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Eukaryotic Cell, April 2008, p. 584-601, Vol. 7, No. 4
1535-9778/08/$08.00+0     doi:10.1128/EC.00426-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Calcineurin-Responsive Zinc Finger Transcription Factor CRZ1 of Botrytis cinerea Is Required for Growth, Development, and Full Virulence on Bean Plants

Institut für Botanik, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, 48149 Münster, Germany,¹ Departamento de Química Aplicada, Facultad de Ciencias Químicas, Universidad del País Vasco (UPV/EHU), Po. de Manuel de Lardizabal 3, 20018 San Sebastián, Spain²

Received 23 November 2007/ Accepted 24 January 2008

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, we showed that the subunit BCG1 of a heterotrimeric G protein is an upstream activator of the Ca²⁺/calmodulin-dependent phosphatase calcineurin in the gray mold fungus Botrytis cinerea. To identify the transcription factor acting downstream of BCG1 and calcineurin, we cloned the gene encoding the B. cinerea homologue of CRZ1 ("CRaZy," calcineurin-responsive zinc finger transcription factor), the mediator of calcineurin function in yeast. BcCRZ1 is able to partially complement the corresponding Saccharomyces cerevisiae mutant, and the subcellular localization of the green fluorescent protein-BcCRZ1 fusion product in yeast cells depends on the calcium level and calcineurin activity. Bccrz1 deletion mutants are not able to grow on minimal media and grow slowly on media containing plant extracts. Hyphal morphology, conidiation, and sclerotium formation are impaired. The cell wall and membrane integrity, stress response (extreme pH, H₂O₂, Ca²⁺, Li⁺), and ability of the hyphae to penetrate the intact plant surface are affected in the mutants. However, BcCRZ1 is almost dispensable for the conidium-derived infection of bean plants. The addition of Mg²⁺ restores the growth rate, conidiation, and penetration and improves the cell wall integrity but has no impact on sclerotium formation or hypersensitivity to Ca²⁺ and H₂O₂. The expression of a set of recently identified BCG1- and calcineurin-dependent genes is also affected in Bccrz1 mutants, confirming that this transcription factor acts downstream of calcineurin in B. cinerea. Since the Bccrz1 mutants still respond to calcineurin inhibitors, we conclude that BcCRZ1 is not the only target of calcineurin.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Gray mold rot, caused by the ascomycete Botrytis cinerea Pers.:Fr. [teleomorph: Botryotinia fuckeliana (de Bary) Whetzel], is an important disease of almost all dicotyledonous plants, including vegetable and fruit crops, flowers, and greenhouse-grown crops. The fungus has developed a flexible infection strategy, including manifold tools for penetrating and overcoming plant defenses. In addition to the secretion of cell wall-lysing enzymes and the production of non-host-selective toxins, e.g., botrydial and botcinolides, B. cinerea is able to induce an oxidative burst by the production of reactive oxygen species (reviewed in references 74 and 83). All these processes must be highly regulated: the fungus needs to recognize the host plant and to find the optimum time for infection, expansion, and reproduction. It is suggested that the sensing of plant signals is managed by heterotrimeric G protein-coupled receptor systems, which transduce the external signal into an intracellular signal mediated via the dissociation of the G subunit from the Gβ dimer and subsequent activation of downstream effector pathways, such as the adenylate cyclase/cyclic AMP (cAMP)/protein kinase A (PKA) cascade (reviewed in reference 25).

The functions of the three different G subunits (BCG1, BCG2, and BCG3) of B. cinerea, and their effects on vegetative growth and virulence, have been investigated in recent years (16, 59). The deletion of bcg1 resulted in severely reduced virulence on bean and tomato, loss of protease secretion, changed colony morphology (59), and loss of botrydial biosynthesis (65). It was shown that this G subunit is not only an activator of the adenylate cyclase BAC, regulating colony morphology via the cAMP level, but also the regulator of a second, cAMP-independent signaling pathway (36, 60). Recently, we demonstrated that the subunit BCG1 acts as an activator of the Ca²⁺/calmodulin-dependent calcineurin phosphatase, inducing the expression of a set of genes, including those of the botrydial biosynthesis gene cluster (61).

Calcineurin is a highly conserved protein, consisting of a catalytic (CNA1/2) and a regulatory (CNB) subunit, which is activated by binding of the Ca²⁺/calmodulin complex when the cytosolic Ca²⁺ level is increased (reviewed in references 13, 19, 38, and 55). In Saccharomyces cerevisiae, calcineurin is dispensable for growth under standard culture conditions but is required for response to environmental stress conditions, such as exposure to several cations (Mn²⁺, Li⁺, and Na⁺), alkaline pH, high temperature, and endoplasmic reticulum stress, and incubation with mating pheromone (-factor). In strains lacking components of the cell wall integrity mitogen-activated protein (MAP) kinase pathway, calcineurin is essential even under standard growth conditions (6, 12, 21, 42, 44, 85). In filamentous fungi, calcineurin seems to be even more important: there are only a few viable knockout mutants of calcineurin, all of them showing severely disturbed vegetative growth. Thus, in Aspergillus fumigatus cna1 deletion mutants, the growth rate, hyphal morphology, sporulation, conidial architecture, and pathogenicity are affected (15, 70). Studies of other fungi, using inducible cna1 antisense constructs or calcineurin inhibitors, such as the immunosuppressive drug cyclosporine or FK506, revealed the requirement for calcineurin in vegetative differentiation (e.g., sclerotial development in Sclerotinia sclerotiorum) (26), cell wall integrity (e.g., cell wall β-1,3-glucan content in S. sclerotiorum) (26), and virulence (e.g., infection structure formation in Magnaporthe grisea and B. cinerea) (75, 76). In the basidiomycete Cryptococcus neoformans, calcineurin is required for growth at an elevated temperature (37°C) and for virulence (18, 48, 71).

So far, the calcineurin-dependent (CND) gene expression program has been extensively characterized only in S. cerevisiae: in response to stress, calcineurin activates the transcription factor CRZ1 ("CRaZy," calcineurin-responsive zinc finger) by docking at the PIISIQ site (the calcineurin-docking domain) and dephosphorylating the serine-rich region (SRR) motif, in a manner similar to the calcineurin-dependent regulation of members of the mammalian NFAT (nuclear factor of activated T cells) transcription factor family (reviewed in reference 2). The dephosphorylation of CRZ1 affects its subcellular localization: when calcineurin is inactive, the phosphorylated CRZ1 protein is distributed throughout the cell. After stimulation by an increase of cytosolic Ca²⁺, CRZ1 rapidly accumulates in the nucleus in a calcineurin-dependent manner due to its increased nuclear import and decreased nuclear export (50, 69). Antagonists of the calcineurin phosphatase are two protein kinases: the cAMP-dependent protein kinase (PKA), which negatively regulates CRZ1 activity by inhibiting its nuclear import (32), and HRR25, a casein kinase 1 homologue that affects nuclear import, export, or both (33).

