Alison M. Ashby

Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape

Using a detached cotyledon test for pathogenicity, a UV-induced, non-pathogenic mutant of Pyrenopeziza brassicae was isolated which was also deficient in extracellular protease production in vitro . The proteolytic activity in the wild type was apparently due to a single cysteine protease with a mol. wt of 34 k, a temperature optimum of 40°C and a pH optimum of 8. When the mutant was crossed with a wild-type isolate of P. brassicae , the non-proteolytic and non-pathogenic traits co-segregated in the resulting progeny. The protease − mutant was transformed with clones from a genomic library of P. brassicae and a transformant obtained which had a single cosmid insert and showed concomitant restoration of pathogenicity and proteolytic activity in vitro . These results suggest that extracellular protease is a pathogenicity determinant of P. brassicae and possible functions for this protease in the disease process are discussed.

Cloning of the mating type loci from Pyrenopeziza brassicae reveals the presence of a novel mating type gene within a discomycete MAT 1‐2 locus encoding a putative metallothionein‐like protein

The mating type loci were cloned from Pyrenopeziza brassicae by chromosome walking from a mating type‐linked polymerase chain reaction (PCR) fragment and shown to be idiomorphic by sequence analysis. The MAT 1‐1 locus is approximately 3.2 kb and contains a single gene encoding a putative high‐mobility group (HMG) domain protein. The MAT 1‐2 locus is approximately 3.9 kb with three open reading frames (ORFs) encoding a putative HMG domain, an α‐1 domain and metallothionein‐like proteins. The putative α‐1 domain ORF on MAT 1‐2 is transcribed in the opposite orientation to the other two transcripts and extends into non‐idiomorphic sequence. This is the first report of sequence analysis of the mating type loci from a discomycete fungus, which has revealed an interesting mating type infrastructure within the MAT 1‐2 locus. Although metallothionein‐like proteins have been implicated in a number of processes in animals and plants, they have not to date been implicated in the mating process of filamentous fungi. Possible roles for metallothionein‐like proteins in the mating process are discussed.

Intra-specific and inter-specific conservation of mating-type genes from the discomycete plant-pathogenic fungi Pyrenopeziza brassicae and Tapesia yallundae

Abstract In previous work, four genes involved in mating-type determination were cloned from reference strains of Pyrenopeziza brassicae; three genes, PAD1, PMT1, and PHB1 (re-named henceforth as MAT-1-1, MAT-1-4, and MAT1-3, respectively), are encoded by the MAT-1 idiomorph, and one gene, PHB2 (re-named MAT-2), by the corresponding MAT-2 idiomorph. To assess MAT gene organisation within field-populations of P. brassicae, 30 field-isolates of both mating-types from different geographical locations were analysed by PCR using primers designed for the MAT genes of P. brassicae. The results indicate that mating-type gene structure and organisation within these isolates is conserved and is consistent with the mating-type designations established by crossing experiments. The four P. brassicae MAT genes were then used as probes against gel blots of the genomic DNA of a discomycete Tapesia yallundae from the same family (Dermateaceae, order Helotiales) and one, Ascobolus stercorarius, from a distantly related family (Ascobolaceae, order Pezizales), in order to determine whether P. brassicae MAT-gene homologs were present. MAT-specific hybridisation signals were obtained with T. yallundae using all four probes. In particular, MAT-1 DNA of T. yallundae gave a strongly hybridising signal with MAT-1-4 (PMT1), a putative metallothionein gene found in the P. brassicae MAT-1 idiomorph but not in any other MAT idiomorph examined to-date. No MAT-specific hybridisation was obtained with A. stercorarius. A fragment of the MAT-2 gene of T. yallundae was obtained by PCR using degenerate primers designed to amplify the high-mobility group (HMG) domain present in other ascomycete MAT genes. Sequencing of this PCR product revealed similarities to MAT HMG domains from other ascomycetes with the greatest degree of similarity exhibited with P. brassicae. The T. yallundae HMG-DNA sequence was shown to co-segregate with mating type (MAT-2) in progeny from a sexual cross.

