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Eukaryotic Cell, February 2007, p. 211-221, Vol. 6, No. 2
1535-9778/07/$08.00+0     doi:10.1128/EC.00153-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

BcSAK1, a Stress-Activated Mitogen-Activated Protein Kinase, Is Involved in Vegetative Differentiation and Pathogenicity in Botrytis cinerea{triangledown}

Nadja Segmüller, Ursula Ellendorf,{dagger} Bettina Tudzynski, and Paul Tudzynski*

Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, D-48149 Münster, Germany

Received 29 May 2006/ Accepted 12 December 2006


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ABSTRACT
 
The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H2O2 and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, {Delta}bcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.


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INTRODUCTION
 
Mitogen-activated protein kinase (MAPK) cascades play an important role in the ability of organisms to perceive changes in their environment and adjust their intracellular activities by controlling expression of genes involved in stress response, cell division, differentiation, cell survival, and apoptosis as a reaction to diverse extracellular triggers (3, 21, 22, 57). MAPKs that specifically transmit environmental stress signals are called stress-activated protein kinases (SAPKs). Members of this MAPK subfamily are the Saccharomyces cerevisiae Hog1, Schizosaccharomyces pombe Sty1, and mammalian p38 and JNK MAPKs. Whereas Hog1 is mainly activated by hyperosmotic stress (6, 48), fission yeast and mammalian SAPKs are responsible for response to multiple stress conditions such as hyperosmolarity, heat shock, UV light irradiation, and oxidative stress (11, 25, 29, 34, 51, 64). Correspondingly, S. cerevisiae hog1 mutants are only sensitive to high osmolarity, whereas sty1 mutants in S. pombe are also sensitive to heat shock and oxidative stress. Like Sty1 of S. pombe, the Hog1 homologue SakA of the filamentous ascomycete Aspergillus nidulans is activated in response to osmotic and oxidative stress, but the sakA null mutant is not sensitive to hyperosmotic stress. This indicates that in this filamentous fungus, the osmostress response is differently regulated. A possible explanation would be the existence of a second stress-activated MAP kinase in A. nidulans, MpkC (17); SakA is mainly involved in general stress (including oxidative stress), signal transduction, and repression of sexual development, and it is required for spore stress resistance and survival (26).

Hog1 homologues also have been studied in several pathogenic fungi, such as Magnaporthe grisea, Colletotrichum lagenarium, Cryphonectria parasitica, and Bipolaris oryzae for their roles in pathogenicity. In M. grisea, the Hog1 homologue Osm1 controls the accumulation of arabitol in response to hyperosmolarity in mycelia but not the accumulation of glycerol for generation of turgor in appressoria (14). Gene replacement mutants of osm1 show growth defects and abnormal hyphal morphology under hyperosmotic stress. However, glycerol accumulation and turgor generation in appressoria are not affected in osm1 mutants; they still form normal functional appressoria and are fully pathogenic. Also, mutants of C. lagenarium defective in the gene osc1 maintained full pathogenicity but were more sensitive to osmostress; in addition, they showed significant resistance to the fungicide fludioxonil (28). In C. parasitica, knockout of the hog1-homologous gene cpmk1 led to osmosensitivity and resulted in several, but not all, hypovirulence-associated changes, such as reduced pigmentation, conidiation, laccase production, cryparin expression, and reduced virulence on chestnut trees compared to the wild-type strain (41). The Hog1 homologue gene srm1 of B. oryzae also plays a role in resistance against osmotic, oxidative, and UV stressors; knockout mutants showed moderate resistance against dicarboximide and phenylpyrrol fungicides but maintained full virulence on rice leaves (35). In the mycoparasite Trichoderma harzianum the Hog1 homologue ThHog1 is primarily involved in osmotic stress response and seems to play a minor part in oxidative stress; deletion of the gene modified mycoparasite properties only for some of the host fungi (13). Thus, the role of this MAPK cascade in filamentous fungi is markedly divergent.

Botrytis cinerea is a ubiquitous plant pathogen causing gray mold disease in a broad variety of crop plants. The fungus is a necrotroph, i.e., it depends essentially on the ability to kill its host cells before colonizing the plant tissue. One important aspect of this special life style is that the fungus has to be resistant to the oxidative burst, one of the early plant defense reactions. Von Tiedemann (62) showed that B. cinerea induces a significant oxidative burst in all analyzed host tissues and that the virulence of the fungus correlates positively with the intensity of this oxidative burst. Govrin and Levine (20) presented evidence that B. cinerea may even need the hypersensitive response of its host plant to achieve full pathogenicity. Therefore, an efficient oxidative stress response system could be essential for this fungus, suggesting a role of the Hog pathway in pathogenicity of Botrytis. Another link to this signaling pathway comes from the observation that B. cinerea produces reactive oxygen species (ROS) in axenic culture and in planta: cytochemical analysis showed the presence of O2– in hyphal tips and H2O2 generation in and around the penetrated cell wall (55). There is further clear evidence for ROS and the involvement of other markers of oxidative stress in tissues attacked by B. cinerea (12, 37, 38, 39, 40). Two potential H2O2-generating enzymes, a superoxide dismutase (BcSOD1) and a glucose oxidase (BcGOD1), were analyzed by Rolke et al. (45). Whereas the GOD plays no essential role in pathogenicity (knockout mutants showed normal virulence), the SOD activity appears to be a virulence factor. This indicates that the H2O2 production via Cu-Zn-SOD from (perhaps) an internal O2– source has impact on virulence. The source for this O2– is unknown; a possible candidate would be an NADPH oxidase, an enzyme used deliberately by higher eukaryotic cells to produce reactive oxygen species. Lara-Ortiz et al. (30) identified an NADPH oxidase-encoding gene in A. nidulans (noxA). This gene encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes (1). In A. nidulans, noxA is regulated by the SAPK SakA (a Hog1 homologue), connecting stress MAPK signaling with the regulation of ROS production.