CRZ1 contains a C2H2 zinc finger motif that binds to the calcineurin-dependent response element in the promoter regions of calcineurin-regulated genes and was shown to be sufficient for Ca²⁺ and CND gene expression in yeast (68). Analyses of global calcineurin/CRZ1-dependent gene expression by performing microarray experiments revealed 153 genes, which are involved in ion homeostasis, cell wall synthesis, vesicle transport, lipid/sterol synthesis, and protein degradation (84). The crz1 phenotype is comparable to that of the calcineurin mutant, and the mutants are also defective in calcineurin-dependent induction of gene expression (42, 68). However, the loss of calcineurin is more severe than loss of CRZ1, suggesting that calcineurin has additional substrates, such as HPH1/HPH2, which are tail-anchored integral membrane proteins localized to the endoplasmic reticulum and are required to promote growth under several stress conditions (27). Signaling via calcineurin can be modulated by a conserved family of calcineurin regulators, termed calcipressins. For example, the RCN1 protein of S. cerevisiae inhibits the protein phosphatase activity of calcineurin and operates as an endogenous feedback inhibitor of calcineurin (35).

The calcineurin phosphatase and the CRZ1 transcription factors have also been investigated in the human pathogen Candida albicans. Calcineurin mutants are hypersensitive to agents that disturb cell membrane integrity, such as azoles and sodium dodecyl sulfate (SDS), and to elevated Na⁺, Li⁺, and Mn²⁺ concentrations and high pH and are strongly attenuated in virulence (4, 11, 57). Like the calcineurin mutants, the deletion mutants of Cacrz1 show increased sensitivity to high cation concentrations and membrane stress caused by SDS and azoles. Due to the fact that cna1 mutants are less virulent than crz1 mutants, it is proposed that CaCRZ1 acts downstream of calcineurin but is not the only signaling effector (34, 49, 58). Furthermore, several Ca²⁺-, CNA-, and CRZ1-dependent genes were identified whose gene products are involved in cell wall organization, cellular organization, cellular transport and homeostasis, cell metabolism, and protein fate (34).

While in S. cerevisiae and C. albicans the calcineurin pathway and its components and functions are well characterized, little is known about the pathway and downstream targets in filamentous fungi. Recently, we showed that the G subunit BCG1 is an upstream regulator of the Ca²⁺- and calcineurin-dependent pathway, as a common set of genes were regulated by both BCG1 and calcineurin (61). Therefore, we wanted to know if the transcription factor BcCRZ1 is a potential downstream effector of calcineurin in B. cinerea.

In this work, we demonstrate that BcCRZ1 is a functional homologue of yeast CRZ1 that is able to partially complement CRZ1 function in the S. cerevisiae mutant, showing a Ca²⁺- and calcineurin-dependent localization pattern in yeast cells. We report that Bccrz1 deletion mutants are severely impaired in vegetative growth and differentiation, such as conidiation and sclerotium formation and cell wall and membrane integrity, as well as virulence. Interestingly, growth, conidiation, and virulence, but not the ability to form sclerotia and to grow in the presence of high H₂O₂ and Ca²⁺ concentrations, can be specifically restored by exposure to higher Mg²⁺ concentrations. Finally, we show that a set of previously identified CND genes are expressed in a similar manner in Bccrz1 mutants, suggesting that BcCRZ1 is indeed a target of the calcineurin activation pathway.

	MATERIALS AND METHODS

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B. cinerea. Strain B05.10 of B. cinerea Pers.:Fr. [Botryotinia fuckeliana (de Bary) Whetz] is a putative haploid strain obtained after benomyl treatment of an isolate from Vitis (52) and is used as a host strain for gene replacement experiments. The bcg1 strain is a knockout mutant for the G protein -subunit-encoding gene bcg1 (59). Wild-type and mutant strains were grown on several complex media: potato dextrose agar (Sigma-Aldrich Chemie, Steinheim, Germany) was supplemented with 10% homogenized leaves of French bean (Phaseolus vulgaris) (PDAB). Grape agar contained undiluted grape juice (100 ml contained on average 0.2 g protein, 15.2 g carbohydrates, and 0.01 g fat) supplemented with 0.1% yeast extract and was adjusted to a final pH of 5. Synthetic complete medium (CM) was made according to the method of Pontecorvo et al. (51). As minimal medium, modified Czapek-Dox (CD) medium (2% sucrose, 0.1% KH₂PO₄, 0.3% NaNO₃, 0.05% KCl, 0.05% MgSO₄·7 H₂O, 0.002% FeSO₄·7 H₂O, pH 5.0) was used. For conidiation, the strains were incubated for 1 week at 21°C under light conditions; for sclerotium formation, they were incubated for 4 weeks at 21°C in darkness. For DNA and RNA minipreparations, mycelium was grown for 3 to 4 days at 20°C on CM agar with a cellulose acetate (cellophane) overlay. Plate assays were performed using CM agar with or without 67 mM MgCl₂ (equivalent to 0.2 osmol/liter due to three osmotically active ions) supplemented with Congo red, calcofluor white, FK506, menadione, fluconazole (Sigma-Aldrich, St. Louis, MO), H₂O₂ (AppliChem GmbH, Darmstadt, Germany), cyclosporine (Calbiochem, Merck KGaA, Darmstadt, Germany), SDS, and Triton X-100 (MP Biomedicals Inc., Solon, OH) as indicated. Protoplasts were generated using Glucanex (Novozymes, Denmark) or β-glucanase (InterSpex Products), added to 15 µg of the linearized vector, and transformed according to the method of Siewers et al. (64). Resistant colonies were transferred to plates containing CM agar complemented with 70 µg/ml of hygromycin B (Invivogen, San Diego, CA) or 70 µg/ml of nourseothricin (Werner-Bioagents, Jena, Germany). Single conidial isolates were obtained by spreading conidial suspensions on CM plates containing 70 µg/ml of hygromycin B. The conidia were germinated, and single colonies were transferred individually to new plates containing the selection marker.

S. cerevisiae. Wild-type strain W303-1A and its derivative MSE104 (crz1) have been described previously (58, 79). Yeast cells were grown either in complete YPD medium consisting of 2% glucose, 2% peptone, and 1% yeast extract or in minimal SD medium containing 2% glucose, 0.67% yeast nitrogen base, and the amino acids, purine, and pyrimidine bases required by the strains. Solid media contained 2% agar. S. cerevisiae strains were transformed by the lithium-acetate procedure as described previously (22). For maintenance of plasmids, yeast transformants were precultured in selective media supplemented with methionine and then transferred to the experimental conditions.

