Current Genetics

, Volume 43, Issue 2, pp 87–95 | Cite as

Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase gpmk1

  • Nicole J. Jenczmionka
  • Frank J. Maier
  • Anke P. Lösch
  • Wilhelm Schäfer
Research Article

Abstract.

To date, only very little is known about the molecular infection mechanisms of the head-blight pathogen of wheat, Fusarium graminearum (teleomorph Gibberella zeae). Here, we report on the isolation and characterization of the Fus3/Pmk1 mitogen-activated protein kinase homologue Gpmk1 from F. graminearum. Disruption of the gpmk1 gene in F. graminearum results in mutants that are reduced in conidial production, are sexually sterile and are fully apathogenic. This leads to the conclusion that gpmk1 is responsible for signal transduction processes taking place during the most important developmental stages in the life cycle of this fungal pathogen. Thus, Δgpmk1 mutants are a useful tool to find other important genes involved in plant-infection mechanisms. Previously, only the trichothecene biosynthesis pathway was identified as a virulence factor in F. graminearum. Hence, Gpmk1 is now the second pathogenicity trait to be known in this important plant pathogen.

Keywords.

MAP kinase Pathogenicity Mating Conidiation 

Introduction

Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch], a homothallic ascomycete, is the main causal agent of the head-blight disease of wheat (Triticum aestivum L.), leading to major crop-yield losses worldwide (Dubin et al. 1997). The infection is initiated by ascospores or macroconidia in wheat florets during the short period (10–20 days) from anthesis through the soft stage of kernel development (Schroeder and Christensen 1963). Conidia germinate and penetrate anthers, followed by penetration of the ovary and the floral bracts, including the glume, the palea and the lemma (Adams 1921). The infected florets become necrotic and are bleached. Underdeveloped kernels lead to a reduction in grain yield (McMullen et al. 1997). Furthermore, grain quality is reduced by the accumulation of trichothecene mycotoxins, such as deoxynivalenol, in the infected tissues (Sutton 1982). F. graminearum is able to produce perithecia carrying heterozygous (outcrossed) or homozygous (selfed) ascocarps (Bowden and Leslie 1999). Outcrossing enables the fungus to have a high genotypic diversity, which allows natural populations to adapt faster to selective pressures, such as cultivar resistance or fungicides (Bowden and Leslie 1999). Therefore, new ways of crop protection have continuously to be developed.

So far, only trichothecene biosynthesis has been identified as a pathogenicity trait of F. graminearum and verified by gene disruption in the trichothecene gene cluster, e.g. the tri5 gene. This gene encodes a trichodiene synthase that catalyzes the first reaction in the trichothecene pathway. F. graminearum mutants with a disrupted tri5 gene were shown to be unable to produce deoxynivalenol and to be drastically reduced in virulence (Bai et al. 2001; Desjardins et al. 1996; Proctor et al. 1995). Yet, a greater understanding of the molecular mechanisms involved in the infection process of F. graminearum is still needed. In general, the first step in plant infection is perception of the host by the fungal pathogen. This is mediated by fungal receptors that recognize physical and/or chemical signals from the plant, leading to responses needed for successful infection, e.g. the development of penetration structures and/or the secretion of cell wall-hydrolyzing enzymes. Plant perception and expression of proteins necessary for infection are linked by signal transduction pathways, which thus are likely to be a crucial factor in disease establishment. In fact, it has been demonstrated that mitogen-activated protein (MAP) kinase-mediated signal transduction pathways play important roles in the pathogenicity of many filamentous fungi (Lev et al. 1999; Mayorga and Gold 1999; Mey et al. 2002; Ruiz-Roldán et al. 2001; Takano et al. 2000; Xu and Hamer 1996; Xu et al. 1998; Zheng et al. 2000). Fungal MAP kinases have regulating functions, ranging across conidiation and conidia germination, appressorium and penetration peg formation, invasive hyphal growth and response to hyperosmotic stress and cell turgor (Xu 2000). Furthermore, MAP kinases have also been reported to play an important role during the regulation of mating (Müller et al. 1999). MAP kinases belong to the family of serine/threonine protein kinases. They are activated by a MAPKKK-MAPKK-MAP kinase cascade. This cascade is conserved in eukaryotic organisms and is involved both in the transduction of a variety of extracellular signals and in the regulation of growth and differentiation processes (Dickmann and Yarden 1999; Nishida and Gotoh 1993; Schaeffer and Weber 1999). This allows MAP kinases to give valuable information about the molecular mechanisms of fungal infection processes.

Using PCR with degenerate primers and thermal asymmetric interlaced PCR (TAIL-PCR), we cloned a MAP kinase, Gpmk1, from the grain pathogen F. graminearum. Targeted disruption of the gpmk1 gene in F. graminearum showed that this MAP kinase plays an essential role in mating and pathogenicity.

