Penetration and colonization of unwounded maize tissues by the maize anthracnose pathogen Colletotrichum graminicola and the related nonpathogen C. sublineolum

The maize anthracnose stalk-rot fungus Colletotrichum graminicola infects its host primarily through wounds in the stalks that are caused by insects. However it also can cause stalk-rot disease without wounding. It is not known how the pathogen enters stalks in the absence of wounds. Studies have suggested that direct invasion through the highly lignified rind tissues is not a viable means of entry. A cytological approach was used to investigate the ability of C. graminicola to penetrate and colonize intact maize stalks. The pathogen had a significant capacity for direct penetration, but this mechanism of infection was much slower and less efficient than penetration through wounds. The fungus breached the lignified rind fibers by passing through small openings in the cell walls via narrow hyphal connections. Epidermal cells and rind fiber cells did not appear to become rotted. Rotting only occurred once the pathogen had penetrated into the pith parenchyma cells. To our surprise the closely related fungus C. sublineolum, which is not normally a pathogen of maize, also was capable of infecting intact maize stalks, although to a lesser degree than C. graminicola. The two species also were observed on intact roots and leaves, and C. sublineolum was incapable of infecting those tissues whereas C. graminicola efficiently colonized both. This suggests the interesting possibility that nonhost resistance to C. sublineolum is conditional and perhaps also tissue-specific.     

Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.     

A chitin synthase with a myosin-like motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity of the maize anthracnose fungus Colletotrichum graminicola

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. DeltaCgChsI and DeltaCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of DeltaCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, DeltaCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, DeltaCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.     

A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize

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.     

MVE1, encoding the velvet gene product homolog in Mycosphaerella graminicola, is associated with aerial mycelium formation, melanin biosynthesis, hyphal swelling, and light signaling

The ascomycete fungus Mycosphaerella graminicola is an important pathogen of wheat that causes Septoria tritici blotch. Despite the serious impact of M. graminicola on wheat production worldwide, knowledge about its molecular biology is limited. The velvet gene, veA, is one of the key regulators of diverse cellular processes, including development and secondary metabolism in many fungi. However, the species analyzed to date are not related to the Dothideomycetes, the largest class of plant-pathogenic fungi, and the function of veA in this group is not known. To test the hypothesis that the velvet gene has similar functions in the Dothideomycetes, a veA-homologous gene, MVE1, was identified and gene deletion mutations (Δmve1) were generated in M. graminicola. All of the MVE1 mutants exhibited consistent pleiotropic phenotypes, indicating the involvement of MVE1 in multiple signaling pathways. Δmve1 strains displayed albino phenotypes with significant reductions in melanin biosynthesis and less production of aerial mycelia on agar plates. In liquid culture, Δmve1 strains showed abnormal hyphal swelling, which was suppressed completely by osmotic stress or lower temperature. In addition, MVE1 gene deletion led to hypersensitivity to shaking, reduced hydrophobicity, and blindness to light-dependent stimulation of aerial mycelium production. However, pathogenicity was not altered in Δmve1 strains. Therefore, the light-signaling pathway associated with MVE1 does not appear to be important for Septoria tritici blotch disease. Our data suggest that the MVE1 gene plays crucial roles in multiple key signaling pathways and is associated with light signaling in M. graminicola.     

Intraspecific comparison and annotation of two complete mitochondrial genome sequences from the plant pathogenic fungus Mycosphaerella graminicola.

The mitochondrial genomes of two isolates of the wheat pathogen Mycosphaerella graminicola were sequenced completely and compared to identify polymorphic regions. This organism is of interest because it is phylogenetically distant from other fungi with sequenced mitochondrial genomes and it has shown discordant patterns of nuclear and mitochondrial diversity. The mitochondrial genome of M. graminicola is a circular molecule of approximately 43,960bp containing the typical genes coding for 14 proteins related to oxidative phosphorylation, one RNA polymerase, two rRNA genes and a set of 27 tRNAs. The mitochondrial DNA of M. graminicola lacks the gene encoding the putative ribosomal protein (rps5-like), commonly found in fungal mitochondrial genomes. Most of the tRNA genes were clustered with a gene order conserved with many other ascomycetes. A sample of 35 additional strains representing the known global mt diversity was partially sequenced to measure overall mitochondrial variability within the species. Little variation was found, confirming previous RFLP-based findings of low mitochondrial diversity. The mitochondrial sequence of M. graminicola is the first reported from the family Mycosphaerellaceae or the order Capnodiales. The sequence also provides a tool to better understand the development of fungicide resistance and the conflicting pattern of high nuclear and low mitochondrial diversity in global populations of this fungus.     

Intraspecific comparison and annotation of two complete mitochondrial genome sequences from the plant pathogenic fungus Mycosphaerella graminicola

The mitochondrial genomes of two isolates of the wheat pathogen Mycosphaerella graminicola were sequenced completely and compared to identify polymorphic regions. This organism is of interest because it is phylogenetically distant from other fungi with sequenced mitochondrial genomes and it has shown discordant patterns of nuclear and mitochondrial diversity. The mitochondrial genome of M. graminicola is a circular molecule of approximately 43,960bp containing the typical genes coding for 14 proteins related to oxidative phosphorylation, one RNA polymerase, two rRNA genes and a set of 27 tRNAs. The mitochondrial DNA of M. graminicola lacks the gene encoding the putative ribosomal protein (rps5-like), commonly found in fungal mitochondrial genomes. Most of the tRNA genes were clustered with a gene order conserved with many other ascomycetes. A sample of 35 additional strains representing the known global mt diversity was partially sequenced to measure overall mitochondrial variability within the species. Little variation was found, confirming previous RFLP-based findings of low mitochondrial diversity. The mitochondrial sequence of M. graminicola is the first reported from the family Mycosphaerellaceae or the order Capnodiales. The sequence also provides a tool to better understand the development of fungicide resistance and the conflicting pattern of high nuclear and low mitochondrial diversity in global populations of this fungus.     

PHL1 of Cercospora zeae-maydis encodes a member of the photolyase/cryptochrome family involved in UV protection and fungal development

DNA photolyases harvest light energy to repair genomic lesions induced by UV irradiation, whereas cryptochromes, presumptive descendants of 6-4 DNA photolyases, have evolved in plants and animals as blue-light photoreceptors that function exclusively in signal transduction. Orthologs of 6-4 photolyases are predicted to exist in the genomes of some filamentous fungi, but their function is unknown. In this study, we identified two putative photolyase-encoding genes in the maize foliar pathogen Cercospora zeae-maydis: CPD1, an ortholog of cyclobutane pyrimidine dimer (CPD) photolyases described in other filamentous fungi, and PHL1, a cryptochrome/6-4 photolyase-like gene. Strains disrupted in PHL1 (Deltaphl1) displayed abnormalities in development and secondary metabolism but were unaffected in their ability to infect maize leaves. After exposure to lethal doses of UV light, conidia of Deltaphl1 strains were abolished in photoreactivation and displayed reduced expression of CPD1, as well as RAD2 and RVB2, orthologs of genes involved in nucleotide excision and chromatin remodeling during DNA damage repair. This study presents the first characterization of a 6-4 photolyase ortholog in a filamentous fungus and provides evidence that PHL1 regulates responses to UV irradiation.     

Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola

We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.     

CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis,regulates cercosporin biosynthesis,fungal development,and pathogenesis

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.     

Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.     

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.     

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.