Fig 1.
The nuclear protein Ros1 contributes to virulence and is essential for spore formation.
(A) Cellular localization of Ros1. FB1PotefRos1mCherry-Pnup107Nup107eGFP expressing a Ros1mCherry fusion protein and the nuclear envelope marker Nup107eGFP were grown to mid exponential phase in YEPSL and analyzed by fluorescence microscopy. The green and red fluorescing signals corresponding to Nup107GFP and Ros1mCherry are shown in the left and middle panels, respectively. The right panel shows an overlay of both signals. The size marker corresponds to 10 μM. (B) The deletion of ros1 attenuates virulence. Wild type strains FB1 and FB2, the ros1 deletion strains FB1Δros1 and FB2Δros1 and the complementation strains FB1Δros1-Ros1 and FB2Δros1-Ros1 were mixed in the indicated combinations and injected into maize seedlings. Disease symptoms were scored 12 days after infection according to [10]. Colors used for disease scores are indicated on the right side. Three independent experiments were performed and the average values are expressed as a percentage of the total number of infected plants (n) given above each column. Error bars indicate standard deviation. Statistically significant differences between FB1 x FB2 and ros1 deletion strains FB1 x FB2 and the complementation strains are indicated by black stars. Statistically significant differences between ros1 deletion strains and the complementation strains are indicated by white stars (one-way ANOVA applying the Tukey-Kramer test[36]) (C) Representative tumors formed after infection of maize seedlings with the indicated combinations of strains are shown 12 days after infection (top panel). In the examples chosen, stem-bending is induced. The ros1 deletion strains induce tumors, but these lack dark coloration typical for tumors containing mature spores. Lower panel: dispersed tumor tissue of the indicated strains was analyzed by light microscopy. Mature teliospores are absent from tumors induced by the ros1 deletion strains. The size bar corresponds to 20 μM.
Fig 2.
ros1 deletion strains fail to aggregate and form spores.
Tumor samples from maize seedlings infected with the wild type strains FB1 x FB2 and the corresponding ros1 deletion strains were collected at 4, 6, 8, 10, 12 dpi, stained with wheat germ agglutinin-Alexa Fluor 488 and analyzed by laser scanning confocal microscopy (bar = 50 μm). In the FB1 x FB2 infection (left column), spore formation starts at 6 dpi with the formation of hyphal aggregates which expand (8 dpi), begin to fragment (10 dpi) and develop into teliospores (12 dpi). In comparison, the ros1 deletion strains are locked in the filamentous state, do not aggregate and do not develop teliospores (right column). The size bar corresponds to 50 μM.
Fig 3.
ros1 is upregulated late after infection.
qRT-PCR analysis of ros1 expression in FB1 and FB2 grown in YEPSL or during plant infection (FB1 x FB2). Axenic culture samples were collected at OD600 = 1.0. Infected plant samples were collected at the time-points indicated below. qRT-PCR analysis was performed using the constitutively expressed ppi gene (UMAG_03726) for normalization. Relative expression was determined using the ΔΔCt method. Values shown are means of three biological replicates. Bars indicate the standard deviation between biological replicates. Asterisks indicate significant differences (paired t-test, p ≤ 0.05).
Fig 4.
Ros1 is required for karyogamy, matrix formation and biomass increase.
(A) Visualization of fungal nuclei. Nuclear status of biotrophic hyphae was assessed by imaging fungal nuclei inside hyphae in tumor tissue. Maize seedlings were infected with a combination of FB1Pnup107Nup107eGFP-Psso1Sso1mCherry x FB2Pnup107Nup107eGFP-Psso1Sso1mCherry or FB1Δros1Pnup107Nup107eGFP-Psso1Sso1mCherry x FB2Δros1Pnup107Nup107eGFP-Psso1Sso1mCherry expressing both the nuclear envelope marker Nup107eGFP (green fluorescence) and the membrane marker Sso1mCherry (red fluorescence). Tumor samples were collected between 6 and 8 dpi and analyzed by confocal microscopy. Only one nucleus is visible in hyphae of FB1Pnup107Nup107eGFP-Psso1Sso1mCherry x FB2Pnup107Nup107eGFP-Psso1Sso1mCherry (upper panel) while FB1Δros1Pnup107Nup107eGFP-Psso1Sso1mCherry x FB2Δros1Pnup107Nup107eGFP-Psso1Sso1mCherry hyphae are dikaryotic (lower panel). White arrows indicate the position of nuclei. Bar = 10 μm. (B) Staining of the mucilaginous matrix. Leaf tumors from maize seedlings infected with the indicated strains were collected at 10 dpi. Samples were fixed and embedded in Epoxy resin. 1–2 μm thick sections were generated and stained with methylene blue-azure II-basic fuchsin. The mucilaginous matrix appears in pink in the FB1 x FB2 infection while no matrix is visible in the tissue infected by FB1Δros1 x FB2Δros1. Blue arrowheads indicate maturing spores, red arrowheads indicate fungal hyphae. Bar = 50 μm. (C) Fungal biomass increase during plant infection. Plants were infected with the indicated strains and samples were collected at 2, 4, 6, 8, 10, and 12 dpi. Relative fungal biomass (grey columns FB1 x FB2; white columns FB1Δros1 x FB2Δros1) was determined by qRT-PCR using U. maydis-specific and maize-specific primers. Columns give mean ratios of fungal DNA to plant DNA from three independent experiments. The ratio in FB1 x FB1 infected plants at 2 dpi was set to 1.0. Error bars indicate standard deviation. Asterisks indicate significant differences (t-test, p ≤ 0.05).
