EUKARYOTIC CELL, July 2010, p. 1029–1038 Vol. 9, No. 71535-9778/10/$12.00 doi:10.1128/EC.00292-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

MoTea4-Mediated Polarized Growth Is Essential for Proper AsexualDevelopment and Pathogenesis in Magnaporthe oryzae�†

Rajesh N. Patkar,1* Angayarkanni Suresh,1 and Naweed I. Naqvi1,2*Fungal Patho-Biology Group, Temasek Life Sciences Laboratory,1 and Department of Biological Sciences, 1 Research Link,

National University of Singapore,2 Singapore 117604, Republic of Singapore

Received 8 October 2009/Accepted 4 May 2010

Polarized growth is essential for cellular development and function and requires coordinated organizationof the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly andbipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in therice 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, aerialhyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispens-able for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerialhyphae and a huge reduction in conidiation. The majority of the tea4� conidia were two celled, as opposed tothe tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coildomains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4� conidia failed to produceproper appressoria and consequently failed to infect the host plants. The tea4� conidia and germ tubes showeddisorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to thewild-type conidia, the tea4� conidia showed aberrant germination, poor cytoplasmic streaming, and persistentaccumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment ofgerminating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorialdevelopment in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actincytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and patho-genic development in Magnaporthe.

Both unicellular and multicellular organisms have the abilityto dynamically reorganize their cytoskeletons in response toenvironmental changes, as well as during polarized growth thatis crucial for proliferation, differentiation, and morphogenesis.Fungal cells represent a perfect example of polarized growththat efficiently responds to environmental cues. The inductionof cell polarity is particularly dramatic in fungi that show di-morphic growth or morphogenic transition and is often asso-ciated with virulence in the pathogenic species (9).

Magnaporthe oryzae, a filamentous ascomycete and the causalagent of cereal blast disease, undergoes vegetative or infec-tious growth in the presence of rich nutrients or a host induc-tive surface, respectively. Under such growth conditions, Mag-naporthe shows unipolar extension of the mycelial tips (or germtubes), and its growth is well coordinated with morphogenicdifferentiation only under asexual or pathogenic development.Polarized growth is induced at several points in M. oryzaeduring the infection cycle: emergence of a germ tube from theconidium, elongation of the germ tube, penetration peg for-mation by the appressorium, both intra- and intercellular ex-tension of the invasive hyphae, and development of aerialhyphae from the mass of invasive hyphae within the host. The

aerial hypha starts to swell at the tip, suggestive of conidio-phore initiation, which includes the hypha (conidiophore stalk)and the swollen tip (conidiophore vesicle), separated by a septumat the neck. The vesicle eventually develops into a mature3-celled conidium (6). In yeasts, polarized growth is regulatedby several proteins in a polarisome complex at the growthzone, which in turn depends on polarity pathways that aretemporally and spatially regulated (12). Therefore, proteinsthat control morphogenic differentiation through cell polaritymight play key roles in microbial pathogenesis and in adapta-tion to new environments.

Members of the Rho family of small GTP-binding proteinsact as pivotal signaling switches and play a key role in mor-phogenesis during pathogenic development in M. oryzae (28,29). In Aspergillus nidulans, cell end marker proteins TeaA(Tea1), TeaR (Mod5), KipA (Tea2), and TeaC (Tea4) havebeen shown to be important for polarized hyphal growth (10,24). Similarly, the Tea1 homolog ClaKel2 has been implicatedin pathogenic differentiation in Colletotrichum lagenarium (18).It has been reported that a cyclin-dependent kinase from theCdk5/Pho85 family plays a key role in regulating polar growthrequired for developing infection structure and virulence in thedimorphic fungal pathogen Ustilago maydis (4).

Other polarity factors, apart from Tea1 and Tea2, includeTea3, Tea4, Tip1, Pom1, and Bud6 (21, 22, 26). Tea1 localizesto the cell tips in a Mod5-dependent manner (21) and is re-quired for the recruitment of Pom1 kinase (2, 25), Bud6 (8),and the formin For3, which nucleates F-actin in Schizosaccha-romyces pombe (7). Importantly, Tea4 mediates the interactionbetween Tea1 and For3, which is essential for F-actin nucle-

* Corresponding author. Mailing address: Fungal Patho-BiologyGroup, Temasek Life Sciences Laboratory, 1 Research Link, NationalUniversity of Singapore, Singapore 117604, Republic of Singapore.Phone: (65)-6872-7493. Fax: (65)-6872-7007. E-mail for NaweedNaqvi: naweed@tll.org.sg. E-mail for Rajesh Patkar: rajesh@tll.org.sg.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 14 May 2010.

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ation in S. pombe (13). However, the role of Tea4 has not beendefined in pathogenic fungi that undergo morphogenic differ-entiation in response to cues from the host or the environment.

In this study, we show that Tea4 in M. oryzae (MoTea4) playsan important role in maintaining polarized growth of aerialhyphae during asexual development and in differentiation ofgerm tubes into appressoria during pathogenic growth. Weanalyze the importance of microtubule and actin cytoskeletalorganization in appressorial development and the effect of theloss of Tea4 function on the organization of the actin cytoskel-eton in M. oryzae. Lastly, MoTea4 function was found to beimportant for asexual differentiation and effective virulence inM. oryzae.

