MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis

The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.     

ABC transporters of the wheat pathogen<Emphasis Type="Italic"> Mycosphaerella graminicola</Emphasis> function as protectants against biotic and xenobiotic toxic compounds

We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin.     

Protein kinase A subunits of the ascomycete pathogen Mycosphaerella graminicola regulate asexual fructification, filamentation, melanization and osmosensing

As in many fungi, asexual reproduction of Mycosphaerella graminicola in planta is a complex process that requires proper differentiation of the infectious hyphae in the substomatal cavities of foliar tissue before pycnidia with conidia can be formed. In this study, we have investigated the role of the cAMP signalling pathway in development and pathogenicity of this pathogen by disruption of the genes encoding the catalytic (designated MgTpk2 ) and regulatory subunit (designated MgBcy1 ) of protein kinase A. The MgTpk2 and MgBcy1 mutants showed altered phenotypes in vitro when grown under different growth conditions. On potato dextrose agar (PDA), MgBcy1 mutants showed altered osmosensitivity and reduced melanization, whereas the MgTpk2 mutants showed accelerated melanization when compared with the M. graminicola IPO323 wild-type strain and ectopic transformants. MgTpk2 mutants also secreted a dark-brown pigment into yeast glucose broth medium. In germination and microconidiation assays, both mutants showed a germination pattern similar to that of the controls on water agar, whereas on PDA filamentous growth of MgTpk2 mutants was impaired. Pathogenicity assays showed that the MgTpk2 and MgBcy1 mutants were less virulent as they caused only limited chlorotic and necrotic symptoms at the tips of the inoculated leaves. Further analyses of the infection process showed that MgTpk2 and MgBcy1 mutants were able to germinate, penetrate and colonize mesophyll tissue, but were unable to produce the asexual fructifications, which was particularly due to inappropriate differentiation during the late stage of this morphogenesis-related process.     

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.     

Sexual reproduction facilitates the adaptation of parasites to antagonistic host environments: Evidence from empirical study in the wheat-Mycosphaerella graminicola system

Most eukaryotes use sexual reproduction to transmit genetic information from generation to generation despite the advantages offered by asexual reproduction. One theory to explain the origin and maintenance of sexual reproduction hypothesises that sexual recombination generates genetic variation that allows faster adaptation to fluctuating and/or stressful environments. We used a combination of ecological, molecular genetic, statistical and experimental evolution approaches to test this hypothesis in an agricultural plant-pathogen system. We inoculated wheat hosts with 10 strains of the fungal pathogen Mycosphaerella graminicola in a field experiment and estimated the contributions of sexual reproduction, asexual reproduction and immigration to the genetic composition of fungal populations sampled from moderately resistant and susceptible hosts through the course of an epidemic cycle. We found that a significant proportion of the M. graminicola population in the late phase of the epidemic originated from sexual reproduction among isolates that had been introduced into the field plots at the beginning of the epidemic. Recombinants were recovered at a higher frequency on the moderately resistant plant host Madsen than on the susceptible host Stephens. By the end of the growing season, we estimated that approximately 13% of the strains sampled from the resistant host were recombinants, compared with 9% in the samples collected from the susceptible host. We also found that pathogen strains originating from the resistant cultivar displayed higher levels of fitness, virulence and fungicide tolerance than those originating from the susceptible cultivar. Our results provide empirical support for the hypothesis that sexual reproduction facilitates the evolution of parasites to overcome host resistance.     

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.     

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.     

Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters

Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.     

A new member of the NY-ESO-1 gene family is ubiquitously expressed in somatic tissues and evolutionarily conserved

Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.     

LEAF CAVITY SIZE DIFFERENTIATION AND WATER RELATIONS IN CAREX ELEOCHARIS

Carex eleocharis leaves contain large intercellular cavities that traverse the length of the leaf above rows of stomata and are roughly constant in size throughout a leaf. Semithin sections (1–2 μm) demonstrate that the substomatal chambers are directly continuous with the intercellular cavities. Leaves of plants inhabiting moist swale regions of the shortgrass steppe, in northeastern Colorado, were found to have larger cavities as compared to leaves of plants inhabiting dry hilltops. Plants collected from a common hilltop site were grown in a controlled environment chamber, and, by manipulating the watering schedule, we obtained water potentials similar to those in the field. Leaves of “well-watered” plants were found to have larger intercellular cavities as compared to “water-stressed” plants. Leaf mesophyll cell sizes did not differ significantly between “well-watered” and “water-stressed” plants, suggesting that cavity size differences are not the result of developmental differences. Leaf cavities were shown to contain gases and to occur along the leaf length above rows of stomata. Additionally, the cavities in unstressed plants were continuous with substomal chambers. It is proposed that the reduction of cavity size is a mechanism to reduce water loss from the leaves during periods of plant water stress.     

