PMC

Expression Profile of CHS Null Mutants in Liquid Culture.
(A) RNA gel blot of RNA isolated from yeast-like cells of U. maydis. All CHS genes are expressed in sporidia.
(B) Quantitative real-time PCR revealed that chs1 and chs4 are transcriptionally upregulated in hyphae of U. maydis (strain AB33). Note that Δchs1 and Δchs4 mutant hyphae are without phenotype, whereas chs7, which is transcriptionally not induced, is of high importance for the filamentous growth of U. maydis. Values are given as means ± se (n = 3).

Effect of the CHS Inhibitor Nikkomycin Z on Wild-Type and CHS Null Mutant Strains.
(A) Incubation of growing cells with 5 μM Nikkomycin Z (NZ), a specific inhibitor of CHS, for 1 h leads to a swelling of the growing tip of the cell (arrows). Interestingly, this effect was also seen in large-budded cells that form septa, as indicated by Calcofluor staining (asterisk). After longer periods of drug treatment, cells formed large vacuoles (arrowheads) and died. Note that these cells did not burst, even when transferred into water, indicating that Nikkomycin Z–mediated inhibition of CHS at the growing cell pole only partially weakened the cell wall. Bars = 10 μm.
(B) Plate growth of wild-type cells was only slightly inhibited at 1 μM Nikkomycin Z. However, at this concentration, growth of Δchs1, Δchs5, Δchs7, and Δmcs1 cells was impaired, whereas Δchs6 mutants did not form any colonies. Because Nikkomycin Z leads to swelling of the tip (see above), the hypersensitivity of Δchs1, Δchs5, Δchs6, Δchs7, and Δmcs1 mutants might reflect activity of these CHSs at the growth region.
(C) Incubation of Δchs6 mutants in 5 μg/mL Nikkomycin Z killed most cells and led to slight swelling of the mother cells. Note that control cells were still viable under these conditions.

Overview of the Importance of CHSs in Different Life Cycle Stages of U. maydis.
The cellular importance of each CHS is reflected by the size of its name.
(A) Haploid yeast-like cells grow by polar budding, and Chs5 is essential for their morphology. Δchs7 cells show an additional cell separation defect, and Δmcs1 mutants are often thicker and show polar swellings, indicating that these CHSs also participate in morphogenesis. Finally, Δchs1, Δchs5, Δchs6, Δchs7, and Δmcs1 are hypersensitive against Nikkomycin Z, which apparently inhibits CHS activity at the growing tip. This finding suggests that these CHSs support the tip growth of yeast-like cells.
(B) Pathogenic development is initiated by the formation of conjugation hyphae. Chs5 and Chs7 are essential for the formation of these tubes, whereas Chs1, Chs3, and Chs4 are of minor importance. However, only Chs7 activity is required for the proper morphology of mating tubes.
(C) Chs7 is essential for the proper morphology and growth of dikaryotic hyphae, whereas ΔChs5 shows a significant delay in the growth of dikaryotic hyphae. Again, Chs6 participates in hyphal growth, but its activity is of minor importance.
(D) Only Chs6, Chs7, and Mcs1 are essential during the infection process, whereas Chs2, Chs3, Chs4, and Chs5 play only minor roles that become crucial when the plant grows under optimal conditions. In part, the pathogenicity of Δchs7 mutants could be restored when solopathogenic strains were used, indicating that the reduced virulence is partially attributable to the described mating defects. By contrast, Mcs1 activity becomes crucial when the fungus enters the plant.