Materials and methods

Fungal strains and culture conditions

The fungal F. graminearum wild-type strain 8/1 was kindly provided by Thomas Miedaner of the Landessaatzuchtanstalt Hohenheim, Germany. The strain was stored at −70 °C as an aqueous conidia suspension. For extraction of DNA and RNA, wild-type or mutant mycelium was obtained from cultures grown in 50 ml complete medium (CM; Leach et al. 1982) for 3 days at 28 °C, at 175 rpm.

Conidiation was induced on SNA agar plates (Nirenberg 1981) at 18 °C under near-UV light and white light (both TL 40 W-33 RS; Philips, Eindhoven, The Netherlands) with a 12-h photoperiod. Conidia were washed from agar plates with sterile water, using a sterile glass rod.

Sexual reproduction of the fungal isolates was induced on carrot agar plates (Klittich and Leslie 1988). Small mycelial plugs from cultures of each parent strain were placed on opposite sides of the agar plate. The plates were incubated at 24 °C under a mixture of near-UV and white light with a 12-h photoperiod. After 7 days, the aerial mycelia were knocked down with 1 ml of sterile 2.5 % Tween 60 solution, using a sterile glass rod, while plates were rotated several times to spread the solution. After further 7 days of incubation under the conditions mentioned above, perithecia were found covering the plate (Bowden and Leslie 1999).

The growth rate of the wild-type strain and several independent mutants were determined on Czapek, a minimal medium lacking amino acid supplements (Raper and Thorn 1949), and on CM agar plates. The inoculated plates were incubated for 7 days in the dark at 28 °C. To determine the amount of biomass produced, the wild-type strain and the mutants were cultivated in liquid Czapek and CM for 3 days on a rotary shaker at 175 rpm, at 28 °C. The mycelium was then separated from the medium by filtration, lyophilized and weighed.

Isolation, cloning and sequencing of a MAP kinase from F. graminearum

Four degenerate primers were designed on the basis of conserved amino acid regions of various MAP kinases (Ruiz-Roldán et al. 2001): primer 1 (5′-CCAICKIGTNGCIACRTAYTC-3′) and primer 4 (5′-AYYTCIGGIGCICKRTAVYA-3′) were designed from the conserved region of domain VIII, primer 2 (5′-GCITAYGGIRTNGTNTG-3′) from the conserved region of domain I and primer 3 (5′-GTIGCNATRAARAARAT-3′) from the conserved region of domain II (Hanks and Quinn 1991). In a first PCR step, a DNA fragment was amplified from F. graminearum strain 8/1 genomic DNA using primers 2 and 4. The 50-µl PCR reaction contained 1.5 mM MgCl2, 0.2 mM each dNTP, 150 pmol of each primer, 100 ng of genomic DNA, 1 unit of Taq polymerase and 1× PCR buffer (Gibco BRL, Paisley, UK). The PCR was initiated by denaturation at 94 °C for 5 min, followed by 40 cycles of 94 °C for 1 min, 40 °C for 1 min and 72 °C for 1 min. The PCR cycling included a final extension step at 72 °C for 10 min and a cooling step to 4 °C. A second nested PCR followed, using primers 1 and 3 and 1 µl of a 1:50 dilution of the first PCR reaction as template. The PCR conditions were as described above. The amplified fragment was cloned into the pGEM-T-vector (Promega, Mannheim, Germany), resulting in the vector pGEM-T::gpmk1, and was sequenced. The 5′ and 3′ ends of the corresponding gene were obtained via TAIL-PCR, using unspecific arbitrary primers and PCR conditions modified after Lui and Whittier (1995). The gene-specific primers for the TAIL-PCR were designed on the basis of the pGEM-T::gpmk1 insert sequence (for the gpmk1 5′ direction: primary PCR with 5′-GAGTGCATCGCCTTGAGGGC-3′, secondary PCR with 5′-CTCCTGCAGTGTTTGTTAGCTCG-3′, tertiary PCR with 5′-GCAAAACAGTGGCGTTTCGTA-3′; for the gpmk1 3′ direction: primary PCR with 5′-GCCATAAGAAGATCACTCCTTTCGA-3′, secondary PCR with 5′-CAGAAGCCCCGAAGTTACGAG-3′, tertiary PCR with 5′-CACACAGGATCTTTCCGACGA-3′). The DNA-fragments thus amplified were purified by elution from an agarose gel, cloned into the pGEM-T-vector (Promega, Mannheim, Germany) and sequenced. In order to confirm the sequence, the complete gene was amplified in two fragments from genomic DNA with Expand (Roche Diagnostics, Mannheim, Germany), cloned into the pCR 2.1-TOPO vector (Invitrogen, Paisley, UK) and sequenced again. The specific primers used were designed on the basis of the fragments amplified via TAIL-PCR [for the gpmk1 5′ gene end: primer 48F (5′-ACCGACCGAACGACGGATTGC-3′) and primer 8R (5′-GCAGCGGATCGCGCAAGACCG-3′); for the gpmk1 3′ gene end: primer 7F (5′-GCGATACTTCAACCACGAGAAC-3′) and primer 38R (5′-GCATAGGGGCCTTGGTTAGGTC-3′)]. The PCR was initiated by denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 sec, 60 °C for 30 sec and 68 °C for 90 sec. The PCR included a final extension step at 68 °C for 5 min and a cooling step to 4 °C.

