Calcineurin, a conserved Ca2+/calmodulin-regulated protein phosphatase, is an important mediator of calcium-dependent signal transduction pathways in many organisms. In Saccharomyces cerevisiae, calcineurin positively regulates transcription in response to stress by dephosphorylating the transcription factor Crz1p. Here we describe the identification, cloning, and function of the gene encoding the Magnaporthe grisea CRZ1 homolog, MgCRZ1. Specifically, we demonstrated that MgCRZ1 partially complemented a yeast Δcrz1 mutant and exhibited Ca2+ and calcineurin activity-dependent cellular localization. Targeted disruption of MgCRZ1 resulted in hypersensitivity to Ca2+. Compared with the wild-type Guy11 strain, the Δcrz1 mutants formed significantly reduced numbers of conidia and a large portion of abnormal appressoria (>50%) that exhibited little or no melanin production. Lipid metabolism was delayed, and the level of turgor pressure within the appressoria declined, thereby notably attenuating mutant pathogenicity. We conclude that MgCRZ1 is essential for growth, development, and full virulence of M. grisea.
Magnaporthe grisea causes blast disease, which is the most destructive of cultivated rice diseases worldwide, and infects a wide range of grass hosts, including barley, wheat, and finger millet (Talbot, 2003). To initiate blast disease, M. grisea undergoes a series of defined morphogenetic steps, leading to the production of numerous appressoria, which are specialized dome-shaped infection cells harboring a complex cell wall structure (Talbot, 2003; Ebbole, 2007). Subsequently, internal glycerol levels rise to about 3 M, and enormous turgor pressure (8 MPa) is generated within the appressorium, providing the invasive force that allows the penetration infection peg to breach the plant cuticle (Howard et al., 1991; Talbot, 2003). The appressorium cell wall is rich in chitin and contains a layer of melanin on the inner side of the wall. Melanin production is a virulence characteristic of many pathogenic fungi (Henson et al., 1999). Several M. grisea mutants, including albino, buff, and rosy, are defective in melanin biosynthesis and are nonpathogenic because their appressoria lack melanin deposition and cannot support high turgor pressure (Chumley & Valent, 1990; Talbot, 2003). During appressorium maturation in these mutants, the lipid bodies mobilize quickly from the conidium to the germ tube apex and to the incipient appressorium on the leaf surface. Subsequently, the lipid bodies coalesce and are absorbed by vacuoles in the appressorium, where they are eventually degraded to fatty acids and glycerol (Weber et al., 2001). Therefore, a requirement for fatty acid β-oxidation and subsequent activation of the glyoxylate cycle and gluconeogenesis has been proposed (Thines et al., 2000; Weber et al., 2001). Consistent with this idea, a recent study has shown that peroxisomal fatty acid β-oxidation is required for appressorium-mediated plant infection (Wang et al., 2007).
Calcineurin is a highly conserved protein phosphatase heterodimer, comprising catalytic (CNA) and regulatory (CNB) subunits that are activated by binding to the Ca2+/calmodulin complex when the cytosolic Ca2+ level is increased (Rusnak & Mertz, 2000; Fox & Heitman, 2002; Cyert, 2003; Kraus & Heitman, 2003). In animals and fungi, calcineurin regulates a variety of physiological processes such as cell cycle progression, polarized growth, and stress adaptation. In the budding yeast Saccharomyces cerevisiae, calcineurin is activated by extracellular stresses such as exposure to high concentrations of Na+, Li+, Mn2+, and OH−, but is dispensable for growth under standard culture conditions (Mendoza et al., 1996; Matheos et al., 1997; Cunningham & Fink, 1996). Deleting CNA1 in the filamentous fungus Aspergillus fumigatus resulted in defective hyphal morphology, sporulation, and pathogenicity (Steinbach et al., 2006). A recent study used a high-throughput RNA silencing system to analyze calcium-signaling proteins such as Ca2+-permeable channels, Ca2+ pumps, Ca2+ exchangers, phospholipase C, calmodulin, calcineurin (regulatory and catalytic subunits), Ca2+ and CAM binding proteins, calreticulin/calnexin, and calpactin heavy chain in M. grisea. Most of these silenced mutants showed growth, sporulation and virulence defects, and the CNA- and CNB-silenced mutants exhibited severe growth defects, including low conidium production, low appressorium formation, and almost no pathogenicity (Nguyen et al., 2008). In the pathogenic basidiomycete Cryptococcus neoformans, calcineurin is required for growth at 37 °C, virulence, and mating (Odom et al., 1997; Fox et al., 2001; Steinbach et al., 2007). Absence of CNA1 affects the colony morphology of Candida albicans in several media (Sanglard et al., 2003). Gene silencing or using the drugs cyclosporin A (CsA) or FK506 suppresses calcineurin activity in other fungi such as Sclerotinia sclerotiorum and Botrytis cinerea, suggesting that calcineurin is required for vegetative differentiation, cell wall integrity, and virulence (Viaud et al., 2003; Harel et al., 2006). Although most research studies have focused on the calcineurin-dependent signal transduction pathway in S. cerevisiae, C. albicans, A. fumigatus, and B. cinerea, little is known about this pathway and its downstream targets in M. grisea.