Here we present evidence that the Hog-homologous stress-activated MAPK BcSAK1 has, in contrast to most of the fungi tested so far, significant impact on pathogenesis, and that it is involved (but probably is not the only player) in the oxidative stress response. On the other hand, again in contrast to other fungi, BcSAK1 is essential for conidiogenesis and influences sclerotial formation; both phenomena (and the reduced pathogenicity) might be linked to a possible role in ROS homoeostasis.


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MATERIALS AND METHODS
 
Fungal strains. B. cinerea Pers.: Fr. (teleomorph Botryotinia fuckeliana [de Bary] Whetz) strain B05.10 (44) was used as a recipient strain for the transformation experiments and as a wild-type control.

Bacterial strains. For the propagation of lambda clones, strain LE 392 F– [hsdR574 (rk– mk+) supE4 lacY1 supE58 {Delta}(lacIZY)6 galK2 galT22 metB1 trpR55 {Delta}(mrr-hsdRMS-mcrBC)] (Stratagene) was used; subcloning was performed in strain TOP10F'– [(lacIq, Tu10, Tetr) merA {Delta}(mrr-hsdRMS-merBC) {Phi}80lac2{Delta}M15 {Delta}lacX74 recA1 {Delta}araD139 {Delta}(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG] (Invitrogen).

Media and culture conditions. B. cinerea strains were grown on complete medium (CM) (42) or potato dextrose agar (containing 10% bean leaves) at 20°C under near UV for conidiation. Testing of stress sensitivity was performed by inoculating strains on CM plates supplemented with H2O2 (Merck), menadione (Sigma), iprodione, fludioxonil (Sigma), and NaCl (Merck) as indicated. Single conidial isolates were obtained by spreading 100 µl of conidial suspension on Gamborg's B5 Medium (Duchefa Biochemie BV, Haarlem, The Netherlands) plates containing 70 µg ml–1 of nourseothricin. Conidia were germinated for 48 h, and single colonies were transferred individually to new plates containing nourseothricin. For DNA isolation, mycelium was grown for 2 to 3 days at 20°C on CM agar with a Cellophane overlay. For RNA and protein isolation, strains were cultivated in 300-ml Erlenmeyer flasks with 100 ml of a defined liquid medium (Czapek Dox iCD) containing 1 g liter–1 NaNO3 instead of 3 g liter–1 and 20 g liter–1 glucose instead of sucrose and were cultivated at pH 5.2. For target gene analysis and phosphorylation assays, media were supplemented with menadione, H2O2, iprodione, fludioxonil, and NaCl as indicated. Fungi were harvested after 2 days.

Standard molecular methods. Fungal genomic DNA was isolated as described by Cenis (8). Lambda DNA was isolated according to the standard method (46). Plasmid DNA was isolated using a plasmid DNA preparation kit (Genomed, Bad Oeynhausen, Germany).

Southern blot analysis. Blots were performed according to Sambrook et al. (46). Hybridization was carried out in 6x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 5x Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), and 50 mM phosphate buffer, pH 6.6, at 65°C for 16 to 20 h in the presence of a random-primed [{alpha}-32P]dCTP-labeled probe. Membranes were washed in 2x SSPE, 0.1% SDS at 65°C before being exposed to an autoradiographic film.

Northern analysis. RNA was isolated from mycelial samples using the RNAgents Total RNA Isolation System (Promega, Mannheim, Germany). Samples of 10 to 15 µg RNA were transferred onto Hybond-N+ filters after electrophoresis on a 1% agarose gel containing formaldehyde according to Sambrook et al. (46). Blot hybridizations were carried out in 0.6 M NaCl, 0.16 M Na2HPO4, 0.06 M EDTA, 1% N-lauroylsarcosine (Sigma), 10% dextran sulfate (Eppendorf AG, Hamburg, Germany), pH 6.5, as described for Southern blots.

Western blot analysis. Liquid cultures were filtered (filter type 595; Schleicher and Schuell) to separate mycelium from culture medium, and the former was lyophilized overnight. For disruption of mycelia, mycelia were ground, using a mortar and pestle, to a fine powder under liquid nitrogen. Buffer containing 50 mM K2HPO4 (pH 7.8) was added to the powder, and these suspensions were slightly shaken on ice for 30 min. After centrifugation at 14,000 rpm for 20 min at 4°C, supernatants of these suspensions were desalted via NAP25 columns (Amersham). Protein concentrations were determined by the method of Lowry et al. (31), and 50 µg was separated on a 10% polyacrylamide gel and blotted onto nitrocellulose membranes. For detection of BcSAK1, anti-Hog1p antibody from Santa Cruz Biotechnology was used (C-terminal anti-Hog1). Phosphorylation of SAK1 in B. cinerea was examined by using a dually phosphorylated p38 antibody (NEB). Antibody binding was visualized using 5-bromo-4-chloro-3-indolylphosphate (Fluka) and nitroblue tetrazolium after binding of an alkaline phosphatase-conjugated secondary antibody.

DNA sequencing. DNA sequencing was performed with automatic sequencer LI-COR 4200 (MWG Biotech, Munich, Germany) using the Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Amersham Pharmacia). For sequence analysis and construction of phylogenetic trees, the program DNA Star (Madison, WI) was used.

