Rtf1 regulates cryptococcal bisexual mating by facilitating H2Bub1 via the HMD domain.

(A) Immunoblot analysis of H2Bub1 in C. deneoformans wild-type XL280, rtf1Δ, and rtf1Δ/RTF1 strains. (B) Immunoblot analysis of H3K4me (including H3K4me1, H3K4me2, and H3K4me3) in C. deneoformans wild-type XL280, rtf1Δ, and rtf1Δ/RTF1 strains. (C) Colony morphology of cells during bisexual mating between indicated strains. The same volume of cells with opposite mating type at OD600 = 3 were mixed, and 3 μL of mixtures were spotted and cultured on V8 media for 2 days at room temperature in dark. (D) Schematic diagram of pheromone-dependent signaling pathway and transcript levels of genes involved in pheromone signaling. The mating cells were prepared and cultured on V8 media following the same protocol as the colony morphology assay. After 24 h, cells were collected for total RNA extraction and qPCR. (E) Domain structure of Rtf1 homologs in indicated eukaryotes. Cn, C. deneoformans, Ca, Candida albicans, Af, Aspergillus fumigatus, Fg, Fusarium graminearum, Nc, Neurospora crassa, Sp, Schizosaccharomyces pombe, Sc, S. cerevisiae, Dm, Drosophila melanogaster, Hs, Homo sapiens. (F and G) Neighbor- joining tree of Rtf1 homologs and their corresponding HMD in indicated eukaryotes. (H) Schematic diagram of overexpressing constructs of Rtf1, HMD domain, and Plus3 domain. The constitutive promoter of TEF1 gene was used to drive gene expression. (I) Immunoblot analysis of H2Bub1 in strains expressing the indicated proteins. (J) Unisexual hyphal formation of indicated strains on V8 media.

The expression of HMD domain alone rescues the downregulation of genes involved in pheromone signaling and filamentous growth due to the deletion of RTF1.

(A) Volcano plots of differentially expressed genes in rtf1Δ, rtf1Δ/HMD, and rtf1Δ/Plus3 relative to wild-type XL280, respectively, during unisexual mating on V8 media. Genes involved in pheromone signaling and filamentous growth were indicated. (B) Reads coverage of indicated gene loci in XL280, rtf1Δ, rtf1Δ/HMD, and rtf1Δ/Plus3 strains. Reads coverage at TEF1 locus served as control. (C) qPCR quantification of transcript levels of indicated genes in XL280, rtf1Δ, rtf1Δ/HMD, and rtf1Δ/Plus3 strains cultured on V8. The transcript level of indicated genes were relative to its transcript level in XL280 cells cultured in YPD.

The HMD domain alone is sufficient to facilitate global H2Bub1 and restore hyphal formation in rtf1Δ strain.

(A) Schematic diagram of constructs expressing mNG-labelled Rtf1 or HMD domain with cell membrane tag (RGS2) or NLS. (B) Fluorescence analysis of sub-cellular localizations of Rtf1 and HMD with RGS2 and NLS. (C) Immunoblot analysis of H2Bub1 and H3K4me in strains expressing the indicated proteins. (D) Colony morphology of indicated strains during unisexual mating on V8. (E) ClustalW multiple amino acid sequence alignment of the HMD domain in the indicated eukaryotes. The E95 and F118 residues in C.de neoformans were indicated with asterisk and dot, respectively. Cn, C. deneoformans, Ca, Candida albicans, Af, Aspergillus fumigatus, Fg, Fusarium graminearum, Nc, Neurospora crassa, Sp, Schizosaccharomyces pombe, Sc, S. cerevisiae, Dm, Drosophila melanogaster, Hs, Homo sapiens. (F and G) The 3D structure of C. deneoformans HMD domain predicted by SWISS-MODEL with the 3D structure of S. cerevisiae HMD domain (5emx) as the template. The conserved E95 residue was indicated in red. (H) Immunoblot analysis of H2Bub1 and H3K4me in strains expressing the indicated proteins. (I) Colony morphology of indicated strains during unisexual mating on V8.

Rtf1 and HMD domain regulate the production of virulence factors in C. neoformans.

(A) Immunoblot analysis of H2Bub1 and H3K4me in strains expressing the indicated proteins in C. neoformans. (B) Capsule production in the indicated strains on capsule-inducing media. The capsule-overproducing strain pas3Δ was used as control [52]. (C) Thermal tolerance of indicated strains. (D) melanin production of indicated strains on L-DOPA media.

Rtf1 and HMD domain regulate cryptococcal pathogenicity in murine models of cryptococcosis.

(A) Schematic diagram of the intranasal and intravenous infection models of cryptococcosis. The inoculum and time for detecting fungal burden in these two infection models were indicated, respectively. (B) The fungal burden of lungs infected by indicated strains through intranasal infection. (C-F) The fungal burden of lungs, brain, kidney, and spleen infected by indicated strains through intravenous infection. (G) The survival curve of animals infected by indicated strains through intravenous infection. The inoculum was the same as the fungal burden assay for intravenous infection. (H) Schematic diagram of the working model depicting the role of Rtf1 and its HMD domain in regulating fungal morphogenesis in C. deneoformans.

(A) RTF1 gene regulates fungal filamentation in C. deneoformans. (B) The neighbor-joining tree of Plus3 domain in the indicated eukaryotes. (C) ClustalW multiple amino acid sequence alignment of Plus3 domain in the indicated eukaryotes. Cn, C. deneoformans, Ca, Candida albicans, Af, Aspergillus fumigatus, Fg, Fusarium graminearum, Nc, Neurospora crassa, Sp, Schizosaccharomyces pombe, Sc, S. cerevisiae, Dm, Drosophila melanogaster, Hs, Homo sapiens.

Overexpression of Plus3 domain alone failed to rescue the downregulation of genes enriched in sexual reproduction, pheromone signaling, and filamentous growth due to RTF1 deletion.

(A) Venn diagram of significantly enriched (p-value < 0.05) GO terms of downregulated genes in rtf1Δ, rtf1Δ/ΗΜD, and rtf1Δ/Plus3 relative to the wild-type XL280 strain on V8. (B) The 19 GO terms of downregulated genes shared by rtf1Δ and rtf1Δ/Plus3 strains.

HMD domain alone successfully restores the expression of MAT2 and MFα1 in rtf1Δ strain.

Strains, plasmids, primers and antibodies used in this study.