Fig 5.
ros1 overexpression inhibits b-induced filamentation and triggers septation and nuclear divisions.
(A) Strain AB33Pcrg1Ros1 expressing bE1 and bW2 from the nitrate-inducible nar1 promoter and ros1 from the arabinose-inducible crg1 promoter was grown in CM supplemented with glucose to an OD600 of 0.6 and then shifted to NM medium containing either glucose (left panel) or arabinose (right panel). In NM + glucose only the b genes are induced whereas in NM + arabinose expression of the b genes and ros1 is induced. Samples were collected 6 h after induction. Nuclei and cell walls were stained with DAPI and calcofluor, respectively, and the morphology of the cells was assessed by DIC and fluorescence microscopy. In the presence of nitrate and glucose a functional bE1/bW2 heterodimer is produced and cells switch to filaments. In NM + arabinose, bE1, bW2 and ros1 are expressed simultaneously. In this case, cells fail to form filaments and become swollen and septated with one nucleus in each section. Bar = 10 μM. (B) AB33Pcrg1Ros1 was pre-incubated for 6 h in NM + glucose to allow cells to filament and then transferred to either NM + glucose (left panel) or NM + arabinose (right panel) for another 6 h. In NM + glucose the cells remained filamentous and contained one nucleus (red arrow). In NM+ arabinose, when ros1 is induced, filaments became septated (yellow arrows) and each section contained one nucleus (red arrows). Nuclei and cell walls were stained as in A. Bar = 10 μm.
Fig 6.
Premature expression of ros1 triggers an early arrest of filamentous growth and induces septation.
Maize seedlings were infected with a combination of FB1 x FB2 (A, B) or FB1Δros1Pmig2-6Ros1 x FB2Δros1Pmig2-6Ros1 strains expressing ros1 from the mig2-6 promoter which is induced two days after colonization (C, D). A and C show macroscopic symptoms on leaves at 8 dpi. Only very small tumors are visible on plants infected with FB1Δros1Pmig2-6Ros1 x FB2Δros1Pmig2-6Ros1. B and D panels show confocal microscopy pictures obtained from 3 dpi plant samples. The fungal cell wall was stained with wheat germ agglutinin-Alexa Fluor 488 (green fluorescence) and the plant cell wall was stained with propidium iodide (red fluorescence). Growth of the strains expressing ros1 prematurely is restricted to a small area surrounding the penetration site and hyphae exhibit an altered morphology. The inserts depict enlargements of biotrophic hyphae displaying increased septation when Ros1 is prematurely expressed. Bar = 10 μm.
Fig 7.
Cellular processes regulated by Ros1.
Functional enrichment analysis of the differentially regulated genes identified by RNA-seq in FB1 x FB2 compared to FB1Δros1 x FB2Δros1 strains 8 days after infection of maize seedlings. Analysis was performed using the FunRich program and FunCat annotations available on the MIPS Ustilago maydis database (http://www.helmholtz-muenchen.de). The category “secreted effectors” was added and refers to all genes encoding secreted proteins without annotated functional domain. The chart shows the proportion of genes belonging to selected functional categories in the up-regulated (red) and down-regulated (blue) gene sets. Overrepresented categories in each up and downregulated gene sets compared to the global gene distribution are indicated with asterisks (p ≤ 0.05).
Fig 8.
Ros1 alters the expression of the effector repertoire late during infection.
