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Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.
Both unicellular and multicellular organisms have the ability to dynamically reorganize their cytoskeletons in response to environmental changes, as well as during polarized growth that is crucial for proliferation, differentiation, and morphogenesis. Fungal cells represent a perfect example of polarized growth that efficiently responds to environmental cues. The induction of cell polarity is particularly dramatic in fungi that show dimorphic growth or morphogenic transition and is often associated with virulence in the pathogenic species (9).
Magnaporthe oryzae, a filamentous ascomycete and the causal agent of cereal blast disease, undergoes vegetative or infectious growth in the presence of rich nutrients or a host inductive surface, respectively. Under such growth conditions, Magnaporthe shows unipolar extension of the mycelial tips (or germ tubes), and its growth is well coordinated with morphogenic differentiation only under asexual or pathogenic development. Polarized growth is induced at several points in M. oryzae during the infection cycle: emergence of a germ tube from the conidium, elongation of the germ tube, penetration peg formation by the appressorium, both intra- and intercellular extension of the invasive hyphae, and development of aerial hyphae from the mass of invasive hyphae within the host. The aerial hypha starts to swell at the tip, suggestive of conidiophore initiation, which includes the hypha (conidiophore stalk) and the swollen tip (conidiophore vesicle), separated by a septum at the neck. The vesicle eventually develops into a mature 3-celled conidium (6). In yeasts, polarized growth is regulated by several proteins in a polarisome complex at the growth zone, which in turn depends on polarity pathways that are temporally and spatially regulated (12). Therefore, proteins that control morphogenic differentiation through cell polarity might play key roles in microbial pathogenesis and in adaptation to new environments.
Members of the Rho family of small GTP-binding proteins act as pivotal signaling switches and play a key role in morphogenesis during pathogenic development in M. oryzae (28, 29). In Aspergillus nidulans, cell end marker proteins TeaA (Tea1), TeaR (Mod5), KipA (Tea2), and TeaC (Tea4) have been shown to be important for polarized hyphal growth (10, 24). Similarly, the Tea1 homolog ClaKel2 has been implicated in pathogenic differentiation in Colletotrichum lagenarium (18). It has been reported that a cyclin-dependent kinase from the Cdk5/Pho85 family plays a key role in regulating polar growth required for developing infection structure and virulence in the dimorphic fungal pathogen Ustilago maydis (4).
Other polarity factors, apart from Tea1 and Tea2, include Tea3, Tea4, Tip1, Pom1, and Bud6 (21, 22, 26). Tea1 localizes to the cell tips in a Mod5-dependent manner (21) and is required for the recruitment of Pom1 kinase (2, 25), Bud6 (8), and the formin For3, which nucleates F-actin in Schizosaccharomyces pombe (7). Importantly, Tea4 mediates the interaction between Tea1 and For3, which is essential for F-actin nucleation in S. pombe (13). However, the role of Tea4 has not been defined in pathogenic fungi that undergo morphogenic differentiation in response to cues from the host or the environment.
In this study, we show that Tea4 in M. oryzae (MoTea4) plays an important role in maintaining polarized growth of aerial hyphae during asexual development and in differentiation of germ tubes into appressoria during pathogenic growth. We analyze the importance of microtubule and actin cytoskeletal organization in appressorial development and the effect of the loss of Tea4 function on the organization of the actin cytoskeleton in M. oryzae. Lastly, MoTea4 function was found to be important for asexual differentiation and effective virulence in M. oryzae.
Magnaporthe oryzae wild-type (WT) strain B157 (field isolate, mat1-2) was obtained from the Directorate of Rice Research (Hyderabad, India). Magnaporthe strains were grown on prune agar (PA) medium or complete medium (CM) as described previously (6, 16, 23). Nucleic acids were isolated from 2-day-old cultures by grinding CM-grown mycelia in liquid nitrogen. Magnaporthe isolates were cultivated on PA medium or CM agar, at 28°C for 1 week, to assess the growth and colony characteristics.
For quantitative analysis of conidiation, colonies were cultivated for 3 days on PA medium in the dark, followed by 4 days of growth under constant illumination at room temperature. Inoculation loops were used to scrape the surface of the colonies in the presence of water, and the fungal biomass was collected in Falcon conical tubes (BD Biosciences, San Jose, CA). Maximum detachment of conidia from mycelia was ensured by thoroughly vortexing the suspension. The suspension was then filtered through two layers of Miracloth (Calbiochem, San Diego, CA), and conidia thus collected were washed twice with and finally resuspended in sterile water containing 100 μg/ml each of streptomycin and carbenicillin. The conidial count for a given colony was estimated using a hemocytometer and reported as the total number of conidia per unit area of the colony.
To test appressorial development, conidia were spot inoculated either on a rice leaf sheath or a cover glass (1000 Deckglaser, 22 mm, no. 1; Thermo Scientific, Germany) and incubated under humid conditions at 25°C for up to 24 h. For pathogenicity tests, droplets (20 μl) of conidial suspension (ca. 500 or 1,000 conidia per droplet) were inoculated on barley leaf explants and incubated under humid conditions at 23°C for up to 5 days (23).