Improved PCR-based assays for pre-symptomatic diagnosis of light leaf spot and determination of mating type of Pyrenopeziza brassicae on winter oilseed rape

An existing PCR-based method for diagnosis of the winter oilseed rape (Brassica napus ssp oleifera) fungal pathogen Pyrenopeziza brassicae (cause of light leaf spot) was improved by the development of a pair of primers (PbN1 and PbN2) for use in nested-PCR reactions. The nested-PCR technique improved the detection of P. brassicae DNA in vitro by three orders of magnitude over that achieved using the first-round PCR primers (Pb1 and Pb2). In controlled environment experiments, the nested-PCR assay detected P. brassicae within infected B. napus leaves before visible light leaf spot symptoms developed and earlier than was possible by incubating infected leaves in polyethylene bags to promote sporulation of P. brassicae. A three-primer PCR technique using the primers PbM-1-3, PbM-2 and Mt3 was developed to distinguish between the two mating types (MAT-1 and MAT-2) of P. brassicae. This technique was able to determine the mating types present within DNA extracted from infected plant tissue, including tissue infected with both mating types together.

Resistance to infection by stealth: Brassica napus (winter oilseed rape) and Pyrenopeziza brassicae (light leaf spot)

Light leaf spot (Pyrenopeziza brassicae) is an important disease on winter oilseed rape crops (Brassica napus) in northern Europe. In regions where economically damaging epidemics occur, resistance to P. brassicae in commercial cultivars is generally insufficient to control the disease without the use of fungicides. Two major genes for resistance have been identified in seedling experiments, which may operate by decreasing colonisation of B. napus leaf tissues and P. brassicae sporulation. Much of the resistance present in current commercial cultivars is thought to be minor gene-mediated and, in crops, disease escape and tolerance also operate. The subtle strategy of the pathogen means that early colonisation of host tissues is asymptomatic, so a range of techniques and molecular tools is required to investigate mechanisms of resistance. Whilst resistance of new cultivars needs to be assessed in field experiments where they are exposed to populations of P. brassicae under natural conditions, such experiments provide little insight into components of resistance. Genetic components are best assessed in controlled environment experiments with single spore (genetically fixed) P. brassicae isolates. Data for cultivars used in the UK Recommended List trials over several seasons demonstrate how the efficacy of cultivar resistance can be reduced when they are deployed on a widespread scale. There is a need to improve understanding of the components of resistance to P. brassicae to guide the development of breeding and deployment strategies for sustainable management of resistance to P. brassicae in Europe.

Development of Pyrenopeziza brassicae apothecia on agar and oilseed rape debris.

The development of apothecia of Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) on oilseed rape debris and compost malt agar was observed by scanning electron and light microscopy. On oilseed rape debris, apothecia developed directly beneath the epidermis as small globular structures of dense mycelium, which protruded through the epidermis as they increased in size. The apices of erumpent immature apothecia then developed small ostiolar openings which increased in diameter to expose the hymenia of mature apothecia containing dome-shaped asci interspersed with filiform paraphyses. On compost malt agar, hyphae of one mating type grew towards hyphae of the opposite mating type 3-4 d after inoculation of conidia onto agar surfaces. The subsequent development of apothecia on compost malt agar was similar to that on oilseed rape debris.

Expression of the E. coli β-glucuronidase gene in the light leaf spot pathogen Pyrenopeziza brassicae and its use as a reporter gene to study developmental interactions in fungi