In S. cerevisiae, C. albicans, A. fumigatus, and B. cinerea, calcineurin regulation of the cellular localization and transcription factor activity of Crz1p is well characterized (Stathopoulos-Gerontides et al., 1999; Santos & de Larrinoa, 2005; Schumacher et al., 2008; Soriani et al., 2008). The phosphorylated form of Crz1p accumulates in the cytosol under resting conditions. Calcineurin dephosphorylates Crz1p and regulates its localization to the nucleus (Stathopoulos-Gerontides et al., 1999; Karababa et al., 2006). The manner in which Crz1p is regulated by calcineurin is similar to that of nuclear factor of activated T cells; as the Ca2+ concentration in the cytosol increases, Crz1p rapidly translocates from the cytosol to the nucleus (Stathopoulos-Gerontides et al., 1999). Crz1p contains a C2H2 zinc finger motif that binds to a 24-bp promoter element known as the calcineurin-dependent response element to activate transcription of its target genes and to direct Ca2+- and calcineurin-dependent gene expression (Matheos et al., 1997; Stathopoulos & Cyert, 1997). Here we identified the CRZ1 homolog of M. grisea, MgCRZ1, and present direct evidence that MgCRZ1 encodes a CRZ1 homologous protein that can partially complement the yeast Δcrz1 mutant that suppresses the Li+ sensitivity in S. cerevisiae. We also demonstrate that the cellular distribution of an MgCRZ1::green fluorescent protein (GFP) fusion protein depends upon the Ca2+ level and calcineurin activity. The mutants of our targeted gene deletion study displayed hypersensitivity to Ca2+, reduced conidiation, aberrant appressorium formation on inductive surfaces, a turgor pressure deficiency, delayed lipid metabolism in conidia and appressoria, and attenuated virulence, suggesting that MgCRZ1 is required for growth and development and is a crucial virulence determinant in this model phytopathogen.
Materials and methods
Homologous sequence alignment and phylogenetic analysis
All known and putative CRZ1 protein sequences were downloaded from the database at the Broad Institute (http://www.broad.mit.edu/annotation/fgi/). Protein sequences were aligned and phylogenetic trees were constructed using clustal w 1.83.
Strains and culture conditions
We used the M. grisea strain Guy11 as our wild-type strain. All strains were cultured on complete medium (CM: 50 mL 20 × nitrate salts, 1 mL trace elements, 10 g d-glucose, 2 g peptone, 1 g yeast extract, 1 g casamino acids, 1 mL vitamin solution, 15 g agar in 1 L distilled water) at 28 °C. Mycelia used for genomic DNA, and total RNA extractions were harvested from cultures grown in liquid CM for 2 days. To promote conidiation, the strain blocks were cultured on straw decoction and corn (SDC) medium (100 g straw, 40 g corn powder, 15 g agar in 1 L distilled water) for 7 days in the dark, followed by 3 days of continuous illumination.
The wild-type strain Y00000 was conserved in our laboratory, and the mutant strain Y15353 was obtained from EUROSCARF (Frankfurt, Germany). The CRZ1 cDNA was amplified and cloned into pYES2 (pYES2:MgCRZ1). After sequence verification, the DNA constructs were introduced into the S. cerevisiaeΔcrz1 mutant using the lithium-acetate method (Gietz et al., 1995). The yeast cells were grown in YPD liquid medium (2% glucose, 2% peptone, and 1% yeast extract) or SD solid medium (2% galactose, 0.17% yeast nitrogen base without amino acids and ammonium sulfate, and 2% agar) supplemented with the amino acids required by the strains, and incubated for 3 or 5 days at 30 °C.
