The cell wall is the first barrier against external adversity and plays roles in maintaining normal physiological functions of fungi. Previously, we reported a nucleosome assembly protein, MoNap1, in Magnaporthe oryzae that plays a role in cell wall integrity (CWI), stress response, and pathogenicity. Moreover, MoNap1 negatively regulates the expression of MoSMI1 encoded by MGG_03970. Here, we demonstrated that deletion of MoSMI1 resulted in a significant defect in appressorium function, CWI, cell morphology, and pathogenicity. Further investigation revealed that MoSmi1 interacted with MoOsm1 and MoMps1 and affected the phosphorylation levels of MoOsm1, MoMps1, and MoPmk1, suggesting that MoSmi1 regulates biological functions by mediating mitogen-activated protein kinase (MAPK) signalling pathway in M. oryzae. In addition, transcriptome data revealed that MoSmi1 regulates many infection-related processes in M. oryzae, such as membrane-related pathway and oxidation reduction process. In conclusion, our study demonstrated that MoSmi1 regulates CWI by mediating the MAPK pathway to affect development and pathogenicity of M. oryzae.

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

Mitogen-activated protein kinase (MAPK) pathways are fundamental to the regulation of biological processes in eukaryotic organisms. The basidiomycete Cryptococcus neoformans, known for causing fungal meningitis worldwide, possesses five MAPKs. Among these, Cpk1, Hog1, and Mpk1 have established roles in sexual reproduction, stress responses, and cell wall integrity. However, the roles of Cpk2 and Mpk2 are less understood. Our study elucidates the functional interplay between the Cpk1/Cpk2 and Mpk1/Mpk2 MAPK pathways in C. neoformans. We discovered that CPK2 overexpression compensates for cpk1Δ mating deficiencies via the Mat2 transcription factor, revealing functional redundancy between Cpk1 and Cpk2. We also found that Mpk2 is phosphorylated in response to cell wall stress, a process regulated by the MAPK kinase (MAP2K) Mkk2 and MAP2K kinases (MAP3Ks) Ssk2 and Ste11. Overexpression of MPK2 partially restores cell wall integrity in mpk1Δ by influencing key cell wall components, such as chitin and the polysaccharide capsule. Contrarily, MPK2 overexpression cannot restore thermotolerance and cell membrane integrity in mpk1Δ. These results suggest that Mpk1 and Mpk2 have redundant and opposing roles in the cellular response to cell wall and membrane stresses. Most notably, the dual deletion of MPK1 and MPK2 restores wild-type mating efficiency in cpk1Δ mutants via upregulation of the mating-regulating transcription factors MAT2 and ZNF2, suggesting that the Mpk1 and Mpk2 cooperate to negatively regulate the pheromone-responsive Cpk1 MAPK pathway. Our research collectively underscores a sophisticated regulatory network of cryptococcal MAPK signaling pathways that intricately govern sexual reproduction and cell wall integrity, thereby controlling fungal development and pathogenicity.IMPORTANCEIn the realm of fungal biology, our study on Cryptococcus neoformans offers pivotal insights into the roles of specific proteins called mitogen-activated protein kinases (MAPKs). Here, we discovered the cryptic functions of Cpk2 and Mpk2, two MAPKs previously overshadowed by their dominant counterparts Cpk1 and Mpk1, respectively. Our findings reveal that these \"underdog\" proteins are not just backup players; they play crucial roles in vital processes like mating and cell wall maintenance in C. neoformans. Their ability to step in and compensate when their dominant counterparts are absent showcases the adaptability of C. neoformans. This newfound understanding not only enriches our knowledge of fungal MAPK mechanisms but also underscores the intricate balance and interplay of proteins in ensuring the organism\'s survival and adaptability.

Endocytosis has been extensively studied in yeasts, where it plays crucial roles in growth, signaling regulation, and cell-surface receptor internalization. However, the biological functions of endocytosis in pathogenic filamentous fungi remain largely unexplored. In this study, we aimed to functionally characterize the roles of EdeA, an ortholog of the Saccharomyces cerevisiae endocytic protein Ede1, in Aspergillus fumigatus. EdeA was observed to be distributed as patches on the plasma membrane and concentrated in the subapical collar of hyphae, a localization characteristic of endocytic proteins. Loss of edeA caused defective hyphal polarity, reduced conidial production, and fewer sites of endocytosis initiations than that of the parental wild type. Notably, the edeA null mutant exhibited increased sensitivity to cell wall-disrupting agents, indicating a role for EdeA in maintaining cell wall integrity in A. fumigatus. This observation was further supported by the evidence showing that the thickness of the cell wall in the ΔedeA mutant increased, accompanied by abnormal activation of MpkA, a key component in the cell wall integrity pathway. Additionally, the ΔedeA mutant displayed increased pathogenicity in the Galleria mellonella wax moth infection model, possibly due to alterations in cell wall morphology. Site-directed mutagenesis identified the conserved residue E348 within the third EH (Eps15 homology) domain of EdeA as crucial for its subcellular localization and functions. In conclusion, our results highlight the involvement of EdeA in endocytosis, hyphal polarity, cell wall integrity, and pathogenicity in A. fumigatus.
OBJECTIVE: Aspergillus fumigatus is a significant human pathogenic fungus known to cause invasive aspergillosis, a disease with a high mortality rate. Understanding the basic principles of A. fumigatus pathogenicity is crucial for developing effective strategies against this pathogen. Previous research has underscored the importance of endocytosis in the infection capacity of pathogenic yeasts; however, its biological function in pathogenic mold remains largely unexplored. Our characterization of EdeA in A. fumigatus sheds light on the role of endocytosis in the development, stress response, and pathogenicity of pathogenic molds. These findings suggest that the components of the endocytosis process may serve as potential targets for antifungal therapy.