Construction of a replacement vector for bcsak1. By use of the primer pairs 5/6 and 7/8 (see Table 1), respectively, the 5'- and 3'-flanking regions of the gene were amplified. The flanking regions were cloned into vector pCR2.1. From this product the 5' and 3' fragments were excised with SacI/XbaI and HindIII/ClaI, respectively, and cloned into the nourseothicin resistance vector pNR1, as indicated in Fig. 2A. The complete replacement fragment was excised with SacI/ClaI and used to transform strain B05.10.


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  		TABLE 1. PCR primers


Figure 2
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  		FIG. 2. Generation of {Delta}bcsak1 mutants. A. Gene replacement strategy for bcsak1. The coding region is boxed; the orientation is indicated by an arrow. The bacterial part of pNR1 is indicated with a thick black line; the nourseothricin resistance cassette (nat1) is in gray. Primer binding sites are indicated by arrowheads (see Table 1). B. Southern blot analysis of transformants. Genomic DNA preparations of the putative deletion mutants ({Delta}bcsak-3, {Delta}bcsak-4, {Delta}bcsak-12, {Delta}bcsak-16, {Delta}bcsak-17, {Delta}bcsak-19, and {Delta}bcsak-27) and wild-type B05.10 (WT) were digested with BamHI, separated in an agarose gel, blotted on a nitrocellulose membrane, and hybridized to the 3' flank of bcsak1. M, molecular size marker.

Complementation of bcsak1 in {Delta}bcsak1-27. To confirm that the phenotype of the {Delta}bcsak1 mutants is due to the deletion of the bcsak1 gene in B. cinerea, the mutant {Delta}bcsak1-27 was complemented. Therefore, a 3.6-kb fragment was amplified by PCR using the primer pair 9/10 (see Table 1). The obtained fragment included the bcsak1 gene together with 1 kb of the bcsak1 promoter and terminator. The sequence of the PCR product was determined to ensure flawlessness of the sequence for complete functionality when transformed in the mutants. For construction of the complementation vector, the plasmid pOLiHP (45) carrying the Escherichia coli hygromycin phosphotransferase gene hph under control of the A. nidulans oliC promoter and trpC terminator was used as a basis vector. The fragment was cloned in pCR-BluntII-TOPO (Invitrogen), cut with HindIII, and cloned into the corresponding site of pOLiHP. By cutting with SmaI, the complementation vector was linearized prior to transformation.

Transformation of B. cinerea. Protocols for protoplast formation and transformation were adapted from ten Have et al. (53) as described by Schulze Gronover et al. (49).

Pathogenicity assays. Intact and prewounded primary leaves of Phaseolus vulgaris L. genotype N90598 [GenBank] (originating from J. D. Kellx, Michigan State University, East Lansing) were inoculated with agar plugs of B. cinerea strains for the standard pathogenicity test as described by Klimpel et al. (27). The infected plants were incubated in a plastic propagator box at 20°C under natural illumination. Disease symptoms were scored 24 and 48 h postinoculation (hpi).

Microscopic analyses. For scanning electron microscopic (SEM) analysis, infection of primary leaves of Phaseolus vulgaris L. was performed as described above. Lesions were excised from inoculated bean leaves 24 and 48 hpi. The samples were aldehyde fixed and dehydrated in an ethanol series as described by Giesbert et al. (19). For scanning electron microscopy, samples were critical-point dried (Emitech K850 Critical Point dryer), gold sputtered (Emitech vacuum sputter device K550x), and examined with a Hitachi S-3000N scanning electron microscope at 15 kV. Micrographs were directly electronically recorded.


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RESULTS
 
bcsak1 encodes a Hog-type stress-activated MAP kinase. An expressed sequence tag library from an axenic culture of B. cinerea strain ATCC 58025 (52) contained a cDNA clone with significant homology to Hog-type MAP kinases, which was used to probe a genomic EMBL3 library of B. cinerea strain SAS56. Hybridizing lambda clones were subcloned and sequenced. A genomic 6-kb KpnI fragment was shown to contain the coding region of a putative stress-activated MAPK gene (termed bcsak1). The bcsak1 sequence of 1,670 bp contains an open reading frame of 1,065 bp interrupted by eight introns; the presence of these introns was confirmed by reverse transcription-PCR analysis (data not shown). The deduced bcsak1 protein product (BcSAK1) consists of 354 amino acids, with an estimated molecular mass of 40.49 kDa and a pI of 5.47. Sequence comparison of BcSAK1 with other MAP kinases revealed the presence of all characteristic conserved subdomains, including the dual phosphorylation site TGY (at positions 171 to 173), a site for threonine and tyrosine phosphorylation required for kinase activation. The BcSAK1 sequence has significant similarity to MAP kinases of filamentous fungi of the Hog clade, such as MapIII (90% identity) from Blumeria graminis, CpMK1 (89% identity) from C. parasitica (41), Osm1 (88% identity) from M. grisea (14), and Os-2 (88% identity) from Neurospora crassa, as shown in Fig. 1.


Figure 1
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  		FIG. 1. Phylogenetic tree of MAP kinase sequences of fungi. Sequence alignment and tree construction were done with ClustalV and by use of MegAlign, a program of the Lasergene package (DNASTAR). GenBank accession numbers (in parentheses) were the following: Aspergillus fumigatus OSM1 (CAD28436); Blumeria graminis MAPI (AAG53654), MAPII (AAG53655), and MAPIII (AAL83917); Botrytis cinerea BMP1 (AAG23132) and BcSAK1 (AM236311); Claviceps purpurea CPMK1 (CAC47939) and CPMK2 (CAC87145); Magnaporthe grisea OSM1 (AAF09475); Neurospora crassa OS-2 (AAK83124); Schizosaccharomyces pombe STY1 (CAA91771); Saccharomyces cerevisiae Fus3 (AAA34613), HOG1 (AAA34680), and SLT2 (CAA41954); Aspergillus nidulans SakA (AAF97243); C. lagenarium MAPK (AAL50116); and C. parasitica CpMK1 (AAO27796).

