Candida auris is an emerging fungal pathogen notable for its intrinsically high resistance to fluconazole, the most prescribed antifungal drug. However, the genetic regulators underlying fluconazole susceptibility in C. auris remain unclear. Here we performed a pooled screen of piggyBac (PB) transposition mutants and identified significant enrichment of mitochondrial genes whose inactivation reduces fluconazole susceptibility. A genome-wide genetic interaction analysis of a mitochondrial gene deletion mutant, pet309Δ, suggests that the vacuolar calcium pump homologue CDT1 (Calcium and Drug Transporter 1) is responsible for its reduced fluconazole susceptibility. Fluconazole induces significant upregulation of CDT1 through the calcineurin signalling pathway. Cdt1, beyond its canonical calcium-pumping function, has evolved another function in mediating fluconazole efflux through its fluconazole-induced, calcineurin- and ATP hydrolysis-dependent plasma membrane localization. In addition, Cdt1 accelerates the evolution of fluconazole resistance or tolerance, and its transcript levels are substantially elevated across resistant clinical isolates. Our findings reveal a neofunctionalized role for Cdt1 in mediating fluconazole efflux in C. auris.

Covid-19 is a new type of epidemic, we performed sentiment classification tasks on Covid-19 tweets using different machine learning models. The famous pre-training models are not trained with the text relevant to COVID-19, a new kind of virus appearing at December 2019. The twitter posts with such a topic also have not been applied to test the performance of existing pre-training models and neural networks well. In our experiment, we used LSTM and Transformer to do the sentiment analysis (quinary classification) with a dataset including those twitter posts and tried different hyperparameters and models to improve the performance of classification. We finally found that the Transformer performs better than LSTM with an extra softmax layer in the encoder part, and the bidirectional transformer with 4 hidden layer and dropout 0.2 provides the best results among all hyperparameters we have tested. With the finetune of BERT,we got the best performance with the accuracy over 85%.

OPINION article Front. Microbiol., 21 August 2023Sec. Microbial Physiology and Metabolism Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1259144

Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome-editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed. Further, a precisely targeted gene deletion via homology-directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc-1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Double genes, Cmwc-1 and Cmvvd, were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non-selective culture conditions, which could permit recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars. RNA Pol III promoters, endogenous tRNA(Pro) of C. militaris, and chimeric AfU6-tRNA(Gly) can be used to improve the efficiency. Polyethylene glycol-mediated protoplast transformation was markedly more efficient than Agrobacterium tumefaciens-mediated transformation of C. militaris. To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding.

True morels (Morchella spp.) are edible mushrooms that are commercially important worldwide due to their rich nutrition and unique appearance. In recent years, outdoor cultivation has been achieved and expanded on a large scale in China. However, the mechanisms of fruiting body development in morels are poorly understood. In this study, the role of mating-type genes in fruiting body development was researched. Fruiting bodies cultivated with different mating-type strains showed no difference in appearance, but the ascus and ascospores were slightly malformed in fruiting bodies obtained from the MAT1-1 strains. The transcript levels of mating-type genes and their target genes revealed that the regulatory mechanisms were conserved in ascomycetes fungi. The silencing of mat1-2-1 by RNA interference verified the direct regulatory effect of mat1-2-1 on its target genes at the asexual stage. When cultivated with the spawn of single mating-type strains of MAT1-1 or MAT1-2, only one corresponding mating-type gene was detected in the mycelial and conidial samples, but both mat1-1-1 and mat1-2-1 were detected in the samples of primordium, pileus, and stipe. An understanding of the mating-type genes' role in fruiting body development in M. sextelata may help to understand the life cycle and facilitate artificial cultivation.

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H 2 O 2 production was high in the hyphae area, while expression of H 2 O 2 -scavenging genes was high in sclerotial cells, leading to a higher H 2 O 2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H 2 O 2 -induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H 2 O 2 -induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.

概述羊肚菌产业规模变化,从栽培品种、栽培季节、制种技术、播种技术、营养袋技术、环境调控技术、病虫害防控技术等方面总结了羊肚菌的生产技术,并介绍了近年来产品形式及产品销售发展变化;最后,探讨分析羊肚菌产业化发展的趋势与前景,提出深入开展基础研究、驯化选育新种类、开展工厂化栽培、加大羊肚菌精深加工等研究方向.

外源营养袋的应用是国内羊肚菌大田栽培成功的关键技术之一,但其作用机理和影响因素一直没有得到充分的解析.本研究首先于室内建立一个外源营养模型,确认模型中外源营养模块可以向外输出营养,且被梯棱羊肚菌菌丝利用.通过模型实验发现,外源营养添加到贫瘠培养基时,菌体生物量的增加显著高于营养丰富的培养基;在梯棱羊肚菌生长的不同阶段添加外源营养,菌核形成位置不同,并影响生物量的增加,在菌核起始阶段添加,主培养基的生物量增加最多;外源营养的添加位置、块数等因素在本模型中没有检测到对生物量的影响.外源营养使用聚丙烯袋包裹和不包裹比较发现,包裹组的主培养基内生物量增速和总量低于无包裹组,而外源营养块内趋势相反,但最终两组之间外源营养块与主培养基内生物量总和没有显著差异.使用滤纸和铝箔包裹外源营养时,对生物量的影响和聚丙烯袋材质没有显著差别.外源营养碳氮比20∶1时,最有利于主培养基生物量的积累.本研究建立的外源营养研究模型得到的结论与大田栽培有较好的一致性,通过模型发现了实际栽培中不容易观察到的现象,有助于对外源营养袋作用机理进行深入研究.