Fungal genomic DNA was extracted using the MasterPure yeast DNA purification kit (Epicentre Biotechnologies). Plasmid DNA was isolated with the QIAprep plasmid miniprep kit (Qiagen, Valencia, CA), and nucleotide sequencing was performed using the ABI Prism BigDye Terminator method (PE Applied Biosystems, CA). Homology searches of DNA/protein sequences were performed using BLAST (1). The domains in the protein sequences were determined using SMART and COILS. The following primers were used to amplify the 5′ and 3′ untranslated regions (UTR) (1 kb and 600 bp, respectively) of the TEA4 gene: MoTea4-5F (5′-GAGAGTGTTaagcttTGACCTCAATCGGTTCCGCGTTC-3′), MoTea4-5R (5′-GAGACTGTTctgcagAGAAATGGGCTATCTTGGGCTAG-3′), MoTea4-3F (5′-GAGAGTGAtctagaACGCCTGCGACGATTCGATTTAG-3′), and MoTea4-3R (5′-GAGAGTGAggtaccCCTAGTGAGTATCTGCCAGGTC-3′). (Lowercase letters in the primer sequences represent the restriction enzyme sites used for cloning.) The amplified 5′ UTR of MoTEA4 was cloned in HindIII/PstI sites of pFGL59 (a modified pCAMBIA1300 vector) to obtain pFGL501. The amplified MoTEA4 3′ UTR was cloned in XbaI/KpnI sites of pFGL501 to obtain pFGL502. Thus, the 5′ and 3′ UTR of TEA4 were ligated sequentially to flank the hygromycin phosphotransferase (HPH1) expression cassette in pFGL59 to obtain the plasmid pFGL502. Cloned TEA4 fragments were confirmed by nucleotide sequence analysis, and pFGL502 was introduced into the WT Magnaporthe strain via Agrobacterium T-DNA-mediated transformation (ATMT). Strains showing replacement of the TEA4 open reading frame (ORF) with the HPH1 cassette were identified by Southern blot analysis.
For genetic complementation, a full-length genomic copy of the S. pombe TEA4 (SpTEA4) ORF (SPBC1706.01) along with 3′ UTR (353 bp) was amplified with SpTea4-ORF-F (5′-GAGAGTGTTccatggAAATTATGGAAAGTC-3′) and SpTea4-3UTR-R (5′-GAGACTGTAgtcgacCACTGCTCGCATTACTGTAC-3′). The amplified PCR product was cloned in NcoI/SalI sites in pFGL275 in order to place the SpTEA4 ORF downstream of the MPG1 promoter and obtain pFGL558. The cloned SpTEA4 ORF and the 3′ UTR fragments were confirmed by restriction analysis, and pFGL558 was introduced into the Magnaporthe tea4Δ strain via ATMT for random insertion. Single-copy integrants were verified by Southern blot analysis.
For C-terminal tagging of Tea4 (MGG_06439.6) with green fluorescent protein (GFP) (Clontech), the last 803 bp of the Tea4 ORF without the stop codon was amplified using MoTea4-TagF (5′-GAGAGTGTTgaattcCTGGACAGTTCAAAGGGATCG-3′) and MoTea4-TagR (5′-GAGAGTGTTggtaccTGCAGCCCCCCTGAGTCTTTG-3′). A GFP fragment of 717 bp was amplified using EGFPF (5′-GAGAGTGTTggtaccATGAGTAAAGGAGAAGAAC-3′) and EGFPR (5′-GAGACTGTTggatccTTATTTGTATAGTTCATCCATG-3′). The 3′ UTR of TEA4 was amplified by using MoTea4-3UTR-F (5′-GAGAGTGActgcagTGTCTGTAGACTTGGTAACTG-3′) and MoTea4-3UTR-R (5′-GAGAGTGAaagcttTCTGTGAAGAGTACACTC-3′). The amplified Tea4 ORF (803 bp) and GFP fragments were digested with EcoRI/KpnI and KpnI/BamHI, respectively, and cloned in EcoRI/BamHI sites of pFGL347 such that the Tea4 and GFP ORF are just upstream of the TrpC terminator in the vector. This recombinant vector was named pFGL541. The amplified 3′ UTR of TEA4 was digested with PstI/HindIII and cloned in respective sites in pFGL541 so that the BAR (bialaphos/phosphinothricin resistance) cassette was flanked by the Tea4-GFP fusion construct and the Tea4 3′ UTR. The resultant recombinant vector, pFGL542, was transferred into the M. oryzae WT strain using the ATMT method. Strains showing replacement of the TEA4 ORF with the TEA4-GFP fusion construct along with the BAR cassette were identified by Southern blot analysis.
