Transcriptome profiling brings new insights into the ethanol stress responses of Spathaspora passalidarum.

Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: • Ethanol stress halts Spathaspora passalidarum metabolism and fermentation • Genes encoding nutrient transporters showed downregulation under ethanol stress • Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.

A unique effector secreted by Pseudozyma flocculosa mediates its biocontrol activity.

BACKGROUND: Pseudozyma flocculosa is a highly efficient biocontrol agent (BCA) of powdery mildews whose mode of action remains elusive. It is known to secrete unique effectors during its interaction with powdery mildews but effectors have never been shown to be part of the arsenal of a BCA. Here, we characterize the role of the effector Pf2826 released by Pseudozyma flocculosa during its tripartite interaction with barley and the pathogen fungus Blumeria graminis f. sp. hordei. RESULTS: We utilized CRISPR-Cas9-based genome editing and confirmed that secreted P. flocculosa effector Pf2826 is required for full biocontrol activity. We monitored the localization of the effector Pf2826 with C-terminal mCherry tag and found it localized around the haustoria and on powdery mildew spores. His-tagged Pf2826 recombinant protein was expressed, purified, and used as bait in a pull-down assay from total proteins extracted during the tripartite interaction. Potential interactors were identified by LC-MS/MS analysis after removing unspecific interactions found in the negative controls. A two-way yeast two-hybrid assay validated that Pf2826 interacted with barley pathogenesis-related (PR) proteins HvPR1a and chitinase and with an effector protein from powdery mildew. CONCLUSIONS: In contrast to the usual modes of action of competition, parasitism, and antibiosis ascribed to BCAs, this study shows that effector pf2826 plays a vital role in the biocontrol activity of P. flocculosa by interacting with plant PR proteins and a powdery mildew effector, altering the host-pathogen interaction.

Identification of the main proteins secreted by Kluyveromyces marxianus and their possible roles in antagonistic activity against fungi.

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.