Targeted disruption and complementation of CRZ1
For constructing the CRZ1 gene replacement construct, we amplified two c. 1-kb flanking sequences of CRZ1 with the primers FL828 and FL829 (FL828: 5′-ATGGATCAGTTGCAGCACCACCAG-3′; FL829: 5′-ACACGAACGGCCTCTCATCAGTGTGTTTAAACGCATGAAGCTACTGTTGGGTTGGT-3′) and FL830 and FL831 (FL830: 5′-ACCAACCCAACAGTAGCTTCATGCGTTTAAACACACTGATGAGAGGCCGTTCGTGT-3′; FL831: 5′-GTAGATCCGTGGAACCATCACAGG-3′). We then amplified a fragment of c. 2 kb consisting of two flanking sequences with the FL828 and FL831 primers and cloned this segment into a pMD19-T vector (Takara Co., Dalian, China). We inserted the last 1.4 kb of a hygromycin-resistance gene cassette, which encodes hygromycin phosphotransferase under the control of the A. nidulans TrpC promoter (Carroll et al., 1994), into the pMD19-T vector between the two flanking sequences to generate the targeted gene replacement vector. The 3.4-kb fragment that was amplified with the FL828 and FL831 primers was transformed into the Guy11 protoplasts. All amplified fragments were verified by sequencing. To generate the complementation of the Δcrz1 mutant, we amplified a fragment of roughly 3.9 kb that included the promoter region and the entire ORF and inserted this segment into the vector pCB1532 containing a sulfonylurea (Sur) resistance gene.
Appressorium formation and turgor assay
The conidia were harvested from 10-day-old cultures, filtered through three layers of lens paper, and resuspended to a concentration of 5 × 104 spores mL−1 in sterile water. Droplets (30 μL) of the conidial suspension were placed on plastic coverslips and incubated under humid conditions at room temperature, and the samples were microscopically observed at intervals. The appressorium turgor was measured using an incipient cytorrhysis (cell collapse) assay and a 1.0–4.0 M glycerol solution (Howard et al., 1991). Droplets (20 μL) of the conidial suspension (5 × 104 spores mL−1) were placed on plastic coverslips and incubated in a humid chamber for 24 h at room temperature. The water surrounding the conidia was removed carefully and then replaced with an equal volume (20 μL) of glycerol in concentrations ranging from 1.0 to 4.0 M. The number of appressoria that had collapsed after 10 min was recorded. The experiments were repeated three times, and >100 appressoria were observed for each replicate.
Lipid droplet observation
The lipid droplets in the germinating conidia and appressoria of the Guy11 and mutants were visualized by staining these tissues with a Nile Red solution consisting of 50 mM Tris/maleate buffer (pH 7.5), 20 mg mL−1 polyvinylpyrrolidone, and 2.5 μg mL−1 Nile Red Oxazone (9-diethylamino-5H-benzo-α-phenoxazine-5-one, Sigma; Weber et al., 1999; Thines et al., 2000; Wang et al., 2007). The conidial suspension and appressoria were prepared as described above. The samples were removed at intervals to observe appressorium formation and lipid mobilization. Within a few seconds, the lipid droplets in the conidia and appressoria began to fluoresce as we observed them under a microscope with an episcopic fluorescence attachment.
We harvested the conidia from 10-day-old cultures, filtered them through three layers of lens paper and resuspended them to 5 × 104 spores mL−1 in sterile water supplemented with 0.2% (w/v) gelatin. We used the leaves from 7-day-old barley (cv. four-arris) seedlings for the cut-leaf assay, for which a 20-μL droplet was placed onto the upper side of the cut leaves maintained on 4% (w/v) water agar plates. The leaves were observed after 3–5 days of incubation at 25 °C. For the spray inoculation, we used 2-week-old seedlings from the blast-susceptible rice variety CO-39. For this assay, we sprayed 5 mL of the conidial suspensions from each treatment evenly onto the plants with a larynx-like sprayer. The inoculated plants were kept in a growth chamber at 25 °C and 90% humidity in the dark for the first 24 h, followed by a 12 h/12 h light/dark cycle exposure. We observed the progression of lesion development daily, documenting lesion growth with photographs and counting them 7–10 days postinoculation (Fang & Dean, 2000).
