Pub Date : 2024-12-01DOI: 10.1016/j.fgb.2024.103944
Jia-Yu Shen , Fei-Hong Mao , Qiwen Wang , Pei-Pei Ou , Ji-Kai Liu , Qunfei Zhao , Qing-Li He
CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.
CRISPR技术已被广泛应用于各种物种的基因编辑，但由于内源启动子未知和DNA传递效率低下，基枝菌的基因操作仍是众所周知的难题。Steccherinum ochraceum 是一种白腐基生真菌，具有丰富的次生代谢产物，在世界范围内发挥着重要的生态作用。为了便于研究赭色链格孢的基因功能，研究人员通过识别高效的内源启动子，并利用农杆菌转化法，成功开发了一套有效的 CRISPR/Cas9 系统。该系统识别并鉴定了两个高效的内源 RNA 聚合酶 II 启动子（Psogpd 和 Psotef1）和一个高效的 RNA 聚合酶 III 启动子（Pu6-d），在 ura3 基因座上的编辑效率为 61.5%。利用这一优化系统，敲除了在 A. oryzae 异源表达系统中可产生 10 种倍半萜的倍半萜基因 A0064，得到了 A0064 基因敲除株 S. ochraceum（ΔA0064）。在 S. ochraceum（ΔA0064）中检测不到菊酯氧化物 A，这表明 A0064 是 S. ochraceum 中负责β-chamigrene（菊酯氧化物 A 的倍半萜骨架）生物合成的唯一酶。这一高效系统将实现对赭色真菌基因的精确靶向和多重编辑，有助于对赭色真菌中参与木质素降解和天然产物生物合成的基因进行功能研究，并为数以万计的大型真菌的基因编辑提供一些有价值的指导。
{"title":"Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum","authors":"Jia-Yu Shen , Fei-Hong Mao , Qiwen Wang , Pei-Pei Ou , Ji-Kai Liu , Qunfei Zhao , Qing-Li He","doi":"10.1016/j.fgb.2024.103944","DOIUrl":"10.1016/j.fgb.2024.103944","url":null,"abstract":"<div><div>CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. <em>Steccherinum ochraceum</em> is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in <em>S. ochraceum</em>, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the <em>Agrobacterium</em>-transformation method. Two efficient endogenous RNA polymerase II promoters (P<em>sogpd</em> and P<em>sotef1</em>) and one efficient RNA polymerase III promoter (P<em>u6-d</em>) were identified and characterized, with an editing efficiency of 61.5 % at the <em>ura3</em> locus. Using this optimized system, the sesquiterpene gene <em>A0064</em>, which could produce 10 possible sesquiterpenes in the heterologous expression system of <em>A. oryzae</em>, was knocked out to obtain <em>A0064</em> knockout strain <em>S. ochraceum</em> (∆A0064). Steperoxide A could not be detected in <em>S. ochraceum</em> (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in <em>S. ochraceum</em>. This efficient system will enable precise targeting and multiplex editing of <em>S. ochraceum</em> genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in <em>S. ochraceum</em>, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103944"},"PeriodicalIF":2.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142734851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hydrophobins are small-secreted proteins with both hydrophobic and hydrophilic regions, enabling the mycelium to break through the air-medium interface by reducing the medium surface tension. Over 20 putative hydrophobin-encoding genes have been predicted in the agaricomycete Pleurotus ostreatus. Three hydrophobin-encoding genes, vmh2, vmh3, and hydph16, were predominantly expressed in the vegetative mycelium. Despite these common properties, we have previously demonstrated the distinct functions of Vmh2 and Vmh3 in environmental stress resistance. In this study, we focused on hydph16 and found that Δhydph16 strains had sparser aerial mycelium than control strains. The cell wall thickness of Δhydph16 strains reduced by 40 % compared to that of control strains, but no significant differences were found in the relative chitin and glucan percentages or relative putative cell wall synthesis-related gene expression levels. Furthermore, unlike vmh2 and vmh3, hydph16 deletion did not change the hydrophobicity of the aerial mycelium. This study is the first to report that the lack of hydrophobin can lead to a significant change in aerial hyphae cell wall formation without altering the major cell wall polysaccharide composition. Additionally, this study revealed multiple roles for Hydph16, distinct from those of other highly expressed hydrophobins, Vmh2 and Vmh3. These results suggested that agaricomycetes, including P. ostreatus, have evolved to possess multiple hydrophobins with different functions.