E. coli. Escherichia coli strain TOP10F' (Invitrogen, Groningen, The Netherlands) and E. coli XL1-Blue (7) were used as hosts for plasmid construction and propagation.

Germination assays. Analysis of nutrient-dependent germination of conidia from the B. cinerea wild-type and mutant strains was done on glass surfaces according to the method of Doehlemann et al. (16).

Pathogenicity assays. Infection assays were performed with conidiospores from 10-day-old grape agar cultures as described previously (59). In addition, agar plugs taken from 3-day-old CM agar cultures were used to inoculate primary leaves of Phaseolus vulgaris. The infected plants were incubated in a plastic propagator box at 20°C under natural illumination conditions. Disease symptoms were scored until 12 days after inoculation.

Microscopic analyses. To study the hyphal morphology, the B. cinerea strains were grown on microscope slides that carried an overlay of CM agar. After incubation for 2 days in a humid chamber at 20°C, the colonies were incubated for 5 min in 1% (wt/vol) calcoflour white solution and then washed with water. The stained colonies were observed by epifluorescence microscopy using a Leica DMRBE microscope with a PixelFly digital camera (PCO Computer Optics GmbH) and Leica filter set A (BP 340 to 380; RKP 400; LP425).

Standard molecular methods. Fungal genomic DNA was isolated as described previously (9). Plasmid DNA was isolated using a plasmid DNA preparation kit (Genomed, Bad Oeynhausen, Germany). For Southern analysis, the fungal DNA was transferred to Hybond N⁺ filters (Amersham Biosciences, Freiburg, Germany) after digestion with restriction enzymes and size separation on a 1% agarose gel according to the method of Sambrook et al. (56). Hybridization was carried out in 6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt's solution, 0.1% SDS, and 50 mM phosphate buffer, pH 6.6, at 65°C in the presence of a random-primed [-³²P]dCTP-labeled probe. The membranes were washed once (2x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA {pH 7.7}], 0.1% SDS) before being exposed to autoradiographic film. Total RNA was isolated from mycelial samples using the Trizol procedure (Invitrogen, Groningen, The Netherlands). Samples (25 µg) of total RNA were transferred to Hybond N⁺ membranes after electrophoresis on a 1% agarose gel containing formaldehyde, according to the method of Sambrook et al. (56). Blot hybridizations were carried out in 0.6 M NaCl, 0.16 M Na₂HPO₄, 0.06 M EDTA, 1% N-lauroylsarcosine (Sigma-Aldrich, St. Louis, MO), 10% dextran sulfate (Eppendorf AG, Hamburg, Germany), 0.01% salmon sperm DNA, pH 6.2, as described for Southern blots; 1 µg of total RNA was taken for cDNA synthesis using the oligo(dT)12-18 primer and SuperScript II reverse transcriptase (Invitrogen, Groningen, The Netherlands) according to the manufacturer's instructions. PCR mixtures contained 25 ng DNA, 5 pmol of each primer, 200 nM concentrations of desoxynucleotide triphosphates, and 1 unit of BioThermDNA polymerase (GeneCraft GmbH, Lüdinghausen, Germany). The reactions started with 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 56 to 65°C, and 1 min at 70°C, and a final 10 min at 70°C. PCR products were cloned into pCR2.1-TOPO (Invitrogen, Groningen, The Netherlands). DNA sequencing was performed with the automatic sequencer Li-Cor 4200 (MWG Biotech, Munich, Germany) using the Thermo Sequenase fluorescence-labeled primer cycle-sequencing kit (Amersham Biosciences). For sequence analysis, Lasergene v6 software (DNAStar, Madison, WI) was used.

Macroarray analysis. The cDNA macroarrays contained B. cinerea cDNAs from three different expressed sequence tag collections. One cDNA library was created from the B. cinerea strain ATCC 58025, a nonsporulating overproducer of abscisic acid under abscisic acid biosynthesis conditions (64); the second library was derived from a suppression subtractive hybridization approach, which was used to identify genes of the wild-type B05.10, whose expression on the host plant P. vulgaris was specifically affected (60); the third library was derived from germinating conidia of B05.10 and early stages of plant infection (L. Kokkelink, unpublished data). In summary, the assembling of 16,525 cDNA sequences by means of the assembly program CAP3 (30) resulted in 1,901 contigs and 3,047 singlets. Thus, the macroarrays altogether contained 4,948 genes (including genes of plant origin). Sequence analysis for the prediction of protein function was done using BlastX at NCBI (1). Radiolabeled cDNA probes were prepared in the presence of 30 µCi [-³³P]dATP using SuperScript II reverse transcriptase as described above. Fungal cultures with the two genotypes (B05.10 and Bccrz1) were repeated three times. Each RNA sample was hybridized once to the macroarrays, making a total of three biological repeats and, due to the two replicates, six values per cDNA clone. The hybridization images from the Typhoon 8600 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were analyzed for initial data quantification using ArrayVision 8.0 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The Excel macro FiRe (find regulons) (http://www.unifr.ch/plantbio/FiRe/main.html) was used to select candidate genes for differential expression based upon their change ratios (5, 20), using standard parameters (lower threshold, 0.5; upper threshold, 2.0). The ratios of the genes (see Table 3) were, in at least five of six cases, below the threshold of 0.5 (down-regulation in Bccrz1) or above the threshold of 2.0 (up-regulation in Bccrz1).

Complementation of S. cerevisiae crz1.

View larger version (29K): [in this window] [in a new window]   FIG. 3. Gene replacement of Bccrz1. (A) Physical maps of Bccrz1, the gene replacement fragment Bccrz1-RF, and the gene locus of a Bccrz1 knockout mutant showing the Bccrz1 open reading frame (gray arrow), the components of the hygromycin resistance cassette (gray boxes), and the flanking regions of Bccrz1 (heavy lines). The small arrows indicate the positions of primers used for cloning the full-length cDNA for yeast complementation (primers 1 and 2), the replacement vector (primers 3 to 6), and the diagnostic PCR analysis of the transformants (primers 7 to 11) (see Materials and Methods). (B) For Southern blot analysis, the genomic DNAs of the wild type (WT) and the mutants were digested with HindIII, blotted, and hybridized to the 5' flank (dotted line in panel A) of the replacement vector pbccrz1. In both mutants, the wild-type fragment, with a size of 2.2 kb, was replaced by a 4.5-kb fragment, resulting from the loss of HindIII restriction sites within the Bccrz1 gene.