Nucleic acid manipulation

Genomic DNA was isolated from lyophilized and ground mycelium using the Puregene genomic isolation kit (Gentra systems, Minneapolis, USA). For Southern hybridization analysis, approximately 3 µg of genomic DNA were digested with appropriate enzymes and subjected to the hybridization protocols of Sambrook and colleagues (1989). A digoxigenin (DIG)-labeled probe was generated with the gene specific primers 7F and 8R, using the pGEM-T::gpmk1 vector as template. DIG-dUTPs (Boehringer Mannheim, Mannheim, Germany) were used for the PCR. The PCR was initiated by denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 sec, 60 °C for 30 sec and 72 °C for 30 sec. The PCR included a final extension step at 72 °C for 3 min and a cooling step to 4 °C.

PCR products were cloned according to Sambrook and colleagues (1989), either into the pGEM-T (Promega, Mannheim, Germany) vector, or using the 2.1 TOPO cloning kit (Invitrogen, Paisley, UK).

DNA sequencing was done by the dideoxynucleotide chain-termination method (Sanger et al. 1977) with the ABI Prism BigDye terminator cycle sequencing ready reaction (Applied Biosystems, Perkin Elmer, Weiterstadt, Germany).

Total RNA was isolated with the peqGOLD RNAPure isolation kit (peqLab Biotechnology, Erlangen, Germany) from mycelium grown in liquid CM and from uninfected and infected (24 h, 48 h, 7 days after inoculation) wheat spikelets, in accordance with the manufacturer's protocols. DNA digestion and first-strand synthesis were accomplished with the DNase I kit (Gibco BRL, Paisley, UK) and the Super Script II kit (Gibco BRL, Paisley, UK). All kits were used according to the manufacturer's instructions.

Transcription analysis

The cDNA from the complete gpmk1 gene was amplified by RT-PCR from the F. graminearum wild-type strain 8/1 RNA in two fragments, using Expand (Roche Diagnostics, Mannheim, Germany) and the following gene-specific primers: for the 5′ gene end: primer 49F (5′-TTTTCGGTCGCACGCTCTCCG-3′) and primer 8R; for the 3′ gene end: primer 7F and primer 53R (5′-CAGGTCGTCTTTTTACAGAGGC-3′). The primer 53R binds to the cDNA strand approximately 29 bp after the stop codon. The PCR was initiated by denaturation at 94 °C for 3 min followed by 30 cycles of 94 °C for 45 sec, 60 °C for 30 sec and 68 °C for 90 sec. The PCR included a final extension step at 68 °C for 5 min and a cooling step to 4 °C. In order to confirm their identity and to locate the position of the introns, the amplified fragments were cloned into the PCR 2.1-TOPO vector (Invitrogen, Paisley, UK) and sequenced. Transcript analysis of the gpmk1 mutant strains were also carried out by various RT-PCRs, using two gene-specific primers: 46F (5′-GATATCCAAGATGTGGTCGGCG-3′) and 47R (5′-AATCCTCCATGGTGGGTGTGCC-3′) and two primers binding to the insert flanking regions of the pAN7-1 vector backbone: panehe5′SM (5′-ACTCGACCTGCAGGCATGCAAGC-3′) and panehe3′SM (5′-TGTCGGGGCTGGCTTAACTATG-3′). The PCR was initiated by denaturation at 94 °C for 5 min followed by 30 cycles of 94 °C for 45 sec, 60 °C for 30 sec and 72 °C for 60 sec. The PCR included a final extension step at 72 °C for 5 min and a cooling step to 4 °C. The DNA sequence between the specific primers contains introns, so that a possible DNA contamination would be visible through the sizes of the amplified products. Wild-type strain cDNA was used as control. RT-PCR was also done with RNA isolated from uninfected and infected (24 h, 48 h, 7 days after inoculation) wheat spikelets using the gpmk1-specific primers 7F and 8R. Control PCRs were done with RNA and genomic DNA from F. graminearum grown on CM.