Δmcs1 Mutant Phenotype of cAMP-Dependent Filaments and Chitin Distribution in b-Dependent Hyphae.
(A) Deletion of adr1, which encodes the catalytic subunit of cAMP-dependent kinase (), results in continuous filamentous growth of the haploid cells. Staining of nuclei with 4′,6-diamidino-2-phenylindole reveals that these hyphae contain numerous nuclei (arrowheads). Occasionally, condensed and closely spaced nuclei are seen (asterisks) that are indicative of a mitotic event, which demonstrates that cAMP-dependent hyphae are not cell cycle arrested. Bar = 20 μm.
(B) Δmcs1 Δadr1 double mutants also form multinucleated hyphae (nuclei indicated by arrowheads). Hyphal morphology is almost normal, although some hyphae appear thicker and less regular (asterisk). Thus, Mcs1 is dispensable for hyphal growth of continuously growing cAMP hyphae. Bars = 20 μm.
(C) Staining of chitin with low amounts of rhodamine-conjugated wheat germ agglutinin weakly labeled the lateral cell walls and a prominent chitin cap at the hyphal apex (SG200, arrows). Note that this chitin cap corresponds well with the localization of Mcs1 (see ). By contrast, this polar chitin cap is absent from Δmcs1 hyphae (SG200ΔMcs1). Bars = 10 μm.
(D) Quantitative line-scan analysis of the intensity of rhodamine-conjugated wheat germ agglutinin along the apical 30 μm of control (SG200) and Δmcs1 (SG200ΔMcs1) hyphae demonstrates that the amount of chitin in the lateral cell wall is reduced and that the apical chitin cap (arrow in top graph) is missing from mutant hyphae. These data suggest that Mcs1 participates in wall synthesis of U. maydis hyphae but that its activity is not crucial for hyphal morphology.

Localization of CHS-GFP/YFP Fusion Proteins in Haploid Yeast-Like Cells and Dikaryotic Hyphae.
(A) GFP or YFP fused to the 3′ end of endogenous CHS genes resulted in fluorescent fusion proteins that localized to septa and in some cases the growing tip of the bud (Msc1-YFP is given as an example). In large-budded cells, fusion protein was detected at the primary septum as well as at the bud tip (arrows), indicating that the cell still expands while it forms septa, a result that was also indicated by the Nikkomycin Z experiments (see ). Bar = 3 μm.
(B) Fluorescent fusion proteins of Chs5, Chs6, Chs7, and Mcs1 localize to the growing bud. Note that Chs7-YFP forms a distinct point at the tip, whereas the other CHSs cover a broader region of the apex. Bar = 3 μm.
(C) All CHSs localize to secondary septa. Note that no signal was seen for Chs1-GFP, although small amounts were detected in protein gel blots (data not shown). Chs2-GFP was not detected on protein gel blots or by microscopy. Bar = 3 μm.
(D) In dikaryotic hyphae, most CHSs localized to the growing tip and septum that separates the living tip cell from the vacuolated older part of the hyphae (Mcs1-YFP is given as an example). Arrows mark the poles of the growing tip cell. Bar = 10 μm.
(E) Fluorescent fusion proteins of Chs5, Chs6, Chs7, and Mcs1 localize to the hyphal apex. Again, Chs7-YFP shows a very pointed localization. Bar = 3 μm.
(F) Similar to the situation in yeast-like cells, all CHS-GFP/YFP fusion proteins are found at the septa. Note that the CHS-GFP/YFP fusion proteins were functional, as they restored the phenotype of Δmcs1 mutants (see also ) or did not cause the typical phenotypes when introduced into the endogenous locus of chs5, chs6, and chs7. Bar = 3 μm.

Defects of Δmcs1 Hyphae in Early Plant Infection.
(A) Series of Z axis projections showing the initial infection state of the wild type (control) and Δmcs1 mutants. One day after infection, wild-type hyphae enter the plant epidermis without obvious morphological differentiation (arrowhead, −10 μm). By contrast, even at 3 d after infection, most Δmcs1 hyphae fail to invade the host tissue or they reach just into the upper layer (arrowhead, −10 μm). Note that Δmcs1 mutants are heavily swollen at the infection site. Distance in the Z-direction is given in micrometers. Bars = 20 μm.
(B) Those hyphae that entered the plant tissue did not continue directed growth but formed large aggregates of rounded and swollen cells. Distance in the Z-direction is given in micrometers. Bar = 20 μm.
(C) Invading mutant hyphae are able to grow inside plant cells (the plant cell wall is indicated by arrowheads) but are thicker than control cells. This indicates that Mcs1 is not needed for invasion but becomes essential for hyphal morphogenesis and directed growth. Bar = 5 μm.
(D) Quantitative analysis of the diameter of control and Δmcs1 hyphae in planta. In the absence of Mcs1, the hyphal diameter is increased significantly, indicating that cells are able to grow but have lost their polarity. Values are given as means ± se (n = 10 to 11 appressoria).
(E) On agar plates, the growth of wild-type FB2 cells, Δmcs1, and Δmcs1 Δchs6 mutants was not different. In the presence of ∼0.0035% (v/v) H₂O₂ oxidative stress, the growth of control cells and mutants was impaired, but again no strong difference between control and mutant strains was detected.

