Mating Types of Ustilago esculenta Infecting Zizania latifolia Cultivars in Japan Are Biased towards MAT-2 and MAT-3.

Zizania latifolia cultivars infected by the endophytic fungus Ustilago esculenta develop an edible stem gall. Stem gall development varies among cultivars and individuals and may be affected by the strain of U. esculenta. To isolate haploids from two Z. latifolia cultivars in our paddy fields, Shirakawa and Ittenkou, we herein performed the sporadic isolation of U. esculenta strains from stem gall tissue, a PCR-based assessment of the mating type, and in vitro mating experiments. As a result, we obtained heterogametic strains of MAT-2 and MAT-3 as well as MAT-2, but not MAT-3, haploid strains. Another isolation method, in which we examined poorly growing small clusters of sporidia derived from teliospores, succeeded in isolating a MAT-3 haploid strain. We also identified the mating types of 10 U. esculenta strains collected as genetic resources from different areas in Japan. All strains, except for one MAT-1 haploid strain, were classified as MAT-2 haploid strains or heterogametic strains of MAT-2 and MAT-3. The isolated strains of MAT-1, MAT-2, and MAT-3 mated with each other to produce hyphae. Collectively, these results indicate that the mating types of U. esculenta infecting Z. latifolia cultivars in Japan are biased towards MAT-2 and MAT-3 and that U. esculenta populations in these Japanese cultivars may be characterized by the low isolation efficiency of the MAT-3 haploid.

Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha.

The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.

Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

The protein phosphatase Siw14 controls caffeine-induced nuclear localization and phosphorylation of Gln3 via the type 2A protein phosphatases Pph21 and Pph22 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14.

Saccharomyces cerevisiae protein phosphatase Ppz1 and protein kinases Sat4 and Hal5 are involved in the control of subcellular localization of Gln3 by likely regulating its phosphorylation state.

A Saccharomyces cerevisiae mutant lacking PPZ1, encoding a serine/threonine protein phosphatase (PPase), is caffeine-sensitive. To clarify the function of Ppz1 in resistance to caffeine, we attempted systematically to identify protein kinase (PKase) whose disruption lead to suppression of caffeine sensitive phenotype of the ∆ppz1 disruptant since disruption of PPZ1 might cause caffeine sensitivity by increasing its phosphorylated substrates and we presumed that disruption of genes for PKase sharing the substrate with Ppz1 could restore the resistance through bypassing necessity for dephosphorylation of substrates. Among the 102 viable pkase disruptions, disruption of either SAT4 or HAL5 suppressed the caffeine sensitivity phenotype and increased expression of ENA1, encoding a P-type ATPase of the ∆ppz1 disruptant. Because increased expression of ENA1 in the ∆ppz1 disruptant was found to be suppressed by disruption of GLN3, localization and phosphorylation of Gln3 in the ∆ppz1 disruptant was compared to that in the ∆ppz1∆sat4 and ∆ppz1∆hal5 double disruptants. Gln3 was found to accumulate in the nucleus in the ∆ppz1 disruptant, and this nuclear localization was abolished by disruption of either SAT4 or HAL5. Interestingly, the level of Gln3 phosphorylation in the ∆ppz1∆sat4 and ∆ppz1∆hal5 disruptants decreased relative to wild type independent of caffeine. From these observations, we conclude that Ppz1 controls Gln3 localization by regulating its phosphorylation state in combination with Sat4 and Hal5.

Deciphering cellular functions of protein phosphatases by comparison of gene expression profiles in Saccharomyces cerevisiae.

Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearson's correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Deltappase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearson's correlation coefficient was >0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Delta ppase phenotypes were found. Notably, Delta sit4 and Delta siw14 disruptants exhibited hygromycin B sensitivity, Delta sit4 and Delta ptc1 disruptants grew slowly on glycerol medium, the Delta ptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Delta ppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes.