Ascomycete Leptosphaeria Maculans Research Articles

Molecular Interactions Between Leptosphaeria maculans and Brassica Species.

Canola is an important oilseed crop, providing food, feed, and fuel around the world. However, blackleg disease, caused by the ascomycete Leptosphaeria maculans, causes significant yield losses annually. With the recent advances in genomic technologies, the understanding of the Brassica napus-L. maculans interaction has rapidly increased, with numerous Avr and R genes cloned, setting this system up as a model organism for studying plant-pathogen associations. Although the B. napus-L. maculans interaction follows Flor's gene-for-gene hypothesis for qualitative resistance, it also puts some unique spins on the interaction. This review discusses the current status of the host-pathogen interaction and highlights some of the future gaps that need addressing moving forward.

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.

Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.

Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations.

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.

Mutations to LmIFRD affect cell wall integrity, development and pathogenicity of the ascomycete Leptosphaeria maculans

Maintaining cell wall integrity is essential for fungal growth and development. We describe two mutants with altered expression of a gene, LmIFRD, from the ascomycete Leptosphaeria maculans. Truncation of the LmIFRD transcript in a T-DNA insertional mutant led to slower germination, less sporulation and loss-of-pathogenicity towards Brassica napus, whereas silencing of the LmIFRD transcript led to increased germination, sporulation and earlier infection. The increased tolerance to cell wall lysing enzymes and cell wall-disrupting compounds of the T-DNA mutant contrasts with decreased tolerance of the silenced mutant and suggests altered cell wall integrity and accessibility to 1,3-linked glucan and chitin. Lectin binding experiments and monosaccharide analysis revealed altered polysaccharide content and structure within the cell wall of the LmIFRD mutants, notably increased 1,3-linked galactose and chitin within the cell wall of the T-DNA mutant. This is the first analysis of monosaccharide linkage composition of cell walls of spores and mycelia for any dothideomycete.

Analysis of EST Libraries from Leptosphaeria maculans and Blackleg Infected Brassica napus

The ascomycete Leptosphaeria maculans is the causal agent of blackleg of crucifers. cDNA libraries were constructed and partially sequenced to increase the amount of L. maculans sequence data available and to survey genes expressed during pathogenesis. Two libraries were built, one from cultured mycelia (LM) and one from Brassica napus leaf tissue infected with L. maculans (ILS). A total of 741 expressed sequence tags (ESTs) were obtained from the LM library, and 1284 were obtained from the ILS library. The vast majority of the ESTs obtained from the ILS library appear to originate from B. napus (70-81% depending on the identification method). Various methods for determining sequence origin in mixed libraries were compared. cDNAs with a putative role in pathogenesis were identified from the ILS library. These and other cDNAs of interest from the LM library are discussed.

Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans.

A vector system was constructed that is designed to decrease the number of transformants required to be screened when looking for gene disruption events in filamentous fungi. This vector was used to mutate two genes, an ATP-binding cassette transporter ( LmABCt4) and a two-component histidine kinase gene ( LmHK1) in the ascomycete Leptosphaeria maculans. The system uses the thymidine kinase gene from the herpes simplex virus as a negative selectable marker. Thymidine kinase expression is regulated by the TrpC regulatory elements from Aspergillus nidulans and should be applicable to other ascomycetous fungi. When thymidine kinase is expressed in the presence of particular thymidine analogues, these analogues are converted to toxic compounds which kill the cell. We also report the transformation of L. maculans using Agrobacterium tumefaciens-mediated DNA delivery.

Sterol composition of mycelia of the plant pathogenic ascomycete Leptosphaeria maculans

Analysis of sterols in mycelia of the ascomycete, Leptosphaeria maculans by gas chromatography–mass spectrometry revealed that ergosterol comprised 95% of the total sterols, with eight other sterols comprising the remaining 5%. Six of these latter sterols were putative precursors of ergosterol and their presence suggested a pathway for ergosterol biosynthesis in this fungus. Ergosterol biosynthesis in fungi is inhibited by the triazole antifungal agent flutriafol. When L. maculans was grown in the presence of flutriafol, ergosterol content decreased while two 14α-methylated sterols, 24-methylene dihydrolanosterol and obtusifoliol, accumulated.

Organisation of ribosomal DNA in the ascomycete Leptosphaeria maculans

In the ascomycete Leptosphaeria maculans tandem repeats of ribosomal DNA (rDNA) are restricted to one or two particular chromosomes of the 15 chromosomes of 19 field isolates examined. Ribosomal DNA can account for size differences of 35% between homologous chromosomes in a particular tetrad. During crossing, no detectable recombination between blocks of tandem repeats, nor changes in their size occur. The organisation of rDNA in L. maculans differs from many other haploid fungi. Firstly, sequence heterogeneity occurs within tandem repeats of rDNA; regularly spaced Sal 1 sites (0.25 Mb apart) are present within a 1.4 Mb block of tandem repeats. Secondly, individual isolates have different-sized rDNA repeats; this variation occurs in the non-transcribed intergenic spacer region. Thirdly, there is a wide range in the copy number of the rDNA repeat (from 56 to 225) amongst only four field isolates examined.

Linear plasmids, pLm9 and pLm10, can be isolated from the phytopathogenic ascomycete Leptosphaeria maculans by pulsed-field gel electrophoresis.

Two linear DNA plasmids (pLm9 and pLm10, sized 9 and 10 kb respectively) were isolated from the phytopathogenic ascomycete Leptosphaeria maculans, using pulsed-field gel electrophoresis. pLm9 and pLm10 are found only in aggressive isolates of L. maculans but, because aggressive and non-aggressive strains appear to be different species, these plasmids are probably not involved in pathogenicity. pLm9 and pLm10 copurify with a mitochondrially-enriched cell fraction, and do not hybridise to chromosomal or mitochondrial DNA, or to each other. Exonuclease digestions suggest that both these molecules contain covalently-bound proteins at their 5' termini. pLm9 hybridises to the RNA polymerase of a linear plasmid from the ascomycete Podospora anserina, and pLm10 hybridises to the DNA polymerase from the same P. anserina plasmid, suggesting that pLm9 and pLm10 encode their own replication and transcription enzymes.

Non-aggressive Strains of the Blackleg Fungus, Leptosphaeria maculans, Are Present in Australia and Can Be Distinguished From Aggressive Strains by Molecular Analysis

Isolates of the pathogenic ascomycete Leptosphaeria maculans have been cultured from blackleg-affected oilseed rape (Brassica napus) stubble from Horsham, Victoria. These isolates are indistinguishable on the basis of morphological characters, but can be classified as either aggressive or non-aggressive by their ability to infect B. napus cultivars Midas and Westar. These aggressive and non-aggressive isolates of L. maculans can be distinguished by molecular techniques including electrophoretic karyotyping, Southern analysis of the ribosomal RNA gene repeat, Random Amplified Polymorphic DNA (RAPD) marker analysis, and pigment production. The presence of aggressive and non-aggressive strains of L. maculans in North America and Europe has been previously described. This is the first report of non-aggressive L. maculans strains isolated from B. napus in Australia.

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans.

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.