[show abstract][hide abstract] ABSTRACT: The cyclic AMP (cAMP) signaling and mitogen-activated protein (MAP) kinase pathways are the most important signal transduction pathways in eukaryotes. In many plant pathogenic fungi they play pivotal roles in virulence and development. Identification and understanding the role of signal transduction pathways in regulation of cellular responses require robust biochemical techniques. Determination of both the phosphorylation status of MAPKs and the intracellular levels of cAMP is required to unravel the function of these pathways during adaptation of fungi to environmental stress conditions or when particular fungal genes are disrupted or silenced. Here we describe protocols to determine the phosphorylation status of three different MAPKs including Fus3, Slt2 and Hog1 as well as a protocol to measure the intracellular levels of cAMP levels. These protocols can be adapted for a wide range of fungi.
Methods in molecular biology (Clifton, N.J.) 01/2012; 835:571-83.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.
[show abstract][hide abstract] ABSTRACT: Mycosphaerella graminicola is the causal agent of septoria tritici blotch, currently the most important disease of wheat in Europe. Despite the recent identification of 15 resistance genes and their potential application in plant breeding, disease control is currently achieved mainly by fungicides. However, fungicide resistance development in natural M. graminicola populations frequently occurs and is a serious concern. Depending on the fungicides this may develop gradually, such as with resistance to azoles, or much more rapidly as was observed for strobilurin fungicides. In order to understand this rapid spread of resistance we have performed a range of crossing experiments that demonstrate that external stress factors hamper disease development but cannot prevent sexual development. As M. graminicola is a heterothallic bipolar pathogen, sexual development requires two mating partners ‐ carrying different mat alleles (mat1‐1 or mat 1‐2) ‐ that both produce female and male organs. We use an in planta crossing protocol that reliably enables the isolation of segregating/mapping populations. The first stress factor that we used was host resistance. Various crosses on a range of cereal hosts indicated that sex always takes place as long as one of the mating partners is virulent. Thus, even an avirulent isolate that does not establish a compatible interaction with the host plant is perfectly able to enter into the sexual process resulting in viable ascospores. As a consequence the genes of such an avirulent isolate are transmitted to subsequent generations. This is fundamentally different from many other host‐pathosystems where avirulent isolates ‐ and their genes ‐ are lost in subsequent generations. We used strobilurin fungicides as a second stress factor by crossing sensitive and resistant isolates under various strobilurin concentrations (3‐ 200%). Although strobilurins prevent disease development of sensitive isolates, and as a consequence minimize biomass, abundant sexual development occurred under all conditions, thus irrespective of the applied strobilurin concentration. Moreover, our results showed that the ‘stressed’ mating partner – the sensitive parent – acted as the preferred paternal partner. Thus, external stress factors on avirulent or sensitive isolates do not preclude the production of M. graminicola spermatia that effectuate viable ascospore production. The fact that the sensitive isolates are preferred paternal donors – and consequently the resistant strains are maternal donors – in the sexual process resulted in major shifts in strobilurin resistance in the segregating populations as the target site for strobilurins is on the mitochondrial genome. A minimal dose of 6% strobilurin already rendered entire populations resistant to these compounds. This explains the rapid pan‐European spread of strobilurin resistance in M. graminicola, likely in temporally and geographically independent occasions, with no loss of nuclear genetic variation. The recently discovered genome plasticity of M. graminicola may contribute to its ability to overcome environmentally adverse conditions.
[show abstract][hide abstract] ABSTRACT: We identified and functionally characterized genes encoding three Galpha proteins and one Gbeta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Galpha(i) and Galpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.
[show abstract][hide abstract] ABSTRACT: Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitotic division following the two meiotic divisions. The transient diploid phase allows for recombination among homologous chromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Because these chromosomes are not present universally in the genome of the organism they can be considered to be dispensable. To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated by crossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towards durum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT, 258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensable chromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size, and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely, with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes. Between 15-20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The two high-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing may require distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification of individual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that the chromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and, less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enabling this versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat fields.
PLoS ONE 02/2009; 4(6):e5863. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have performed a genome-wide analysis of the mimp family of miniature inverted-repeat transposable elements, taking advantage of the recent release of the F. oxysporum genome sequence. Using different approaches, we detected 103 mimp elements, corresponding to 75 nonredundant copies, half of which are located on a single small chromosome. Phylogenetic analysis identified at least six subfamilies, all remarkably homogeneous in size and sequence. Based on high sequence identity in the terminal inverted repeats (TIRs), mimp elements were connected to different impala members. To gain insights into the mechanisms at the origin and amplification of mimps, we studied the potential of impala to cross-mobilize different mimps, native but also created de novo by inserting a short DNA segment between two TIRs. Our results show that TIR sequences are the main requirement for mobilization but that additional parameters in the internal region are likely to influence transposition efficiency. Finally, we show that integration site preference of native versus newly transposed mimps greatly varies in the host genomes used in this study.
Journal of Molecular Evolution 12/2008; 67(6):631-42. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.
Fungal Genetics and Biology 10/2008; 45(12):1552-61. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transposon mutagenesis was applied to generate Mutants in Fusarium graminearum. The mimp1/impala system originally identified in F. oxysporum proved very promising for mutagenesis as the transposon and reinserted at high frequency in (the vicinity) of genes. A collection of mutants was screened for growth, for pathogenicity and for perithecia production. Several mutants blocked in one or more functions were obtained. The wild-type phenotype of one such mutant Could be restored by complementation with a non-disrupted copy of the gene. In addition reinsertions occurred on each of the four chromosomes of F. graminearum, making this system it powerful tool in the functional analyses of the > 10,000 genes predicted in the F graminearum genome.
Cereal Research Communications 36 (2008) Suppl. B. 01/2008;
[show abstract][hide abstract] ABSTRACT: The mimp1 element previously identified in the ascomycete fungus Fusarium oxysporum has hallmarks of miniature inverted-repeat transposable elements (MITEs): short size, terminal inverted repeats (TIRs), structural homogeneity, and a stable secondary structure. Since mimp1 has no coding capacity, its mobilization requires a transposase-encoding element. On the basis of the similarity of TIRs and target-site preference with the autonomous Tc1-like element impala, together with a correlated distribution of both elements among the Fusarium genus, we investigated the ability of mimp1 to jump upon expression of the impala transposase provided in trans. Under these conditions, we present evidence that mimp1 transposes by a cut-and-paste mechanism into TA dinucleotides, which are duplicated upon insertion. Our results also show that mimp1 reinserts very frequently in genic regions for at least one-third of the cases. We also show that the mimp1/impala double-component system is fully functional in the heterologous species F. graminearum, allowing the development of a highly efficient tool for gene tagging in filamentous fungi.
[show abstract][hide abstract] ABSTRACT: Transposon mutagenesis was applied to generate mutants in Fusarium graminearum. The mimp1/impala system originally identified in F. oxysporum proved very promising for mutagenesis as the transposon and reinserted at high frequency in (the vicinity) of genes. A collection of mutants was screened for growth, for pathogenicity and for perithecia production.Several mutants blocked in one or more functions were obtained. The wild-type phenotype of one such mutant could be restored by complementation with a non-disrupted copy of the gene. In addition reinsertions occurred on each of the four chromosomes of F. graminearum, making this system a powerful tool in the functional analyses of the > 10,000 genes predicted in the F. graminearum genome