Cuticle penetration assay
The conidial suspensions for each treatment were prepared as described above. Droplets (20 μL) of the suspensions were placed on strips of onion epidermis, incubated under humid conditions at room temperature for 24 h, and observed microscopically for elaboration of the penetration hyphae.
Characterization of the M. grisea CRZ1 homologs
The blastp program was used to search the M. grisea genome database (http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/Blast.html) using S. cerevisiae CRZ1 as the query. Thirty-one protein sequences produced significant alignments (P=7.4e−44 to 5.2e−5). All 31 sequences contained two conserved zinc finger (zf-C2H2 domain) motifs; of these, the annotated hypothetical protein MGG_05133.5 (MgCRZ1; P=7.4e−44) was selected for further analysis. MgCRZ1 is a putative peptide of 727 amino acids with low identity to known CRZ1 proteins in various organisms, including S. cerevisiae (19%), A. fumigatus (36%), B. cinerea (49%), C. albicans (18%), and C. neoformans (15%). The sequence identities are similar primarily in the two C2H2 zinc finger domains in the C-terminal end of the proteins. The proteins were aligned and a phylogenetic tree constructed (see Supporting Information, Fig. S2a). The M. grisea MgCRZ1 sequence did not contain the exact calcineurin-docking domain (PxIxIT-related motif) of S. cerevisiae (see Fig. S1). Furthermore, Southern blot analysis showed only one copy of CRZ1 in the entire M. grisea genome (see Fig. S3a).
MgCRZ1 suppressed Li+ sensitivity of S. cerevisiaeΔcrz1 mutants
In previous studies, deletion of CRZ1 in S. cerevisiae resulted in heightened sensitivity of this yeast strain to many cations, including Na+, Li+, Mn2+, and Ca2+, as well as hydroxyl anions. The S. cerevisiae cells showed a growth defect when cultured in media containing only a moderate concentration of these ions (Matheos et al., 1997; Stathopoulos & Cyert, 1997). To explore whether MgCRZ1 can rescue the yeast Δcrz1 mutant, we transformed the MgCRZ1 cDNA under the control of the GAL1 promoter into the yeast Δcrz1 mutant strain. The transformants grew in the presence of high concentrations of Na+ (0.8–1.2 M) and Mn2+ (6–10 mM) and exhibited no physical differences compared with the wild-type strain. However, in the plates supplemented with high Li+ concentrations (0.1–0.2 M), the MgCRZ1-transformed cells showed improved growth, indicating that the MgCRZ1 gene could endure Li+ stress in this yeast Δcrz1 mutant (see Fig. S2b). These results suggested that the MgCRZ1 gene encoded a CRZ1-homologous protein that could partially complement the Δcrz1 mutant, which suppressed the Li+ sensitivity in S. cerevisiae.
Targeted disruption of MgCRZ1
To determine the functionality of the predicted transcription factor MgCRZ1 in M. grisea, we generated mutants with CRZ1 gene deleted. The targeted gene deletion vector pMD19-T::CRZ1 was constructed by replacing part of the CRZ1 ORF with the hph gene cassette and introduction into the wild-type strain. The hygromycin-resistant colonies were isolated and confirmed, using PCR and Southern blots, that two positive isolates contained the MgCRZ1 deletion sequence (see Fig. S3a). Further confirmation of the targeted deletions for two Δcrz1 isolates was obtained by reverse transcriptase-PCR to amplify fragments within the deleted region of the gene. As expected, the cDNAs encoding CRZ1 could not be amplified from the Δcrz1 mutants (see Fig. S3b).
MgCRZ1 mutant is more sensitive to calcium
In previous studies, stress-sensitive Δcrz1 mutants were functionally characterized in S. cerevisiae, A. fumigatus, and B. cinerea (Stathopoulos & Cyert, 1997; Schumacher et al., 2008; Soriani et al., 2008). To clarify whether MgCRZ1 possesses the same stress sensitivity, we tested the wild-type Guy11 and mutant strains on plates with different kind of stresses, such as osmotic stress (sorbitol), oxidative stress (H2O2), and concentrations of the cations Na+, Li+, Mn2+, and Ca2+, which were sufficient to affect growth of M. grisea. Consistent with the previous results, the Δcrz1 mutants showed a higher sensitivity to Ca2+ (0.2–0.6 M) than did the wild-type strain. However, the mutants did not alter their responses to other stress conditions (Na+, Li+, and Mn2+) or to oxidative stress (H2O2; Fig. 1). These results revealed that MgCRZ1 is required for Ca2+ stress tolerance in M. grisea.