Generation of {Delta}bcsak1 mutants. For a detailed functional analysis, bcsak1 deletion mutants were created using a replacement approach. To yield the replacement vector p{Delta}SAK1, the 5'- and 3'-flanking regions of the gene (453 and 828 bp, respectively) were amplified and cloned into the nourseothricin resistance vector pNR1 (Fig. 2A) (for details, see Materials and Methods). The replacement fragment was excised with SacI and ClaI and used to transform strain B05.10. Nourseothricin-resistant transformants were tested by PCR for homologous integration of the replacement fragment. The primer pair 1/2 (binding within the resistance cassette and outside the replacement fragment, respectively; see Fig. 2A) yielded a fragment of 1.3 kb only if the fragment had been integrated by double crossover at the homologous site. Of the 30 transformants obtained, 16 showed this diagnostic band. All these transformants were heterokaryons (as is normal in B. cinerea), since PCR with primer pair 3/4 yielded the fragment specific to the wild type (766 bp). Seven of the primary transformants were subjected to one round of single-spore isolation. Homokaryotic derivatives have been obtained from all seven primary transformants showing only the diagnostic fragment and lacking the wild-type fragment (data not shown). They were named {Delta}bcsak1-3, {Delta}bcsak1-4, {Delta}bcsak1-12, {Delta}bcsak1-16, {Delta}bcsak1-17, {Delta}bcsak1-19, and {Delta}bcsak1-27. The PCR data were confirmed by Southern blot analyses (Fig. 2B): BamHI-digested total DNA of the seven mutants and the recipient strain B05.10 (wild type) were blotted and hybridized with the labeled 3' flank (see Fig. 2A). Whereas the wild type showed a hybridizing band of about 10 kb, in the seven mutants this fragment was replaced by a 4.5-kb fragment as expected. Only transformant {Delta}bcsak1-4 seems to contain an additional ectopic integration.

BcSAK1 affects vegetative differentiation and tolerance against oxidative and osmotic stress. The {Delta}bcsak1 mutants showed a complete lack of conidiation on all media tested; microscopical analyses showed that they developed normal aerial hyphae but neither conidiophores nor mature conidia. In addition, BcSAK1 has an impact on sclerotia formation: {Delta}bcsak1 mutants developed sclerotia earlier and were more abundant than the wild-type strain (Fig. 3). Formation of microconidia is not impaired in the mutants. The growth rate of one mutant, {Delta}bcsak1-27, was exemplarily tested on CM media containing different stress-inducing components, like oxidants, salt, and fungicides; the data are summarized in Fig. 4. The response to hyperosmotic stress was determined based on growth in the presence of 0.8 M, 1 M, and 1.5 M NaCl. The mutant was significantly more osmosensitive than the wild type. Thus, after 4 days of incubation on CM medium containing 0.8 M NaCl, the colony diameters of the {Delta}bcsak1-27 mutant were about 28% of that of the wild-type strain. This growth defect becomes stronger with increasing amounts of NaCl. During incubation on CM medium containing 1.5 M NaCl, no growth is detectable for the mutant any more (Fig. 4), suggesting that BcSAK1 is involved in osmotic stress response. Additionally, the transformant was tested for sensitivity to oxidative stress based on the growth in the presence of 5 mM and 10 mM H2O2 as well as 250 µM and 500 µM menadione (generation of O2–). Growth was significantly reduced when the transformant was exposed to 5 mM and 10 mM H2O2. Compared to the wild type, the growth rate was about 45% during exposition to 5 mM H2O2 and 21% during exposition to 10 mM H2O2. On the other hand, sensitivity to menadione (at 250 and 500 µM) seemed to be unchanged. Obviously, deletion of bcsak1 does influence oxidative stress resistance caused by H2O2 but not by menadione.


Figure 3
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  		FIG. 3. Impact of BcSAK1 on sclerotia formation. Mutants and the wild type were incubated for 8 weeks on malt medium at 21°C in darkness. Arrows indicate sclerotia.


Figure 4
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  		FIG. 4. Growth rates of mutant {Delta}sak1-27 and wild-type strain B05.10 on CM media containing different stressors. The values are averages from at least six colonies.

Because of the impact of BcSAK1 homologues on fungicide resistance (especially against phenylpyrrols) in other filamentous fungi, like C. lagenarium (28), plate assays with iprodione (dicarboximide), dicloran (aromatic hydrocarbon), and fludioxonil (phenylpyrrol) were performed. While the transformant and the wild type showed no significant difference in resistance to dicloran (1 µg ml–1) and fludioxonil (0.5 and 1 µg ml–1; no growth of the wild type and the transformants was detectable), the transformants were significantly more resistant to iprodione (0.5 and 1 µg ml–1). Whereas the transformant was still able to grow on media containing 1 µg ml–1 iprodione, no growth was detectable for the wild type. These data indicate that BcSAK1 has no impact on resistance against phenylpyrrols and aromatic hydrocarbon fungicides but does against dicarboximides.

Expression of bcsak1 and activation of BcSAK1 in axenic culture and in planta. To investigate the role of BcSAK1 in osmotic, fungicide, and oxidative stress responses in more detail, expression of the gene under various stress conditions was studied (Fig. 5). Osmotic stress was generated with 0.8 M and 1 M NaCl, fungicide stress was generated with 5 and 10 µg ml–1 iprodione as well as with 10 and 50 µg ml–1 fludioxonil, and oxidative stress was generated with H2O2 (5 mM and 10 mM) and menadione (250 µM and 500 µM). In the wild-type B05.10, bcsak1 was upregulated during oxidative stress mediated by H2O2 (5 and 10 mM), fungicide stress, and osmotic stress mediated by NaCl, whereas expression of bcsak1 was not affected by oxidative stress mediated by menadione, which matches with the results of the plate assay (Fig. 4). These data indicate that bcsak1 expression depends on the mode of oxidative stress in B. cinerea.