综述了羊肚菌菌核的形成过程、影响因素及其在栽培中的形成特征,并结合目前我国羊肚菌大田栽培的实际情况,探讨了菌核在栽培中的应用,为羊肚菌的栽培提供参考.

茯苓Wolfiporia cocos是我国药食两用的传统中药,本文对茯苓的分类地位、交配型、生活史、遗传育种、菌核栽培以及组学相关研究进展进行了总结,对有关专利进行了分析.以研究及生产现状为基础,分析了茯苓在基础生物学方面研究的不足、菌核栽培中存在的相关问题,重点分析了交配型研究、子实体诱导形成和生活史研究等重要方面,并对未来茯苓生物学研究和科学栽培进行了展望.

Phosphatidate phosphatases play essential roles in lipid metabolism by converting phosphatidic acid to diacylglycerol. Here, we have investigated the roles of a phosphatidate phosphatase, Pah1, in the fungal pathogen Candida albicans. Deleting PAH1 causes multiple phenotypes, especially severe hyphal defects, increased sensitivity to cell wall stress, and reduced virulence in mice. By qPCR, we detected a significant downregulation of hyphal-specific genes including two key hyphal-promoting genes UME6 and HGC1. Overexpression of UME6 in pah1Δ/Δ cells largely restored the hyphal growth, indicating that the reduced expression of UME6 is primarily responsible for the hyphal defects. We also detected decreased expression of three hyphal-promoting transcription factors EFG1, FLO8, and CPH1 in pah1 mutants, consistent with the reduced expression of UME6. Furthermore, the pah1Δ/Δ mutant exhibited increased sensitivity to cell wall stress. During systemic infection of mice, the mutant showed significantly impaired ability to colonize the kidney and to kill the host. Together, C. albicans PAH1 plays an important role in hyphal growth, adaptability to environmental stresses, and virulence. Thus, Pah1 could be targeted for the development of new antifungal drugs.

Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0-7620 kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was similar to 500 t in 2015-2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.

Morels are some of the most highly prized edible and medicinal mushrooms, and the outdoor cultivation has been achieved in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and the number of sclerotia indicates the spawn quality during cultivation. However, the sclerotial formation and differentiation mechanisms are poorly understood. In this study, the sclerotial formation process of Morchella importuna and the effects of reactive oxygen species on scerotial formation were studied. Scerotial formation was defined as five distinctive phases, hypha early, hyphal growth, sclerotial initiation, development, and maturation. The mycelia in the sclerotium-forming area were swollen, darkened, and dense with sclerotial formation, but hydrogen peroxide accumulated in the region lacking sclerotial formation. The expression of all six genes for superoxide dismutases tested increased with sclerotial maturation. A difference in hydrogen peroxide concentration of 20 mM could promote the sclerotial initiation and induce expression of sod genes. The MAPK signaling pathway was activated, and they passed the signal from an area of high oxidative stress to a low area to initiate sclerotial formation. An understanding of the sclerotial formation mechanisms in M. importuna may help to understand the life cycle and facilitate the fruiting body cultivation.

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans. The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans. We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.

Edible fungi are now the fifth largest crops in China and the annual output in China has accounted for more than 70%of the total of the world .Here, we review the important progress including name revision , breakthrough in artificial cultivation and genomics of edible and medicinal fungi .The names of the important species are corrected as Ganoderma lingzhi, Auricularia heimu er, Auricu al ria oc rnea, Sanghuangporus sanghuang and Ophiocordyceps sinensis, respectively .Some species have been cultivated successfully for the first time throughout the world including Ophio-cordyceps sinensis, Morchella spp., Phlebopus portentosus and Taiwanouf ngusc amphro atus.Genome sequencing and annotation have been completed for most of the common species .The fundamental research and industry development promote each other .

The protein kinase Rad53 and its orthologs play a fundamental role in regulating the DNA damage checkpoint in eukaryotes. Rad53 is activated by phosphorylation in response to DNA damage and deactivated by dephosphorylation after the damage is repaired. However, the phosphatases involved in Rad53 deactivation are not entirely understood. In this study, by investigating the consequences of overexpressing SDS22, a gene encoding a regulatory subunit of the PP1 phosphatase Glc7, in the human fungal pathogen Candida albicans, we discovered that Sds22 plays an important role in Rad53 dephosphorylation and thus the deactivation of the DNA damage checkpoint. Sds22 cellular levels increase when cells are exposed to DNA damaging agents and decrease after removing the genotoxins. Depletion of Glc7 has similar phenotypes. We provide evidence that Sds2 acts through inhibitory physical association with Glc7. Our findings provide novel insights into the mechanisms for the control of DNA damage checkpoint. Furthermore, SDS22 overexpression reduces C. albicans virulence in a mouse model of systemic infection, suggesting potential targets for developing antifungal drugs.

Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.

To study the function of CaPpt1, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Ppt1 did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and salt-induced stress. We also found that Ppt1 was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that ppt1Δ cells and wild-type cells showed similar G2/M arrest profiles when exposed to DNA damage stress. Ppt1 was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in ppt1Δ cells under DNA damage stress. We suggest that Ppt1 plays important roles in response to various stress conditions in C. albicans.