Aberrant protein N-glycosylation impacts upon infection-related growth transitions of the haploid plant-pathogenic fungus Mycosphaerella graminicola.

The ascomycete fungus Mycosphaerella graminicola is the causal agent of Septoria Tritici Blotch disease of wheat and can grow as yeast-like cells or as hyphae depending on environmental conditions. Hyphal growth is however essential for successful leaf infection. A T-DNA mutagenesis screen performed on haploid spores identified a mutant, which can undergo yeast-like growth but cannot switch to hyphal growth. For this reason the mutant was non-pathogenic towards wheat leaves. The gene affected, MgAlg2, encoded a homologue of Saccharomyces cerevisiae ScAlg2, an alpha-1,2-mannosyltransferase, which functions in the early stages of asparagine-linked protein (N-) glycosylation. Targeted gene deletion and complementation experiments confirmed that loss of MgAlg2 function prevented the developmental growth switch. MgAlg2 was able to functionally complement the S. cerevisiae ScAlg2-1 temperature sensitive growth phenotype. Spores of ΔMgAlg2 mutants were hypersensitive to the cell wall disrupting agent Calcofluor white and produced abnormally hypo-N-glycosylated proteins. Gene expression, proteome and glycoproteome analysis revealed that ΔMgAlg2 mutant spores show responses typically associated with the accumulation of mis-folded proteins. The data presented highlight key roles for protein N-glycosylation in regulating the switch to hyphal growth, possibly as a consequence of maintaining correct folding and localization of key proteins involved in this process.

Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat.

Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

Identification and characterisation of Mycosphaerella graminicola secreted or surface-associated proteins with variable intragenic coding repeats.

Pathogenic micro-organisms have been suggested to vary the number of intragenic repeats present within secreted or cell membrane/cell wall-associated proteins in order to manipulate host immune responses. We have identified a number of genes predicted to encode secreted proteins possessing internal tandem repeats in the genome sequence of Mycosphaerella graminicola (isolate IPO323), a wheat leaf-specific fungal pathogen and causal agent of Septoria tritici blotch disease. Twenty-three M. graminicolaTandem Repeat Proteins (MgTRPs) were subject to further analysis. Many MgTRPs varied in the number of intragenic repeats between isolates and almost all were expressed. Peak gene expression was frequently observed towards the end of the symptomless phase of wheat leaf colonisation which typically lasts for 8-10 days after inoculation. In contrast, with one exception, increased expression of the majority of MgTRPs was not detected during interactions with resistant host genotypes. Repeat number differences detected in genomic DNA were retained in different transcript sizes produced during plant infection by different isolates. One in planta expressed MgTRP was found to reside within a approximately 6 kb region that appears to be absent from a number of tested isolates and also from individual members of a modern field population. Sequence analysis of another in planta expressed MgTRP from six isolates highlighted the potential for structural changes which may occur as a consequence of varying internal repeat numbers and provided support for repeat variation occurring as a consequence of intragenic recombination. These data provide new insights into the genetic variation which exists within M. graminicola populations at the level of in planta expressed secreted/surface-associated proteins which are candidate effectors in the host-pathogen interaction.

Molecular characterization and functional analysis of MgNLP, the sole NPP1 domain-containing protein, from the fungal wheat leaf pathogen Mycosphaerella graminicola.

Analysis of the fully sequenced genome of the wheat leaf-specific fungal pathogen Mycosphaerella graminicola identified only a single gene encoding a member of the necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein family (NLP). NLP proteins have frequently been shown to trigger cell death and the activation of defense signaling reactions in dicotyledonous plants. However, complete loss-of-function reverse genetics analyses for their importance in the virulence of eukaryotic plant pathogens are generally lacking. Real-time quantitative polymerase chain reaction on MgNLP demonstrated the gene to be specifically expressed in planta. Peak expression was observed during the immediate presymptomatic phase of colonization of a susceptible host genotype. This was followed by a dramatic decrease during disease lesion formation which, in this system, exhibits characteristics of host programmed cell death (PCD). No comparable peak in transcript levels was seen during an incompatible interaction with a host genotype exhibiting gene-for-gene-based disease resistance. Heterologously expressed MgNLP protein induced necrotic cell death and the activation of defense-related genes when infiltrated into Arabidopsis leaves but not in leaves of a susceptible wheat genotype. MgNLP infiltration also failed to stimulate wheat mitogen-activated protein kinase activities. Finally, targeted deletion of M. graminicola MgNLP caused no detectable reduction in plant pathogenicity or virulence, suggesting that this protein is not a major virulence determinant during fungal infection of its host plant. To our knowledge, this represents the first complete loss-of-function analysis of NLP in a eukaryotic plant pathogen and we discuss our findings in the context of possible functions for NLP in pathogens which only infect monocotyledonous plants.

The use of conventional and quantitative real-time PCR assays for Polymyxa graminis to examine host plant resistance, inoculum levels and intraspecific variation.

* A real-time PCR protocol based on 18S rDNA sequences was developed to provide a specific, sensitive and quantitative assay for the root-infecting virus vector Polymyxa graminis. * The assay was calibrated with zoospore suspensions and inoculated roots and then shown to work with naturally infected plant roots and infested soil. Both the temperate P. graminis ribotypes previously described are detected but are not distinguished. DNA from related plasmodiophorids and from a range of fungi and plants was not detected. * Different genotypes of Triticum were grown in a soil infested with P. graminis and Soil-borne cereal mosaic virus (SBCMV). The genotypes differed in susceptibility to P. graminis, the least susceptible being the Triticum monococcum accession K-58505. * Conventional PCR assays and sequencing of amplified rDNA fragments showed that P. graminis isolates infecting wheat were mostly, but not exclusively, of ribotype II. Ribotype II was clearly associated with SBCMV transmission and seems to occur preferentially on wheat whereas ribotype I is mostly associated with barley.