All genes encoding secreted effectors without predicted functional domain are listed and their up regulation or downregulation by Ros1 is indicated. Based on the RNA-seq data comparing gene expression in FB1 x FB2 and the corresponding ros1 deletion strains 8 days after infection of maize seedlings and the ChIP-seq data from FB1Δros1-Ros1HA x FB2Δros1-Ros1HA at the same time point, effector genes were sorted in 5 different categories presented as histograms: directly downregulated, indirectly downregulated, directly upregulated, indirectly upregulated and not differentially regulated by Ros1. The percentage (Y axis) represented by each category is calculated relative to the total number of effector genes without functional domains (320 genes based on [47] and secretion prediction by SignalP 4.1 [48]). Colors refer to the fold change of transcripts in wild type compared to ros1 deletion strains in the RNA-seq analysis; the darker the color the higher the fold change. Downregulated effector genes are indicated in blue, upregulated effector genes are indicated in red, effector genes not regulated by Ros1 are indicated in yellow. All genes names or identification numbers are listed; genes which have been characterized or mentioned in previous publications are indicated in bold characters. Leaf-specific effector genes without functional domains [13] showing virulence defects after seedling infection are shown in white, tassel-specific effector genes without functional domains [13] are shown in green. cmu1 [8] and three other differentially regulated effector genes (UMAG_03615 [10], UMAG_11763, UMAG_01130 [13]) are not included in this figure as they contain a functional domain.
Fig 9.
Ros1 binds to the ros1 promoter region.
(A) The graph generated with the CLC Genomics Workbench 7.5 software (CLC bio) shows the ChIP-seq read distribution in the genomic region containing ros1 in output DNA from the sample where maize was infected with FB1Δros1-Ros1HA x FB2Δros1-Ros1HA (Output Ros1HA) and in the control sample where the infection was done with FB1Δros1-Ros1 x FB2Δros1-Ros1 (output Ros1) Open reading frames are represented by yellow arrows. Peaks with significant peak shape scores are indicated in the ChIP peaks lane by dark red arrows. The location of the fragment (WT-probe) used as a probe for EMSA is indicated by a black arrow. (B) Sequence of the probe fragment used for EMSA assays. Putative binding sites (m1, m2 and m3) for Ros1 are boxed and mutations introduced in the respective sites are indicated (mut-m1, mut-m2). (C) In vitro binding of Ros1WOPR-His to the ros1 promoter. Ros1WOPR-His expressed and purified from E. coli was used in EMSA assays with the probe shown in B (WT-probe). When incubated with Ros1WOPR-His, the WT-probe was shifted and this could be competed by addition of a non-labeled WT-probe (competitor). Ros1WOPR-His did not bind a probe of the same length corresponding to a part of the ros1 coding sequence (ORF-probe). Probes mut-m1 and mut-m2 harboring mutations in motifs 1 and 2 are also bound by Ros1WOPR-His, but the interaction results in a less pronounced shift than observed for the WT-probe and an even smaller shift when both motifs are mutated (mut-m1+2). Binding to mutated probes could also be efficiently competed by adding a non-labeled WT-probe.
Fig 10.
Two transcription factors targeted by Ros1 are involved in the regulation of spore development.
Maize seedlings were inoculated with the indicated strains. Typical tumors induced by wild type and corresponding deletion strains are shown in the left panels. Tumor tissue was collected at 12 dpi, dispersed in water and spore structures were observed with a light microscope (middle panels). Enlarged parts of the middle panel are depicted in the right panel. Bar = 10 μm.
Fig 11.
Model for Ros1 function in the control of late development of U. maydis.
The model shows the processes repressed (blue box) and induced (red box) by Ros1 during the late development of U. maydis. Ros1 inhibits filamentous growth of the dikaryon by downregulating elements of the bE/bW regulatory cascade. It also represses 128 effector genes (126 of the 320 predicted effector genes [47, 48] without functional domain, cmu1 [8] and UMAG_01130 [13]) (blue box). Concomitantly, Ros1 induces teliospore formation (Red box). Ros1 triggers karyogamy followed by proliferation of the diploid and matrix formation. This coincides with the upregulation of genes involved in cell cycle, DNA processing and protein synthesis. The gelatinization of the cell wall and subsequent fragmentation of sporogenous hyphae might involve cell wall loosening and membrane lipid synthesis. The two Ros1 induced transcription factors UMAG_02775 and UMAG_01390 regulate spore maturation following the fragmentation stage. Secondary metabolism, lipid storage as well as 70 late effectors are induced by Ros1. Yellow = cytoplasm, black line = cell wall, blue / red discs = nuclei, brown dots = matrix.