MATERIALS AND METHODS

Fungal strains and growth conditions. Magnaporthe oryzae wild-type (WT)strain B157 (field isolate, mat1-2) was obtained from the Directorate of RiceResearch (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 my-celia in liquid nitrogen. Magnaporthe isolates were cultivated on PA medium orCM 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 onPA medium in the dark, followed by 4 days of growth under constant illumina-tion at room temperature. Inoculation loops were used to scrape the surface ofthe colonies in the presence of water, and the fungal biomass was collected inFalcon conical tubes (BD Biosciences, San Jose, CA). Maximum detachment ofconidia from mycelia was ensured by thoroughly vortexing the suspension. Thesuspension was then filtered through two layers of Miracloth (Calbiochem, SanDiego, CA), and conidia thus collected were washed twice with and finallyresuspended in sterile water containing 100 �g/ml each of streptomycin andcarbenicillin. The conidial count for a given colony was estimated using a hemo-cytometer and reported as the total number of conidia per unit area of thecolony.

To test appressorial development, conidia were spot inoculated either on a riceleaf 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. Forpathogenicity tests, droplets (20 �l) of conidial suspension (ca. 500 or 1,000conidia per droplet) were inoculated on barley leaf explants and incubated underhumid conditions at 23°C for up to 5 days (23).

Molecular biology, gene deletion, and complementation analyses. Fungalgenomic DNA was extracted using the MasterPure yeast DNA purification kit(Epicentre Biotechnologies). Plasmid DNA was isolated with the QIAprep plas-mid miniprep kit (Qiagen, Valencia, CA), and nucleotide sequencing was per-formed using the ABI Prism BigDye Terminator method (PE Applied Biosys-tems, CA). Homology searches of DNA/protein sequences were performed usingBLAST (1). The domains in the protein sequences were determined usingSMART and COILS. The following primers were used to amplify the 5� and 3�untranslated regions (UTR) (1 kb and 600 bp, respectively) of the TEA4gene: 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 therestriction enzyme sites used for cloning.) The amplified 5� UTR of MoTEA4 wascloned in HindIII/PstI sites of pFGL59 (a modified pCAMBIA1300 vector) toobtain pFGL501. The amplified MoTEA4 3� UTR was cloned in XbaI/KpnI sitesof pFGL501 to obtain pFGL502. Thus, the 5� and 3� UTR of TEA4 were ligatedsequentially to flank the hygromycin phosphotransferase (HPH1) expressioncassette in pFGL59 to obtain the plasmid pFGL502. Cloned TEA4 fragmentswere confirmed by nucleotide sequence analysis, and pFGL502 was introducedinto the WT Magnaporthe strain via Agrobacterium T-DNA-mediated transfor-mation (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. pombeTEA4 (SpTEA4) ORF (SPBC1706.01) along with 3� UTR (353 bp) was ampli-fied 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 andobtain pFGL558. The cloned SpTEA4 ORF and the 3� UTR fragments wereconfirmed by restriction analysis, and pFGL558 was introduced into the Magna-porthe tea4� strain via ATMT for random insertion. Single-copy integrants wereverified by Southern blot analysis.

Plasmid constructs for Tea4-GFP and Abp1-RFP. For C-terminal tagging ofTea4 (MGG_06439.6) with green fluorescent protein (GFP) (Clontech), the last 803bp of the Tea4 ORF without the stop codon was amplified using MoTea4-TagF(5�-GAGAGTGTTgaattcCTGGACAGTTCAAAGGGATCG-3�) and MoTea4-TagR (5�-GAGAGTGTTggtaccTGCAGCCCCCCTGAGTCTTTG-3�). A GFPfragment of 717 bp was amplified using EGFPF (5�-GAGAGTGTTggtaccATGAGTAAAGGAGAAGAAC-3�) and EGFPR (5�-GAGACTGTTggatccTTATTTGTATAGTTCATCCATG-3�). The 3� UTR of TEA4 was amplified by usingMoTea4-3UTR-F (5�-GAGAGTGActgcagTGTCTGTAGACTTGGTAACTG-3�) and MoTea4-3UTR-R (5�-GAGAGTGAaagcttTCTGTGAAGAGTACACTC-3�). The amplified Tea4 ORF (803 bp) and GFP fragments were digested withEcoRI/KpnI and KpnI/BamHI, respectively, and cloned in EcoRI/BamHI sitesof pFGL347 such that the Tea4 and GFP ORF are just upstream of the TrpCterminator in the vector. This recombinant vector was named pFGL541. Theamplified 3� UTR of TEA4 was digested with PstI/HindIII and cloned in respec-tive 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. oryzaeWT strain using the ATMT method. Strains showing replacement of the TEA4ORF with the TEA4-GFP fusion construct along with the BAR cassette wereidentified 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 amplifiedusing 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 ofthe 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 wasdigested with PstI/HindIII and cloned in respective sites in pFGL347 to obtainpFGL543. The fragments of the Abp1 ORF and RFP were digested with EcoRI/KpnI and KpnI/BamHI, respectively, and cloned in EcoRI/BamHI sites of pFGL543to obtain pFGL544, which was transferred to WT M. oryzae. Southern blot analysiswas performed to confirm successful gene replacement and single-copy integrations.