Figure 5
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  		FIG. 5. Expression of bcsak1 in axenic culture. Wild-type B05.10 was incubated in 1.5% malt medium for 24 h and moved to CD medium, pH 5.2, for 24 h. Subsequently mycelia were shifted in CD medium containing H2O2, menadione, NaCl, iprodione, and fludioxonil, as indicated, for 1 h. RNA was isolated and used for a Northern blot hybridization with a bcsak1 probe. As a loading control the blot was hybridized with a B. cinerea rDNA probe.

Because of the loss of sporulation and the defect in pathogenicity of the {Delta}bcsak1 mutants, expression of bcsak1 was studied in wild-type strain B05.10 before and during sporulation (after 48 h and 3, 4, and 7 days incubation): no sporulation-correlated induction of bcsak1 expression could be observed (data not shown). In planta expression studies using RNA isolated from infected bean plants at 17, 24, and 48 hpi revealed an increased expression of bcsak1 in the oldest tissue; this is probably due to an increasing ratio of fungus versus plant material in the tissue as shown by the control hybridization using the B. cinerea actA gene (data not shown).

Since the bcsak1 transcript level is only of limited relevance, the activation (phosphorylation) of the BcSAK1 protein was determined using a Western approach. Osmotic stress was generated with sodium chloride (0.8 M and 1 M), and oxidative stress was generated with H2O2 (5 mM and 10 mM, respectively) and menadione (250 µM and 500 µM). Additionally, the effect of fungicides on activation of BcSAK1 was tested using iprodione (5 and 10 µg ml–1) and fludioxonil (10 and 50 µg ml–1). Total protein samples were separated on an SDS-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. The presence of the BcSAK1 protein was monitored using an antibody raised against the C terminus of yeast Hog1 (anti-Hog1p). Anti-Hog1p detected four bands in protein extracts of wild-type B05.10. One band of about 40 kDa was lacking in extracts of the {Delta}bcsak1 mutant, strongly suggesting that this band represents the bcsak1 gene product, BcSAK1 (data not shown). Phosphorylation of this band was monitored using a specific anti-phospho-p38 antibody (see Materials and Methods); it clearly detected a corresponding band in extracts of mycelia of the wild type. Increased phosphorylation of BcSAK1 was detected in mycelia treated with 0.8 M NaCl, confirming the Northern blot data and the growth tests (Fig. 6). H2O2 at higher concentrations (5 and 10 mM) caused activation of BcSAK1 (whereas 0.5, 1.0, and 2.0 mM had no effect; data not shown). Interestingly, BcSAK1 was activated by menadione (250 µM and 500 µM), although we observed neither upregulation of the bcsak1 transcript in the Northern analysis nor a growth effect of menadione in the plate assay (Fig. 4). The anti-Hog1p control showed that the amount of BcSAK1 differed under the various conditions: under osmotic and oxidative stress, the amount of BcSAK1 protein is only slightly increased compared to the control (Czapek Dox medium), whereas higher fungicide concentrations seem to induce the synthesis of more BcSAK1. If this is taken into account, the slight increase in phosphorylated BcSAK1 in the fungicide samples might not be significant. However, these results strongly suggest that BcSAK1 is involved in osmotic stress response and in oxidative stress response mediated by higher concentrations of H2O2 and menadione.


Figure 6
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  		FIG. 6. Phosphorylation of BcSAK1 under oxidative, fungicide, and osmotic stresses. Phosphorylation of BcSAK1 was monitored using anti-phospho-p38, and the total amount of BcSAK1 protein was determined with anti-Hog1p (see Materials and Methods). For a description of stress conditions, see the legend to Fig. 5.

To analyze if the BcSAK1-mediated stress response pathway plays a role during infection, phosphorylation of BcSAK1 in planta was tested at four time points, 17, 24, and 48 hpi and 3 days post inoculation (dpi). As a control, a protein extract of an untreated leaf of P. vulgaris was used. The anti-phospho-p38 antibody detected a signal in all samples from infected leaves, proving that BcSAK1 is activated already at early stages of infection and during the whole infection process. No signal was detected in the uninfected control. The signal intensity correlated roughly with the amount of BcSAK1 protein (as estimated with anti-Hog1p, though due to cross-hybridizing plant proteins this quantification is of limited relevance), i.e., it increased with the amount of fungal biomass in the infected tissue, confirming the Northern blot data (data not shown). Finally, activation of BcSAK1 was tested during formation of conidia. Mycelia were harvested after 48 h and 3, 4, and 7 days of incubation, respectively. There was no increase in phosphorylation of BcSAK1 visible in protein samples of these cultures, i.e., a clear correlation between conidiation and activation of BcSAK1 could not be established (data not shown).

Effect of BcSAK1 on expression of oxidative stress response genes. To study the role of BcSAK1 in oxidative stress response in more detail, expression of various potential "target" genes was studied in Northern blot experiments using mycelia grown under various stress conditions. Several typical oxidative stress response genes (bccatA, bccat2, and bccatC, encoding catalases [47, 59]; bcgstI and bcgstII, encoding glutathione S-transferases [43, 50]; and bcsod1, encoding a superoxide dismutase [45]) as well as two NADPH oxidase genes probably involved in the generation of O2– (bcnoxA and bcnoxB; N. Segmüller and P. Tudzynski, unpublished data) were tested for induction under these conditions in the wild type and the mutant (Fig. 7).