Similarly, to tag Abp1 (MGG_06358.6) with red fluorescent protein (RFP) (mDsRed; Clontech), the last 1 kb of the ORF without the stop codon was amplified using primers MoAbp1-ORF-F (5′-GAGAGTGTTgaattcCAGAGGACCGGAGGCGATGAC-3′) and MoAbp1-ORF-R (5′-GAGAGTGTggtaccCTGATCAAGCTCTACGTAG-3′). A DsRed fragment of 688 bp was amplified using DsRedF (5′-GAGAGTGTTggtaccATGGACAACACCGAGGACGTC-3′) and DsRedR (5′-GAGAGTGTTggatccCTACTGGGAGCCGGAGTGGC-3′). The 3′ UTR of the ABP1 genomic region (932 bp) was amplified by using MoAbp1-3UTR-F (5′-ACCCAActgcagGTCTAACATGGCTTC-3′) and MoAbp1-3UTR-R (5′-GAGAGTGTTaagcttAGGGATTACAACTTCCAC-3′). The amplified Abp1 3′ UTR was digested with PstI/HindIII and cloned in respective sites in pFGL347 to obtain pFGL543. The fragments of the Abp1 ORF and RFP were digested with EcoRI/KpnI and KpnI/BamHI, respectively, and cloned in EcoRI/BamHI sites of pFGL543 to obtain pFGL544, which was transferred to WT M. oryzae. Southern blot analysis was performed to confirm successful gene replacement and single-copy integrations.
To express either the SH3 (MoTea41-405) or the coiled-coil (MoTea4406-979) domain of MoTea4 individually in Magnaporthe, constructs were made to replace the full-length wild-type copy of MoTEA4 with the truncated MoTEA4 as follows. To express the SH3 domain alone, the last 1-kb stretch of the sequence encoding the N-terminal half of MoTea4 was amplified using primers MoTea4SH3F (5′-GAGAGTGTTgtcgacCGCCCGCCGCGCATTACAC-3′) and MoTea4SH3R (5′-GAGAGTGTTacgcgtCCTTCTCTTCATGGCAG-3′). The 3′ UTR of the MoTEA4 genomic region (1 kb) was amplified by using MoTea4-3UTR-F (5′-GAGAGTGTTgttaacTAGACTTGCATCACACAG-3′) and MoTea4-3UTR-R (5′-GAGAGTGTTgggcccGTGTGGCACAATGTGGCC-3′). An amplified 1-kb fragment of the sequence encoding the N-terminal half of MoTea4 was digested with SalI/MluI and cloned in respective sites in pFGL557 (hygromycin resistance) to obtain pFGL581. The fragment of the 3′ UTR of MoTEA4 was digested with HpaI/ApaI and cloned in respective sites of pFGL581 to obtain pFGL582. Similarly, to express the coiled-coil domain alone, 1 kb of the 5′ UTR of MoTEA4 was amplified using MoTea4-5UTR-F (5′-GAGAGTGTTgtcgacTGACCTCAATCGGTTCCG-3′) and MoTea4-5UTR-R (5′-GAGAGTGTTacgcgtGGTCGCTTGAGCCTGAGC-3′). The first 1 kb of the sequence encoding the C-terminal half of MoTea4 was amplified using MoTea4-CCF (5′-GAGAGTGTTgttaacACCAAGACTGTAGCCTTC-3′) and MoTea4-CCR (5′-GAGAGTGTTgggcccATCCCTTTGAACTGTCCAG-3′). An amplified 1-kb fragment of the 5′ UTR of MoTEA4 was digested with SalI/MluI and cloned in respective sites in pFGL557 to obtain pFGL579. The 1-kb fragment of the sequence encoding the C-terminal half of MoTea4 was digested with HpaI/ApaI and cloned in respective sites of pFGL579 to obtain pFGL580. The plasmids pFGL580 and pFGL582 were introduced into WT M. oryzae, and the gene replacement events were identified by locus-specific PCR. PCR primers used to screen transformants obtained from pFGL582 were 582LS5′F (5′-CTTCAGGCAGGAGCAAGATG-3′), Hph5′out (5′-CAGAAACTTCTCGACAGACG-3′), DsRed3′out (5′-GTGGAGCAGTACGAGCACGC-3′), and 582LS3′R (5′-TGAGAGCGTCACGAAGCACG-3′). Similarly, for transformants obtained using plasmid pFGL580, the following primers were used: 580LS5′F (5′-CTGGAGATCCAAGTGGGTAG-3′), Hph5′out (5′-CAGAAACTTCTCGACAGACG-3′), DsRed3′out (5′-GTGGAGCAGTACGAGCACGC-3′), and 580LS3′R (5′-TGACAACCTCTGTTGCTGCC-3′).
For transmission electron microscopy (TEM) analysis, fresh conidia were harvested, thoroughly washed in sterile distilled water, fixed overnight at 4°C in glutaraldehyde (2.5%, vol/vol) in 0.1 M phosphate buffer (pH 7.2), and processed for TEM as described previously (23). Bright-field and epifluorescence microscopy analyses were performed with an Olympus IX71 or BX51 microscope (Olympus, Tokyo, Japan) using a Plan-Apochromat 100×/1.45 objective or UPlan FLN 60×/1.25 objective and the appropriate filter sets. Images were captured with a Photometrics CoolSNAP HQ camera (Tucson, AZ) and processed using MetaVue (Universal Imaging, PA) and Adobe Photoshop 7.0.1 (Mountain View, CA).