The plant cell wall provides a strong yet flexible barrier to protect cells from the external environment. Modifications of the cell wall, either during development or under stress conditions, can induce cell wall integrity responses and ultimately lead to alterations in gene expression, hormone production, and cell wall composition. These changes in cell wall composition presumably require remodelling of the secretory pathway to facilitate synthesis and secretion of cell wall components and cell wall synthesis/remodelling enzymes from the Golgi apparatus. Here, we used a combination of live-cell confocal imaging and transmission electron microscopy to examine the short-term and constitutive impact of isoxaben, which reduces cellulose biosynthesis, and Driselase, a cocktail of cell-wall-degrading fungal enzymes, on cellular processes during cell wall integrity responses in Arabidopsis. We show that both treatments altered organelle morphology and triggered rebalancing of the secretory pathway to promote secretion while reducing endocytic trafficking. The actin cytoskeleton was less dynamic following cell wall modification, and organelle movement was reduced. These results demonstrate active remodelling of the endomembrane system and actin cytoskeleton following changes to the cell wall.

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum.

Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin (quasimodo2), cellulose (cellulose synthase3 je5), and xyloglucan (xyloglucan xylosyltransferase1 and 2), to probe whether changes in the expression of cell wall-related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin- and cellulose-deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan-deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load-bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.

Autophagy is a central biodegradation pathway critical in eliminating intracellular cargo to maintain cellular homeostasis and improve stress resistance. At the same time, the key component of the mitogen-activated protein kinase cascade regulating cell wall integrity signaling MoMkk1 has an essential role in the autophagy of the rice blast fungus Magnaporthe oryzae. Still, the mechanism of how MoMkk1 regulates autophagy is unclear. Interestingly, we found that MoMkk1 regulates the autophagy protein MoAtg9 through phosphorylation. MoAtg9 is a transmembrane protein subjected to phosphorylation by autophagy-related protein kinase MoAtg1. Here, we provide evidence demonstrating that MoMkk1-dependent MoAtg9 phosphorylation is required for phospholipid translocation during isolation membrane stages of autophagosome formation, an autophagic process essential for the development and pathogenicity of the fungus. In contrast, MoAtg1-dependent phosphorylation of MoAtg9 negatively regulates this process, also impacting growth and pathogenicity. Our studies are the first to demonstrate that MoAtg9 is subject to MoMkk1 regulation through protein phosphorylation and that MoMkk1 and MoAtg1 dichotomously regulate autophagy to underlie the growth and pathogenicity of M. oryzae.IMPORTANCEMagnaporthe oryzae utilizes multiple signaling pathways to promote colonization of host plants. MoMkk1, a cell wall integrity signaling kinase, plays an essential role in autophagy governed by a highly conserved autophagy kinase MoAtg1-mediated pathway. How MoMkk1 regulates autophagy in coordination with MoAtg1 remains elusive. Here, we provide evidence that MoMkk1 phosphorylates MoAtg9 to positively regulate phospholipid translocation during the isolation membrane or smaller membrane structures stage of autophagosome formation. This is in contrast to the negative regulation of MoAtg9 by MoAtg1 for the same process. Intriguingly, MoMkk1-mediated MoAtg9 phosphorylation enhances the fungal infection of rice, whereas MoAtg1-dependant MoAtg9 phosphorylation significantly attenuates it. Taken together, we revealed a novel mechanism of autophagy and virulence regulation by demonstrating the dichotomous functions of MoMkk1 and MoAtg1 in the regulation of fungal autophagy and pathogenicity.

Histone acetyltransferase (HAT)-mediated epigenetic modification is essential for diverse cellular processes in eukaryotes. However, the functions of HATs in the human pathogen Aspergillus fumigatus remain poorly understood. In this study, we characterized the functions of MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST)-family histone acetyltransferase something about silencing (Sas3) in A. fumigatus. Phenotypic analysis revealed that loss of Sas3 results in significant impairments in colony growth, conidiation, and virulence in the Galleria mellonella model. Subcellular localization and Western blot analysis demonstrated that Sas3 localizes to nuclei and is capable of acetylating lysine 9 and 14 of histone H3 in vivo. Importantly, we found that Sas3 is critical for the cell wall integrity (CWI) pathway in A. fumigatus as evidenced by hypersensitivity to cell wall-perturbing agents, altered cell wall thickness, and abnormal phosphorylation levels of CWI protein kinase MpkA. Furthermore, site-directed mutagenesis studies revealed that the conserved glycine residues G641 and G643 and glutamate residue E664 are crucial for the acetylation activity of Sas3. Unexpectedly, only triple mutations of Sas3 (G641A/G643A/E664A) displayed defective phenotypes similar to the Δsas3 mutant, while double or single mutations did not. This result implies that the role of Sas3 may extend beyond histone acetylation. Collectively, our findings demonstrate that MYST-family HAT Sas3 plays an important role in the fungal development, virulence, and cell wall integrity in A. fumigatus.
OBJECTIVE: Epigenetic modification governed by HATs is indispensable for various cellular processes in eukaryotes. Nonetheless, the precise functions of HATs in the human pathogen Aspergillus fumigatus remain elusive. In this study, we unveil the roles of MYST-family HAT Sas3 in colony growth, conidiation, virulence, and cell wall stress response in A. fumigatus. Particularly, our findings demonstrate that Sas3 can function through mechanisms unrelated to histone acetylation, as evidenced by site-directed mutagenesis experiments. Overall, this study broadens our understanding of the regulatory mechanism of HATs in fungal pathogens.

Grifola frodosa polysaccharides, especially β-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. KEY POINTS: •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.