Plasmid constructs for MoTea41–405 and MoTea4406–979. To express either theSH3 (MoTea41-405) or the coiled-coil (MoTea4406-979) domain of MoTea4 indi-vidually in Magnaporthe, constructs were made to replace the full-length wild-type copy of MoTEA4 with the truncated MoTEA4 as follows. To express theSH3 domain alone, the last 1-kb stretch of the sequence encoding the N-terminalhalf of MoTea4 was amplified using primers MoTea4SH3F (5�-GAGAGTGTTgtcgacCGCCCGCCGCGCATTACAC-3�) and MoTea4SH3R (5�-GAGAGTGTTacgcgtCCTTCTCTTCATGGCAG-3�). The 3� UTR of the MoTEA4 genomicregion (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 thesequence encoding the N-terminal half of MoTea4 was digested with SalI/MluIand cloned in respective sites in pFGL557 (hygromycin resistance) to obtainpFGL581. The fragment of the 3� UTR of MoTEA4 was digested with HpaI/ApaI and cloned in respective sites of pFGL581 to obtain pFGL582. Similarly, toexpress the coiled-coil domain alone, 1 kb of the 5� UTR of MoTEA4 wasamplified 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 halfof MoTea4 was amplified using MoTea4-CCF (5�-GAGAGTGTTgttaacACCAAGACTGTAGCCTTC-3�) and MoTea4-CCR (5�-GAGAGTGTTgggcccATCCCTTTGAACTGTCCAG-3�). An amplified 1-kb fragment of the 5� UTR ofMoTEA4 was digested with SalI/MluI and cloned in respective sites in pFGL557to obtain pFGL579. The 1-kb fragment of the sequence encoding the C-terminalhalf of MoTea4 was digested with HpaI/ApaI and cloned in respective sites ofpFGL579 to obtain pFGL580. The plasmids pFGL580 and pFGL582 were in-troduced into WT M. oryzae, and the gene replacement events were identified bylocus-specific PCR. PCR primers used to screen transformants obtained frompFGL582 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

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primers were used: 580LS5�F (5�-CTGGAGATCCAAGTGGGTAG-3�),Hph5�out (5�-CAGAAACTTCTCGACAGACG-3�), DsRed3�out (5�-GTGGAGCAGTACGAGCACGC-3�), and 580LS3�R (5�-TGACAACCTCTGTTGCTGCC-3�).

Staining protocols, drug treatments, and microscopy. For transmission elec-tron microscopy (TEM) analysis, fresh conidia were harvested, thoroughlywashed 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 describedpreviously (23). Bright-field and epifluorescence microscopy analyses were per-formed with an Olympus IX71 or BX51 microscope (Olympus, Tokyo, Japan)using a Plan-Apochromat 100�/1.45 objective or UPlan FLN 60�/1.25 objectiveand the appropriate filter sets. Images were captured with a Photometrics Cool-SNAP HQ camera (Tucson, AZ) and processed using MetaVue (UniversalImaging, PA) and Adobe Photoshop 7.0.1 (Mountain View, CA).

Calcofluor white (CFW; Sigma-Aldrich) was used at 3 �g/ml (in 100 mMTris-HCl buffer, pH 9.0, containing Triton X-100 at a dilution of 1:1,000) tovisualize the cell wall and septa in the aerial hyphae, conidiophores, and conidiaof the respective strains. For staining of aerial hyphae and conidiophores, colo-nies were incubated with a suitable volume of the CFW solution (enough tocover the surface of the colonies) in the dark at room temperature for 10 min andthen washed several times and subjected to epifluorescence microscopy using therecommended filter sets. Colonies were assessed at 6 to 9 h or at 12, 24, or48 h postphotoinduction to study aerial hyphae or conidiophores, respec-tively. FM4-64 was used at a concentration of 10 �M in the medium. Cells wereincubated for 10 min and washed. Methyl benzimidazole-2-yl-carbamate (MBC;Sigma-Aldrich) was used at a final concentration of 1 �M, diluted from a stocksolution of 1 mg/ml in ethanol. Latrunculin A (LatA; Biomol) was used at a finalconcentration of 10 �M (stock solution, 10 mM in dimethyl sulfoxide). Cyto-plasmic streaming was documented by live imaging of growing germ tubes of theWT or tea4� strain, and the resultant differential interference contrast (DIC)images were stacked using MetaMorph software (Molecular Devices).

RESULTS

Identification of a Tea4 ortholog in M. oryzae. We identi-fied an ortholog of the S. pombe TEA4 gene in M. oryzae(MGG_06439) and named it MoTEA4. Although the overallsequence similarity to the S. pombe Tea4 protein was low, theconserved SH3 domain showed significant identity to the SH3domain of MoTea4. Likewise, MoTea4 showed similarity toBud14 from Saccharomyces cerevisiae. An apparent differencein the domain organization of the Tea4-like proteins fromfilamentous fungi was the presence of a coiled-coil motif inaddition to the SH3 domain. Overall, the yeast and fungalTea4 orthologs showed 26 to 42% sequence identity with asimilar architecture in filamentous fungi (Fig. 1).

MoTea4 localization in M. oryzae. To study the subcellularlocalization, 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-GFPstrain showed WT-like phenotypes in terms of conidial countand morphology, appressorium formation, and the ability tocause infection. Although weak in fluorescence signal, theMoTea4-GFP localized to the tips of the vegetative and aerialhyphae 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 somediffused cytosolic signal, localized away from the tips in theconidiophores (Fig. 2C). The MoTea4-GFP punctae were ob-served in mature conidia, predominantly along the septa, butwere also enriched in the terminal cell at the time of germi-nation (Fig. 2D). The MoTea4-GFP foci did not localize at thetips of the early germ tubes but appeared at the apex of theappressorium initials (Fig. 2E). To test whether MoTea4-GFPlocalization at the hyphal tips depends on microtubules or theactin cytoskeleton, we used the destabilizing agent MBC orLatA, respectively. Treatment of the vegetative hyphae with 1�M MBC for 30 min caused MoTea4-GFP fluorescence todivide into more than one puncta at the tips and/or to partiallydiffuse into the cytoplasm (see Fig. S5 in the supplementalmaterial), whereas the LatA-treated (10 �M, 30 min) vegeta-tive hyphae showed significantly diminished MoTea4-GFP flu-orescence at the tips (see Fig. S5). Our observations suggestthat the localization of MoTea4-GFP in the vegetative hyphaedepends on the microtubule cytoskeleton. However, the sta-bility of the MoTea4-GFP foci at the hyphal tips most likelydepends on the F-actin cytoskeleton. Next, we examined thelocalization of MoTea4-GFP with respect to the Spitzenkorpervesicles and stained the vegetative hyphae of the MoTEA4-GFP strain with FM4-64. MoTea4-GFP partially colocalizedwith the Spitzenkorper vesicles at the apex of the hyphae (Fig.2F). Based on its intracellular distribution, we infer thatMoTea4-GFP likely marks the growth zones at the cell tipsduring polarized growth in M. oryzae during vegetative andasexual development.