Transformation-mediated gene disruption

For the transformation of F. graminearum a vector was needed that could be linearized in the center of the MAP kinase-specific sequence. Therefore, a unique restriction site had to be generated in the center of the MAP kinase-specific sequence. This was accomplished by inverse PCR (Ochman et al. 1988). An exchange of two base pairs in the gpmk1 gene led to the generation of a PauI restriction site. In addition, two bases were deleted to the right and left of the PauI restriction site, to accomplish a frame-shift during translation of the gene. Primers 11 (5′-GTGCATCCGCGCGCAGGATTTTCCGACGACCACTGCCAG-3′) and 12 (5′-GAAAATCCTGCGCGCGGATGCACGGTGCATATCCGTCTC-3′) were used. The pGEM-T::gpmk1 vector was used as template for the inverse PCR. The PCR was initiated by denaturation at 94 °C for 4 min, followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 75 °C for 3,5 min. The PCR included a final extension step at 74 °C for 10 min and a cooling step to 4 °C. The use of the proofreading polymerase DeepVent (New England Biolabs, Frankfurt am Main, Germany) led to a blunt-ended PCR product that was ligated with T4-ligase (Fermentas, St. Leon-Rot, Germany) and cloned. The specific MAP kinase insert was cleaved from the pGEM-T vector by digestion with restriction enzyme PvuII and cloned into the fungal transformation vector, pAN7-1 (Punt et al. 1987), which contains the hygromycin-resistance gene, hph, as a selective marker. The resulting plasmid, pKOgpmk1, was linearized at the unique PauI restriction site, which is located in the MAP kinase insert. This linearized construct was used to transform protoplasts of F. graminearum wild-type strain 8/1. Protoplast formation and transformation were performed according to Royer et al. (1995), with the only difference that a linearized transformation vector was used. The resulting mutants were cultivated on CM plates containing 100 µg hygromycin B/ml and were single-conidiated. The single-conidiated isolates were used for further tests. A Southern hybridization was done to test for mutants that had inserted the transformation vector by homologous recombination.

Pathogenicity tests

The wheat cultivar Nandu (Lochow-Petkus, Bergen-Wohlde, Germany) was used for all virulence assays. Plants were grown in 11-cm pots at 20 °C with a 16-h photoperiod (8,000 lx) and 70% relative humidity. Wheat spikes at anthesis were point-inoculated with the F. graminearum wild-type strain and all independent mutants by placing a droplet (10 µl) of conidia suspension (5×104 conidia/ml) or 10 µl of water within the palea and lemma of two basal florets of one spikelet in the middle of the wheat spike tested (modified after Pritsch et al. 2001). To obtain a moist surrounding for the infection, the plants were sprayed with water before inoculation with the fungus. The inoculated spikes were enclosed in small plastic bags during the first 3 days to ensure a high humidity for infection and to prevent cross-contamination by different F. graminearum isolates. The inoculated plants were incubated in a growth chamber at 21 °C with a 16-h photoperiod (8,000 lx). The plant assays were evaluated 3 weeks after inoculation with the fungus.

Results

Isolation and characterization of a MAP kinase gene from F. graminearum

A PCR strategy was used to amplify a fragment of a putative MAP kinase from F. graminearum genomic DNA. In two PCR rounds with four degenerate primers, designed from conserved gene regions of various MAP kinases, a PCR product of 490 bp was amplified. Sequencing of this PCR product revealed significant similarities to several fungal MAP kinases. The sequence was then used to design gene-specific primers for a TAIL-PCR. The DNA fragments amplified in three nested PCR rounds using various unspecific arbitrary primers and three specific primers were purified, cloned and sequenced. Alignment of the amplified TAIL-PCR-sequences from the 5′ and 3′ direction made it possible to find the complete gene sequence with flanking regions. Comparisons of the genomic DNA sequence with the cDNA sequence revealed the gpmk1 gene to have an open reading frame of 1,065 bp, encoding a 355-amino acid protein (Fig. 1). Three introns of 60 bp, 57 bp and 64 bp were found. Comparison of the Gpmk1 amino acid sequence with database entries revealed high similarities to other fungal MAP kinases, such as Fmk1 of F. oxysporum (983% identity; Di Pietro et al. 2001), Pmk1 of Magnaporthe grisea (97.2% identity: Xu and Hamer 1996), Cmk1 of Colletotrichum langenarium (96.9% identity; Takano et al. 2000), Cpmk1 of Claviceps purpurea (95.2% identity; Mey et al. 2002), Ptk1 of Pyrenophora teres (92.4% identity; Ruiz-Roldán et al. 2001) and Chk1 of Cochliobolus heterostrophus (91% identity; Lev et al. 1999). In the deduced amino acid sequence, the TEY sequence (a site for threonine-tyrosine phosphorylation required for kinase activation) was found (Kültz 1998). Furthermore, the Gpmk1 protein sequence contained all 11 conserved protein kinase domains (Hanks and Quinn 1991). Southern blot analysis of genomic DNA, digested with several restriction enzymes, indicated that this MAP kinase gene is present as single copy in the genome of F. graminearum (data not shown). A 355-bp gpmk1 gene fragment was used as a probe. Transcription analysis of gpmk1 from F. graminearum wild type grown in CM and from infected and uninfected wheat spikelets indicated the gene to be constitutively expressed. In wheat infection, the transcripts were first detectable 48 h after inoculation, when the fungus had grown far enough to be detected by PCR (Fig. 2). Recently, the gpmk1 gene has also been sequenced by M. Urban and K. Hammond-Kosack from the Cereal Technology Group from Monsanto, UK, and can be found under the accession number AF448230.
Fig. 1.