Figure 7
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  		FIG. 7. Expression of potential target genes of BcSAK1. RNA from mycelia incubated under various stress conditions (see the legend to Fig. 5) was blotted and hybridized to labeled fragments of various B. cinerea genes (for details, see the text).

The results correspond to the ambivalent activation pattern of BcSAK1 under different oxidative stress conditions: the transcriptional response was not uniform. Whereas the gstII, catC, and sod genes show no difference in expression between the mutant and the wild type, other ROS-responsive genes are obviously affected by BcSAK1. In the mutant, gstI is induced by almost all oxidative stress stimuli, whereas in the wild type activation of the gene is almost exclusively mediated by H2O2. The two catalase genes catA and cat2 are also differently regulated in the mutant and the wild type: whereas catA is induced in the mutant with menadione and strongly with NaCl, the gene is not expressed at all in the wild type. In contrast to the mutant, cat2 is highly induced only during H2O2-mediated oxidative stress in the wild type. These data indicate that induction of cat2 by H2O2 is mediated by BcSAK1, whereas induction of catA by menadione (and NaCl) is repressed by BcSAK1. The two nox genes tested under the same conditions have the same expression level in the mutant and the wild type, suggesting that BcSAK1 has no impact on NADPH oxidase gene expression under the tested conditions. A general stress protein gene used as a control (hsp30) was strongly induced under all applied stress conditions in the {Delta}bcsak1 mutant but only under some conditions in the wild type, indicating a role of BcSAK1 in general stress control.

BcSAK1 is essential for pathogenicity. Because of the lack of conidia, mycelial plugs of three {Delta}bcsak1 mutants ({Delta}bcsak1-12, {Delta}bcsak1-17, and {Delta}bcsak1-27) were used to infect primary leaves (both intact and wounded) of young bean plants. Lesion diameters were determined at 24 and 48 hpi. None of the three transformants showed any signs of infection when they were incubated on intact bean leaves (Fig. 8A). However, they were able to infect wounded plants, although formation of lesions/colonization of plant tissue (at least for transformants {Delta}bcsak1-12 and {Delta}bcsak1-27) was significantly slower than that of the wild type (Fig. 8B). This differential behavior of the three mutants is unexpected (they should be isogenic, since they contain no additional ectopic copies of the vector) but not unusual (therefore, in such analyses more than one mutant should be tested). There could be additional defects not detectable with PCR and Southern blot analyses leading to the altered virulence on wounded plants. To be sure that the mutant with the strongest phenotype ({Delta}bcsak1-27) has no additional defect, it was used for the complementation test, which fully restored the wild-type phenotype (see below). This proves that the strong virulence defect of this mutant is only due to inactivation of bcsak1, indicating that the milder phenotype of {Delta}bcsak1-17 is due to some additional (perhaps a suppressor) defect.


Figure 8
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  		FIG. 8. Pathogenicity test on bean leaves. A. Comparison of the wild type (WT), mutants {Delta}bcsak1-12 (T12) and {Delta}bcsak1-27 (T27), and complemented transformant CMP9 ({Delta}27C9) on unwounded leaves. B. Compilation of infection tests using three different {Delta}bcsak1 mutants and wounded (w) and unwounded leaves. The values are averages from at least six lesions each (from three different plants).

To study this pathogenicity defect of the {Delta}bcsak1 mutants in detail, electron scanning microscope analyses were performed. Bean plants were infected with agar plugs of wild-type B05.10 and mutant {Delta}bcsak1-27. Samples were taken at 24 and 48 hpi, fixed, and analyzed. Whereas wild-type hyphae grew directly from the agar plug onto the plant surface, formed branches and appressoria-like structures, and penetrated the plant at 24 hpi (Fig. 9), the mutant behaved differently: it grew much slower, with long, almost unbranched hyphae, and even at 48 hpi it did not form appressoria-like structures and did not penetrate the plant (Fig. 9). The {Delta}bcsak1 mutants are obviously defective in early stages of penetration, i.e., already in appropriate branching and formation of appressoria-like structures.


Figure 9
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  		FIG. 9. SEM analyses of infected bean leaves. Upper panel, B05.10 at 24 dpi; arrows indicate branched hyphae and an appressorium-like structure, respectively. Lower panel, {Delta}bcsak1-27 at 48 dpi; the arrow indicates an unbranched hypha (for details, see Materials and Methods).

Complementation of bcsak1 mutants restores conidiogenesis and pathogenicity. To confirm the correlation between the phenotype of the {Delta}bcsak1 mutants and the knockout of the gene, a complementation approach was performed. An intact gene copy including 1 kb of the promoter and terminator regions, respectively, was cloned into the hygromycin resistance vector pOLIHP, and the linearized complementation fragment was transformed into the {Delta}bcsak1-27 mutant. Hygromycin-resistant colonies were isolated and analyzed for complete integration of the fragment. Nine transformants contained the full wild-type gene copy (tested by PCR and Southern blot analyses; data not shown). These complemented transformants showed the wild-type phenotype: conidial production and growth were normal, and the transformants were able to infect unwounded plant tissue (Fig. 8A), proving that the strong phenotype of this transformant is exclusively due to the knockout of bcsak1 in B. cinerea.


