ws-eds-1 accession inoculated by Emoy-2 isolate), H2O2 staining occurred within the vascular tissue. No staining was observed on cell wall at site of fungal penetration. Major H2O2 accumulation was detected on resistant accession of La-er following inoculation with the avirulent isolate Emoy-2 which caused a rapid hypersensitive reaction (HR). Striking and highly localised H2O2 staining was observed in the plant cell walls undergoing the HR, at sites of wall alterations and papilla formation around the penetration point and adjacent cells. These sites were previously found as sites of active lignin deposition as revealed by staining with phloroglucinol. Early appearance of H2O2 was coincident with the development of the HR. The earliest time for observation of H2O2 in epidermal cells undergoing the HR was 18 hr after inoculation. In the intermediates extensive deposition of papillae was identified as the main mechanism of resistance. Less marked accumulation of H2O2 was observed at sites with cell wall alterations. Intense staining was only observed in cells undergoing the HR and within the adjacent cells. In conclusion, highly localised accumulation of H2O2 at reaction sites suggest that production of H2O2 may be critical to development of resistance as manifested by the HR, accumulation of phenolic and cell wall lignification during incompatible interactions between Arabidopsis and Peronospora.

mlo -Resistance to Barley Powdery Mildew: Instability After Stress
Keith Stewart and Sarah Gurr
Department of Plant Sciences, University of Oxford, OX1 3RB

mlo-resistance is currently the only durable and effective resistance mechanism in spring barley to the powdery mildew pathogen Blumeria (Erysiphe) graminis f. sp. hordei. It mediates a broad-spectrum resistance to virtually all known pathogen isolates, and is used as the primary resistance mechanism in the vast majority of the European spring barley crop. The cellular manifestation of mlo-resistance is that of the rapid formation of localised cell wall appositions papillae which prevent fungal penetration of the host epidermal cells.

mlo-resistance breakdown has been observed under field, glasshouse and laboratory conditions. It is a temporary phenomenon, which follows the relief of drought stress1,2. More recently, the relief of drought induced chemically by osmotica has also been demonstrated to result in the breakdown of mlo-resistance. Additionally, mlo-resistance is unstable following the relief of exposure to low temperatures (+4C). Following the relief of salt stress, no resistance breakdown is observed on mlo-resistant varieties and a reduced infection frequency is observed in susceptible barley genotypes.

Extensive physiological studies, have ruled out any significant difference in the stress tolerance of mlo-resistant and Mlo-susceptible barley varieties as being responsible for mlo-resistance breakdown. These studies include continuous, non-invasive measures of change in leaf thickness following the relief of stress and cryoscopic osmometry of cell sap extracts.

Current investigations focus on the effects of the induction and relief of abiotic stresses on the expression of the Mlo-gene, together with other defence genes induced by abiotic and biotic stresses. Results from this work will be presented here.

Baker SJ, Newton AC, Crabb D, Guy DC Jefferies RA, Mackerron DKL, Thomas WTB, Gurr SJ. 1998. Temporary partial breakdown of mlo-resistance in spring barley by sudden relief of soil water stress under field conditions: the effects of genetic background and mlo allele. Plant Pathol. 47:401-410.

Baker SJ, Newton AC, Gurr SJ. 2000. Cellular characteristics of temporary partial breakdown of mlo-resistance in barley to powdery mildew. Phys. Mol. Plant Pathol.56:1-11.

Effects of fungicide seed and spray treatments on the progress of septoria leaf blotch (Mycosphaerella graminicola) on winter wheat
Wanzhong Tan, Bruce Fitt
IACR-Rothamsted, Harpenden, Herts AL5 2JQ