Deletion of M. oryzae TEA4 and its effect on vegetativeand asexual development. We generated an MoTEA4 dele-tion strain (hereafter called the tea4� strain; relevant geno-type, TEA4::HPH1) in Magnaporthe and analyzed it duringvegetative, asexual, and pathogenic development of the fungus.The colony growth of the tea4� strain was comparable to thatof the WT, except that the tea4� colony appeared white, asopposed to gray in the WT (Fig. 3A). The vegetative hyphae ofboth the WT and tea4� strains showed a normal straight mor-phology. However, unlike that of the WT, the vegetative hy-phae of the tea4� strain showed bundling toward the base,away from the tips (Fig. 3B). Similarly, both the WT and thetea4� vegetative hyphae showed normal branching (Fig. 3B,inset) and septation (see Fig. S4 in the supplemental material).To assess the asexual development in the tea4� strain, westudied the growth and morphology of the aerial hyphae andconidiophores stained with calcofluor white. Unlike vegetativehyphae, most of the conidiophores of the tea4� strain showeda zigzag morphology (Fig. 3C; see also Fig. S6B in the supple-mental material) and aberrant cell wall deposits. Furthermore,the majority of the tea4� conidiophores failed to initiateconidiation and did not show proper swelling at the tips evenafter 24 h of photoinduction (Fig. 3C). All of the conidiawere harvested from the WT or tea4� strain and quantified

FIG. 1. Identification of the Tea4 ortholog in M. oryzae. Sche-matic representation and similarity indices of Tea4-related proteinsfrom M. oryzae (MoTea4, MGG_06439.6), A. nidulans (AnTeaC,AN1099.3), S. pombe (SpTea4, SPBC1706.01), and S. cerevisiae(ScBud14, YAR014C). A highly conserved SH3 domain (black) anda coiled-coil domain (gray) are depicted. Sequence identity and similaritysearches were carried out using BLAST (1), and the domains were iden-tified using SMART (http://smart.embl-heidelberg.de/) and COILS (http://www.ch.embnet.org/software/COILS_form.html).

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to study the ability of the mutant to perform asexual differ-entiation. The number of conidia per square centimeter offungal growth was (106.9 � 3.5) � 102 in the WT, whereas itwas (0.63 � 0.09) � 102 in the tea4� strain (Fig. 3D; P �0.005). Moreover, the tea4� conidia were smaller than the WTconidia (15 �m versus 21 �m) and morphologically aberrantcompared to those produced by the WT. In contrast to thetricellular pyriform conidia in the WT, the tea4� conidia weremostly two celled and appeared spindle shaped (Fig. 3E). Ap-proximately 80% (n � 300) of the tea4� conidia had oneseptum, while only 4% of the WT conidia had one septum (Fig.3F; P � 0.005). We substantiated this result by analyzing theultrastructure of the tea4� conidia under TEM. The electronmicrograph of the mutant conidia showed only one septum,with a few exceptional cases where an attempt at making asecond septum was evident. However, the second septum wasaberrant and incomplete (see Fig. S2 in the supplemental ma-

terial). WT conidia invariably showed two completely devel-oped septa.

Next, we expressed the S. pombe TEA4 gene in the Magna-porthe tea4� strain to check if it could suppress the mutantdefects during asexual development and to ascertain whetherthe two orthologs share any functional similarity. The comple-mented strain showed a marginal suppression of mutant de-fects, with a 5- to 6-fold increase in the total number of conidiacompared to that in the tea4� strain (see Fig. S1A in thesupplemental material). Similarly, the percentage of 3-celledconidia increased from 20% to �40% (n 300) in the com-plemented strain (see Fig. S1B). Further, we replaced thefull-length MoTEA4 gene with the truncated allele encodingeither the SH3 or the coiled-coil domain in the MagnaportheWT. Although the strains expressing the SH3 domain aloneshowed an approximate 4-fold increase in total conidiationcompared to that in the tea4� strain, the conidiation was not

FIG. 2. Subcellular localization of MoTea4-GFP during Magnaporthe development. Epifluorescence microscopy-based localization of MoTea4-GFP during different stages of asexual development (vegetative hyphae [A], aerial hyphae [B], conidiophores [C], and conidia [D]) or pathogenicdevelopment (appressorium initials [E]) in an M. oryzae strain expressing the MoTea4-GFP fusion protein. The MoTea4-GFP puncta werepredominant at the tips of vegetative and aerial hyphae, in the subapical region of conidiophores, and in the septa of mature conidia (A to E,arrows). Fungal structures in some panels were outlined (dashed lines) using Adobe Photoshop 7. (F) Partial colocalization of Tea4-GFP and theSpitzenkorper vesicles (stained with FM4-64) in the vegetative hyphae of the MoTea4-GFP strain were visualized using epifluorescence micros-copy. The arrow indicates the tip of the hypha in a corresponding bright-field image. Bars, 5 �m.