Co-transformation using the hygromycin-resistant plasmid pAN7-1 for transformation selection was used to transform spores of both mating types of the light leaf spot pathogen P. brassicae with the chimeric β-glucuronidase (GUS) vector pNOM102. 78 and 90% of the hygromycin-resistant transformants of strains JH26 (MAT1-2) and NH10 (MAT1-1) respectively expressed GUS activity and 30% of these transformants showed high levels of GUS activity. Quantitative analysis of GUS expression by measurement of the rate of hydrolysis of the fluorogenic substrate 4-methylumbelliferyl glucuronide revealed that the wild type produces no detectable activity and that 15 transformants of each mating type displayed a 500-fold range of activity. Ten of the transformants were analysed further to establish mitotic stability of GUS, the ability to pathogenize the plant and an ability to undergo sexual morphogenesis when crossed with a compatible mating-type partner. All of the transformants tested were fertile when crossed with a compatible mating-type partner. Two out of the transformants tested had lost their ability to express GUS activity on continuous sub-culturing. All of the NH10 (MAT1-1) GUS transformants were able to produce extracellular proteolytic activity and were able to pathogenize the plant. However three JH26 (MAT1-2) GUS transformants displayed reduced proteolytic activity and one (JH26pNOM102/13) produced no extracellular protease in vitro and had lost the ability to produce pathogenic symptoms in planta. GUS transformants of P. brassicae were shown to stain blue with the chromogenic substrate 5-bromo-4-chloro-3-indolyl β- d -glucuronide. This staining reaction enabled differentiation between both spores and mycelium during sexual development when a wild type of either mating type was crossed with a GUS transformant of the opposite mating type. Such preliminary studies demonstrate the relative usefulness of the GUS reporter system for the analysis of an otherwise indistinguishable mating interaction and suggest that it will be a valuable reporter system for analysis of the temporal and spatial expression of developmentally regulated genes in this economically important plant pathogen.

Transformation of the fungusPyrenopeziza brassicae, cause of light leaf spot of brassicas, and complementation of mutants using a genomic library

Abstract An efficient gene transfer system is a prerequisite for the molecular genetic analysis of pathogenicity and other genes of plant pathogens. A transformation procedure for the fungus Pyrenopeziza brassicae was therefore devised. Three plasmids, encoding hygromycin resistance (pAN7-1, pAN7-2) or phleomycin resistance (pAN8-1), were used to transform conidial protoplasts of P. brassicae in the presence of calcium chloride and polyethylene glycol. Transformation arose due to integration of transforming DNA, apparently at random sites, and multiple integration events were common. The frequency of transformation was variable but similar to that reported for other phytopathogenic fungi (up to 20 μg −1 DNA) and was increased when homologous DNA was included in the vector. The pathogenicity of the transformants was unchanged by transformation and, when reisolated from inoculated host tissue, the transformants were found to have retained their antibiotic resistance. The transformation technique was used to complement adeninerequiring and extracellular enzyme-deficient, UV-induced mutants to prototrophy and extracellular protease production, respectively, with cosmids from a genomic library of the fungus.

Purification and biochemical characterisation of Psp1, an extracellular protease produced by the oilseed rape pathogen Pyrenopeziza brassicae

Abstract An extracellular serine protease, designated Pyrenopeziza brassicae serine protease 1 (Psp1), was purified by a combination of ammonium sulphate precipitation and fast protein liquid chromatography (FPLC) to yield a single 34 kDa band on SDS–PAGE which showed proteolytic activity following renaturation. The N-terminal sequence of the purified protein was similar to that of several fungal serine proteases, with the highest identity (84% over 13aa) with PrtA from Aspergillus nidulans. The effect of protease inhibitors on Psp1 confirmed that it is a member of the subtilisin (S8) family of serine proteases. Characterisation of Psp1 activity on a range of synthetic di- tri- and oligo-peptide substrates indicated mixed trypsin/chymotrypsin specificity. A renaturable 34 kDa protease, corresponding to Psp1, was observed in cotyledons 7 days and 13 days post infection with P. brassicae.

Cloning and expression studies during vegetative and sexual development of Pbs1, a septin gene homologue from Pyrenopeziza brassicae.

A septin gene homologue designated Pyrenopeziza brassicae septin 1 (Pbs1) has been identified and cloned from the plant pathogenic fungus Pyrenopeziza brassicae and its expression analysed. Pbs1 is present in both mating types and in a single copy within each genome and is transcribed in proportionate levels during both vegetative and sexual growth.