Tryptase

BACKGROUND MAPK/HOG signaling pathway plays a key role in the response of yeast to external hyperosmotic stress. Over the past few decades, the regulation mechanism for this pathway in the robust yeast,Saccharomyces cerevisiae, has been elucidated. However, the weak ability of the biotechnical workhorse,Pichia pastoris, in surviving hyperosmotic stress suggests a unique regulatory mechanism needing further investigation. RESULT Here, we identified crucial genes in the MAPK/HOG pathway ofP. pastorisand investigated their effects on cell growing in osmotically stressed environments by knocking out these genes using a novel CRISPR/Cas9 system. Using real-time polymerase chain reaction (RT-PCR) and yeast two-hybrid assay, transcription factors Hot1, Msn4 and Sko1 were demonstrated to be regulated by Pbs2 and Hog1 either at mRNA or protein level. We also examined the subcellular localization of these transcription factors, reflecting their translocation between cytoplasm and nucleus. The transcriptions of putative osmo-responsive genes were then studied by RT-PCR. We found the induction of glycerol-related genes, such asGT1andGPD1, was marginal when cells experienced high osmolarity. The ability ofP. pastoristo increase intracellular glycerol level was determined and found to be much weaker than that inS. cerevisiae. By contrast, stress-induced damage repair genes, includingCTT1andHSP12, were dramatically increased. CONCLUSION We conclude thatP. pastoriscould barely balance hyperosmotic stress by increasing intracellular glycerol concentrations, and stress-induced damage repair is still an important mechanism forP. pastorissurvival under hyperosmotic stress. This study demonstrates a description of the MAPK/HOG pathway inP. pastorisand provides a trigger for improving its robustness. (c) 2020 Society of Chemical Industry (SCI)

The extent of mycorrhizal fungi colonization is an important factor for determining the function of mycorrhizal fungi in fungi-host interaction, and quantifying the extent of mycorrhizal fungi colonization is a fundamental and essential task for researchers engaged in mycorrhizal studies. Intersect methods, such as the gridline intersect and magnified intersections methods, are accurate and objective, and are widely used to assess the colonization status of arbuscular mycorrhizal (AM) fungus. However, no convenient procedures or add-ins for Excel spreadsheets have been developed to simplify these methods. Here, we propose a procedure using the Visual Basic for Application (VBA) program in Excel that is based on the magnified intersections method, which we refer to as VBA-AMF (arbuscular mycorrhizal fungi). Time-saving and convenience are the two most prominent advantages of the VBA-AMF procedure, as it enables researchers to compute the colonization rate of AM fungi in roots, and consequently the extent of root colonization by AM fungi. VBA-AMF can also be modified to measure the status of other fungal colonizations in plant roots following the same strategy.