Replacement of Bccrz1. For construction of a gene replacement vector for Bccrz1, the plasmid pOliHP (53), carrying the E. coli hygromycin B phosphotransferase gene hph under the control of the Aspergillus nidulans oliC promoter and trpC terminator, was used as a basal vector. The gene flanks were amplified by PCR with primers, derived from the genomic sequence of B. cinerea B05.10 (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html), containing artificial restriction sites for further cloning (Table 1; see Fig. 3A). An 840-bp fragment was amplified from the Bccrz1 promoter region using primers 3 and 4, and a 740-bp fragment of the 3'-untranslated region was generated as a second flank using primers 5 and 6. Both PCR products were cloned into pCR2.1-TOPO; isolated with KpnI/SalI and HindIII/SacI, respectively; and then cloned into the corresponding restriction sites of pOliHP, creating pbccrz1. For transformation, the replacement cassette was isolated with KpnI and SacI. The hygromycin B-resistant transformants were analyzed by PCR for homologous integration using primers 7 to 10 and were purified by single-spore isolation and screened by PCR for the absence of the crz1 wild-type allele using primers 11 and 2. For the Southern blot analysis, the genomic DNAs of the wild-type B05.10 and the Bccrz1 transformants were digested with HindIII and hybridized to the 5' flank of Bccrz1. The approach showed that the transformants had undergone homologous integration into the Bccrz1 locus and that they did not contain additional copies of the gene replacement fragment (see Fig. 3B).

Complementation of Bccrz1 with Bccrz1 cDNA. To verify that the phenotype of Bccrz1 was due to the deletion of Bccrz1 in B. cinerea, the mutant Bccrz1-4 was complemented. For this, a 1-kb promoter fragment of Bccrz1 was amplified by PCR using primers 12 and 13 (Table 1) containing artificial restriction sites for SmaI and XbaI, respectively. The promoter was fused to the XbaI/ClaI-digested Bccrz1 cDNA fragment (see "Complementation of S. cerevisiae crz1" above) by insertion of both fragments into pbcniaD, creating pbccrz1-Com. pbcniaD contains the noncoding 5' and 3' regions of BcniaD, encoding nitrate reductase, to mediate homologous integration of complementation constructs at the BcniaD locus (61). The nourseothricin resistance cassette, containing nat1 from Streptomyces noursei under the control of the A. nidulans oliC promoter, was amplified using pNR1 as a template (40) and primers 14 (with an additional ClaI restriction site) and 15. The resulting PCR fragment was digested with ClaI (a second ClaI site within the resistance cassette) and ligated into the ClaI-digested pbccrz1-Com, creating pbccrz1-Com+. Prior to transformation, the plasmid was linearized by digestion with SacI. The nourseothricin-resistant transformants were analyzed by PCR for the presence of a complete Bccrz1 copy using primers 12 and 16. The homologous integration of the Bccrz1 complementation constructs at the BcniaD locus was confirmed by Southern analysis; after digestion of the genomic DNAs of the transformants (Bccrz1, Com-7, and Com-9) and B05.10 with XbaI and hybridization with the BcniaD-5' flank, the BcniaD wild-type fragment was replaced by a smaller fragment, due to the additional XbaI restriction site within the complementation fragment (data not shown).

	RESULTS

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Identification of the B. cinerea CRZ1 orthologue. A similarity search using the CRZ1 protein sequence of S. cerevisiae and the program BlastP (1) in the protein database of the B. cinerea B05.10 genome project (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html) revealed 18 protein sequences that produced significant alignments (E values from 3e–43 to 6e–5) with a length of approximately 110 amino acids when standard parameters were used. Even though all these proteins contained two conserved zinc finger motifs (zf-C2H2 type), the respective domain of the annotated hypothetical protein BC1G_00093.1 appeared to be the best candidate, with an E value of 3e–43 and an amino acid identity of 64%, in contrast to the second hit, which displayed an E value of 5e–18 and an amino acid identity of only 41%. Thus, we investigated the first candidate gene in more detail and named it Bccrz1. The open reading frame of Bccrz1 contains 2,151 bp and one intron with a size of 51 bp, resulting in a protein with 716 amino acids. The protein sequence shows low rates of overall amino acid identity to the known CRZ1 proteins (32% to S. cerevisiae CRZ1, 30% to C. albicans CRZ1, 35% to Torulaspora delbrueckii CRZ1, and 28% to Schizosaccharomyces pombe PRZ1). An alignment of these protein sequences and of the putative CRZ1 sequence of B. cinerea is shown in Fig. 1, demonstrating the low level of similarity between the different sequences, except for the two highly conserved zinc finger domains in the C-terminal part of the protein. However, other known features of the CRZ1 protein of S. cerevisiae, such as the SRR domain in the N-terminal part or the calcineurin-docking domain (Fig. 1), were not found in the B. cinerea CRZ1 sequence. Since the similarities between the putative B. cinerea crz1 and the known yeast homologues were extremely low, we first expressed the Bccrz1 gene in the S. cerevisiae crz1 mutant to verify that we had cloned the functional homologue of yeast CRZ1.

View larger version (48K): [in this window] [in a new window]   FIG. 1. Alignment of known CRZ1 protein sequences of different yeasts and the putative CRZ1 orthologue of B. cinerea: C. albicans (XP_716600) (49), S. cerevisiae (P53968) (42), S. pombe (Q09838) (29), T. delbrueckii (AAZ04388) (28), and B. cinerea (BC1G_00093.1) (Botrytis cinerea Database [http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html]). Sequence alignment was done with ClustalW of the HUSAR sequence analysis package (http://genome.dkfz-heidelberg.de/) using standard parameters. The amino acids indicated by asterisks are conserved in all of the proteins shown. The amino acids of the zinc finger C2H2 domains are shaded gray, and the invariant cysteines and histidines within the zinc finger regions are shaded black. The SRR of S. cerevisiae CRZ1 is underlined, and the identified calcineurin (CN)-docking domains of S. cerevisiae and T. delbrueckii are indicated.

BcCRZ1 suppresses Na⁺ sensitivity of S. cerevisiae crz1 mutants.

View larger version (50K): [in this window] [in a new window]   FIG. 2. Heterologous expression of Bccrz1 in yeast. (A) Bccrz1 complementation of a crz1 mutation in S. cerevisiae. Yeast cells were grown to saturation in minimal medium without methionine and washed with sterile water, and serial 10-fold dilutions were dropped on YPD plates supplemented with 1.2 M NaCl or 1.2 M KCl. The plates were incubated at 30°C, and cell growth was recorded after 48 to 72 h. Bccrz15 and Bccrz16 are two independent yeast transformants. S. cerevisiae wild type (WT) and crz1 were transformed with the empty vector. (B) BcCRZ1 localization in yeast cells is regulated by Ca2+ and calcineurin. GFP-BcCRZ1-transformed yeast cells were grown overnight at 30°C in minimal methionine-free medium with NH4Cl (5 g/liter) as a nitrogen source. Exponential-phase cells were observed in a confocal microscope (Olympus Fluoview FV500) using an argon laser (488 nm) before and immediately after addition of Ca2+ (0.2 M CaCl2) to the culture. The cells were treated with FK506 (3 µg/ml) for 45 min before Ca2+ induction. The images were processed with Fluoview software version 5.