MgCRZ1 is required for normal appressorium shape and conidiogenesis of M. grisea
The conidia produced by the Δcrz1 mutant were morphologically indistinguishable from the wild-type Guy11 conidia. However, the conidiation of the Δcrz1 mutants was reduced to 15.5% of that of Guy11 (Fig. 2a and b). At 24 h postincubation, over 50% of the germinated conidia produced abnormal appressoria that displayed little or no melanin, and numerous large lipid droplets were observed in the conidia, germ tubes, and abnormal appressoria during appressorium maturation (Fig. 2c).
MgCRZ1 is essential for full virulence
To investigate the role of MgCRZ1 in pathogenesis, conidial suspensions were placed or sprayed onto two susceptible hosts, barley and rice (cv. CO-39) seedlings. A pathogenicity assay showed that the virulence of the Δcrz1 mutants was remarkably reduced. Eight days postinoculation, the typical lesions seen in the Δcrz1 mutants were greatly reduced compared with those in the wild-type Guy11 (Fig. 3c). Most of the lesions in the wild-type strain coalesced later, whereas those in the Δcrz1 mutant remained small and isolated (Fig. 3b). Following inoculation with the Δcrz1 mutant conidial suspension, the barley samples exhibited very slight lesions, whereas the wild-type Guy11 induced very apparent lesions (Fig. 3a). Consistent with these results, the onion epidermal penetration assay revealed that the wild-type Guy11 appressoria could successfully penetrate the onion epidermis and spread rapidly within 24 h, whereas most of the mutant appressoria failed to penetrate and spread through the onion cells (Fig. 3d), indicating that the Δcrz1 mutant could attenuate M. grisea pathogenicity. These data demonstrated that MaCRZ1 was essential for full virulence.
MgCRZ1 is involved in lipid metabolism
During appressorium development, a vast number of large lipid droplets appeared in the appressoria and conidia of the mutants. Those lipid droplets were transported rapidly from the conidia to the nascent appressoria during appressorium development. To determine whether impairment of MgCRZ1 could affect mobilization and degradation of the lipid droplets, we assayed the intracellular lipid stores with Nile Red staining during appressorium morphogenesis of the Δcrz1 mutants. In the wild-type Guy11 strain, the lipid droplets were transported to the appressorium within 6 h. During appressorium maturation, we observed large lipid deposits within the appressoria, where they coalesced and were absorbed into the vacuoles. Lipid degradation occurred rapidly during appressorium maturation (8 h), and the fully melanized appressoria were formed 12–24 h after germination and were almost devoid of lipid droplets. Interestingly, we observed bright, large lipid droplets in the mutants during the late stages of appressorium formation. Compared those in the wild-type Guy11 strain, the lipid droplet mobilization was decelerated in the mutants, and the droplets were more evenly distributed throughout conidia and germ tubes. Even after 48 h, some undegraded lipid droplets remained in the conidia, germ tubes, and appressoria (Fig. 4). These results indicated that impairment of CRZ1 function prevented the appressorium from undergoing lipolysis, and affected the capability of the appressorium to penetrate the host cuticle.
Measurement of appressorium turgor pressure in the Δcrz1 mutant
Appressorium-mediated penetration requires very high internal turgor pressure to facilitate sufficient generation of the mechanical force required to breach the rice leaf cuticle (Wang et al., 2007). Appressorium turgor can be measured with an incipient cytorrhysis assay, which uses hyperosmotic concentrations of a solute to collapse the appressoria, thereby allowing empirical estimation of their internal solute concentration and turgor (Howard et al., 1991; De Jong et al., 1997). As described above, about 50% of the germinated conidia of the mutants produced abnormal appressoria. To determine whether the turgor pressure of these abnormal appressoria was altered compared with that of the wild-type appressoria, we performed incipient cytorrhysis. Surprisingly, we found that over 50% of the abnormal appressoria did not collapse even in a very high (5 M) concentration of glycerol solution, whereas we observed no significant changes in appressorium turgor in the normal appressoria of the Δcrz1 mutant and of the wild-type strain (Fig. 5a and b).