Alignment of the predicted amino acid sequence encoded by the gpmk1 gene to several MAP kinases. Alignment was performed with the CLUSTAL W 1.8 program. Identical amino acids are indicated as white letters on a black background. Similar residues are shown on a gray background. Gaps introduced for the alignment are indicated by hyphens. Protein kinase domains are indicated by Roman numerals (Hanks and Quinn 1991). The TEY sequence required for kinase activation is marked by asterisks (Kültz 1998). The GenBank accession numbers for Fusarium graminearum Gpmk1, F. oxysporum Fmk1, Magnaporthe grisea Pmk1, Colletotrichum langenarium Cmk1, Claviceps purpurea Cpmk1, Pyrenophora teres Ptk1 and Cochliobolus heterostrophus Chk1 are AF448230, AF286533, U70134, AJ318517, AF174649, AF272831 and AF178977, respectively

Fig. 2.

Expression of gpmk1 during plant infection and in vitro, as shown via reverse transcription-polymerase chain reaction (RT-PCR). First-strand cDNA generated from total RNA isolated from uninfected wheat spikelets (lane 1), infected wheat spikelets at 24 h (lane 2), 48 h (lane 3) and 7 days (lane 4) and mycelium grown on complete medium (lane 5) was used as template for PCR with the gene-specific primers, F7 and R8. Lane M pUC 19 DNA/Mps1 marker. Lane C PCR with genomic DNA as template performed as control

Disruption of the gpmk1 MAP kinase in F. graminearum

A disruption of the gpmk1 gene was carried out to determine the function of this MAP kinase in the life cycle of F. graminearum. The disruption vector pKOgpmk1 was constructed from the pAN7-1 vector (Punt et al. 1987) and contained a hygromycin B resistance gene cassette as a selective marker and an internal fragment of the gpmk1 gene (Fig. 3A). The transformation vector was linearized and introduced into the F. graminearum wild-type strain 8/1 by a single crossover at the homologous locus. The transformants were selected on medium containing hygromycin B. A Southern blot analysis with the internal MAP kinase fragment as probe was done to identify the number of homologous integrations (Fig. 3B). From several transformations, a total of five mutants carrying a disrupted gpmk1 gene were further analyzed and showed the same phenotype in subsequent experiments.
Fig. 3A, B.

Disruption of the F. graminearum gpmk1 gene. A Disruption vector pKOgpmk1 and the gpmk1 locus. The vector was constructed by cloning an internal 490-bp fragment of the gpmk1 gene (gray-striped bar) into the pAN7-1 vector (Punt et al. 1987), next to the hygromycin B resistance gene hph (black bar). The vector was linearized at the unique PauI restriction site, introduced into the gpmk1 internal fragment via inverse PCR (Ochman et al. 1988). The gpmk1 gene was interrupted by homologous recombination of the vector through a single crossover. White lined bars show the homologous regions in the wild-type gpmk1 locus. Methionine and stop codons are indicated by inverted black triangles. B Southern blot analysis of genomic DNA from the F. graminearum wild-type strain (WT), the gpmk1-disruption mutants (lanes 1–5) and one ectopic integration transformant (E). DNA was digested with EcoRV. The blot was probed with the 355-bp internal fragment of the gpmk1 gene contained in the transformation vector, pKOgpmk1. gpmk1-disruption mutants lack the wild-type hybridization signal of 1 kb. Instead, the signal has shifted in size in the transformation vector, to 7,752 bp. The ectopic integration transformant shows the wild-type hybridization signal and a second signal, indicating a random integration of the transformation vector in the genome

