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1. Protein kinases MpkA and SepH transduce crosstalk between CWI and SIN pathways to activate protective hyphal septation under echinocandin cell wall stress

   https://doi.org/10.1128/msphere.00641-24  

   Doan, Alexander G; Schafer, Jessica E; Douglas, Casey M; Quintanilla, Matthew S; Morse, Meredith E; Edwards, Harley; Huso, Walker D; Gray, Kelsey J; Lee, JungHun; Dayie, Joshua K; et al (December 2024, mSphere)

   Park, Hee-Soo (Ed.)

   ABSTRACT This study investigates a previously unreported stress signal transduced as crosstalk between the cell wall integrity (CWI) pathway and the septation initiation network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific. Our findings suggest that this is due largely to CWI–SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of β-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery of the previously unknown crosstalk between the CWI and SIN pathways not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin-resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work, we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also describe, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. This work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin-resistant species. 

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2. Callose deposition is essential for the completion of cytokinesis in the unicellular alga, Penium margaritaceum

   https://doi.org/10.1242/jcs.249599  

   Davis, Destiny J.; Wang, Minmin; Sørensen, Iben; Rose, Jocelyn K.; Domozych, David S.; Drakakaki, Georgia (December 2020, Journal of Cell Science)

   null (Ed.)

   Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a β-1,3 glucan accumulates at later stages of cell plate development presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, P. margaritaceum. Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition by Endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants. 

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3. Unusual Boronium Salt Shows Antifungal Activity Comparable to a Commercial Quaternary Ammonium Disinfectant

   https://doi.org/10.1002/slct.202104344  

   Ravine, Terrence_J; Soltani, Mohammad; Davis, Jr., James_H; Salter, Edward_A; Wierzbicki, Andrzej (March 2022, ChemistrySelect)

   Abstract Previously, a boronium salt possessing a terminal benzyl group was reported to have greater antibacterial activity than a commercial quaternary ammonium disinfectant solution againstEscherichia coli,Pseudomonas aeruginosa, andStaphylococcus aureus. Results of the current study indicate that the same boronium salt without a benzyl group, exhibited equal or better antifungal activity against actively growingCandida albicansyeast andAspergillus fumigatusmold when compared to the same quat disinfectant. This same compound also displayed antifungal activity against dormantA. fumigatusspores comparable to the quat disinfectant. In contrast, the boronium ion with a benzyl group was 4–16X less effective than either the non‐benzylated form or quat disinfectant for all 3 fungal test conditions. The observation that the boronium salt without a benzyl group exhibited substantial antifungal activity in the current study but did not display any antibacterial activity in the previous study is of particular interest. This finding represents a flip‐flop outcome from the previous bacterial testing. It suggests that the presence of a terminal benzyl group greatly influences the boronium ion's ability to interact with fungal membranes. 

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4. Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-glucan Synthase that Functions during Plant Cell Wall Synthesis.

   https://doi.org/doi: 10.1105/tpc.19.00637  

   *Yang J., *Bak G. (May 2020, The plant cell)

   In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6. 

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5. Staining and automated image quantification of callose in Arabidopsis cotyledons and leaves.

   Mason, KN; Ekanayake, G; Heese, A (June 2020, Methods in cell biology)

   Anderson, CT; Elizabeth S. Haswell, ES; null (Ed.)

   Callose is a β-1,3-glucan polysaccharide that is deposited at discrete sites in the plant cell wall in response to microbial pathogens, likely contributing to protection against pathogen infection. Increased callose deposition also occurs in response to the 22-amino acid peptide flg22, a pathogen-associated molecular pattern (PAMP) derived from bacterial flagellin protein. Here, we provide protocols for callose staining using aniline blue in cotyledon and leaf tissue of the model plant Arabidopsis thaliana. Aniline blue stain utilizes a fluorochrome that complexes with callose for its visualization by microscopy using an ultraviolet (UV) filter. For robust quantification of callose deposits, we outline an automated image analysis workflow utilizing the freely available Fiji (Fiji Is Just ImageJ; NIH) software and a Trainable Weka Segmentation (TWS) plugin. Our methodology for automated analysis of large batches of images can be easily adapted to quantify callose in other tissues and plant species, as well as to quantify fluorescent structures other than callose. 

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