Pathogenicity of CHS Null Mutants.
(A) Infection of maize plants with compatible control or CHS mutant strains led to tumor formation in most inoculated plants and eventually killed the host. By contrast, compatible Δmcs1 mutant strains showed no symptoms, indicating that the deleted CHS is of great importance for plant infection.
(B) The solopathogenic strain SG200ΔChs5 showed ∼60% of the virulence of control strains, and SG200ΔChs7 was able to infect ∼30% of all plants. However, in both mutants, the resulting tumors were much smaller than those of control plants.
(C) Quantitative analysis of at least two experiments counting infected plants that formed tumors 14 to 16 d after infection revealed that Chs1 to Chs4 activity was of no or minor importance for plant infection (black bars), whereas Δchs6, Δchs7, and Δmcs1 were nonpathogenic (black bars). Values are given as means ± se (n = 2 to 3 experiments and >63 plants). Under improved plant growth conditions, the virulence of control strains was attenuated (data not shown) and Δchs4 mutants also did not induce tumor formation (gray bars).
(D) In the solopathognic strain SG200ΔChs7, virulence was partially restored, indicating that the mating defect of Δchs7 mutants is largely responsible for the reduced pathogenicity, Values are given as means ± se (n = 2 to 3 experiments and >55 plants). However, pathogenicity was at 25.7%, which argues for important roles of Chs7 in planta. SG200ΔMcs1 was still nonpathogenic, and virulence was restored by expression of a Mcs1-GFP fusion protein.
(E) Fourteen days after infection, tumors of plants infected with control cells and Δchs1 to Δchs5 were harvested and their fresh weight per plant was determined. Although CHS mutants formed tumors, the size of these tumors was drastically reduced, suggesting that all CHSs participate in pathogenic development.

Sequences of CHSs in U. maydis.
(A) Comparison of the eight CHSs that were identified in the genomic sequence of U. maydis (see Methods for URL). Fragments of UmChs1 and UmChs2 were published by Bowen et al. (1992). Partial sequence information for UmChs3, UmChs4 UmChs5, and Umchs6 was reported previously (, ). In addition, the U. maydis genome encodes a class IV CHS (Chs7, hypothetical protein UM05480; and the myosin-CHS Mcs1, hypothetical protein UM3204_1). Dendrograms are based on the complete amino acid sequence (see Supplemental Table 1 online for sequence alignment). Af, Aspergillus fumigatus; An, Aspergillus nidulans; Ao, Aspergillus oryzae; Aq, Ampelomyces quisqualis; Bg, Blumeria graminis; Ca, Candida albicans; Ci, Coccidioides immitis; Ed, Exophiala dermatiditis; Gg, Glomerella graminicola; Mg, Magnaporthe grisea; Nc, Neurospora crassa; Pb, Paracoccioides brasiliensis; Pc, Phanerochaete crysosporium; Rm, Rhizopus microsporus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. Note that only the 3′ ends of all CHSs were experimentally determined; the start of all genes is predicted based on comparison with published CHS gene sequences from other species.
(B) Domain organization of the CHSs from U. maydis. The predicted gene products share 15 to 73% sequence identity in the central CHS core region containing parts that most likely provide the enzymatic activity. In addition, numerous transmembrane domains are predicted. Mcs1 contains an N-terminal domain that shows significant homology with the motor domain of myosins. The domain organization of Chs2p from S. cerevisiae is given for comparison. aa, amino acids.