Subcellular localization of MgCRZ1
Fungal CRZ1 homologs are translocated from the cytoplasm to the nucleus following calcineurin-mediated dephosphorylation (Matheos et al., 1997; Stathopoulos & Cyert, 1997; Stathopoulos-Gerontides et al., 1999; Hirayama et al., 2003; Karababa et al., 2006; Soriani et al., 2008). To analyze the subcellular distribution of CRZ1 in M. grisea, we transformed an MgCRZ1-GFP fusion construct into the Δcrz1 mutant strain. Two transformants were verified by PCR and Southern blotting (data not shown). In the absence of Ca2+ and any form of stress, the MgCRZ1::GFP construct localized in the cytosol of the cells and was apparently excluded from nuclei. However, after we supplemented the media with Ca2+, the MgCRZ1::GFP construct rapidly translocated to the nuclei, as shown by the colocalization of the GFP and DAPI fluorescence signals in the cells (Fig. 6). In contrast, when treated with Ca2+ and the calcineurin inhibitor CsA, nuclear translocation was blocked, and the reporter protein was localized predominantly in the cytosol (Fig. 6). These results indicated that MgCRZ1 was subjected to an active nuclear import and export mechanism that depended on the increase of cytosolic Ca2+; this process was blocked by a specific inhibitor of calcineurin activity, thus implicating calcineurin as the provider of the Ca2+ signal.
We identified and characterized a calcineurin-dependent transcription factor, MgCRZ1, from M. grisea. Several lines of evidence supported our assumption that MgCRZ1 encoded a functional homolog of the S. cerevisiae transcription factor CRZ1. First, using blastp to analyze known CRZ1 and homologous proteins in S. cerevisiae and C. albicans, we identified one putative CRZ1 homolog MGG_05133.5 in the M. grisea genome, which contained two conserved C2H2-type zinc finger motifs. Secondly, MgCRZ1 expression in the yeast Δcrz1 mutant could rescue the growth defect under high Li+ concentrations. In previous studies, yeast Δcrz1 mutant cells exhibited higher sensitivity to many ions, including Na+, Li+, Mn2+, and Ca2+ cations and hydroxyl anions (Stathopoulos & Cyert, 1997). However, in our study, the yeast cells transformed with MgCRZ1 were only sensitive to Na+, Mn2+, and Ca2+. Independent studies conducted by Santos & de Larrinoa (2005) and Schumacher (2008), using complementation of the yeast Δcrz1 mutants of C. albicans CRZ1 (CaCRZ1) and B. cinerea CRZ1 (BcCRZ1), respectively, showed that CaCRZ1 could suppress the yeast sensitivity to Na+, Mn2+, and Ca2+, whereas BcCRZ1 could only suppress the Na+ sensitivity. These results suggested that the transcription factor CRZ1 may play different roles and perform general functions in yeasts and filamentous fungi. In yeast, there is a highly conserved calcineurin-docking domain (PIISIQ; Roy et al., 2007), but no obvious docking domain was found in the M. grisea CRZ1 homolog. However, the subcellular localization assay revealed that MgCRZ1::GFP fusion protein translocation to the nuclei was regulated by Ca2+ in a calcineurin-dependent manner, which was consistent with the results in C. albicans, A. fumigatus, and B. cinerea (Santos & de Larrinoa, 2005; Schumacher et al., 2008; Soriani et al., 2008). In summary, all of our results reported here corroborate those of previous studies and demonstrate that MgCRZ1 is the functional homolog of yeast CRZ1.
To further elucidate the role of MgCRZ1 in M. grisea, we used targeted gene deletion manipulation methods during different developmental stages in the Δcrz1 mutant. We determined that disruption of MgCRZ1 caused pleiotropic effects and revealed the involvement of the calcineurin/CRZ1 signaling pathway in vegetative growth, appressorium formation, lipid metabolism, and pathogenicity in M. grisea.
