Clp1 Interacts with bW and Rbf1 in Vitro and in Vivo.
(A) In vitro expression and coimmunoprecipitation of Myc-tagged Clp1 (~48 kD) and HA-tagged Cib1 (~61 kD), bW (~40 kD), and Rbf1 (~35 kD). In the negative control (Clp1_Myc), no unspecific binding of Myc-tagged Clp1 to the αHA-antibody was detected after coincubation with lysate. By contrast, after coincubation with HA-tagged Cib1, bW, or Rbf1, the Myc-tagged Clp1 protein was coimmunoprecipitated, demonstrating that the Clp1 protein physically interacts with Cib1, bW, and Rbf1 in vitro. Proteins were labeled with biotin, detected with streptatividin-conjugated alkaline phosphatase, and visualized with NBT/BCIP. Asterisks indicate HA-tagged proteins, and arrows indicate the Myc-tagged Clp1 protein.
(B) BiFC analysis of Clp1-protein interactions. Clp1 fused to NG (N-terminal part of eGFP) was coexpressed with bW1, Cib1, Rbf1, or a nuclear localization sequence (NLS) from VP16 (negative control) fused to CG (C-terminal part of eGFP). A nuclear localized GFP signal was detected in strains coexpressing Clp1 with bW1, Cib1, or Rbf1. By contrast, only background fluorescence was observed in the negative control. For visualization of nuclei, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI). Bars = 10 μm.

cib1 Is Required for Proliferation in Planta.
Infected leaves were stained with Chlorazole Black E 3 DAI. The solopathogenic strain SG200 (a1 mfa2; bE1/bW2) penetrates the leaf surface via appressorium-like structures (A) and continues to proliferate in planta (B). Clamp cells of proliferating SG200 hyphae (C), indicative of active mitosis. Deletion of cib1 (D) or clp1 (G) in SG200 does not interfere with formation of appressoria, but proliferation of fungal hyphae is blocked ([E] and [H]) and clamp cells are not observed ([F] and [I]). Arrows mark appressorium-like structures ([A], [B], and [D] to [I]) or clamp cells (C). Bars = 20 μm.

Developmental Regulation of Cib1 Protein Expression and cib1 Splicing.
(A) cib1 gene expression was monitored by replacing the cib1 ORF with 3xeGFP (UKH140, a1 b1; P_cib1::3xeGFP). Shown is the morphology (differential interference contrast [DIC]) and eGFP expression of cells grown in axenic culture. Bar = 10 μm.
(B) A functional Cib1:3eGFP fusion protein was expressed under the control of the native promoter in strain USA1 (a1 mfa2; bE1/bW2; cib1:3xeGFP). Fungal hyphae were stained 24 h after inoculation with calcofluor to visualize appressorium-like structures. Nuclear localized GFP fluorescence was observed in fungal hyphae only after penetration of the leaf surface. Bar = 10 μm.
(C) Gene expression analysis of cib1 mRNA derivatives using qRT-PCR. Gene expression is shown relative to the lowest expression value. RNA samples were isolated from strain FB1 (a1b1) during growth in liquid MM-NG, from strain SG200 (a1 mfa2 bE1/bW2) during filamentous growth on solid Potato Dextrose medium containing charcoal (CC), and from leaf tissue infected with strain SG200 2, 4, and 8 DAI. Actin and eIF2b were used for normalization. Shown are the mean values of two technical replicates. Error bars represent the sd. The experiment was repeated three times with similar results.
(D) Molecular ratio of unspliced to spliced cib1 mRNA derivatives. The ratio of unspliced and spliced cib1 mRNA was obtained by calculating the absolute number of mRNA molecules by RT-PCR, using serial dilutions with standardized concentrations of 500-bp cib1 mRNA fragments with (unspliced) or without intron (spliced) for calibration. Calibration curves were calculated using the Bio-Rad Icycler software (unspliced r² = 0.969; spliced r² = 0.975); the absolute concentration of unspliced und spliced cib1 mRNA molecules from U. maydis strains grown in axenic culture or during pathogenic development were calculated by plotting the CT values of the RT-PCR analysis against the calibration curves. Samples for calibration were done in triplicate and samples for absolute quantification of gene expression in duplicates. The experiment was repeated three times with similar results.

Induced Coexpression of clp1 and rbf1 Results in Reduced rbf1-Dependent Filament Formation and Repression of the a Mating-Type Genes.
(A) Strains UKH156 (a1 Δb::P_nar1:rbf1) and UKH164 (a1 Δb::P_nar1:rbf1; P_crg1:clp1) were grown in nitrate minimal medium supplemented with glucose (clp1 not expressed) or arabinose (clp1 expressed) as sole the carbon source. In UKH156, the nitrate-induced expression of rbf1 leads to filament formation; the arabinose-induced coexpression of clp1 in UKH164 results in reduced filament formation.
(B) Quantification of filament formation in strains UKH156 and UKH164 12 h after induction of rbf1 with or without clp1 as shown in (A). N = number of cells counted.
(C) qRT-PCR analysis of mfa1 and pra1 gene expression in response to rbf1 or clp1 induction. Strains UKH156 and UKH164 and, as a control, JB1 (a1 Δb) were grown for 12 h in nitrate minimal medium supplemented with arabinose. Shown are the mean values of two technical replicates. The experiment was repeated twice with similar results. Actin and eIF2b were used for normalization. Error bars represent the sd. Expression was calculated relative to the lowest expression value.