Calcofluor white (CFW; Sigma-Aldrich) was used at 3 μg/ml (in 100 mM Tris-HCl buffer, pH 9.0, containing Triton X-100 at a dilution of 1:1,000) to visualize the cell wall and septa in the aerial hyphae, conidiophores, and conidia of the respective strains. For staining of aerial hyphae and conidiophores, colonies were incubated with a suitable volume of the CFW solution (enough to cover the surface of the colonies) in the dark at room temperature for 10 min and then washed several times and subjected to epifluorescence microscopy using the recommended filter sets. Colonies were assessed at 6 to 9 h or at 12, 24, or 48 h postphotoinduction to study aerial hyphae or conidiophores, respectively. FM4-64 was used at a concentration of 10 μM in the medium. Cells were incubated for 10 min and washed. Methyl benzimidazole-2-yl-carbamate (MBC; Sigma-Aldrich) was used at a final concentration of 1 μM, diluted from a stock solution of 1 mg/ml in ethanol. Latrunculin A (LatA; Biomol) was used at a final concentration of 10 μM (stock solution, 10 mM in dimethyl sulfoxide). Cytoplasmic streaming was documented by live imaging of growing germ tubes of the WT or tea4Δ strain, and the resultant differential interference contrast (DIC) images were stacked using MetaMorph software (Molecular Devices).
We identified an ortholog of the S. pombe TEA4 gene in M. oryzae (MGG_06439) and named it MoTEA4. Although the overall sequence similarity to the S. pombe Tea4 protein was low, the conserved SH3 domain showed significant identity to the SH3 domain of MoTea4. Likewise, MoTea4 showed similarity to Bud14 from Saccharomyces cerevisiae. An apparent difference in the domain organization of the Tea4-like proteins from filamentous fungi was the presence of a coiled-coil motif in addition to the SH3 domain. Overall, the yeast and fungal Tea4 orthologs showed 26 to 42% sequence identity with a similar architecture in filamentous fungi (Fig. 1).
To study the subcellular localization, we generated a strain expressing the MoTea4-GFP fusion protein under its native promoter by replacing the WT TEA4 with a MoTEA4-GFP allele. The MoTEA4-GFP strain showed WT-like phenotypes in terms of conidial count and morphology, appressorium formation, and the ability to cause infection. Although weak in fluorescence signal, the MoTea4-GFP localized to the tips of the vegetative and aerial hyphae as mostly single punctate structures. Such MoTea4-GFP foci localized close to the periphery at the hyphal apex (Fig. 2A and B). The MoTea4-GFP punctae, along with some diffused cytosolic signal, localized away from the tips in the conidiophores (Fig. 2C). The MoTea4-GFP punctae were observed in mature conidia, predominantly along the septa, but were also enriched in the terminal cell at the time of germination (Fig. 2D). The MoTea4-GFP foci did not localize at the tips of the early germ tubes but appeared at the apex of the appressorium initials (Fig. 2E). To test whether MoTea4-GFP localization at the hyphal tips depends on microtubules or the actin cytoskeleton, we used the destabilizing agent MBC or LatA, respectively. Treatment of the vegetative hyphae with 1 μM MBC for 30 min caused MoTea4-GFP fluorescence to divide into more than one puncta at the tips and/or to partially diffuse into the cytoplasm (see Fig. S5 in the supplemental material), whereas the LatA-treated (10 μM, 30 min) vegetative hyphae showed significantly diminished MoTea4-GFP fluorescence at the tips (see Fig. S5). Our observations suggest that the localization of MoTea4-GFP in the vegetative hyphae depends on the microtubule cytoskeleton. However, the stability of the MoTea4-GFP foci at the hyphal tips most likely depends on the F-actin cytoskeleton. Next, we examined the localization of MoTea4-GFP with respect to the Spitzenkörper vesicles and stained the vegetative hyphae of the MoTEA4-GFP strain with FM4-64. MoTea4-GFP partially colocalized with the Spitzenkörper vesicles at the apex of the hyphae (Fig. 2F). Based on its intracellular distribution, we infer that MoTea4-GFP likely marks the growth zones at the cell tips during polarized growth in M. oryzae during vegetative and asexual development.
