BSPP Presidential Meeting 2003

Plant Pathogen Genomics - From Sequence To Application

PH Gregory Talk Abstracts

PH1 Partial sequencing of an endophytic Fusarium oxysporum that mediates complex plant defense for Lycopersicon esculentum Mill

Idress H. Attitalla

Uppsala University, Evolutionary Biology Centre, Department of Molecular Evolution, Norbyvägen 18C, SE-752 36 Uppsala, Sweden.Tel. +46 18 471 6430; Fax: +46 18 4716404; e-mail: Idress.Attitalla@ebc.uu.se

The plant defense of a susceptible tomato (Lycopersicon esculentum Mill.) cultivar involved a complex plant-pathogen-endophyte relation mediated by an endophytic fungus Fo-(IMI 386351). A study of that plant defense and the involved three-organism relationship was complemented by an analysis of the molecular phylogenetics of Fo-(IMI 386351). The results show that plants inhabited by Fo-(IMI 386351) had a considerably enhanced capacity to deter fusarium wilt caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici. The results also indicate that the endophyte was able to induce dose-dependent systemic resistance in the tomato cultivar, and was able to promote plant growth, which can contribute to the overall ability of a plant to defend itself against pathogens.

PH2 Gene expression profiles in Blumeria graminis

Maike Both*, George Dimopoulos**, Mike Csukai# and Pietro Spanu*

*Imperial College, London, **Johns Hopkins University, Baltimore, # Syngenta Ltd., Jealott’s Hill

Blumeria graminis f. sp. hordei (Bgh) is the causal agent of barley powdery mildew, an economically significant disease that can cause yield losses of up to 40%. Bgh is an obligate biotrophic plant pathogen; it can only complete its life cycle on a living host. The asexual life cycle of Bgh on the host proceeds in a highly ordered fashion. The conidiospore produces a primary germ tube and subsequently an appressorial germ tube, which forms the appressorium. By a combination of physical force and enzymatic action, the appressorium sends a penetration peg into the plant cell, where a haustorium starts to develop. Once the nutrient supply is established, the fungus grows by spreading mycelium over the plant leaf surface. We have developed microarrays to study global gene expression in Bgh. The arrays are about 3500 sequenced and annotated cDNAs derived from Bgh libraries. We probe the arrays with fluorescent cDNA from RNA extracted from the fungus at 8 different stages of development, from the ungerminated conidium to a fully sporulating, mature colony. Expression analysis is examined with Genedata ExpressionistTM software and reveals patterns of expression that identify some gene clusters that are likely to play key roles at various developmental stages. This data allows us to propose some models for metabolic and signalling pathways that regulate and control development of infection in this obligate biotrophic pathogen.

PH3 Sequence analysis of the entire RNA genome of sugarcane yellow leaf luteovirus of an Indian isolate

R.K.Gaur1, G.P.Rao1, Maneesha Singh1 and Axel T. Lehrer2

1 Sugarcane Research Station, Kunraghat, Gorakhpur-273008, UP, India
2 Hawaii Biotech. Inc, 99-193 Aiea Heights Drive, 200, Aiea, Hawaii, USA

The complete nucleotide sequence of the single standard RNA genome of an Indian isolate of sugarcane yellow leaf luteovirus (SCYLV-IND) was determined. The RNA genome is 5899 nucleotides long having six open reading frames (ORFs). These ORFs encodes multifunction protein (ORF 1, 72.5 kDa), RdRp (ORF 2, 64.4 kDa), capsid protein (ORF3, 21.8), putative movement protein p17 (ORF 4, 16.6 kDa), putative aphid transmission factor (ORF 5, 52.1 kDa) and ORF 0 (30.2 kDa) with unknown function. The phylogeny of the RdRp of sequence strongly suggests that SCYLV contains a sobemovirus-like rather than a carmovirus-like polymerase gene that included SCYLV in the Polerovirus genus. Comparison of the coat protein and 17 kDa sequence revealed that SCYLVis also closely related to viruses in the genus Luteovirus. Based on this consideation it is supposed that the 5’ coding blocks of SCYLV is more similar to that of the genus Polerovirus while its 3’ coding block is closest to that of the genus Luteovirus. This is the first molecular characterization of SCYLV from India.