Induced Expression of clp1 Blocks Pheromone-Induced Conjugation Tube Formation and Cell Cycle Arrest in an rbf1-Dependent Manner.
(A) Strains JB1 (a1 Δb), UVO151 (a1 Δb; P_crg1:clp1), and UVO151Δrbf1 (a1 Δb; Δrbf1 P_crg1:clp1) were treated with synthetic a2 pheromone (2.5 μg/mL) for 6 h in MM-NA. DMSO, which was used as solvent for the a2 pheromone, served as negative control. Strains JB1 and UVO151Δrbf1 respond to pheromone treatment with the formation of conjugation tubes (arrows). By contrast, cells of strain UVO151 do not form conjugation tubes, but continue to grow by budding, indicating that the cell cycle is not arrested upon pheromone treatment in that strain. Bar = 15 μm.
(B) Pheromone-dependent gene expression in strains JB1 (a1 Δb), UVO151 (a1 Δb; P_crg1:clp1), and UVO151Δrbf1 (a1 Δb; Δrbf1; P_crg1:clp1). Cells were treated as described above, and gene expression of the a mating-type locus genes mfa1 and pra1 and of the pheromone response factors prf1 and rbf1 was determined using qRT-PCR. Shown are mean values of two technical replicates. Error bars represent the sd. Actin and eIF2b were used for normalization. The experiment was repeated three times with similar results.

Ectopic Expression of rbf1 and clp1 Is Sufficient for the Initial Steps of Pathogenic Development.
Mixtures of strains UKH184 (a1 Δb::P_lga2:rbf1) and UKH186 (a2 Δb::P_lga2:rbf1) (A) or UKH178 (a1 Δb::P_lga2:rbf1; P_lga2:clp1) and UKH180 (a2 Δb::P_lga2:rbf1; P_lga2:clp1) (B) were coinoculated into 7-d-old maize seedlings. Pictures of leaves were taken 6 DAI; fungal development was investigated by Chlorazole Black E staining of infected leaf samples 4 DAI. Infection of maize plants with a mixture of strains UKH184 and UKH186 expressing rbf1 under the control of the lga2 promoter resulted in local chlorosis. Strains were able to form appressoria and to penetrate the host plant; however, development was arrested immediately or shortly after penetration by formation of enlarged bulbous cells. By contrast, infection with strains UKH178 and UKH180, expressing both rbf1 and clp1 under the control of the lga2 promoter, led to more severe disease symptoms, as enhanced formation and spreading of chlorotic areas and anthocyanin production. Fungal progression after plant penetration was not restricted to single plant cells, but hyphae continued to spread throughout the leaf. The formation of bulbous cells was observed as well; however, these structures did not terminate hyphal growth. Arrows indicate the point of plant penetration. Bars = 20 μm.

Nuclear Distribution during Pathogenic Development of U. maydis Strains Expressing rbf1 and rbf1/clp1.
(A) Nuclear distribution in infection structures of a mixture of UKH184GN (a1 Δb::P_lga2:rbf1 P_{mig2_5}:NLS-3eGFP) and UKH186GN (a2 Δb::P_lga2:rbf1 P_{mig2_5}:NLS-3eGFP) expressing nuclear-localized GFP under the control of the plant-induced mig2-5 promoter. Calcofluor staining was used to visualize fungal growth on the leaf surface. Bulbous structures were formed directly after penetration of the host plant and invariably contained only the two nuclei of the dikaryotic hyphae formed after cell fusion. No progression of fungal development was observed at later time points of infection.
(B) Mixtures of UKH178GN (a1 Δb::P_lga2:rbf1; P_lga2:clp1 P_{mig2_5}:NLS-3eGFP) and UKH180GN (a2 Δb::P_lga2:rbf1; P_lga2:clp1 P_{mig2_5}:NLS-3eGFP), expressing nuclear-localized GFP during in planta growth. Hyphae infected the plant via appressorium-like structures, continued to proliferate inside the plant, and contained multiple nuclei. Hyphae were often found to contain uneven numbers and irregularly spaced nuclei. At 3 DAI, morphologically abnormal fungal hyphae (top panel, 3 DAI) or bulbous structures (bottom panel, 3 DAI) were frequently found that accumulated an increasing number of nuclei over time.
Bars = 20 μm.

Model for the Coordinated Action of the Mating Types to Regulate Different Stages of Pathogenic Development of U. maydis.
(A) Recognition of a compatible mating partner (Mfa) by the a-encoded pheromone/receptor (Pra) triggers signaling cascades that culminate in the activation of the high mobility group transcription factor Prf1 via differential phosphorylation by Protein Kinase A and the mitogen-activated protein kinase Kpp2 (not shown). Activated Prf1 in turn induces the expression of the a locus genes mfa and pra, the b locus genes bE and bW, as well as rbf1. Activation of the a pathway leads to formation of conjugation tubes and G2 cell cycle arrest.
(B) After cell fusion, compatible bE/bW proteins dimerize and further induce the expression of rbf1. High levels of rbf1 ensure the maintenance of cell cycle arrest, induce filamentous growth, and facilitate the initial stages of plant infection.
(C) After penetration of the host plant, Clp1 expression interferes with a- and b-dependent gene regulation via interaction with bW and Rbf1. The Clp1/Rbf1 interaction effectively represses the a-dependent pathway, whereas Clp1/bW interaction blocks b-dependent gene regulation. Since Clp1 is regulated by the bE/bW heterodimer, a negative autoregulatory feedback loop is established. The consequences of these regulatory cross-connections are (1) the downregulation of rbf1 after plant penetration and (2) the counteraction of the a- and b-induced cell cycle arrest. Further proliferation requires in addition the expression of the Cib1 protein, which is subject to developmentally regulated splicing.