We generated an MoTEA4 deletion strain (hereafter called the tea4Δ strain; relevant genotype, TEA4::HPH1) in Magnaporthe and analyzed it during vegetative, asexual, and pathogenic development of the fungus. The colony growth of the tea4Δ strain was comparable to that of the WT, except that the tea4Δ colony appeared white, as opposed to gray in the WT (Fig. 3A). The vegetative hyphae of both the WT and tea4Δ strains showed a normal straight morphology. However, unlike that of the WT, the vegetative hyphae of the tea4Δ strain showed bundling toward the base, away from the tips (Fig. 3B). Similarly, both the WT and the tea4Δ vegetative hyphae showed normal branching (Fig. 3B, inset) and septation (see Fig. S4 in the supplemental material). To assess the asexual development in the tea4Δ strain, we studied the growth and morphology of the aerial hyphae and conidiophores stained with calcofluor white. Unlike vegetative hyphae, most of the conidiophores of the tea4Δ strain showed a zigzag morphology (Fig. 3C; see also Fig. S6B in the supplemental material) and aberrant cell wall deposits. Furthermore, the majority of the tea4Δ conidiophores failed to initiate conidiation and did not show proper swelling at the tips even after 24 h of photoinduction (Fig. 3C). All of the conidia were harvested from the WT or tea4Δ strain and quantified to study the ability of the mutant to perform asexual differentiation. The number of conidia per square centimeter of fungal growth was (106.9 ± 3.5) × 102 in the WT, whereas it was (0.63 ± 0.09) × 102 in the tea4Δ strain (Fig. 3D; P < 0.005). Moreover, the tea4Δ conidia were smaller than the WT conidia (15 μm versus 21 μm) and morphologically aberrant compared to those produced by the WT. In contrast to the tricellular pyriform conidia in the WT, the tea4Δ conidia were mostly two celled and appeared spindle shaped (Fig. 3E). Approximately 80% (n > 300) of the tea4Δ conidia had one septum, while only 4% of the WT conidia had one septum (Fig. 3F; P < 0.005). We substantiated this result by analyzing the ultrastructure of the tea4Δ conidia under TEM. The electron micrograph of the mutant conidia showed only one septum, with a few exceptional cases where an attempt at making a second septum was evident. However, the second septum was aberrant and incomplete (see Fig. S2 in the supplemental material). WT conidia invariably showed two completely developed septa.
Next, we expressed the S. pombe TEA4 gene in the Magnaporthe tea4Δ strain to check if it could suppress the mutant defects during asexual development and to ascertain whether the two orthologs share any functional similarity. The complemented strain showed a marginal suppression of mutant defects, with a 5- to 6-fold increase in the total number of conidia compared to that in the tea4Δ strain (see Fig. S1A in the supplemental material). Similarly, the percentage of 3-celled conidia increased from 20% to ~40% (n = 300) in the complemented strain (see Fig. S1B). Further, we replaced the full-length MoTEA4 gene with the truncated allele encoding either the SH3 or the coiled-coil domain in the Magnaporthe WT. Although the strains expressing the SH3 domain alone showed an approximate 4-fold increase in total conidiation compared to that in the tea4Δ strain, the conidiation was not comparable to that of the WT (see Fig. S1C). However, the number of 2-celled conidia decreased significantly (from ~80% in the tea4Δ strain) to 32% in the strain expressing the coiled-coil domain (see Fig. S1D in the supplemental material). These findings indicate that the SH3 and the coiled-coil domains of MoTea4 are necessary for its function in conidiation in Magnaporthe. We conclude that MoTea4-mediated polarized growth is important for proper differentiation of conidiophores and conidia during asexual development in Magnaporthe.
We studied infection-related development in the tea4Δ strain, both in vitro and in planta, to assess the role of MoTea4 in the pathogenesis of M. oryzae. In in vitro assays, infection-related morphogenesis (appressorial development) was studied using an inductive hydrophobic glass surface. A striking polarity-related defect was apparent in germ tube emergence in the tea4Δ strain on the inductive surface. A random germination pattern with more than one germ tube per cell in a conidium was observed in the tea4Δ strain (Fig. 4A). In some cases, terminal cells in the tea4Δ conidia developed two germ tubes, which were never found in the WT conidia during pathogenic development (Fig. 4A). The majority of the tea4Δ conidia failed to develop appressoria on the artificial inductive surface in comparison to the WT conidia. While 85.4% ± 2.0% of the WT conidia formed appressoria, only 9.2% ± 0.7% of the mutant conidia were capable of appressorium formation (Fig. 4B; P < 0.005). The mutant conidia developed long, multiseptate, and branched germ tubes in contrast to the WT conidia, where mostly a single, short, and nonseptate germ tube per conidium developed into an appressorium (Fig. 4C). A vast majority of the tea4Δ germ tubes initiating appressoria (a swollen germ tube tip) failed to complete the process, and fresh germ tubes emerged from the immature appressorium-like structures (Fig. 4C). To assess whether appressorium development depends on the number of cells per conidium, we quantified the ability to form appressoria in the 2-celled and 3-celled tea4Δ conidia. About 87% of the 3-celled tea4Δ conidia failed to develop appressoria, whereas nearly 10% of the 2-celled mutant conidia formed such infection structures (see Fig. S7 in the supplemental material). These results indicate that the loss of MoTea4 function leads to a failure in proper pathogenic differentiation that is likely due to the loss of regulation of polarized growth during conidial germination and appressorium initiation.
We examined the sensitivity of the Magnaporthe WT toward the microtubule-destabilizing agent MBC. Approximately 70% of the untreated WT conidia developed appressoria, while 20% formed appressorium initials within 6 h postinfection (hpi) (Fig. 5A). Although none of the MBC-treated WT conidia formed immature appressoria in 6 h, about 40% showed appressorium initials (Fig. 5A). Importantly, the MBC-treated WT showed significant recovery at 20 hpi, at which point 78% of conidia developed into mature appressoria (Fig. 5B).