PH4 Antioxidants in Blumeria graminis and Magnaporthe grisea

Catherine Henderson

University of Oxford

Phytopathogenic fungi elicit a range of defence responses in their hosts, the oxidative burst being one of the most rapid. Reactive oxygen species (ROS), in particular H2O2, accumulate at sites of pathogen invasion and are thought to have a number of functions including being directly antimicrobial, signalling, and bringing about the cross-linking of cell wall proteins. I am interested in the pathogen’s perspective on this defensive action. Since it is faced with .O2- and H2O2 during invasion of host tissue, the evolution of reactive oxygen detoxifying systems may arm it with a major selective advantage. My work compares antioxidant strategies in both the genetically intractable obligate biotroph Blumeria graminis, and the more amenable Magnaporthe grisea. I will discuss use of a combination of immunolocalisation, real-time RT-PCR, mutant analysis and a novel H2O2 scavenging assay to investigate the role of a secreted catalase, CATB, and how I am now extending the work to encompass further putative antioxidant genes.

PH5 The dual role of peroxisomes in Magnaporthe grisea pathogenicity

Marilou Ramos-Pamplona, Shanthi Soundararajan and Naweed Isaak Naqvi

Fungal Genomics, Temasek Life Sciences Laboratory. 1 Research Link The National University of Singapore. Singapore 117604.

During a screen of random T-DNA insertional mutants in Magnaporthe grisea, TMP6-2 was identified as a nonpathogenic isolate with nonmelanized and aberrant appressoria. Flanking sequence tag data revealed that the T-DNA insertion in TMP6-2 occurred in PEX6, a gene essential for peroxisome biogenesis. Peroxisomes are ubiquitous organelles with key metabolic functions such as fatty acid metabolism. Further studies conducted in TMP6-2 and a pex6D strain confirmed defects in peroxisome-related functions such as inability to utilize complex fatty acids as sole carbon source; and mislocalization of GFP tagged with a peroxisomal targeting signal (PTS1). Detailed characterization of pex6D indicated that acetyl-CoA generated by peroxisomal activity in appressoria is essential for melanin biosynthesis. Deposition of a melanin layer underneath the appressorial cell wall enables the appressorium to maintain a high turgor pressure needed to penetrate the host epidermis. Thin-section EM revealed that pex6D appressoria lacked melanin completely and are unable to penetrate host surfaces. Genetic complementation of the pex6D with a full length PEX6 gene fully restored appressorial melanization and pathogenicity. Phenotypic defects such as aberrant appressoria and the inability to plug septal pores under hypotonic conditions could be attributed directly to the loss of peroxisome-derived Woronin bodies in the pex6D strain.

PH6 Bacterial Communication: the Role of luxS in a Plant and an Animal Pathogen

Sarah Coulthurst

Department of Biochemistry, University of Cambridge, 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. The role of luxS has been investigated in the plant pathogen Erwinia carotovora subsp. carotovora (Ecc) and the opportunistic human pathogen Serratia marcescens. Production of AI-2 activity was detected and the luxS locus identified and sequenced in Ecc ATTn10 and S. marcescens ATCC274. luxS mutants were constructed in both strains and their phenotypes were examined. Compared to the wild type, the luxS mutant of S. marcescens ATCC274 exhibited decreased prodigiosin and haemolysin production and decreased virulence in a C. elegans model. In Ecc ATTn10, a proteomic comparison of the luxS mutant with the wild type using 2D-DiGE technology was performed. This identified a limited set of proteins whose abundance is altered in the luxS mutant compared to the wild type. It was also demonstrated 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 species. Therefore luxS regulates virulence and antibiotic production in S. marcescens, 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.

PH7 Investigating the role of trehalose metabolism in pathogenicity of the rice blast fungus Magnaporthe grisea

Joanna M. Jenkinson and Nicholas J. Talbot

School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4JQ, United Kingdom.