Morphology of Yeast-Like CHS Null Mutants.
(A) The deletion of most CHSs had no or only minor effects on the shape of haploid yeast-like cells. Δchs6 mutant mother cells were thicker (arrow), and a small portion of Δmcs1 cells showed swelling of the bud tip and, occasionally, also of the mother cell. Bar = 5 μm.
(B) Δchs5 mutants grow irregularly (see inset) and often lose the neck constriction that separates the mother and the daughter cells. Furthermore, ∼11% of all cells formed cell chains, indicating that the activity of Chs5 is essential for the proper morphology of yeast-like cells. Bars = 5 μm.
(C) Deletion of the newly identified chs7 led to a significant swelling of mother cells (see inset) and resulted in a cell separation defect. Bars = 5 μm.
(D) The phenotype of Δchs5 mutants was in contradiction to previous reports (). However, RNA gel blot analysis confirmed the absence of the chs5 message in deletion strains. Moreover, the morphology phenotype could be rescued by expression of Chs5 from a self-replicating plasmid (ΔChs5 + pCHS5), whereas the empty plasmid (ΔChs5 + empty plasmid) was without effect. Bars = 5 μm.
(E) Chitin staining with rhodamine-conjugated wheat germ agglutinin demonstrates that chitin is deposited at the growing bud (control, arrows). In addition, the bud scar is detected (control, arrowhead). In Δchs5 mutants, chitin staining is strongly reduced and no buds are detected (ΔChs5). Instead, chitin accumulates in patches at the cell poles (ΔChs5, arrows). Surprisingly, Δchs7 mutants had almost no chitin staining in the growing buds (ΔChs7, arrows) but an unusually strong staining of the mother cell wall. This indicates that the absence of this CHS leads to defects in chitin depositioning or maturation of the cell wall. Bar = 5 μm.
(F) Quantitative analysis of the rhodamine wheat germ agglutinin signal intensity in the lateral cell wall demonstrates that deletion of chs5 reduced the chitin content (ΔChs5), whereas deletion of Chs7 increased the wheat germ agglutinin signal (ΔChs7). Values are given as means ± se (n = 12 to 18).

The Role of CHS in the Formation and Morphology of Conjugation and Dikaryotic Hyphae.
(A) In medium supplemented with synthetic pheromone, ∼80% of wild-type cells form long conjugation hyphae (see [B], control). Under the same conditions, Δchs1, Δchs3, Δchs4, and Δchs6 mutants showed significantly fewer hyphae, whereas the activity of Chs5 and Chs7 is crucial for tube formation. Values are means ± se (n = 2 to 3 experiments, and at least 100 cells per experiment were analyzed).
(B) After 11 h of pheromone treatment, some Δchs5 conjugation hyphae were found that showed almost normal morphology. By contrast, Δchs7 mating tubes grew irregularly, had additional septa, and were much thicker. Bar = 10 μm.
(C) Crossing of compatible strains on charcoal-containing plates resulted in the formation of a fuzzy colony that is covered by white aerial hyphae (the genotype of control strains is indicated as an example). The formation of these filaments was attenuated in Δchs5 and Δchs6 strains and completely abolished in crossings of compatible Δchs7 mutants (arrows). Solopathogenic SG200 mutants form filaments without fusion to a mating partner, which results in a white colony (bottom row). However, SG200ΔChs5 and SG200ΔChs7 still did not form hyphae, indicating that both CHSs are important for hyphal growth.
(D) After 2 d on charcoal, compatible Δchs5 and Δchs6 strains form fuzzy colonies, indicating that filament formation is delayed in these mutants. Note that Δchs6 × Δchs6 colonies did not become white and fuzzy. Insets show larger magnifications of the colony edge.
(E) Control hyphae of crossings of compatible wild-type strains (a1b1 × a2b2) and solopathogenic SG200 are indistinguishable from Δchs5 (Δchs5 × Δchs5), Δchs6 (SG200ΔChs6), and Δmcs1 (SG200ΔMcs1) mutant hyphae. Only Δchs7 mutants show severely altered morphology (SG200ΔChs7). Bar = 30 μm.
(F) Deletion of mcs1 and chs6 led to minor defects in the morphology of yeast-like cells. Occasionally in Δmcs1, the growing bud was swollen (ΔMcs1, arrow) and the mother cells were slightly thicker. By contrast, deleting both class V CHSs led to more drastic swellings of the Δmcs1 Δchs6 mutants (ΔMcs1ΔChs6, arrow). Note that this mutant phenotype varied between experiments and that extreme examples are shown. Bar = 3 μm.
(G) In contrast with yeast-like cells, deletion of mcs1 and chs6 did not affect the morphology of dikaryotic hyphae, indicating that both class V CHSs have minor roles in hyphal growth ex planta. Bar = 10 μm.