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comparable to that of the WT (see Fig. S1C). However, thenumber of 2-celled conidia decreased significantly (from �80% inthe tea4� strain) to 32% in the strain expressing the coiled-coildomain (see Fig. S1D in the supplemental material). These find-ings indicate that the SH3 and the coiled-coil domains ofMoTea4 are necessary for its function in conidiation inMagnaporthe. We conclude that MoTea4-mediated polar-ized growth is important for proper differentiation of con-idiophores and conidia during asexual development in Mag-naporthe.

Impaired pathogenic development in the tea4� strain. Westudied infection-related development in the tea4� strain, bothin vitro and in planta, to assess the role of MoTea4 in the

pathogenesis of M. oryzae. In in vitro assays, infection-relatedmorphogenesis (appressorial development) was studied usingan inductive hydrophobic glass surface. A striking polarity-related defect was apparent in germ tube emergence in thetea4� strain on the inductive surface. A random germinationpattern with more than one germ tube per cell in a conidiumwas observed in the tea4� strain (Fig. 4A). In some cases,terminal cells in the tea4� conidia developed two germ tubes,which were never found in the WT conidia during pathogenicdevelopment (Fig. 4A). The majority of the tea4� conidiafailed to develop appressoria on the artificial inductive surfacein comparison to the WT conidia. While 85.4% � 2.0% of theWT conidia formed appressoria, only 9.2% � 0.7% of the

FIG. 3. Phenotypic analysis of the tea4� mutant. (A) Characteristics of 7-day-old WT or tea4� colonies on PA medium. (B) Morphology ofvegetative hyphae of the WT or tea4� strain grown on PA medium for 48 h. The arrow indicates bundling of the mutant hyphae away from thetips. Hyphal branching is highlighted in the inset. (C) Asexual differentiation in the WT or tea4� strain. The morphologies of the CFW-stainedaerial hyphae and conidiophores are shown for the indicated strains. The arrows highlight the zigzag morphology, whereas the arrowhead depictsa lack of swelling at the tips of conidiophores in the tea4� strain. Bars, 5 �m. (D) Conidial count of the WT and tea4� strains grown on PA mediumfor 7 days. Data represent the means � standard errors of the mean (SEM) from three independent experiments. (E) Conidial morphology of theWT or tea4� strain. Conidia from the indicated strains were harvested from 7-day-old colonies and stained with calcofluor white. Bars, 10 �m.(F) Bar graph depicting quantification of bi- and tricellular conidia in the WT or tea4� strain; n 300 conidia each from three independentreplicates of the experiment.

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mutant conidia were capable of appressorium formation (Fig.4B; P � 0.005). The mutant conidia developed long, multisep-tate, 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 vastmajority of the tea4� germ tubes initiating appressoria (aswollen germ tube tip) failed to complete the process, andfresh germ tubes emerged from the immature appressorium-like structures (Fig. 4C). To assess whether appressoriumdevelopment depends on the number of cells per conidium,we quantified the ability to form appressoria in the 2-celledand 3-celled tea4� conidia. About 87% of the 3-celled tea4�conidia failed to develop appressoria, whereas nearly 10% ofthe 2-celled mutant conidia formed such infection structures(see Fig. S7 in the supplemental material). These results indi-cate that the loss of MoTea4 function leads to a failure inproper pathogenic differentiation that is likely due to the lossof regulation of polarized growth during conidial germinationand appressorium initiation.

MoTea4, cytoskeletal organization, and appressorial devel-opment. We examined the sensitivity of the Magnaporthe WTtoward the microtubule-destabilizing agent MBC. Approximately70% of the untreated WT conidia developed appressoria, while20% formed appressorium initials within 6 h postinfection (hpi)(Fig. 5A). Although none of the MBC-treated WT conidiaformed immature appressoria in 6 h, about 40% showed appres-sorium initials (Fig. 5A). Importantly, the MBC-treated WTshowed significant recovery at 20 hpi, at which point 78% ofconidia developed into mature appressoria (Fig. 5B).

Next, we studied the sensitivity of the M. oryzae WT tothe actin-destabilizing agent LatA. Unlike the untreated WTconidial germination response described above, none of theLatA-treated WT conidia developed appressoria and �35% ofthem showed appressorium initials at 6 hpi (Fig. 5A). Further-more, the LatA-treated WT did not show significant recoveryat 20 hpi, and hardly 8% of conidia developed into appressoria(Fig. 5B). Interestingly, the LatA-treated WT conidia showedpolarity defects reminiscent of the tea4� mutant conidia, withmore than one germ tube emerging from a single cell in aconidium (Fig. 5B, arrowheads). Based on these observationsfrom studies of the WT, we propose that an intact F-actincytoskeleton, but not the microtubules, is required for properappressorial development in Magnaporthe.

Next, we analyzed the role of MoTea4 in F-actin organiza-tion by characterizing the WT and tea4� strains expressing theAbp1-RFP fusion protein to aid the visualization of F-actin.We studied asexual development, conidial germination, andappressorium development to test the effect of TEA4 deletionon the actin cytoskeleton at these developmental stages. Westudied the distribution of F-actin patches in the aerial hyphaeand the conidiophores of the WT or the tea4� strain expressingthe Abp1-RFP protein. The aerial hyphae of the WT showedenrichment 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 ofthe developing conidia (see Fig. S6A in the supplemental ma-terial). However, the tea4� aerial hyphae showed aberrant andreduced F-actin patches. The developing tea4� conidia showedF-actin aggregates within the vesicles (see Fig. S6A). MatureWT conidia showed the cortical F-actin patches (Abp1-RFPfoci) along the septa and at the cortex. Such cortical patcheswere significantly reduced in numbers in the tea4� strain, andmost of the Abp1-RFP signal was diffused and likely vacuolar(Fig. 5C). In germinating WT conidia, the F-actin patches were