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DISCUSSION
 
MAPK cascades have been shown to be involved in many essential aspects of fungal pathogenesis (for reviews, see references 58 and 68). The Fus3-homologous cascade especially has turned out to be essential for pathogenicity in a wide range of fungi (32, 66) without affecting vegetative properties like growth rate or conidiation. Also, in B. cinerea deletion of the fus3-homologous gene bmp1 led to full loss of pathogenicity; the mutant was obviously unable to penetrate (72). A second conserved class of MAPK, homologous to yeast Slt2, was also identified in a wide variety of fungi, but its function has been studied only in a few systems, e.g., in M. grisea (67), F. graminearum (23), and Claviceps purpurea (33); it is also essential for pathogenesis. In contrast to the fus3 mutants, mutants of slt2 homologues also have defects in vegetative differentiation and cell wall structure, which could explain their reduced virulence. In contrast, the Hog-type MAPK cascade does not seem to have major effects on vegetative differentiation and pathogenicity in the so-far studied systems: conidiation in most {Delta}hog-like mutants is normal, and at least in the model systems M. grisea and C. lagenarium the hog mutants are fully pathogenic (14, 28). The only exception so far is C. parasitica, where {Delta}cpmk1 mutants show hypovirulence-like symptoms (41), including reduced pigmentation and conidiation. In A. nidulans, {Delta}sakA mutants have a defect in conidiospore viability; asexual spores are highly sensitive to oxidative and heat shock stress and lose viability upon storage (25). In the model organisms N. crassa and C. lagenarium (28, 70), the hog-homologous pathway is responsible for increased resistance against phenylpyrrol fungicides like fludioxonil and is involved in response to hyperosmotic stress. Also, for B. cinerea BcSAK1 it has been shown that the protein is phosphorylated during fludioxonil treatment (28).

In B. cinerea, {Delta}bcsak1 mutants have a block in conidiogenesis, produce more sclerotia, are resistant to iprodione, and are completely apathogenic, by far the strongest phenotype observed for this type of mutant. Cytological analyses showed that the mutants are unable to penetrate, probably because of an inability to form penetration structures. The role of the apical swellings of B. cinerea hyphae in cuticle penetration is not yet well understood: they lack the septum which is typical for genuine appressoria, and there are reports of penetration by B. cinerea hyphae without any morphological changes (56). The {Delta}bcsak1 mutants might help to identify the processes involved before and during penetration in B. cinerea. The impact of BcSAK1 on the development of spreading lesions has to be studied in more detail, especially since the mutants investigated are not uniform in this special feature. Biological variability of supposedly isogenic strains has been noted previously in filamentous fungi (7) and in transformation experiments (63), although it is rarely published, i.e., the one-off transformants are normally skipped. Also, in Botrytis cinerea this phenomenon is familiar for groups working with transgenic strains (Jan van Kan, personal communication). This biological variability is possibly due to epigenetic changes elicited by the general "stress" applied by the protoplast-mediated transformation procedure or even possibly to ectopic integrations of (partial) vector sequences not detected by our Southern blot analyses. This is the reason why it is essential to analyze several independent mutants and to perform complementation experiments. Since we were able to fully restore the wild-type phenotype in the mutant {Delta}bcsak1-27 (with slow secondary lesion formation in wounded plant tissue), BcSAK1 also has a clear impact on this process. However, in this paper we would like to focus on the phenotypes common to all mutants analyzed: they produce no conidia but more sclerotia, and they are unable to infect unwounded tissue.

The unique impact of BcSAK1 on vegetative differentiation (sclerotia and conidia) will now be studied in more detail. So far we were unable to link phosphorylation of BcSAK1 directly with conidial formation; however, this could be due to the limited degree of synchrony in conidial formation and a highly localized activation of the enzyme. BcSAK1 is clearly involved in this process, as proven by the knockout and by the regain of function in the complemented transformants. The effect could also be more indirect, e.g., via a slight modification of the redox status. In Candida albicans, Hog1 is involved in the transition from yeast to filamentous growth and in chlamydospore formation (2). Recent evidence indicates that this influence on differentiation is caused by cross-talk to the MAPK cascade involved in cell wall biogenesis (16). Phosphorylation of the Slt2-homologous MAPK in C. albicans requires the presence of Hog1p under some conditions. It could be tested if this is also the case in B. cinerea by analyzing the reciprocal effects of mutations in the BcSAK1 pathway and the Slt2 homologue pathway. Interesting effects were observed in A. nidulans: sakA mutants are not able to form septa, leading to the inability to maintain turgor pressure, which affects cell expansion. In addition, the mutants show disproportionate accumulation of mitotic nuclei (24). {Delta}bcsak1 mutants of B. cinerea seem to have normal septa in vegetative hyphae (data not shown), but a defect in septa formation during conidiation cannot be excluded. Additionally, disturbances in mitotic cell division, like in A. nidulans, could play a role in conidia formation. It is only possible to speculate about the role of BcSak1 in formation of sclerotia. In Sclerotinia sclerotiorum it could be shown that the cyclic AMP-dependent signaling pathway is involved in sclerotia formation (9). Additionally, an ERK-type MAPK (smk1) and a small GTPase (ssRas) are necessary for sclerotial development in this fungus (8). Loss of ras activity and increases of endogenous and exogenous cyclic AMP levels block MAPK activation and the formation of sclerotia. For B. cinerea it was shown by Doehlemann et al. (15) that bmp1 mutants do not form sclerotia, suggesting a role of this MAPK similar to that in S. sclerotiorum. Maybe an inappropriate activation of the ERK-homologous MAPK BMP1 in the {Delta}bcsak1 mutant leads to an upregulation of sclerotia development-dependent genes in B. cinerea. The striking differences in effects on pathogenicity between M. grisea, C. lagenarium, and B. cinerea might reflect their different pathogenic strategies: M. grisea and C. lagenarium start the infection with a biotrophic phase, i.e., they avoid plant defense reactions, whereas the necrotroph B. cinerea provokes plant defense reactions and probably even uses them for its infection process. Therefore, B. cinerea might depend more on the stress-signaling system to cope with the different stress situations it has to face, especially in the early stages of infection. The finding that several genes responsible for ROS detoxification are probably regulated by BcSAK1 agrees with the fact that oxidative stress is probably the major stress occurring in the early in planta situation. Since B. cinerea seems to use an "oxidative attack" to facilitate penetration of the cuticle and the cell wall (54, 55) and the virulence of the fungus correlates positively with the intensity of the "oxidative burst" (62), the finding that the hog-homologous cascade is obviously not involved in the regulation of NADPH oxidases (in contrast to A. nidulans [29]) is unexpected and has to be studied in more detail.