Next, we studied the sensitivity of the M. oryzae WT to the actin-destabilizing agent LatA. Unlike the untreated WT conidial germination response described above, none of the LatA-treated WT conidia developed appressoria and ~35% of them showed appressorium initials at 6 hpi (Fig. 5A). Furthermore, the LatA-treated WT did not show significant recovery at 20 hpi, and hardly 8% of conidia developed into appressoria (Fig. 5B). Interestingly, the LatA-treated WT conidia showed polarity defects reminiscent of the tea4Δ mutant conidia, with more than one germ tube emerging from a single cell in a conidium (Fig. 5B, arrowheads). Based on these observations from studies of the WT, we propose that an intact F-actin cytoskeleton, but not the microtubules, is required for proper appressorial development in Magnaporthe.
Next, we analyzed the role of MoTea4 in F-actin organization by characterizing the WT and tea4Δ strains expressing the Abp1-RFP fusion protein to aid the visualization of F-actin. We studied asexual development, conidial germination, and appressorium development to test the effect of TEA4 deletion on the actin cytoskeleton at these developmental stages. We studied the distribution of F-actin patches in the aerial hyphae and the conidiophores of the WT or the tea4Δ strain expressing the Abp1-RFP protein. The aerial hyphae of the WT showed enrichment of the F-actin patches (Abp1-RFP) at the tips, which continued to mark the swollen tips of the conidiophores. The F-actin patches were later observed along the periphery of the developing conidia (see Fig. S6A in the supplemental material). However, the tea4Δ aerial hyphae showed aberrant and reduced F-actin patches. The developing tea4Δ conidia showed F-actin aggregates within the vesicles (see Fig. S6A). Mature WT conidia showed the cortical F-actin patches (Abp1-RFP foci) along the septa and at the cortex. Such cortical patches were significantly reduced in numbers in the tea4Δ strain, and most of the Abp1-RFP signal was diffused and likely vacuolar (Fig. 5C). In germinating WT conidia, the F-actin patches were enriched at the tips of the germ tubes and later uniformly along the periphery of the developing appressoria (Fig. 5C). In contrast, the tea4Δ germ tubes showed aberrant F-actin aggregates, with a significantly reduced number of patches at the tips (Fig. 5C; see also Fig. S8 in the supplemental material). To substantiate these findings, we studied the effect of LatA on the WT M. oryzae strain expressing the Abp1-RFP fusion protein. In the LatA-treated Abp1-RFP strain, the F-actin patches were rarely seen at the apex of the germ tubes. In addition, aggregates of F-actin (Abp1-RFP) were evident along the LatA-treated germ tubes (Fig. 5D). Our observations indicate that the actin cytoskeleton plays an important role in asexual development and pathogenic differentiation and that the loss of MoTea4 function likely disrupts the proper organization of the actin cytoskeleton in M. oryzae.
We tested whether F-actin organization is involved in cytoplasmic streaming in Magnaporthe. During appressorial development, LatA-treated WT germlings showed a significantly slow and restricted movement of cytoplasmic content. Next, we analyzed the effect of MoTea4 deletion on cytoplasmic streaming in M. oryzae. In WT germlings, a rapid and bidirectional movement of intracellular particles was observed (see Video S1 in the supplemental material), while the tea4Δ strain showed a relatively slow and highly constrained movement of the cytoplasmic particles, especially in the germ tubes (see Video S2). As a marker for bulk cytoplasmic streaming, we studied lipid droplet (LD) mobilization in the WT and tea4Δ germlings (germinating conidia). In the WT, the germlings showed bulk movement of the LDs from the conidia to the germ tubes, followed by accumulation in the incipient appressoria. In contrast, the tea4Δ germlings showed accumulation of LDs in conidia at 6 hpi (see Fig. S3 in the supplemental material). Taken together, we propose that MoTea4 is required for proper organization of the F-actin cytoskeleton, which may be involved in cytoplasmic streaming and bulk mobilization of cellular content during appressorium development in Magnaporthe.
We examined appressorial development on a rice leaf sheath where, as on an inductive glass surface (Fig. 4A and and4C),4C), the tea4Δ conidia showed polarity-related defects in germ tube emergence and failed to form proper appressoria. Furthermore, the majority of the tea4Δ appressoria were mostly irregular in shape and size compared to the WT appressoria (Fig. 6A). Next, we assessed the virulence of the WT and tea4Δ strains on barley leaf explants. The WT Magnaporthe strain started developing disease symptoms at 3 days postinoculation (dpi), and typical disease lesions appeared at 5 dpi. However, significant symptoms of effective virulence of the tea4Δ strain were not evident even after 7 dpi, except for marginal browning at the site of inoculation (Fig. 6B). Further, we examined the tea4Δ strain to assess whether the malformed appressoria were capable of host penetration. In the WT, 61% ± 5% of the appressoria developed penetration hyphae after 40 hpi, whereas none of the tea4Δ appressoria showed penetration hyphae within the host tissue (Fig. 6C and D). Taken together, these findings led us to conclude that MoTea4 function is required for pathogenesis, likely through proper development and function of the infection structures in M. oryzae.