{"title":"Physiological function of hydrophobin Hydph16 in cell wall formation in agaricomycete Pleurotus ostreatus","authors":"Junxian Han , Moriyuki Kawauchi , Yuki Terauchi , Kenya Tsuji , Akira Yoshimi , Chihiro Tanaka , Takehito Nakazawa , Yoichi Honda","doi":"10.1016/j.fgb.2024.103943","DOIUrl":"10.1016/j.fgb.2024.103943","url":null,"abstract":"<div><div>Hydrophobins are small-secreted proteins with both hydrophobic and hydrophilic regions, enabling the mycelium to break through the air-medium interface by reducing the medium surface tension. Over 20 putative hydrophobin-encoding genes have been predicted in the agaricomycete <em>Pleurotus ostreatus</em>. Three hydrophobin-encoding genes, <em>vmh2</em>, <em>vmh3</em>, and <em>hydph1</em>6, were predominantly expressed in the vegetative mycelium. Despite these common properties, we have previously demonstrated the distinct functions of Vmh2 and Vmh3 in environmental stress resistance. In this study, we focused on <em>hydph16</em> and found that Δ<em>hydph16</em> strains had sparser aerial mycelium than control strains. The cell wall thickness of Δ<em>hydph16</em> strains reduced by 40 % compared to that of control strains, but no significant differences were found in the relative chitin and glucan percentages or relative putative cell wall synthesis-related gene expression levels. Furthermore, unlike <em>vmh2</em> and <em>vmh3</em>, <em>hydph16</em> deletion did not change the hydrophobicity of the aerial mycelium. This study is the first to report that the lack of hydrophobin can lead to a significant change in aerial hyphae cell wall formation without altering the major cell wall polysaccharide composition. Additionally, this study revealed multiple roles for Hydph16, distinct from those of other highly expressed hydrophobins, Vmh2 and Vmh3. These results suggested that agaricomycetes, including <em>P. ostreatu</em>s, have evolved to possess multiple hydrophobins with different functions.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103943"},"PeriodicalIF":2.4,"publicationDate":"2024-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142755797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-16DOI: 10.1016/j.fgb.2024.103941
TingTing Hu , Jishou Wu , Zixuan Lin , Yi Lin , Lin Lin , Wei Wei , Dongzhi Wei
Glutamine synthetase (GS) is a critical enzyme in nitrogen metabolism regulation and plays an essential role in the metabolic pathways involved in microbial growth and development. Penicillium brevicompactum, known for its rich repertoire of secondary metabolites, including mycophenolic acid (MPA), lacks research on the regulatory mechanisms of GS within this species. This study aimed to investigate the influence of GS on the growth, sporulation, and secondary metabolism of P. brevicompactum to elucidate the biological function of GS in this organism. We identified the glutamine synthetase gene (PbgsA) from P. brevicompactum and constructed PbgsA gene-overexpression and gene-silencing transformants. The impact of PbgsA on growth and sporulation was evaluated, revealing that PbgsA gene-overexpression transformants exhibited enhanced growth and significantly increased the expression levels of sporulation pathway genes (brlA, abaA, and wetA). Additionally, PbgsA gene-overexpression transformants produced higher MPA yields, with a maximum of 4.78 g/L, representing a 54.19 % increase compared to the wild type (WT). Conversely, PbgsA gene-silencing transformants showed reduced MPA production, with a minimum yield of 1.13 g/L, a 63.55 % decrease relative to the WT. Transcriptional analysis of the MPA biosynthetic gene cluster indicated that PbgsA exerted regulatory effects on certain biosynthetic pathway genes, such as mpaA and mpaB. This study demostrated the potential positive regulatory role of glutamine synthetase PbgsA in the growth, spore development, and secondary metabolism of P. brevicompactum, which provided a new strategy for genetic regulation in filamentous fungal.