Generation of Bccrz1 mutants. To investigate the function of the putative BcCRZ1 transcription factor in B. cinerea, we created Bccrz1 deletion mutants using a replacement approach in which the whole open reading frame of Bccrz1 in the wild-type strain B05.10 was replaced by a hygromycin resistance cassette (Fig. 3A) (for details, see Materials and Methods). The homologous integration events at the Bccrz1 locus were detected for several transformants by diagnostic PCR. Homokaryotic knockout strains were obtained after one round of single-spore isolation and subsequent screening by PCR and Southern blot analysis for the absence of the Bccrz1 wild-type allele. Two transformants, termed Bccrz1-4 and Bccrz1-10, which had undergone homologous integration into the Bccrz1 locus without additional ectopic integrations of the replacement cassette into their genomes, were chosen for further studies (Fig. 3B).

Bccrz1 mutants are impaired in growth, morphology, conidiation, and sclerotium formation. Vegetative growth of the Bccrz1 deletion mutants on CM was severely affected, associated with significantly reduced conidiation (Fig. 4A). In contrast to the wild type, only small numbers of brightly colored conidia were observed, even after extended incubation for 2 to 3 weeks on CM agar under light conditions. The spores were morphologically indistinguishable from those of the wild type and showed normal germination on glass surfaces, induced by glucose or fructose (data not shown). To overcome the strong growth defect of the Bccrz1 deletion mutants, we tested several media containing host plant-derived compounds. On PDAB, the Bccrz1 mutant showed an improved growth rate, similar to that of the wild type, but neither aerial hyphae, conidiophores, nor mature conidia were formed. Moreover, after a 2-week incubation period under light, the mycelium became orange (data not shown) and was no longer able to grow when transferred to fresh medium. Interestingly, conidiation, regular colony morphology, and formation of aerial hyphae were improved to similar extents on medium containing undiluted grape juice (Fig. 4A) or vegetable (V8) juice (data not shown). However, the growth rate was still significantly reduced, resulting in the formation of small colonies compared to the wild type after 7 days of incubation. On CD minimal medium, the mutants were not able to grow at all (Fig. 5A).

View larger version (75K): [in this window] [in a new window]   FIG. 4. Impact of BcCRZ1 on growth, morphology, and development. (A) Growth, conidiation, and sclerotium formation on different media. The Bccrz1 mutant and the wild type (WT) were incubated for 1 week under light for conidiation and for 4 weeks at 21°C in darkness for sclerotium formation on CM, PDAB, and grape juice agar. (B) Hyphal morphology of Bccrz1 in comparison to the wild type. The strains were grown for 2 days on CM-overlaid microscope slides and then stained with calcoflour white and observed by epifluorescence microscopy. Negative prints are shown.

View larger version (44K): [in this window] [in a new window]   FIG. 5. Effect of Mg2+ supplementation on growth and development of Bccrz1 mutants. (A) Addition of MgCl2 specifically restores the growth rate and conidiation of Bccrz1. The strains were grown for 1 week on minimal agar (CD) containing 0.2 osmol/liter KCl (0.1 M), 0.2 osmol/liter NaCl (0.1 M), 0.2 osmol/liter CaCl2 (0.07 M), and 0.2 osmol/liter MgCl2 (0.07 M). WT, wild type. (B) Addition of MgCl2 does not restore sclerotium formation of Bccrz1. The mutant and the wild type were incubated on CM without or with 0.2 osmol/liter (0.07 M) MgCl2 for 4 weeks at 21°C in darkness.

Additional Mg²⁺ restores growth and conidiation, but not sclerotium formation. In contrast to S. cerevisiae CRZ1, which is essential only under elevated stress conditions and dispensable under standard culture conditions, B. cinerea CRZ1 was necessary to promote growth and normal vegetative differentiation on minimal medium, even without exposure to environmental stresses. To identify the nutritional basis for the growth defect of Bccrz1 mutants on minimal (CD) medium, we tested different compounds, such as amino acids, vitamins, salts, and microelements, in supplementation experiments for the ability to rescue the growth defect. Although we found slightly improved growth of the Bccrz1 mutants caused by different salts and amino acids in high concentrations, only the addition of MgCl₂ to the medium restored the wild-type phenotype with respect to growth rate and conidiation (Fig. 5A). To establish whether the remedial effect of MgCl₂ was specifically due to the Mg²⁺ ions and not merely to the osmotic effect of the salt, we tested other salts consisting of monovalent (NaCl and KCl) and divalent (CaCl₂) cations in concentrations leading to equivalent osmolarities (0.2 osmol/liter) in the different media. As shown in Fig. 5A, the addition of neither KCl, NaCl, nor CaCl₂ had an effect comparable to that of the MgCl₂ supplement on growth and conidiation, confirming the specific role of Mg²⁺ ions for growth of the Bccrz1 mutants. The fact that the addition of MgCl₂ to the medium did not overcome the sclerotium formation defect (Fig. 5B) suggests that not all features of the Bccrz1 phenotype can be explained by Mg²⁺ starvation.

Bccrz1 mutants are affected in cell wall and membrane integrity. Because the growth of the Bccrz1 mutant was improved on media with high osmolarity caused by different compounds, such as salts and amino acids, we examined the impacts of different osmolarities (0 to 1.6 osmol/liter) of KCl and the osmotic protectant sorbitol on radial growth of the Bccrz1 mutants in more detail. Previously, it had been shown that the cell wall integrity SLT2-type MAP kinase (BMP3) in B. cinerea is essential for hypo-osmotic stress tolerance (54). Therefore, we included the bmp3 mutant in our experiment for a direct comparison. While the bmp3 mutants showed growth rates almost equivalent to that of the wild type on CM with slightly increased KCl concentrations (0.2 osmol/liter), the Bccrz1 mutants grew poorly at low osmolarities. The growth of the Bccrz1 mutant was improved to almost the growth rates of the wild-type and the bmp3 strain only by osmolarities higher than 0.8 osmol/liter. Higher osmolarities caused by sorbitol enhanced the growth rate of the Bccrz1 mutant to an extent similar to that observed for KCl (Fig. 6A). In addition to the growth rate, the conidiation of the Bccrz1 mutants was partially restored by higher osmolarities, caused by either KCl or sorbitol (data not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 6. Impact of BcCRZ1 on cell wall integrity. (A) High-osmolarity conditions caused by salts or sugars improve the growth rates of Bccrz1 mutants. The wild type (WT), the Bccrz1 mutant, and the bmp3 mutant (bmp3 encodes the cell wall integrity MAP kinase) were grown on CM containing different amounts of KCl or sorbitol. Colony diameters were measured after 3 days of incubation. The values are averages from at least six colonies. (B) Hypersensitivity of Bccrz1 to treatment with cell wall-degrading enzymes. Conidiospores of the strains were incubated in liquid CM with or without 67 mM MgCl2 for 24 h. The washed mycelia were incubated for 1.5 h in osmotically stabilized solution containing Glucanex or β-glucanase. Protoplasts were counted microscopically, and the numbers of protoplasts per mg dry weight were determined. The data are the means of four replications. The error bars indicate standard deviations. (C) Expression of genes whose products are involved in cell wall biosynthesis. Wild-type B05.10 and Bccrz1 mutants were cultivated as described for panel B. The Northern blot was hybridized to radioactively labeled probes of the glucan synthase (BC1G_14034.1)- and 1,3-β-glucanosyltransferase (BC1G_14030.1)-encoding genes. Actin (BcactA) and rRNA were used as loading controls.