The rice blast pathogen M. oryzae undergoes a specialized development manifested by spatiotemporally regulated polarized growth, and this process is closely associated with pathogenesis. Studying the role of cell polarity involved in infection-related morphogenesis will help to provide insight into the cell biology of fungal development. In addition, the molecular analysis of polarized growth may lead to the identification of targets for new antifungal agents. Here, we studied the role of a cell end marker protein, MoTea4, in the morphogenesis of M. oryzae and observed that its function is important not only for morphogenic differentiation during asexual development but also for pathogenesis.
MoTea4 is a protein containing an SH3 domain that is found in proteins which regulate the actin cytoskeleton, Myo3p, Myo5p, Abp1p, Bzz1p, Bbc1p, Rvs167p, Lsb1p to Lsb4p, and Sla1p (14). Although overall sequence identity between SpTea4, AnTeaC, and MoTea4 is low, the SH3 domain is highly conserved in these proteins. MoTea4 shows similarity to SH3 domain-containing proteins in fungi other than S. pombe and A. nidulans, like Penicillium marneffei (PMAA_097510), Cryptococcus neoformans (CNBE_001580), and Talaromyces stipitatus (TSTA_043520). Interestingly, Tea4 only from filamentous fungi showed a coiled-coil motif in addition to the SH3 domain. It has been shown that Pea2, a coiled-coil-domain-containing protein, localizes to the sites of polarized growth and is required for efficient mating and bipolar budding in S. cerevisiae (27). It is possible that the coiled-coil domain in Tea4 of filamentous fungi might play an additional albeit important role in the relatively complex morphogenesis of these fungi.
We observed that MoTea4 plays an important role during growth or morphogenic transitions required for asexual and pathogenic development. For instance, MoTea4 is not essential for viability or growth of vegetative mycelia but is required for proper asexual development. MoTea4 localizes to the regions of active growth or morphogenesis, namely, the tips of vegetative and aerial hyphae, conidiophores, and appressorium initials. In agreement with the MoTea4 distribution, most of the tea4Δ aerial hyphae showed aberrant morphology and did not form conidia. Moreover, the tea4Δ conidia lacked one septum and did not develop appressoria on an inductive surface. Surprisingly, growth, morphology, and septation in the vegetative hyphae of the tea4Δ strain were similar to that of the WT except for the unusual hyphal bundling in the mutant. In Magnaporthe oryzae, the majority of the cells (conidial, hyphal, and germ tube) are mononuclear. Nuclear division during conidiation in Magnaporthe, especially formation of a 3-celled conidium from a single conidiophore vesicle, is not yet well understood. It is not clear whether a single nucleus in the conidiophore vesicle undergoes two rounds of division prior to septum formation or if each nuclear division is followed by a septum formation. Mid1, an aniline-like protein in S. pombe, marks the cortex at the cell median for actomyosin ring assembly and subsequent septum formation. Similar to that of S. pombe, septation in Magnaporthe follows every nuclear division, forming mononucleate hyphal compartments. However, there is no ortholog for Mid1 in Magnaporthe, suggesting that either such a protein is poorly conserved or some other protein(s) regulates the placement of the division septa in filamentous fungi. Deletion of TEA4 in Magnaporthe leads to the loss of a septum in most of the conidia, suggesting that such polarity determinants are important regulators of septum formation during asexual development. An additional role for SpTea4 in preventing septum formation at the cell ends in S. pombe has been shown earlier (11). Indeed, we found that most of the 2-celled tea4Δ conidia showed excess septal deposits at the tips of the terminal cells (Fig. 3E, rightmost panels). Similarly, deletion of TEAC in Aspergillus led to an impairment in coordination between mitosis and septation, resulting in short and anucleate compartments in the vegetative hyphae (10). Thus, an additional role for MoTea4 in regulation of septation in Magnaporthe needs further investigation.
It has been shown that ClaKel2p, a Tea1 homolog in Colletotrichum lagenarium, is involved in polarized growth and localizes to the tips of growing hyphae, to germ tubes, and weakly to the periphery of the appressoria. The clakel2 mutant formed abnormal appressoria on glass slides; however, mutant hyphal growth on potato dextrose agar medium and conidiation, though with a reduced amount of spores, was similar to that of the wild type (18). In M. oryzae, Zheng et al. (28) have shown that Magnaporthe grisea Rho3 (MgRho3) is dispensable for vegetative hyphal growth but not for plant infection, as the appressoria formed by the ΔMgrho3 strain are morphologically abnormal and defective in plant penetration. However, it is important to note that unlike in Magnaporthe, the deletion of TEAC in A. nidulans leads to significantly reduced vegetative growth (10) and a likely decrease in conidiation. It appears that different molecular machineries regulate the polarized growth and sporulation of filamentous fungi and yeasts: in filamentous fungi and in hyphal growth in Candida, polarized growth is mediated by the Spitzenkörper vesicles, while it is controlled by the polarisome complex in the budding process of Candida and Saccharomyces (5). Expression of SpTea4 in the tea4Δ strain of Magnaporthe only marginally rescued the mutant defects (see Fig. S1A and B in the supplemental material). Further structure-function analysis of MoTea4 revealed the importance of both the SH3 and the coiled-coil domains in proper conidiation in Magnaporthe. Based on these findings and the partial rescue of the tea4Δ defects by SpTea4, we propose that MoTea4 has most likely diverged significantly from yeast Tea4 and is adapted for an important function(s) during asexual development in M. oryzae. Similarly, it is also likely that the polarized growth and morphogenesis underlying vegetative and pathogenic development in fungi is controlled by different molecular mechanisms with some common polarity determinants.