FIG. 4. Effect of TEA4 deletion on pathogenic development in M.oryzae. (A) Schematic representations, with corresponding bright-fieldand epifluorescence images, of the conidial germination patterns in theWT and tea4� strains, respectively. Conidia from the WT or the tea4�strain were allowed to germinate on an inductive surface for 16 h andstained with calcofluor white (only tea4� germlings). Arrows indicategerm tubes. Scale bars represent 5 �m (WT) and 10 �m (tea4� mu-tant). (B) Bar graph representing quantification of the number ofconidia developing appressoria in the WT or tea4� strain. Data rep-resent the means � SEM from three independent experiments.(C) Pathogenic development in the indicated strains was observedafter 16 hpi using DIC optics. The arrows show emergence of germtube-like structures from malformed appressoria. Bars, 5 �m.

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enriched at the tips of the germ tubes and later uniformly alongthe periphery of the developing appressoria (Fig. 5C). In con-trast, the tea4� germ tubes showed aberrant F-actin aggre-gates, with a significantly reduced number of patches at the tips(Fig. 5C; see also Fig. S8 in the supplemental material). Tosubstantiate these findings, we studied the effect of LatA on

the WT M. oryzae strain expressing the Abp1-RFP fusion pro-tein. In the LatA-treated Abp1-RFP strain, the F-actin patcheswere rarely seen at the apex of the germ tubes. In addition,aggregates of F-actin (Abp1-RFP) were evident along theLatA-treated germ tubes (Fig. 5D). Our observations indicatethat the actin cytoskeleton plays an important role in asexual

FIG. 5. MoTea4 regulates F-actin organization in M. oryzae. Effect of MBC or latrunculin A on early pathogenic development in the WT.Two-hour-old WT germlings were treated with the indicated destabilizing agent for 30 min, washed, and observed after 6 hpi (A) or 20 hpi (B).(A) The data represent the means from two independent assays. (B) Arrowheads indicate multiple germ tubes arising from the terminal cell ofa conidium. Bars, 5 �m. (C) Localization of the Abp1-RFP fusion protein in the WT or tea4� strain of Magnaporthe. Abp1-RFP localized to septaland cortical regions in mature WT conidia (upper panels, arrows) and to the tips of germinating WT conidia (lower panel, arrows). In the tea4�conidia, Abp1-RFP showed diffuse localization in the vacuoles (upper panels) and rarely to the tips of germ tubes (lower panel, asterisks). Thearrowheads indicate Abp1-RFP aggregates in the germ tubes and malformed appressorium initials in the tea4� strain. Bars, 5 �m. (D) Effect ofLatA on F-actin organization during pathogenic development in the Magnaporthe WT. Two-hour-old WT (Abp1-RFP) germlings were treated withLatA for 30 min, washed, and observed after 5 hpi. The arrow indicates an F-actin aggregate, and the asterisk indicates rare apical patches. Bar,10 �m.

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development and pathogenic differentiation and that the lossof MoTea4 function likely disrupts the proper organization ofthe actin cytoskeleton in M. oryzae.

MoTea4, F-actin organization, and cytoplasmic streaming.We tested whether F-actin organization is involved in cytoplas-mic streaming in Magnaporthe. During appressorial develop-ment, LatA-treated WT germlings showed a significantly slowand restricted movement of cytoplasmic content. Next, we an-alyzed the effect of MoTea4 deletion on cytoplasmic streamingin M. oryzae. In WT germlings, a rapid and bidirectional move-ment of intracellular particles was observed (see Video S1 inthe supplemental material), while the tea4� strain showed arelatively slow and highly constrained movement of the cyto-plasmic particles, especially in the germ tubes (see Video S2).As a marker for bulk cytoplasmic streaming, we studied lipiddroplet (LD) mobilization in the WT and tea4� germlings(germinating conidia). In the WT, the germlings showed bulkmovement of the LDs from the conidia to the germ tubes,followed by accumulation in the incipient appressoria. In con-trast, the tea4� germlings showed accumulation of LDs inconidia at 6 hpi (see Fig. S3 in the supplemental material).Taken together, we propose that MoTea4 is required forproper organization of the F-actin cytoskeleton, which may beinvolved in cytoplasmic streaming and bulk mobilization ofcellular content during appressorium development in Magna-porthe.

Impaired virulence in the tea4� strain of Magnaporthe. Weexamined appressorial development on a rice leaf sheath where,as on an inductive glass surface (Fig. 4A and 4C), the tea4�conidia showed polarity-related defects in germ tube emer-gence and failed to form proper appressoria. Furthermore, themajority of the tea4� appressoria were mostly irregular inshape and size compared to the WT appressoria (Fig. 6A).Next, we assessed the virulence of the WT and tea4� strains onbarley leaf explants. The WT Magnaporthe strain started de-veloping disease symptoms at 3 days postinoculation (dpi), andtypical disease lesions appeared at 5 dpi. However, significantsymptoms of effective virulence of the tea4� strain were notevident even after 7 dpi, except for marginal browning at thesite of inoculation (Fig. 6B). Further, we examined the tea4�strain to assess whether the malformed appressoria were ca-pable of host penetration. In the WT, 61% � 5% of the ap-pressoria developed penetration hyphae after 40 hpi, whereasnone of the tea4� appressoria showed penetration hyphaewithin the host tissue (Fig. 6C and D). Taken together, thesefindings led us to conclude that MoTea4 function is requiredfor pathogenesis, likely through proper development and func-tion of the infection structures in M. oryzae.