Transcription analysis of the Δgpmk1 mutant strains were carried out by RT-PCR with Δgpmk1 mutant cDNA, using a gene specific primer and a corresponding pAN7-1 primer. A 711-bp PCR product from the 5′ gene end to the inserted vector and a 1,019-bp PCR product starting from the pAN7-1 vector to the 3′ gene end were amplified (data not shown). This indicates that the transcription of the MAP kinases is interrupted by the inserted transformation vector. The 856-bp fragment for genomic DNA and the 675-bp fragment for cDNA of the gpmk1 wild-type gene region, amplified by two specific primers flanking the inserted vector, could not be amplified under the applied conditions. This also reveals the gene to be interrupted. All PCR products from cDNA showed the spliced fragment sizes, which excludes any contamination of the cDNA with genomic DNA.

gpmk1-deficient mutants are unable to produce aerial hyphae on minimal media

Growth assays were carried out with the F. graminearum wild-type strain and all five independent Δgpmk1 mutants on solid complete (CM) and minimal (Czapek) media. On CM plates, the mutants showed a comparable growth rate, culture color and morphology to the wild-type strain (data not shown), as the wild-type strain grows with very little aerial mycelia on CM plates. However, on Czapek plates, the colony morphology of the Δgpmk1 mutants differed drastically from the wild-type strain. F. graminearum wild type characteristically grows with thick, white aerial mycelia on Czapek plates. In comparison, the gpmk1-deficient mutants are drastically reduced in forming aerial hyphae (Fig. 4). The mutants grow under the surface of the solid medium with few long hyphae. These are multiple-branched with short hyphae that produce conidiophores carrying conidia. Neither autolysis of the central part of the colony nor sensitivity towards high osmolarity could be observed with the gpmk1-deficient mutants (data not shown). Growth assays in liquid CM and Czapek revealed the gpmk1-deficient mutants to have approximately the same biomass production as the wild-type strain in both media tested (data not shown).
Fig. 4.

Colony morphology during growth on Czapek medium, showing the F. graminearum wild-type strain (a) and a mutant carrying an interrupted copy of the gpmk1 gene (b). The pictures were taken at 7 days post-inoculation

The gpmk1-deficient mutants from F. graminearum are reduced in conidiation

Initially, the amount of produced conidia was determined from the F. graminearum wild-type strain and all Δgpmk1 mutants inoculated plainly on SNA plates. Microscopy of the culture plates revealed that the Δgpmk1 mutants produced conidia under the surface of the solid SNA medium and there were only very few conidia on the surface. In addition, the conidia were not formed into typical large, round bundles on the conidiophore, which were distinctly distributed over the SNA plate. Only three to four conidia were produced per conidiophore, and these were found under the surface throughout the whole plate. In order to include the conidia from the Δgpmk1 mutants produced under the surface of the solid medium, the fungus was grown on SNA covered with cellophane foil as a physical barrier. Under these conditions, conidiation increased 10- to 20-fold. Nevertheless, the Δgpmk1 mutants showed a significant reduction in conidial production, compared with the wild-type strain: 8-fold on SNA without cellophane and 4-fold on SNA with cellophane (Fig. 5). The ectopic integration transformand exhibited a conidial production comparable with the wild-type strain. The viability of the conidia produced by the Δgpmk1 mutants was 90–100 %, which is comparable with the viability of the conidia from the wild-type strain.
Fig. 5A–C.

Conidial production on SNA medium (Nirenberg 1981) without (gray bars) and with (white bars) cellophane foil. A F. graminearum wild-type strain, B gpmk1-disruption mutants, C one ectopic integration transformand. Conidia were counted at 16 days post-inoculation (n=4). The values given for the gpmk1-deletion mutants are the average of 5 independent mutants

Gpmk1-deficient mutants are sexually sterile

The fertility of the Gpmk1-deficient mutants was determined by comparing the amount of perithecia produced by the mutants on carrot agar medium with the amount produced by the F. graminearum wild-type strain. All five independent disruption mutants were selfed three times. In all experiments, the Δgpmk1 mutants were unable to form perithecia. The wild-type strain produced an average of 15 perithecia/plate. The ectopic integration transformand produced 20 perithecia/plate (Fig. 6).
Fig. 6.

Perithecial development on carrot agar in F. graminearum wild-type (a) vs gpmk1 mutants (b). Perithecia can be seen as black structures on the surface of the plate inoculated with the wild-type strain. The black arrows mark some of the perithecia that can be seen. The pictures were taken at 15 days post-inoculation