On naturally infected winter wheat (cv. Riband), different septoria leaf blotch (Mycosphaerella graminicola) epidemics were established in 1999/2000 by using fungicide seed treatment (fluquinconazole) and spray treatments (azoxystrobin) at GS32 and/or GS39. Data on % leaf areas which were senesced (LAS), affected by leaf blotch (LAB) or covered by M. graminicola pycnidia (LAP) and green leaf area (GLA, cm2) were collected weekly from GS31 (19 April) to GS85 (12 July) and grain yield (t/ha) was recorded at harvest. Seed treatment effectively reduced septoria blotch on leaves 5 and 4 before GS34, and maintained GLA of these leaves, but these effects were not observed on leaves 3 to 1 (flag leaf). The sprays at GS32 and/or GS39 decreased septoria blotch after GS57 and decreased septoria blotch on the upper 3 leaves, thus greatly reducing losses in grain yield. Septoria leaf blotch epidemics followed different patterns on different leaves and in different treatments. The Gompertz, logistic and exponential functions fitted well to the data for the progress of epidemics on leaves 1, 2 and 3 and to those of most treatments on leaf 4. The integrated areas under progress curves for LAS, LAB, LAP and GLA on leaves were all related to each other. Grain yield was correlated negatively with integrated LAB and LAP, and positively with integrated GLA, on the upper 3 leaves. The yield-disease models established through regression on integrated LAS, LAB and GLA on leaves 1, 2 and 3 alone were significant, but the best yield-disease models were those established with the totals of integrated LAB and GLA, respectively, of the top 4 leaves of the plants.

Expression of elicitor responsive genes in rice plants
Shigeru Tanabe, Mitsuo Okada, Eiichi Minami and Naoto Shibuya
Natl. Inst. Agrobiol. resources, Tsukuba, Japan, 305-8602.

N-acetylchitooligosaccharides elicit a set of defence responses in suspension-cultured rice cells (Oryza sativa cv Nipponbare). We have reported that mRNAs for elicitor responsive genes, EL2, EL3 and PAL, transiently accumulate by elicitor treatment. In this study, we investigated the expression of these genes in rice plant using northern blot analysis and in situ hybridization.

In excised rice leaves, and incubated in (GlcNAc)7 solution, EL2 and EL3 mRNAs were shown to accumulate 15 minutes after treatment with elicitor in the mesophyl cells. These results suggested that the expression of both genes in rice plants were regulated in a similar manner to the suspension-cultured rice cells. It was indicated by 125I-APEA-(GlcNAc)8 transport assay that the expression of both genes in the excised leaves in response to elicitor results from diffusion of elicitor into leaf tissue through the vascular bundles.

In intact plants, 125I-APEA-(GlcNAc)8 applied to the root of rice seedlings was not sucked up to leaves. EL2 mRNA accumulated transiently in root exodermis cells and middle part of root cap, but not in the leaves. On the contrary, PAL mRNA accumulated in the leaves but not in the roots. These results strongly indicate that EL2 is expressed in the cells exposed to the elicitor, whereas PAL is induced by systematic signals from N-acetylchitooligosaccharides in rice seedlings.

Avirulence and virulence functions of effector proteins produced by Pseudomonas syringae
George Tsiamis1, Rob Jackson2, Alan Vivian2 and John Mansfield1
Department of Biology, Imperial College at Wye, Ashford, Kent TN25 5AH, UK
Department of Biological Sciences, University of the West of England, Coldharbor Lane, Bristol BS16 1QY, UK

Numerous avirulence (avr) genes have been cloned by function from pathovars of P. syringae. In many cases the avr function has been shown to be associated with gene-specific interaction with a matching resistance (R) gene in the responding plant. The first virulence (vir) gene, designated virPphA was cloned for its ability to restore virulence to plasmid deficient strains of P.s. pv. phaseolicola; VirPphA is located in a pathogenicity island. Dual function was assigned to virPphA following discovery that it regulates induction of the HR in soybean. The avr gene avrPphF, which matches the R1 gene for resistance to halo-blight in bean cv. Red Mexican was found to have cultivar and gene specific virulence activity in bean cv. Tendergreen. In the absence of the PAI containing virPphA, avrPphF also elicits a strong HR in cv. Canadian Wonder which is fully susceptible to all wild-type strains of P.s. pv. phaseolicola. A gene masking the activity of avrPphF in Canadian Wonder was identified to be avrPphC which was initially cloned for ability to cause the HR in soybean. An intriguing web of avr and vir gene interactions has emerged which adds complexity to the basic gene-for-gene interaction. Models illustrating how effector and receptor proteins may interact are presented.