DISCUSSION

The rice blast pathogen M. oryzae undergoes a specializeddevelopment manifested by spatiotemporally regulated polar-ized growth, and this process is closely associated with patho-genesis. Studying the role of cell polarity involved in infection-related morphogenesis will help to provide insight into the cellbiology of fungal development. In addition, the molecularanalysis of polarized growth may lead to the identificationof targets for new antifungal agents. Here, we studied the roleof a cell end marker protein, MoTea4, in the morphogenesis of

M. oryzae and observed that its function is important not onlyfor morphogenic differentiation during asexual developmentbut also for pathogenesis.

MoTea4 is a protein containing an SH3 domain that is foundin proteins which regulate the actin cytoskeleton, Myo3p,Myo5p, Abp1p, Bzz1p, Bbc1p, Rvs167p, Lsb1p to Lsb4p, andSla1p (14). Although overall sequence identity betweenSpTea4, AnTeaC, and MoTea4 is low, the SH3 domain is highlyconserved in these proteins. MoTea4 shows similarity to SH3domain-containing proteins in fungi other than S. pombe andA. nidulans, like Penicillium marneffei (PMAA_097510), Cryp-tococcus neoformans (CNBE_001580), and Talaromyces stipi-tatus (TSTA_043520). Interestingly, Tea4 only from filamen-tous fungi showed a coiled-coil motif in addition to the SH3

FIG. 6. MoTea4 is required for pathogenesis in M. oryzae. (A) Riceleaf sheaths, inoculated with conidia from the indicated strains, wereobserved after 24 h using bright-field optics. The arrowhead indicatesan irregularly shaped appressorium in the tea4� mutant. The arrowpoints at an aberrantly sized appressorium. Bars, 10 �m. (B) Barleyleaf explants inoculated with 1,000 conidia per droplet from the indi-cated strain were incubated at 22°C and photographed after 6 days.Disease reaction was analyzed based on visible disease lesions andspread at the site of inoculation. (C) Quantification of WT or tea4�appressoria developing penetration hyphae within the host leaf tissue.Data represent the means � SEM from two independent experiments.(D) Barley leaf explants inoculated with WT or tea4� conidia andobserved after staining with acid fuchsin at 40 hpi. The arrowheadindicates an invasive hypha, whereas the arrows indicate mutant ap-pressoria that lack penetration hyphae. Bars, 10 �m.

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domain. It has been shown that Pea2, a coiled-coil-domain-containing protein, localizes to the sites of polarized growthand is required for efficient mating and bipolar budding in S.cerevisiae (27). It is possible that the coiled-coil domain in Tea4of filamentous fungi might play an additional albeit importantrole in the relatively complex morphogenesis of these fungi.

We observed that MoTea4 plays an important role duringgrowth or morphogenic transitions required for asexual andpathogenic development. For instance, MoTea4 is not essen-tial for viability or growth of vegetative mycelia but is requiredfor proper asexual development. MoTea4 localizes to the re-gions of active growth or morphogenesis, namely, the tips ofvegetative and aerial hyphae, conidiophores, and appressoriuminitials. In agreement with the MoTea4 distribution, most ofthe tea4� aerial hyphae showed aberrant morphology and didnot form conidia. Moreover, the tea4� conidia lacked oneseptum and did not develop appressoria on an inductive sur-face. Surprisingly, growth, morphology, and septation in thevegetative hyphae of the tea4� strain were similar to that of theWT except for the unusual hyphal bundling in the mutant. InMagnaporthe oryzae, the majority of the cells (conidial, hyphal,and germ tube) are mononuclear. Nuclear division duringconidiation in Magnaporthe, especially formation of a 3-celledconidium from a single conidiophore vesicle, is not yet wellunderstood. It is not clear whether a single nucleus in theconidiophore vesicle undergoes two rounds of division prior toseptum formation or if each nuclear division is followed by aseptum formation. Mid1, an aniline-like protein in S. pombe,marks the cortex at the cell median for actomyosin ring assem-bly and subsequent septum formation. Similar to that of S.pombe, septation in Magnaporthe follows every nuclear divi-sion, forming mononucleate hyphal compartments. However,there is no ortholog for Mid1 in Magnaporthe, suggesting thateither such a protein is poorly conserved or some other pro-tein(s) regulates the placement of the division septa in fila-mentous fungi. Deletion of TEA4 in Magnaporthe leads to theloss of a septum in most of the conidia, suggesting that suchpolarity determinants are important regulators of septum for-mation during asexual development. An additional role forSpTea4 in preventing septum formation at the cell ends in S.pombe has been shown earlier (11). Indeed, we found thatmost of the 2-celled tea4� conidia showed excess septal depos-its at the tips of the terminal cells (Fig. 3E, rightmost panels).Similarly, deletion of TEAC in Aspergillus led to an impairmentin coordination between mitosis and septation, resulting inshort and anucleate compartments in the vegetative hyphae(10). Thus, an additional role for MoTea4 in regulation ofseptation in Magnaporthe needs further investigation.

