Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation.

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.

Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.

Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

ChMob2 binds to ChCbk1 and promotes virulence and conidiation of the fungal pathogen Colletotrichum higginsianum.

BACKGROUND: Mob family proteins are conserved between animals, plants and fungi and are essential for the activation of NDR kinases that control crucial cellular processes like cytokinesis, proliferation and morphology. RESULTS: We identified a hypomorphic allele of ChMOB2 in a random insertional mutant (vir-88) of the hemibiotrophic ascomycete fungus Colletotrichum higginsianum. The mutant is impaired in conidiation, host penetration and virulence on Arabidopsis thaliana. ChMob2 binds to and co-localizes with the NDR/LATS kinase homolog ChCbk1. Mutants in the two potential ChCbk1 downstream targets ChSSD1 and ChACE2 show defects in pathogenicity. The genome of C. higginsianum encodes two more Mob proteins. While we could not detect any effect on pathogenicity in ΔChmob3 mutants, ChMob1 is involved in conidiation, septae formation and virulence. CONCLUSION: This study shows that ChMob2 binds to the conserved NDR/LATS Kinase ChCbk1 and plays an important role in pathogenicity of Colletotrichum higginsianum on Arabidopsis thaliana.

Interplay of the RNA Exosome Complex and RNA-Binding Protein Ssd1 in Maintaining Cell Wall Stability in Yeast.

Yeast cell wall stability is important for cell division and survival under stress conditions. The expression of cell-wall-related proteins is regulated by several pathways involving RNA-binding proteins and RNases. The multiprotein RNA exosome complex provides the 3'→5' exoribonuclease activity that is critical for maintaining the stability and integrity of the yeast cell wall under stress conditions such as high temperatures. In this work, we show that the temperature sensitivity of RNA exosome mutants is most pronounced in the W303 genetic background due to the nonfunctional ssd1-d allele. This gene encodes the RNA-binding protein Ssd1, which is involved in the posttranscriptional regulation of cell-wall-related genes. Expression of the functional SSD1-V allele from its native genomic locus or from a centromeric plasmid suppresses the growth defects and aberrant morphology of RNA exosome mutant cells at high temperatures or upon treatment with cell wall stressors. Moreover, combined inactivation of the RNA exosome catalytic subunit Rrp6 and Ssd1 results in a synthetically sick phenotype of cell wall instability, as these proteins may function in parallel pathways (i.e., via different mRNA targets) to maintain cell wall stability. IMPORTANCE Stressful conditions such as high temperatures can compromise cellular integrity and cause bursting. In microorganisms surrounded by a cell wall, such as yeast, the cell wall is the primary shield that protects cells from environmental stress. Therefore, remodeling its structure requires inputs from multiple signaling pathways and regulators. In this work, we identify the interplay of the RNA exosome complex and the RNA-binding protein Ssd1 as an important factor in the yeast cell wall stress response. These proteins operate in independent pathways to support yeast cell wall stability. This work highlights the contribution of RNA-binding proteins in the regulation of yeast cell wall structure, providing new insights into yeast physiology.

Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1.

Invasive infections caused by the opportunistic pathogen Candida glabrata are treated with echinocandin antifungals that target ß-1,3-glucan synthase, an enzyme critical for fungal cell wall biosynthesis. Echinocandin resistance develops upon mutation of genes (FKS1 or FKS2) that encode the glucan synthase catalytic subunits. We have analyzed cellular factors that influence echinocandin susceptibility and here describe effects of the post-transcriptional regulator Ssd1, which in S. cerevisiae, can bind cell wall related gene transcripts. The SSD1 homolog in C. glabrata was disrupted in isogenic wild type and equivalent FKS1 and FKS2 mutant strains that demonstrate echinocandin resistance (MICs ˃ 0.5 µg/mL). A reversal of resistance (8- to 128-fold decrease in MICs) was observed in FKS1 mutants, but not in FKS2 mutants, following SSD1 deletion. Additionally, this phenotype was complemented upon expression of SSD1 from plasmid (pSSD1). All SSD1 disruptants displayed susceptibility to the calcineurin inhibitor FK506, similar to fks1∆. Decreases in relative gene expression ratios of FKS1 to FKS2 (2.6- to 4.5-fold) and in protein ratios of Fks1 to Fks2 (2.7- and 8.4-fold) were observed in FKS mutants upon SSD1 disruption. Additionally, a complementary increase in protein ratio was observed in the pSSD1 expressing strain. Overall, we describe a cellular factor that influences Fks1-specific mediated resistance and demonstrates further differential regulation of FKS1 and FKS2 in C. glabrata.

Cell Wall Biogenesis Protein Phosphatase CrSsd1 Is Required for Conidiation, Cell Wall Integrity, and Mycoparasitism in Clonostachys rosea.

Cell wall biogenesis protein phosphatases play important roles in various cellular processes in fungi. However, their functions in the widely distributed mycoparasitic fungus Clonostachys rosea remain unclear, as do their potential for controlling plant fungal diseases. Herein, the function of cell wall biogenesis protein phosphatase CrSsd1 in C. rosea 67-1 was investigated using gene disruption and complementation approaches. The gene-deficient mutant ΔCrSsd1 exhibited much lower conidiation, hyphal growth, mycoparasitic ability, and biocontrol efficacy than the wild-type (WT) strain, and it was more sensitive to sorbitol and Congo red. The results indicate that CrSsd1 is involved in fungal conidiation, osmotic stress adaptation, cell wall integrity, and mycoparasitism in C. rosea.

Serum 1,3-ßD-Glucan assay in the diagnosis of invasive fungal disease in neonates.

Invasive fungal disease is a significant cause of morbidity and mortality in the neonate. The current study aims to assess the 1, 3-ßD-Glucan (BG) assay in a prospective analysis in neonates with suspected fungaemia. A multicentre, prospective cohort study was conducted in Johannesburg, South Africa. The study included 72 neonates with clinically suspected late onset sepsis who were at high risk of fungaemia. A BG assay was performed on each patient and correlated with a sepsis classification based on the full blood count, C-reactive protein and blood culture results as no fungaemia, possible fungaemia, probable fungaemia or definite fungaemia. Sensitivity and specificity of the BG assay at levels of 60 pg/mL are 73.2% and 71.0% respectively and at levels of 80 pg/mL are 70.7% and 77.4% respectively. Positive and negative predictive values at 60 pg/mL are 76.9% and 66.7% respectively and at 80 pg/mL are 80.6% and 66.7% respectively. The area under the receiver operating curve is 0.753. The BG assay is a useful adjunct to the diagnosis of invasive fungal disease in neonates. It does, however, need to be considered in the context of the clinical picture and supplementary laboratory investigations.