The heterothallic ascomycete Magnaporthe grisea causes rice blast disease of cultivated rice. The potentially devastating effects of this disease are determined by the ability of the fungus to mechanically penetrate the plant cuticle and epidermis. The force necessary for cuticle penetration is due to the enormous hydrostatic turgor generated within the appressorium as a result of the high concentrations of compatible solutes, notably glycerol, which accumulate in appressoria. Glycogen, lipid and trehalose are the most likely potential precursors for glycerol biosynthesis in M. grisea as they represent the most abundant storage products in spores of the fungus. The aim of this project is to understand the role of trehalose synthesis and metabolism in the infection cycle of Magnaporthe grisea. Trehalose is synthesized by the trehalose 6 phosphate synthase complex (T6PS), and we have shown that TPS1, encoding the catalytic sub-unit of T6PS, is required for pathogenicity of M. grisea and appressorium turgor generation (Foster et al., 2003). However, the subsequent predicted breakdown of trehalose, which would be required for glycerol synthesis, is dispensable for appressorium turgor generation, and, therefore, it seems more likely that trehalose accumulation contributes to appressorium function, either due to trehalose acting in a protective capacity, or by accumulating as a solute to contribute to turgor. TPS1 also, however, exhibits a regulatory effect on glycolysis and tps1 mutants are unable to grow on glucose and a range of other simple sugars. We speculate that trehalose-6-phosphate may be able to act as a signalling molecule for glycolytic control in M. grisea and that this mechanism is perturbed by loss of TPS1. Using a combination of gene functional analysis, cell biology and metabolite profiling, we have been examining the mechanisms by which T6PS exerts control over glycolysis, and how this impacts upon appressorium function in M. grisea

Reference:

Foster, A.J.Jenkinson, J.M., Talbot, N.J. (2003 Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225-235.

PH8 Symptomless Stagonospora nodorum - the 'disappearance' of glume blotch of wheat

Sarah Bearchell

University of Reading and Rothamsted Research 

Surveys of wheat diseases have been conducted in England and Wales since 1970.  The data show that symptoms of glume blotch (Stagonospora nodorum) were extremely common in the 1970s but are now rare.  Conversely, symptoms of leaf blotch (Mycosphaerella graminicola) were almost absent at the start of the period but this is now the most damaging foliar wheat disease in England and Wales.  They have effectively swapped in prevalence. One reason suggested for this change in incidence is the increase of a symptomless form of S. nodorum.  The presence of the pathogen in symptomless leaves was confirmed by spore washing and ELISA at GS38.  Both methods yielded similar results, suggesting that they measure the same aspect of the fungal population.  There are several possible explanations for the presence of this prolonged symptomless phase and future avenues of research are discussed.

PH9 On the origins of Verticillium isolates associated with crucifers

Emily Clewes

Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, Harper Adams University College, Newport, Shropshire, TF10 8NB

Isolates of the plant pathogenic soil-borne wilt fungal genus Verticillium have been studied at HRI to better understand the nature and origins of those which generally infect crucifers only. All Verticillium crucifer isolates produce microsclerotia and the majority produce long conidia with a high nuclear DNA content (V. dahliae var. longisporum). The long-spored isolates are interspecific hybrids that are genetically amphihaploid. The parents were thought to be V. dahliae and V. albo-atrum. The long-spored isolates were previously divided into two groups (α and β) (Collins et al., 2002). Sequence analysis of the 5S rRNA intergenic region (IGR), a partial sequence of the β-tubulin gene and mitochondrial cytochrome B gene, shown that group β may be further divided, allowing for a third group of crucifer isolates, γ. Furthermore, the 5S rRNA IGR and the β-tubulin provide direct evidence that long-spored amphihaploid isolates are hybrid. PCR amplification of these regions produced two amplicons for the long-spored crucifer isolates whereas a single amplicon was produced for haploid isolates. Sequence analysis of these regions suggests that one parent in all crosses was akin to V. dahliae however, although the other ‘parent’ of the amphihaploids was more like V. albo-atrum it was very distinct from all isolates studied to this time.

Additional short talk

ATR13: An avirulence gene from Peronospora parasitica

Rebecca L. Allen, Peter Bittner-Eddy, Laura Grenville, Anne P Rehmany, Sharon Hall and Jim Beynon
HRI Wellesbourne, Warwick, UK

The obligate biotrophic oomycete Peronospora parasitica is an important pathogen of brassica crop species in which it causes downy mildew. We are studying the interaction of this pathogen with another natural host, Arabidopsis, in order to understand the molecular mechanisms involved in pathogenicity and plant responses. We have cloned several resistance genes from Arabidopsis including the RPP13 gene that recognises pathogen isolate Maks9. This gene causes a highly localised HR and the resistance is independent of known signalling pathways. We decided, therefore, to clone the complementary avirulence gene from Maks9 in order to understand its role in pathogenicity and the nature of RPP13 resistance. We will describe the isolation and structure of ATR13.