It has been shown that ClaKel2p, a Tea1 homolog in Colle-totrichum lagenarium, is involved in polarized growth and lo-calizes to the tips of growing hyphae, to germ tubes, and weaklyto the periphery of the appressoria. The clakel2 mutant formedabnormal appressoria on glass slides; however, mutant hy-phal growth on potato dextrose agar medium and conidia-tion, though with a reduced amount of spores, was similar tothat of the wild type (18). In M. oryzae, Zheng et al. (28) haveshown that Magnaporthe grisea Rho3 (MgRho3) is dispensablefor vegetative hyphal growth but not for plant infection, as theappressoria formed by the �Mgrho3 strain are morphologicallyabnormal and defective in plant penetration. However, it is

important to note that unlike in Magnaporthe, the deletion ofTEAC in A. nidulans leads to significantly reduced vegetativegrowth (10) and a likely decrease in conidiation. It appears thatdifferent molecular machineries regulate the polarized growthand sporulation of filamentous fungi and yeasts: in filamentousfungi and in hyphal growth in Candida, polarized growth ismediated by the Spitzenkorper vesicles, while it is controlled bythe polarisome complex in the budding process of Candida andSaccharomyces (5). Expression of SpTea4 in the tea4� strain ofMagnaporthe only marginally rescued the mutant defects (seeFig. S1A and B in the supplemental material). Further struc-ture-function analysis of MoTea4 revealed the importance ofboth the SH3 and the coiled-coil domains in proper conidia-tion in Magnaporthe. Based on these findings and the partialrescue of the tea4� defects by SpTea4, we propose thatMoTea4 has most likely diverged significantly from yeast Tea4and is adapted for an important function(s) during asexualdevelopment in M. oryzae. Similarly, it is also likely that thepolarized growth and morphogenesis underlying vegetativeand pathogenic development in fungi is controlled by differentmolecular mechanisms with some common polarity determi-nants.

Germ tube emergence and extension were defective in thetea4� strain of M. oryzae. The WT strictly shows only one germtube, per terminal or basal cell of the conidium, that goes on todifferentiate into an appressorium. The tea4� strain failed toform appressoria, likely due to its inability to sustain polarizedgrowth during such early stages of the pathogenic phase. It isinteresting to note that some of the tea4� germ tubes showedan attempt to develop appressoria; however, fresh germ tubesemerged from those immature appressorium-like structures.This is a unique defect and has not been seen in any Magna-porthe mutant. It would be interesting to analyze this transitionin detail to uncover the (Tea4-dependent) mechanisms thatsuppress vegetative growth during pathogenic differentiation.

In filamentous fungi such as A. nidulans and N. crassa, hy-phal tip elongation is a continuous and indefinite process (15,17). However, pathogenic filamentous fungi like M. oryzaeshow morphogenic transitions from polarized to nonpolarizedgrowth and vice versa during the pathogenic life cycle. Polar-ized growth is regulated during the cell cycle in single-cellyeasts, such as S. cerevisiae and S. pombe (22), with tight spa-tiotemporal control of actin assembly. In S. pombe, Tea4 me-diates the interaction between Tea1 and a formin, For3, thatacts as a site for rapid nucleation and assembly of actin fila-ments and network (13). The function of Tea1p and Tea4p isdependent on their localization to the cell end, which in turn isdependent on microtubules in S. pombe (7, 20). Interestingly,the M. oryzae WT showed a significant recovery from MBCtreatment and successfully formed appressoria. Similarly, in C.lagenarium, the localization of ClaKel2p is not dependent onmicrotubules during pathogenic development, and MBC treat-ment does not affect appressorium formation (18). However,LatA-treated germinating WT Magnaporthe conidia did notrecover and failed to develop appressoria, indicating that theappressorial development was dependent on an intact F-actincytoskeleton and not on microtubules. It is also worth notingthat the phenotype of LatA-treated germinating WT conidiawas similar to that of the untreated tea4� strain. Importantly,the Abp1-RFP (F-actin) localization pattern appeared to be

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similar to that of MoTea4-GFP in mature conidia in Magna-porthe, and conversely, deletion of TEA4 disrupted F-actinorganization in mature and germinating conidia. The similaritybetween the disorganized F-actin cytoskeleton (Abp1-RFP) inthe tea4� strain and that of the LatA-treated WT supports ourhypothesis that a properly organized actin cytoskeleton, thoughnot crucial for vegetative growth, is essential for successfulpathogenic differentiation and that MoTea4 plays an impor-tant role in regulating polarized growth in M. oryzae.

It has been inferred that an actin-myosin-based motor sys-tem is required for cytoplasmic streaming in plant cells (19).Magnaporthe shows rapid cytoplasmic streaming during ap-pressorial development. The LatA-treated WT germlingsshowed poor cytoplasmic streaming, indicating that the F-actincytoskeleton is likely important in the bulk movement of cyto-plasm in Magnaporthe. Loss of MoTea4 function in M. oryzaeled to disorganization of the F-actin cytoskeleton, and hence, itis likely that germinating tea4� conidia were affected in propercytoplasmic streaming. Only 8% of the conidia of the tea4�strain, however, formed appressoria, which were aberrantlyshaped and not functional. Such appressoria did not developpenetration hyphae, and as a consequence, the tea4� mutantcould not penetrate the host cells and failed to cause disease.It has been proposed that the actin cytoskeleton plays an im-portant role in the appressorial function (penetration of thehost tissue) (3). Our observations indicate that an impairedF-actin organization within the appressoria likely leads to lossof pathogenesis in the tea4� mutant.

In summary, we showed that MoTea4 function is importantfor polarized growth and in establishing a proper F-actin cytoskel-eton required for the pathogenic development in M. oryzae. Fu-ture studies will be aimed at deciphering the role of the coiled-coil domain in MoTea4 function. Interaction studies to identifythe MoTea4 binding partner(s) in Magnaporthe will certainlybe helpful in understanding the relatively complex regulationof polarized growth in this important fungal pathogen.

ACKNOWLEDGMENTS

This work was funded by intramural research funds from the Te-masek Life Sciences Laboratory (Singapore) and the Singapore Mil-lennium Foundation.

We thank the Fungal Patho-Biology Group and the Cell BiologyForum (TLL) for helpful discussions and suggestions.

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