Gene encoding a c-type cyclin in Mycosphaerella graminicola is involved in aerial mycelium formation, filamentous growth, hyphal swelling, melanin biosynthesis, stress response, and pathogenicity

Mycosphaerella graminicola is an important wheat pathogen causing Septoria tritici blotch. To date, an efficient strategy to control M. graminicola has not been developed. More significantly, we have a limited understanding of the molecular mechanisms of M. graminicola pathogenicity. In this study, we attempted to characterize an MCC1-encoding c-type cyclin, a gene homologous to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out mutants were generated via Agrobacterium tumefaciens-mediated transformation. All of the MCC1 mutants showed consistent multiple phenotypes. Significant reductions in radial growth on potato dextrose agar (PDA) were observed in all of the MCC1 mutants. In addition, MCC1 gene-deletion mutants produced less aerial mycelium on PDA, showed delayed filamentous growth, had unusual hyphal swellings, produced more melanin, showed an increase in their stress tolerance response, and were reduced significantly in pathogenicity. These results indicate that the MCC1 gene is involved in multiple signaling pathways, including those involved in pathogenicity in M. graminicola.     

Ornithine decarboxylase of Stagonospora (Septoria) nodorum is required for virulence toward wheat

A knockout strain of Stagonospora (Septoria) nodorum lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the S. nodorum ODC gene was isolated by polymerase chain reaction using degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the S. nodorum ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Sporulation of the transformants was reduced suggesting abberant regulation of the S. nodorum gene in A. nidulans. Transformation-mediated gene replacement was used to create strains which were auxotrophic for putrescine and lack ODC coding sequences. Pathogenicity studies on these mutants showed that they are greatly reduced in virulence compared with non-disrupted transformants. This confirms that the strains carrying an ODC disruption cannot obtain sufficient polyamines from the host plant for normal growth and, thus, that fungal ODC may be a suitable target for chemical intervention.     

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.     

长江流域禾谷镰孢菌群部分菌株系统发育学、产毒素化学型及致病力研究

从采集自长江流域引起小麦赤霉病的禾谷镰孢菌群(Fusarium graminearum clade)菌株中选取了31株,扩增并测定了这些菌株的EF-1α(translation elongation factor)、PHO(phosphate permease)基因序列,利用相关软件进行了系统发育分析。对这些菌株的产毒素化学型进行了分子检测。同时,用两个小麦品种(扬麦158和安农8455)测定了菌株的致病力。系统发育分析表明绝大多数菌株与F.asiaticum聚为一枝,只有一个菌株11027与F.graminearum聚类。30株F.asiaticum中有24株产脱氧雪腐镰孢菌烯醇(deoxynivalenol,DON)和3-乙酰脱氧雪腐镰孢菌烯醇(3-acetyldeoxynivalenol,3-AcDON),另外6株产雪腐镰孢菌烯醇(Nivalenol,NIV)。一株F.graminearum菌株11027产脱氧雪腐镰孢菌烯醇(DON)和15-乙酰脱氧雪腐镰孢菌烯醇(15-acetyldeoxynivalenol,15-AcDON)。在扬麦158上,菌株间的致病力分化较为明显,产NIV毒素的菌株致病力普遍较弱,强致病力的菌株都产3-AcDON毒素。结果表明在我国长江流域,产3-AcDON毒素的F.asiaticum是引起小麦赤霉病的优势种群,中抗赤霉病的小麦品种扬麦158可以有效评价菌株的致病力强弱。     

Ornithine decarboxylase knockout in Tapesia yallundae abolishes infection plaque formation in vitro but does not reduce virulence toward wheat.

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.     

Correlation of cytological and biochemical parameters with resistance and tolerance to Mycosphaerella graminicola in wheat

This study investigated the infection process of Mycosphaerella graminicola and enzyme activities related to reactive oxygen species (ROS) or oxylipin biosynthesis in four French wheat cultivars with variable resistance to M.?graminicola infection. At field level, cultivars Caphorn, Maxyl and Gen11 were susceptible, whereas Capnor showed high levels of quantitative resistance. Moreover, Capnor and Gen11 were tolerant, i.e., their yield was less affected by infection compared to non-tolerant Maxyl and Caphorn. These four cultivars were inoculated under laboratory conditions with the M.?graminicola IPO323 reference strain. Cytological and biochemical responses were studied on collected first plantlet leaves and several features discriminated between cultivars. However, resistance and tolerance had no impact on the fungal infection process. Levels of lipoxygenase (LOX), peroxidase (PO) and glutathione-S-transferase (GST) activities were also compared with regard to cultivar resistance or tolerance to M.?graminicola. LOX, PO and GST activities did not discriminate resistance and tolerance profiles, although a low level of PO in inoculated and non-inoculated plants could be associated with tolerance. In addition, cell necrosis correlated positively with LOX in non-tolerant cultivars, while mycelia surrounding stomata were positively correlated with PO in the resistant cultivar. GST activity presented correlations between cytological and biochemical parameters only for susceptible cultivars. Stomatal and direct penetration were positively correlated with GST activity in the susceptible non-tolerant cultivars, while these correlations were negative in the tolerant cultivar. When combining cytological and biochemical observations with resistance and tolerance profiles, for each cultivar and at each time point, cultivars could be classified in tight accordance with their previous field characterisation. Moreover, tolerance allowed us to distinguish susceptible cultivars when both biochemical and cytological parameters were considered together.     

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.