Since the Bccrz1 mycelium exhibited higher sensitivity only to glucanases and not to chitinases (data not shown), we suggested that the major impact of BcCRZ1 was on the glucan composition and not on the chitin content of the cell wall. In S. cerevisiae, calcineurin acts via CRZ1 as a positive regulator of the gene FKS2, encoding the key enzyme for synthesis of the β-1,3-glucan polymer, β-1,3-glucan synthase (85). In contrast to yeasts, in which two similar genes (FKS1 and FKS2) encode catalytic subunits of the glucan synthase complex and the deletion of both is lethal (31), the B. cinerea genome contains only a single gene (the protein is annotated as BC1G_14034.1). The expression of this gene was not altered in the Bccrz1 mutant under standard conditions in comparison to the wild type but was slightly down-regulated in Bccrz1 with MgCl₂ supplementation, suggesting a deregulation of gene expression in response to Mg²⁺ treatment (Fig. 6C). The expression of another gene (the protein is annotated as BC1G_14030.1) encoding a protein with similarity to the 1,3-β-glucanosyltransferase Gel4 of A. fumigatus, a glucan-elongating enzyme, was slightly affected by the Bccrz1 deletion independently of Mg²⁺ availability (Fig. 6C). In addition, the findings that the Bccrz1 mutants were less sensitive to the cell wall-interfering compound calcoflour white and that the wild type in the presence of this compound also responded to Mg²⁺ supplementation with decreased sensitivity (Table 2) support the suggestion that Mg²⁺ ions are able to stabilize impaired cell walls.

View this table: [in this window] [in a new window]   TABLE 2. Effects of stress conditions on growth rates of wild-type B05.10 and Bccrz1 strainsa

Bccrz1 mutants have altered stress responses. S. cerevisiae and C. albicans crz1 mutants were reported to be hypersensitive to several types of stress, including exposure to high cation concentrations (58, 68). In order to examine the stress responses of the Bccrz1 mutants, we performed plate assays using CM and MgCl₂-supplemented CM as basal media and applied different kind of stresses, such as pH stress (pH 3 and pH 9), osmotic stress (1.2 M sorbitol), oxidative stress caused by 10 mM H₂O₂ or 500 µM menadione, and concentrations of the cations Na⁺, Li⁺, Mn²⁺, and Ca²⁺ (1.0 M NaCl, 30 mM LiCl, 20 mM MnCl₂, and 0.4 M CaCl₂) that were sufficient to affect the growth rate of B. cinerea (Table 2). The responses of Bccrz1 mutants to osmotic stress (sorbitol and NaCl) and to oxidative stress caused by menadione, which leads to the generation of O₂^– ions, were not altered. However, the mutants were more sensitive to other stress conditions, such as extreme pH values and growth in the presence of high Li⁺ and Ca²⁺ concentrations, as well as oxidative stress caused by H₂O₂, than the wild type and the untreated mutant (CM control). Mg²⁺ supplementation restored wild-type-like growth at pH 9 and in the presence of Li⁺ but did not improve growth rates at pH 3 and during exposure to Ca²⁺ and H₂O₂. Interestingly, Mg²⁺ addition increased the tolerance of the wild type for Li⁺ and Mn²⁺, and the Bccrz1 mutant was less sensitive to Mn²⁺ than the wild type. These results indicate that BcCRZ1 is involved in oxidative-stress resistance and the regulation of ion homeostasis.

BcCRZ1 is required for full virulence on bean leaves, tomato fruits, and apricots. So far, no deletion mutants of the Ca²⁺/calcineurin signaling pathway have been generated and characterized in B. cinerea or related fungi. To study the role of BcCRZ1 in pathogenic development, we performed virulence assays on primary leaves of bean plants, using conidia or mycelia as an inoculum. Surprisingly, the conidia of the mutant germinated in a wild-type-like manner even in minimal medium, and the mutant was able to complete the whole infection cycle in planta, starting with the appearance of primary lesions after 2 days, formation of spreading lesions after 3 days, and soft rot of the whole plant after 7 days, associated with the production of numbers of conidia equivalent to the wild type (Fig. 7A). However, the lesion diameters were slightly reduced (approximately 20%) 2, 3, and 4 days postinoculation in comparison to the wild type, demonstrating that BcCRZ1 is required for full virulence.

View larger version (84K): [in this window] [in a new window]   FIG. 7. Virulence assays on different host plants. (A) Virulence of Bccrz1 conidia on bean plants. Primary leaves were inoculated with 7-µl droplets of conidial suspensions (2 x 105/ml in Gamborgs B5 plus 2% glucose) of the wild type (WT) and the two Bccrz1 mutants. The lesion diameters were determined after 2, 3, and 4 days of incubation (dpi). Mean values were calculated from 26 lesions. The error bars indicate standard deviations. (B) Virulence of Bccrz1 mycelium on intact and wounded bean plants. Leaves were inoculated with agar plugs (CM with or without 67 mM MgCl2) colonized by nonsporulating, 3-day-old mycelia of the wild type, the Bccrz1 mutant, and the complemented mutant Com-7. The leaves were wounded with a needle prior to the inoculation. (C) Virulence of Bccrz1 conidia and mycelia on intact and wounded tomato fruits (left) and apricots (right). Precultivation of the wild-type strain and the two Bccrz1 mutants for conidium- and mycelium-derived infection (A and B). The photographs were taken 4 days postinoculation (dpi). Infected areas are highlighted by dashed lines.