Gpmk1 is essential for pathogenicity

In order to determine whether Gpmk1 participates in signal transduction pathways, which are essential for pathogenicity on wheat spikes, plant inoculation tests were performed. Wheat spikes were point-inoculated with conidia suspensions of five independent gpmk1-disruption mutants and the F. graminearum wild-type strain 8/1 as control and then monitored for 3 weeks. After 4 days, the first symptoms of wild-type strain infection could be seen by obvious growth of aerial mycelia on the point-inoculated spikelet. The wheat spikes browned rapidly after 6–7 days on the inoculated and surrounding spikelets. Bleaching of the infected spikes was obvious 2–3 weeks after inoculation. The spikes inoculated with the Δgpmk1 mutant conidial suspensions showed slight growth of aerial mycelia on the surface of the inoculated spikelet 4 days after inoculation. No further signs of infection, like browning or bleaching of the wheat spike, occurred throughout the 3 weeks of monitoring (Fig. 7A). At 3 weeks after plant inoculation, the wheat spikes were removed from the plant and were tested for kernel development (Fig. 7B). All spikelets of the spike inoculated with wild-type conidia contained small, brown, degenerated kernels typical for head-blight disease. Inoculation with the Δgpmk1 mutant had no effect on kernel development. The inoculated spike exhibited normal, fully developed kernels. Even the kernel of the inoculated spikelet developed normally. This seems to indicate that the conidia germinate in the spikelet, but do not infect the plant.
Fig. 7A, B.

Pathogenicity assay on wheat spikes. Wheat spikes were point-inoculated with the F. graminearum wild-type strain. A Infected wheat spikes at 21 days after inoculation, using the wild-type strain (a), gpmk1-disruption mutants (b) and water as a control (c). B Kernels isolated from wheat spikes infected with the F. graminearum wild-type strain (a), with one mutant strain carrying an interrupted copy of the gpmk1 gene (b), or inoculated with water as control (c)

Discussion

In this report, we describe the isolation and characterization of the MAP kinase gene gpmk1 from the fungal wheat pathogen F. graminearum, the causal agent of the head-blight disease of wheat. According to the amino acid sequence, Gpmk1 belongs to the yeast extracellular signal-regulated kinase 1 subgroup of MAP kinases (Kültz 1998). These are yeast- and fungus-specific extracellular signal-regulated kinases, whose main function is the transduction of extracellular signals. That enables them, like in the case of Gpmk1, to act as signal transmitters between the perception of the plant and the expression of genes needed for infection. Gpmk1 transcripts are present during both vegetative growth and the infection process on the plant, indicating that gpmk1 is expressed constitutively. Hence, the Gpmk1 MAP kinase from F. graminearum might be regulated in a posttranscriptional mode. This mode of regulation has already been proposed for the pathogenicity MAP kinase Ptk1 from P. teres (Ruiz-Roldán et al. 2001) and is widely described for other protein kinases (Hunter 1995).

On minimal medium, the gpmk1 deletion mutants show altered vegetative growth in comparison with the F. graminearum wild type. The Δgpmk1 mutants are incapable of forming aerial hyphae, but show no obvious reduction in growth rate and cell yield. This phenotype is also observed in Δchk1 mutants from C. heterostrophus and in Δmps1 mutants from M. grisea (Lev et al. 1999; Xu et al. 1998). In contrast to the Δchk1 and Δmps1 mutants, the F. graminearum MAP kinase mutants do not undergo autolysis in the central part of the colony. Therefore, drastic structural changes in the fungal cell wall do not seem to be the reason for the failure of the Δgpmk1 mutants to penetrate the plant tissue.

In the following, three possible regulatory effects of Gpmk1 from F. graminearum are discussed. In recent years, hydrophobins have been shown to play an important role during attachment of the fungus to the plant cuticle, infection court preparation and topological signaling (Schäfer 1994). It has been shown that hydrophobins are essential for the development of aerial hyphae, fruiting bodies (Wessel et al. 1991) and full virulence (Talbot et al. 1993). These phenotypes are also found in the gpmk1-deficient mutants of F. graminearum. Similar to M. grisea mutants deleted in the hydrophobin gene mpg1 (Talbot et al. 1993, 1996), the MAP kinase gpmk1-deficient mutants of F. graminearum exhibit a reduced conidial production in general and a reduced amount of conidia per conidiophore. The few conidia made by the Δgpmk1 mutants are found under the agar, again indicating that the mycelia exhibit a diminished capacity for growing in an aerial phase. Nevertheless, those conidia produced by the Δgpmk1 mutants are completely viable. Therefore, it seems likely that no major process of conidial production has been disturbed, as was described for the Δptk1 MAP kinase mutants from P. teres (Ruiz-Roldán et al. 2001). The P. teres mutants form abnormally elongated conidiophores with no conidia at their tips. All these phenotypes could indicate a possible defect of the F. graminearum Δgpmk1 mutants in the production or secretion of hydrophobins. However, a regulatory effect of Gpmk1 on hydrophobins still needs to be shown.