Germ tube emergence and extension were defective in the tea4Δ strain of M. oryzae. The WT strictly shows only one germ tube, per terminal or basal cell of the conidium, that goes on to differentiate into an appressorium. The tea4Δ strain failed to form appressoria, likely due to its inability to sustain polarized growth during such early stages of the pathogenic phase. It is interesting to note that some of the tea4Δ germ tubes showed an attempt to develop appressoria; however, fresh germ tubes emerged from those immature appressorium-like structures. This is a unique defect and has not been seen in any Magnaporthe mutant. It would be interesting to analyze this transition in detail to uncover the (Tea4-dependent) mechanisms that suppress vegetative growth during pathogenic differentiation.
In filamentous fungi such as A. nidulans and N. crassa, hyphal tip elongation is a continuous and indefinite process (15, 17). However, pathogenic filamentous fungi like M. oryzae show morphogenic transitions from polarized to nonpolarized growth and vice versa during the pathogenic life cycle. Polarized growth is regulated during the cell cycle in single-cell yeasts, such as S. cerevisiae and S. pombe (22), with tight spatiotemporal control of actin assembly. In S. pombe, Tea4 mediates the interaction between Tea1 and a formin, For3, that acts as a site for rapid nucleation and assembly of actin filaments and network (13). The function of Tea1p and Tea4p is dependent on their localization to the cell end, which in turn is dependent on microtubules in S. pombe (7, 20). Interestingly, the M. oryzae WT showed a significant recovery from MBC treatment and successfully formed appressoria. Similarly, in C. lagenarium, the localization of ClaKel2p is not dependent on microtubules during pathogenic development, and MBC treatment does not affect appressorium formation (18). However, LatA-treated germinating WT Magnaporthe conidia did not recover and failed to develop appressoria, indicating that the appressorial development was dependent on an intact F-actin cytoskeleton and not on microtubules. It is also worth noting that the phenotype of LatA-treated germinating WT conidia was similar to that of the untreated tea4Δ strain. Importantly, the Abp1-RFP (F-actin) localization pattern appeared to be similar to that of MoTea4-GFP in mature conidia in Magnaporthe, and conversely, deletion of TEA4 disrupted F-actin organization in mature and germinating conidia. The similarity between the disorganized F-actin cytoskeleton (Abp1-RFP) in the tea4Δ strain and that of the LatA-treated WT supports our hypothesis that a properly organized actin cytoskeleton, though not crucial for vegetative growth, is essential for successful pathogenic differentiation and that MoTea4 plays an important role in regulating polarized growth in M. oryzae.
It has been inferred that an actin-myosin-based motor system is required for cytoplasmic streaming in plant cells (19). Magnaporthe shows rapid cytoplasmic streaming during appressorial development. The LatA-treated WT germlings showed poor cytoplasmic streaming, indicating that the F-actin cytoskeleton is likely important in the bulk movement of cytoplasm in Magnaporthe. Loss of MoTea4 function in M. oryzae led to disorganization of the F-actin cytoskeleton, and hence, it is likely that germinating tea4Δ conidia were affected in proper cytoplasmic streaming. Only 8% of the conidia of the tea4Δ strain, however, formed appressoria, which were aberrantly shaped and not functional. Such appressoria did not develop penetration hyphae, and as a consequence, the tea4Δ mutant could not penetrate the host cells and failed to cause disease. It has been proposed that the actin cytoskeleton plays an important role in the appressorial function (penetration of the host tissue) (3). Our observations indicate that an impaired F-actin organization within the appressoria likely leads to loss of pathogenesis in the tea4Δ mutant.
In summary, we showed that MoTea4 function is important for polarized growth and in establishing a proper F-actin cytoskeleton required for the pathogenic development in M. oryzae. Future studies will be aimed at deciphering the role of the coiled-coil domain in MoTea4 function. Interaction studies to identify the MoTea4 binding partner(s) in Magnaporthe will certainly be helpful in understanding the relatively complex regulation of polarized growth in this important fungal pathogen.
This work was funded by intramural research funds from the Temasek Life Sciences Laboratory (Singapore) and the Singapore Millennium Foundation.
We thank the Fungal Patho-Biology Group and the Cell Biology Forum (TLL) for helpful discussions and suggestions.
†Supplemental material for this article may be found at http://ec.asm.org/.
Published ahead of print on 14 May 2010.