In previous studies, ion-sensitive mutants have been functionally characterized in S. cerevisiae and in A. fumigatus. These studies demonstrated sensitivity of these strains to various ions, including Na+, Li+, Mn2+, and Ca2+, and to high pH (Matheos et al., 1997; Stathopoulos & Cyert, 1997; Soriani et al., 2008). Recent research in B. cinerea reported that Mg2+ ions could restore the growth defect of Bccrz1 mutants but that they remained hypersensitive to extreme pH values, high concentrations of Li+ and Ca2+, and oxidative stress caused by H2O2 in CM- and MgCl2-supplemented CM media. However, the mutants were not altered in their responses to other osmotic stressors such as sorbitol, Na+, and oxidative stress caused by menadione (Schumacher et al., 2008). Consistent with these results, the Δcrz1 mutants examined here displayed a higher sensitivity to Ca2+ compared with that exhibited by the wild type. However, under other ionic conditions, such as high Na+, Li+, and Mn2+ exposure, the mutants and wild-type strains exhibited unremarkable differences (data not shown), indicating that the function of CRZ1 plays diverse transcriptional roles in different fungi.
Melanin is a virulence factor for many kinds of pathogenic fungi, and reported melanin-deficient mutants do not generate turgor and possess leaky appressoria, and thus are nonpathogenic (Howard et al., 1991; Henson et al., 1999; Skamnioti & Gurr, 2007). In this report, we demonstrated that the crz1-deleted mutants dramatically lost their ability to infect rice leaves and cause disease symptoms, indicating that MgCRZ1 was a crucial determinant of full virulence. Full virulence of MgCRZ1 may be attributed to the lack of functional appressoria that prevented the penetration to the host cuticle, because more than half (50%) of the abnormal appressoria that appeared at 24 h postinoculation were mostly melanin deficient. However, we did find that no obvious alternation in the transcript of melanin biosynthetic genes such as VCX1, PMR1, PMR2, PMC1, and CHSB (data not shown), revealing that MgCRZ1 does not regulate the transcription of these genes. We also observed that Δcrz1 mutant was more sensitive to cell wall-degrading enzyme by evaluating protoplast production of wild-type, Δcrz1 mutant and complement strains treated with cell wall-degrading enzyme, suggesting the impairment of cell wall integrity of Δcrz1 mutant (see Fig. S4). Meanwhile, the turgor measurement assay showed similar data (about 50% noncollapsed) to the rate of aberrant appressoria. In glycerol solution at 5 M or higher, the turgor pressure of the normal appressoria in the Δcrz1 mutant strains was not distinct from that of the wild-type Guy11 strain. These data supported our hypothesis that melanin-deficient appressoria were collapsed similarly by hyperosmotic glycerol but could reinflate quickly upon incubation in glycerol solution, whereas the wild-type appressoria could not reinflate even after prolonged incubation in glycerol (De Jong et al., 1997). An unexpected result in this study was that the mobilization and degradation of the lipid droplets was delayed in the conidia and appressoria of the Δcrz1 mutants. Lipid droplets are one component of the conidium, and these lipids mobilized rapidly from the conidium to the appressorium during appressorium maturation, which was under the control of the Pmk1 MAP kinase signaling pathway in M. grisea (Thines et al., 2000). A consequence of lipolysis in the appressorium is the generation of fatty acid and glycerol (Thines et al., 2000; Wang et al., 2007). Recent research has demonstrated that the involvement of lipid metabolism in the process of appressorium-mediated plant infection is required for peroxisomal fatty acid β-oxidation and triacylglycerol lipase activity in M. grisea (Thines et al., 2000; Kadotani et al., 2004; Wang et al., 2007). As CRZ1 plays a role in many diverse cellular processes (Zhu et al., 2001), we propose that the calcineurin-dependent expression of M. grisea MgCRZ1 may exhibit overlapping functions with intracellular lipases. Further work is required to elucidate these exact interaction mechanisms.
Our results suggest that the transcription factor MgCRZ1 plays a role in the calcineurin-dependent signaling pathway and is an important virulence determinant involved in the growth, development, and full virulence capacity of M. grisea.
We gratefully acknowledge funding from a National Basic Research Program of China (grant no. 2006CB101901, Z.Z.), one Natural Science Foundations of China (grant no. 30771394 to X.Z.), and New Century Excellent Scholar Project of Ministry of Education of China (grant no. NCET-07-0442 to Z.Z.). We thank Professor Z.Y. Wang in Zhejiang University for the gift of the plasmids pCB1532 and pCB1003.
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