An inability of filamentous fungi to form aerial hyphae has also been shown to have a drastic effect on the formation of sexual fruiting bodies, e.g. the deletion of the Δchk1 MAP kinase from C. heterostrophus (Lev et al. 1999) or the mpg1 hydrophobin from M. grisea. Hence, the sexually sterile phenotype of the Δgpmk1 mutants from F. graminearum could also be explained by an obvious reduction of aerial mycelia, as exhibited by these mutants. A participation of the Gpmk1 MAP kinase in the production and secretion of mating pheromones could also be possible. Thus, disruption of the Gpmk1 pathway would make the fungus unable to sense the mating partner. As a result, no sexual reproduction processes would be initiated. This regulatory effect has been shown for the Kpp2 MAP kinase of Ustilago maydis (Müller et al. 1999). Wolf and Mirocha (1973) postulated zearalenone to play a role as sex pheromone in F. graminearum, helping the mating partners to find each other. Nevertheless, the importance of pheromones for the mating processes of F. graminearum and the regulatory role of Gpmk1 in this pathway still need to be examined in detail.

A deletion of the Gpmk1 MAP kinase drastically affects the invasive growth of F. graminearum on wheat spikes. The Δgpmk1 mutants are incapable of colonizing the plant tissue and are therefore completely apathogenic, as the mutants do not even infect the inoculated spikelet. To date, only one virulence factor, trichothecene production, has been identified in F. graminearum. Deletion of the tri5 gene leads to mutants that are not able to produce any trichothecenes and show a reduction in virulence (Proctor et al. 1995). The Δtri5 mutants are able to infect the inoculated spikelet, but cannot progress the infection over the complete wheat spike (Bai et al. 2001). This makes the Δgpmk1 mutants the first completely apathogenic F. graminearum mutants to be reported. A regulatory effect in plant infection processes has been shown for other Pmk1 homologous MAP kinases, with the difference that their inability to infect the host derives from a hindered appressorial production. This mode of penetration is not relevant for the non-appressoria-forming pathogen F. graminearum (Pritsch et al. 2000). These fungi rely on other penetration mechanisms, e.g. the enzymatic digestion of the plant cell wall. This makes necessary the secretion of hydrolyzing enzymes, like cutinases, cellulases, amylases and pectinases. For Claviceps purpurea, another highly organ-specific, non-appressoria-forming fungal pathogen, activation of cell wall-degrading enzymes by the Cpmk1 MAP kinase has been discussed. A similar apathogenic phenotype was found in mutants defective in polygalacturonase activity (Mey et al. 2002; Oeser et al. 2002). Furthermore, it was shown that an endopolygalacturonase gene is under the transcriptional control of the Kss1 MAP kinase pathway in Saccharomyces cerevisiae (Madhani et al 1999). Therefore, regulation of cell wall-degrading enzyme production via the MAP kinase pathway is also highly possible for F. graminearum. Experiments concerning the secretion of cell wall-degrading enzymes by the Δgpmk1 mutants are currently being conducted in our group.

All mentioned phenotypes were found in five independent gpmk1-disruption mutants and not in a mutant with an ectopicly integrated transformation vector. This demonstrates that the phenotypes are specifically due to the loss of the Gpmk1 function. The pleiotropic phenotype of the gpmk1-disruption mutants indicates that the F. graminearum pathogenicity MAP kinase is involved in several developmental pathways, each partially responsible for different extracellular signals. Comparison of the various MAP kinase deficient mutants reveals that different fungal plant pathogens seem to use conserved pathways to regulate diverse infection processes. Different signals are sensed and different effector proteins are activated. Therefore, each pathogen gives new insights to the regulation of plant infection and needs to be examined in detail. To date, not much is known about the signals necessary for the activation of MAP kinases taking part in fungal pathogenicity of F. graminearum and about the genes that are transcribed due to the activation of this pathway. Nevertheless, apathogenic signal transduction mutants are a powerful tool for studying processes essential for pathogenicity. Further characterization of the gpmk1-deletion mutants and expression pattern comparisons between the wild-type strain and the gpmk1-deletion mutants will help to solve the open questions. This will lead to a better understanding of the infection mechanisms of this important plant pathogen and could, in the long term, enable us to find new targets for F. graminearum-specific fungicides.

Notes

Acknowledgements.

We are grateful to K. Quester and M. Andermann for technical assistance and to B. Witt for critical reading of the manuscript. The F. graminearum strain 8/1 was generously provided by T. Miedaner of the Landessaatzuchtanstalt Hohenheim, Germany. This work was supported by the BASF Aktiengesellschaft, Crop Protection Research, Ludwigshafen, Germany.

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Nicole J. Jenczmionka
    • 1
  • Frank J. Maier
    • 1
  • Anke P. Lösch
    • 1
  • Wilhelm Schäfer
    • 1
  1. 1.Department of Applied Molecular Biology of Plants III (AMPIII)Institute for General Botany, Universität HamburgHamburgGermany

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