Bccrz1 mutants still respond to the calcineurin inhibitors cyclosporine and FK506. Cyclosporine and FK506 are inhibitors of calcineurin and act in B. cinerea by binding to the specific peptidyl-prolyl isomerases cyclophilin A (BCP1) and FKBP12 (BcPIC5), respectively (23, 75), and subsequent binding of these protein-drug complexes to the active site of calcineurin. If CRZ1 is the only substrate for calcineurin, we would expect increased resistance of the Bccrz1 mutants to these inhibitors, because the substrate for calcineurin is missing. However, if there are more targets of calcineurin activity, the mutants should still respond to these inhibitors with retarded growth. In order to explore this hypothesis, we performed plate assays using the inhibitors cyclosporine and FK506 with and without Mg²⁺ supplementation (Fig. 8). Treatment with these inhibitors had a profound impact on the growth of both the wild type and the Bccrz1 mutant, resulting in the formation of small, compact, sporulating colonies after 3 weeks of incubation. Without Mg²⁺, the growth retardation of the mutant was similar to that of the wild type. However, when the medium was supplemented with Mg²⁺, the mutant appeared to be more resistant to both inhibitors but still responded with significantly reduced growth to the inhibition of calcineurin (Fig. 8). Hence, the presence of other signaling effectors of calcineurin besides BcCRZ1 is proposed for B. cinerea.

View larger version (92K): [in this window] [in a new window]   FIG. 8. Effects of calcineurin inhibitors on growth of the wild type (WT) and the Bccrz1 mutants. The strains were grown on CM without or with 67 mM MgCl2 containing cyclosporine (CsA) (0.5 µg/ml) or FK506 (0.05 µg/ml). The photographs were taken after 3 weeks of incubation.

To study the effect of Mg²⁺on BcCRZ1-dependent gene expression, we cultivated the strains on CM supplemented with 67 mM MgCl₂ and tested the expression of several chosen target genes by Northern blot analyses (Fig. 9). To confirm our recent finding that BCG1 is an upstream regulator of calcineurin signaling (61), we included the bcg1 mutant in our expression studies. Different expression patterns of BcCRZ1-dependent genes were found. One group of genes whose expression was dependent on calcineurin and the G subunit BCG1 but was unaffected by Mg²⁺ (Table 3 and Fig. 9) included the genes for botrydial biosynthesis (e.g., Bcbot1/CND5) and for the production of another as yet unknown secondary metabolite with polyketide structure (e.g., P450-1) (61). Another group represented genes whose expression was found to be dependent on calcineurin activity but unaffected by the bcg1 deletion, as well as Mg²⁺ supplementation. As examples, CND12, exhibiting similarity to the calcium ion transporter VCX1-encoding gene of S. cerevisiae, and CND4, without significant homology to known genes, are shown (Fig. 9). A third group comprised genes whose expression was affected in bcg1, as well as in Bccrz1, strains. Interestingly, the expression of these genes (e.g., contig 1310 and contig 1002) can be significantly increased in the Bccrz1 mutants through Mg²⁺ supplementation, confirming the impact of Mg²⁺ on gene expression in Bccrz1. Also, the expression of some genes, e.g., the endopolygalaturonase 1-encoding gene Bcpg1/CND2, is independent of BCG1 but down-regulated in Bccrz1 without Mg²⁺ and more highly expressed with Mg²⁺ addition. Moreover, the up-regulation of protease-encoding genes (contig 934, contig 902, contig 1515, and contig 783) was noticeable in Bccrz1 (Table 3).

View larger version (83K): [in this window] [in a new window]   FIG. 9. Expression of several BcCRZ1-dependent genes in the wild-type (WT) and mutant strains. The wild-type B05.10, the bcg1 and Bccrz1 mutants, and the complemented mutant Bccrz1 Com-7 were grown for 3 days on CM agar with or without 67 mM MgCl2. The Northern blot was hybridized to radioactively labeled probes of several BcCRZ1-dependent genes (listed in Table 3). rRNA was used as a loading control.

Complementation of Bccrz1 mutants restores the wild-type phenotype. To confirm that the severe phenotype of Bccrz1 is exclusively caused by the inactivation of Bccrz1, a complementation approach was used. For this, the mutant Bccrz1-4 was transformed with the vector pBccrz1-Com+, containing the cDNA sequence of Bccrz1 under the control of the native Bccrz1 promoter, and the gene flanks of BcniaD, encoding nitrate reductase, for the targeted integration of the construct at the respective gene locus. Putative complemented transformants, selected based on their noureothricin resistance, were analyzed by PCR for the complete integration of the Bccrz1 copy. For two transformants, Com-7 and Com-9, the homologous integration event at the BcniaD locus was confirmed (data not shown). Both transformants showed full restoration of the wild-type phenotype with respect to vegetative growth, conidiation, sclerotium formation, virulence on bean plants (Fig. 7B), and gene expression (Fig. 9). Moreover, the growth rates of the complemented mutants in the presence of extreme pH values, LiCl, CaCl₂, and H₂O₂ were comparable to those of the wild type (data not shown). These data clearly show that the described phenotype of the Bccrz1 mutant is caused by the targeted inactivation of the corresponding gene.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study is the first to report the identification and characterization of the putative calcineurin-responsive transcription factor from a filamentous fungus and describes the involvement of the calcineurin/CRZ1 signaling pathway in vegetative growth, differentiation, expression of secondary metabolite genes, cell and membrane integrity, the response to different environmental stress conditions, and virulence in the plant pathogen B. cinerea.

BcCRZ1 acts as a downstream target of (yeast) calcineurin. We identified one putative CRZ1 homologue in the B. cinerea genome that displayed significant similarity to the C-terminal DNA-binding domain (containing two C2H2-type zinc finger motifs) of the S. cerevisiae CRZ1 protein. Several pieces of evidence supported our suggestion that the identified B. cinerea protein BC1G_00093.1 indeed encodes the functional homologue of the yeast calcineurin-regulated transcription factor CRZ1. First of all, the transformation of the S. cerevisiae crz1 mutant with the full-length cDNA of Bccrz1 under the control of the yeast MET25 promoter showed that the B. cinerea gene functionally complemented the yeast mutation during growth under high Na⁺ concentrations. However, the BcCRZ1-transformed yeast cells were still sensitive to Mn²⁺ and Ca²⁺. In contrast, the complementation of crz1 yeast cells with the C. albicans crz1 gene (Cacrz1) led to suppression of sensitivity to Na⁺, Mn²⁺, and Ca²⁺ (58), indicating that the CRZ1 transcription factor possibly has distinct, as well as common, functions in yeasts and filamentous fungi. We propose that the suppression of the Na⁺ sensitivity by expressing Bccrz1 in crz1-defective yeast cells is based on the BcCRZ1-mediated transcriptional activation of the gene ENA1, encoding the ATP-driven ion pump, which is induced and activated by toxic concentrations of Na⁺ and Li⁺, promoting the extrusion of these ions from the cell (41, 43). The partial restoration of the wild-type phenotype by the B. cinerea homologue suggests the potential conservation of the CRZ1-binding motifs in the respective promoter sequence(s). A key factor of CRZ1 regulation by dephosphorylation is the targeting of calcineurin to the conserved PXIXIT motif (the calcineurin-docking domain). Although the calcineurin-docking domains are highly conserved in yeast and mammalian calcineurin targets (NFAT transcription factor family), no obvious docking domain was found in the sequence of the B. cinerea CRZ1 homologue. However, the localization studies of a GFP-BcCRZ1 fusion protein in yeast cells clearly show that the subcellular distribution of BcCRZ1 is regulated by Ca²⁺ in a calcineurin-dependent manner: activation of calcineurin by Ca²⁺ addition to the cells promotes the transport of the cytosolic GFP-BcCRZ1 into the nucleus, whereas calcineurin inhibition by treatment with FK506 impairs the accumulation of GFP-BcCRZ1 in the nucleus. Similar results were reported for C. albicans CRZ1. The GFP-CaCRZ1 fusion protein in S. cerevisiae cells showed the same localization pattern, and CaCRZ1 undergoes posttranslational modifications due to calcineurin activity (34, 58). Taken together, these data confirm our suggestion that BcCRZ1 is indeed a target of the calcineurin phosphatase and the functional homologue of yeast CRZ1.

