Graminicola

Abstract: 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.

Abstract: We have previously shown that the beneficial filamentous fungus Trichoderma virens secretes the highly effective hydrophobin-like elicitor Sm1 that induces systemic disease resistance in the dicot cotton (Gossypium hirsutum). In this study we tested whether colonization of roots by T. virens can induce systemic protection against a foliar pathogen in the monocot maize (Zea mays), and we further demonstrated the importance of Sm1 during maize-fungal interactions using a functional genomics approach. Maize seedlings were inoculated with T. virens Gv29-8 wild type and transformants in which SM1 was disrupted or constitutively overexpressed in a hydroponic system or in soil-grown maize seedlings challenged with the pathogen Colletotrichum graminicola. We show that similar to dicot plants, colonization of maize roots by T. virens induces systemic protection of the leaves inoculated with C. graminicola. This protection was associated with notable induction of jasmonic acid- and green leaf volatile-biosynthetic genes. Neither deletion nor overexpression of SM1 affected normal growth or development of T. virens, conidial germination, production of gliotoxin, hyphal coiling, hydrophobicity, or the ability to colonize maize roots. Plant bioassays showed that maize grown with SM1-deletion strains exhibited the same levels of systemic protection as non-Trichoderma-treated plants. Moreover, deletion and overexpression of SM1 resulted in significantly reduced and enhanced levels of disease protection, respectively, compared to the wild type. These data together indicate that T. virens is able to effectively activate systemic disease protection in maize and that the functional Sm1 elicitor is required for this activity.

Abstract: Cellular aspects of the pathogenesis of Mycosphaerella graminicola in a susceptible and resistant wheat cultivar were studied by light microscopy and scanning and transmission electron microscopy. Experiments were designed as time-sequence studies in two replications with sampling dates at 12-, 24-, and 48-h postinoculation (hpi), and 4-, 8-, 10-, 12-, 14-, and 16-days postinoculation (dpi). A separate experiment was performed to quantify the mycelial biomass in cultivars Shafir and Kavkaz/K4500 1.6.a.4 at the aforementioned intervals using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for M. graminicola. The germination frequency of M. graminicola conidia was high in both compatible and incompatible interactions, but the infection frequency was low. Infection was strictly stomatal, but appeared to be a random process since many germ tubes crossed stomata without penetrating them. Some germ tubes formed branched structures close to or on top of stomata. These structures were small compared with the size of stomata, were formed irregularly, and were not significantly correlated with successful penetrations of the host. Multiple penetrations of stomata occurred regularly. Hyphae of M. graminicola were already observed in the substomatal cavities at 12 hpi and, at 48 hpi, hyphae had reached the nearest mesophyll cells. In the compatible response, colonization was fairly limited until 8 dpi. Hyphae grew intercellularly and in close contact with the mesophyll cells. During the 10- to 12-dpi interval, extensive host cell death occurred, which induced further colonization and, eventually, pycnidium formation in substomatal cavities. Initial and further colonization had marked effects on the number and size of the chloroplasts in the compatible interaction. Nevertheless, leaves remained green until approximately 10 dpi. The resistance response was primarily characterized by very limited colonization, mostly in the vicinity of the substomatal cavity. Quantification of the mycelial mass with ELISA revealed similar mycelial quantities in cultivars Shafir and Kavkaz/K4500 1.6.a.4 until 8 dpi. After 8 days, the mycelial quantity developed exponentially in 'Shafir,' but did not significantly increase in 'Kavkaz/K4500 1.6.a.4.'

Abstract: The Fertile Crescent represents the center of origin and earliest known place of domestication for many cereal crops. During the transition from wild grasses to domesticated cereals, many host-specialized pathogen species are thought to have emerged. A sister population of the wheat-adapted pathogen Mycosphaerella graminicola was identified on wild grasses collected in northwest Iran. Isolates of this wild grass pathogen from 5 locations in Iran were compared with 123 M. graminicola isolates from the Middle East, Europe, and North America. DNA sequencing revealed a close phylogenetic relationship between the pathogen populations. To reconstruct the evolutionary history of M. graminicola, we sequenced 6 nuclear loci encompassing 464 polymorphic sites. Coalescence analyses indicated a relatively recent origin of M. graminicola, coinciding with the known domestication of wheat in the Fertile Crescent around 8,000-9,000 BC. The sympatric divergence of populations was accompanied by strong genetic differentiation. At the present time, no genetic exchange occurs between pathogen populations on wheat and wild grasses although we found evidence that gene flow may have occurred since genetic differentiation of the populations.