Growth tests, transcript analyses, and phosphorylation assays clearly showed that BcSAK1 is involved in osmotic and oxidative, and perhaps also in general, stress response. Interestingly, there is a great variability in the expression of genes responsible for the detoxification of ROS concerning the comparison of expression patterns in the wild type and the mutant and expression of the genes during different types of oxidative stress. gstII, catC, and bcsod1 seem not to be regulated via BcSAK1, whereas other genes, such as gstI, cat2, and catA, are differently expressed in the mutants compared to the wild type. Altogether it seems that BcSAK1 is partially involved in regulation of genes encoding ROS scavenging enzymes and that regulation via BcSAK1 depends on the type of oxidative stress.

Regulation of bcsak1 expression and phosphorylation of BcSAK1 during oxidative stress seems to be different, indicating that both processes are controlled by different upstream components (see below). Whereas upregulation of bcsak1 expression is mediated only by high concentration of H2O2, phosphorylation of BcSAK1 is additionally detectable during treatment with menadione, supporting the hypothesis that BcSAK1 signaling depends on the type of stress applied. The role of the hog-type MAPK cascade in the oxidative stress response in filamentous fungi has not been studied thoroughly so far. To our knowledge, only in A. nidulans has a clear role of SAKA in oxidative stress response been demonstrated (26). There could be alternative systems involved, such as in the yeast MAPK-activated protein kinases (RcK1/2 [5]) and the Sty1-independent transcription factor Pap1 in S. pombe, which is directly activated by ROS (61).

The upstream components of the hog-like cascade in filamentous fungi are largely unknown. The best investigated systems are Ustilago maydis (17) and M. grisea (71), where the MAPK kinases (and scaffold proteins) have been functionally analyzed. In M. grisea, interaction of a Ras homologue with this cascade has been demonstrated, but the linkage to a receptor system has not yet been established (71). In yeast, the histidine kinase Sln1 and the protein ShoI are involved in signal perception of the hog cascade. In contrast to other fungal systems, Sln1 regulates Hog1 negatively due to osmotic stress, whereas ShoI regulates Hog1 positively, e.g., in response to heat stress (65). In N. crassa the histidine kinase Os1 is responsible for the activation of Osm1 (encoding the Hog homologue in this fungus), indicating a positive transcriptional regulation (36). A positive regulation of SAPKs by histidine kinases has also been shown in C. heterestrophus and A. nidulans (18, 69); in C. heterestrophus, the group III histidine kinase Dic1p (homologous to Os1 of N. crassa) has been shown to activate the Hog homologue BmHog1 during osmotic and fungicide stress. In A. nidulans, several enzymes are proposed for the activation of the corresponding cascade: SakA is probably activated by a combined system of activators including histidine kinases NikA and TcsA (18). An sln1 homologous gene is present in B. cinerea, but its deletion had no phenotype (Y. C. Arenas, A. Schouten, and J. van Kan, personal communication). A better candidate for a receptor of the stress cascade is the histidine kinase BOS1. The gene was originally identified through fungicide-resistant mutants (10). It was recently deleted (60), and the {Delta}bos mutant has a phenotype similar to that of {Delta}bcsak1: it does not sporulate, and it is osmosensitive and resistant to iprodione. However, there are also some differences between the mutants, e.g., {Delta}bos still can penetrate, though the spreading of secondary lesions is retarded, as it is in {Delta}bcsak1. These differences could also result from the different genetic background, since bos1 was deleted in a different strain. Whether or not BOS1 really is upstream of the BcSAK1 cascade has to be studied in detail, e.g., by phosphorylation experiments under different stress conditions and generation of double mutants. However, as in A. nidulans it is possible that BcSAK1 is activated (and induced) by several partners, depending on the type of stress. This would explain the different levels of response to osmotic versus oxidative stress. Candidates for these upstream components of BcSAK1 are, e.g., the histidine kinase BcHK1 (N. Segmüller and P. Tudzynki, unpublished), a homologue of SpMAK2/3, the Sty1-activating histidine kinase in S. pombe, and the small GTP-binding protein BcRAS1 (L. Kokkelink and B. Tudzynski, unpublished data).


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ACKNOWLEDGMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (Tu50/13).

We thank B. Williamson (Dundee) for critical reading; M. Viaud and S. Fillinger (Versailles), J. Delgado (Sevilla), and J. van Kan (Wageningen) for sharing unpublished data and for discussions; K. B. Tenberge for helpful advice in the electron microscopy technology; and S. Gergs for excellent technical support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, D-48149 Münster, Germany. Phone: 49-251-83 2 4998. Fax: 49-251-83 2 1601. E-mail: tudzyns{at}uni-muenster.de. Back

{triangledown} Published ahead of print on 22 December 2006. Back

{dagger} Present address: Laboratory of Phytopathology, Wageningen University, P.O. Box 80 25, NL-6700 EE Wageningen, The Netherlands. Back


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Eukaryotic Cell, February 2007, p. 211-221, Vol. 6, No. 2
1535-9778/07/$08.00+0     doi:10.1128/EC.00153-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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