Functions of CRZ1 in B. cinerea. The BcCRZ1 deletion caused a pleiotropic phenotype characterized by abnormal vegetative growth (reduced growth and altered hyphal morphology) and differentiation (reduced conidiation and sclerotium formation). The strength of the phenotype depends on the cultivation conditions: while almost no growth has been observed on synthetic minimal medium, supplemented media, such as CM or grape juice agar, were found to promote growth. In addition, the cell wall and membrane integrity of the mutant was affected, leading to higher susceptibility to cell wall-degrading enzymes and the anionic detergent SDS, which could be the explanation for the pleiotropic phenotype mentioned above. Interestingly, the MAP kinase mutant (bmp3) showed significant similarities to the Bccrz1 strain in regard to the reduction of conidiation and sclerotium formation and poor growth in vitro under low-osmolarity conditions (54), indicating that different signaling pathways might regulate these differentiation processes. As discussed below in more detail, almost all features of the Bccrz1 mutant could be restored by specific growth conditions, such as feeding with additional MgCl₂, demonstrating that the mutant responds to its environment in a different manner than the wild type, which obviously did not respond to the MgCl₂ supplementation.

Due to the sensitivity of the Bccrz1 mutants to glucanases and the decreased expression of a gene involved in the elongation of glucan chains, we suppose that mainly the glucan backbone of the fungal cell wall is affected by the Bccrz1 deletion. However, an influence of calcineurin and BcCRZ1 on chitin synthesis cannot be excluded, since four chitin synthase promoters were shown to be activated by exogenous Ca²⁺ in a calcineurin/CaCRZ1-dependent manner in C. albicans (46). The fact that the inhibition of calcineurin in the closely related fungus S. sclerotiorum resulted in reduction of the β-1,3-glucan content and hypersensitivity to cell wall-degrading enzymes and the glucan synthase inhibitor caspofungin (26) corroborates our hypothesis. Previous studies in S. cerevisiae and C. albicans have elucidated a role for calcineurin signaling in response to membrane stress caused by detergents and antifungal azoles, such as fluconazole (4, 10, 11, 17, 34, 49, 58). This function seems to be conserved in B. cinerea, because the Bccrz1 mutant is more sensitive to SDS in an Mg²⁺-independent manner and to fluconazole in an Mg²⁺-dependent manner.

Other characteristics of the Bccrz1 mutant, such as sensitivity to oxidative stress caused by H₂O₂, in either the presence or the absence of Mg²⁺, differ from the findings in S. cerevisiae, in which hypersensitivity to H₂O₂ was observed only for calcineurin mutants (45). A correlation between oxidative-stress response and Ca²⁺ signaling was demonstrated for the expression of the yeast phospholipid hydroperoxide glutathione peroxidase GPX2, a part of the antioxidant system protecting cells from oxidative stress. The H₂O₂-induced expression of GPX2 was found to be strictly regulated by the transcription factor YAP1 and the response regulator SKN7. In addition, the expression of GPX2 was inducible by Ca²⁺ in a calcineurin- and CRZ1-dependent manner (72, 73). SKN7 has been shown to be a multicopy enhancer of calcineurin- and CRZ1-dependent transcription in yeast, proposing a model in which SKN7 regulates calcineurin signaling through the stabilization of CRZ1 via a direct protein-protein interaction (82). Due to the fact that Bccrz1 mutants are hypersensitive to H₂O₂, it is most likely that similar interconnections between Ca²⁺-dependent signaling and oxidative-stress response also exist in B. cinerea.

The response of the B. cinerea crz1 mutants to the cations Mn²⁺ and Li⁺ is complex and differs from the yeast system. Although the B. cinerea mutants were more sensitive to Li⁺ when Mg²⁺ was missing, the mutants appeared to be more resistant to Mn²⁺ ions than the wild type, and moreover, the resistance of the wild type to Mn²⁺ and Li⁺ was increased by Mg²⁺ supplementation. The fact that the stress-activated MAP kinase BcSAK1 mutants were more sensitive to NaCl than the wild type (62) suggests that NaCl tolerance is mainly regulated via this MAP kinase pathway in B. cinerea. However, overexpression of the crz1 gene from the salt-tolerant yeast T. delbrueckii in S. cerevisiae enhanced tolerance for Na⁺ in wild-type cells and suppressed sensitivity to Mn²⁺, Na⁺, and Li⁺ in crz1 and calcineurin mutants, but surprisingly, Tdcrz1 mutants were insensitive to high Na⁺ and more tolerant of Li⁺ than wild-type cells (28). All these data show that the calcineurin/CRZ1 signaling pathway has some conserved functions, such as the regulation of cell wall/membrane integrity and the general stress response and some fungus-specific functions, reflecting the evolution of calcineurin/CRZ1 signaling output due to the adaptation of the organisms to their environmental niches.

Pathogenicity assays with the B. cinerea mutants showed that BcCRZ1 (and probably also calcineurin) is required for full virulence of B. cinerea on several host systems, such as bean plants, detached tomato leaves, tomato fruits, and apricots. While the transcription factor seems to be almost dispensable for the conidium-derived infection program, BcCRZ1 is essential for penetration of hyphae, indicating distinct modes of penetration of the plant surface by freshly germinated spores and by growing mycelium. The regulatory systems governing the different penetration methods are still unclear, although the phenomenon of different infection properties due to inoculation with conidia or mycelium has been observed previously (61). We cannot exclude the possibility that this effect is due to the impaired cell wall integrity of Bccrz1 mutants, since bmp3 mutants (loss of cell wall integrity MAP kinase) were also defective in mycelium-derived infection (data not shown), while spores were still able to infect the plant (54). Moreover, B. cinerea chitin synthase (Bcchs1 or Bcchs3a) mutants exhibiting reduced chitin contents in their cell walls were also shown to be affected in virulence (66, 67). The fact that Mg²⁺