{"title":"The impact of glutamine synthetase PbgsA on the growth, conidiation and mycophenolic acid production of Penicillium brevicompactum","authors":"TingTing Hu , Jishou Wu , Zixuan Lin , Yi Lin , Lin Lin , Wei Wei , Dongzhi Wei","doi":"10.1016/j.fgb.2024.103941","DOIUrl":"10.1016/j.fgb.2024.103941","url":null,"abstract":"<div><div>Glutamine synthetase (GS) is a critical enzyme in nitrogen metabolism regulation and plays an essential role in the metabolic pathways involved in microbial growth and development. <em>Penicillium brevicompactum</em>, known for its rich repertoire of secondary metabolites, including mycophenolic acid (MPA), lacks research on the regulatory mechanisms of GS within this species. This study aimed to investigate the influence of GS on the growth, sporulation, and secondary metabolism of <em>P. brevicompactum</em> to elucidate the biological function of GS in this organism. We identified the glutamine synthetase gene (<em>PbgsA</em>) from <em>P. brevicompactum</em> and constructed <em>PbgsA</em> gene-overexpression and gene-silencing transformants. The impact of PbgsA on growth and sporulation was evaluated, revealing that <em>PbgsA</em> gene-overexpression transformants exhibited enhanced growth and significantly increased the expression levels of sporulation pathway genes (<em>brlA</em>, <em>abaA</em>, and <em>wetA</em>). Additionally, <em>PbgsA</em> gene-overexpression transformants produced higher MPA yields, with a maximum of 4.78 g/L, representing a 54.19 % increase compared to the wild type (WT). Conversely, <em>PbgsA</em> gene-silencing transformants showed reduced MPA production, with a minimum yield of 1.13 g/L, a 63.55 % decrease relative to the WT. Transcriptional analysis of the MPA biosynthetic gene cluster indicated that PbgsA exerted regulatory effects on certain biosynthetic pathway genes, such as <em>mpaA</em> and <em>mpaB</em>. This study demostrated the potential positive regulatory role of glutamine synthetase PbgsA in the growth, spore development, and secondary metabolism of <em>P. brevicompactum</em>, which provided a new strategy for genetic regulation in filamentous fungal.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103941"},"PeriodicalIF":2.4,"publicationDate":"2024-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142669729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-07DOI: 10.1016/j.fgb.2024.103940
Effie Demos , Sofia Dimou , Claudio Scazzocchio , George Diallinas
Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the uapA gene, albeit we also obtained uapA extragenetic mutants affecting secretion and growth pleiotropically or leading on apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.
{"title":"Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis","authors":"Effie Demos , Sofia Dimou , Claudio Scazzocchio , George Diallinas","doi":"10.1016/j.fgb.2024.103940","DOIUrl":"10.1016/j.fgb.2024.103940","url":null,"abstract":"<div><div>Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus <em>Aspergillus nidulans</em> several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the <em>uapA</em> gene, albeit we also obtained <em>uapA</em> extragenetic mutants affecting secretion and growth pleiotropically or leading on apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103940"},"PeriodicalIF":2.4,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142632912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-31DOI: 10.1016/j.fgb.2024.103939
Susana Hidalgo-Vico , Daniel Prieto , Rebeca Alonso-Monge , Elvira Román , Corinne Maufrais , Christophe d’Enfert , Jesús Pla
Candida albicans normally colonizes the human gastrointestinal tract as a commensal. Studying fungal factors involved in colonizing the mammalian gastrointestinal tract requires mouse models with altered microbiota. We have obtained strains of C. albicans through microevolution in the mouse gut for a prolonged period (one year) that display a substantial increase in fitness in this niche. These strains show resistance to bile salts, an increase in their adhesion to the intestinal mucosa, and are unable to filament in response to serum. Genetic analysis revealed some alterations, mainly a triploidy of chr7, a whole chr6 homozygosis, and an SNP in the FLO8 gene (located in the chr6), resulting in a truncated protein version. A wild type FLO8 gene complemented filamentation and bile salt sensitivity but showed an intermediate fitness phenotype in colonization. Alterations in bile salt sensitivity were also evident in bmt mutants, defective in β-mannosylation, and transcriptional targets of Flo8, suggesting a link between the fungal cell wall and mammalian gut colonization via the Flo8 transcriptional regulator.
{"title":"Candida albicans strains adapted to the mouse gut are resistant to bile salts via a Flo8-dependent mechanism","authors":"Susana Hidalgo-Vico , Daniel Prieto , Rebeca Alonso-Monge , Elvira Román , Corinne Maufrais , Christophe d’Enfert , Jesús Pla","doi":"10.1016/j.fgb.2024.103939","DOIUrl":"10.1016/j.fgb.2024.103939","url":null,"abstract":"<div><div><em>Candida<!--> <!-->albicans</em> normally colonizes the human gastrointestinal tract as a commensal. Studying fungal factors involved in colonizing the mammalian gastrointestinal tract requires mouse models with altered microbiota. We have obtained strains of <em>C.<!--> <!-->albicans</em> through microevolution in the mouse gut for a prolonged period (one year) that display a substantial increase in fitness in this niche. These strains show resistance to bile salts, an increase in their adhesion to the intestinal mucosa, and are unable to filament in response to serum. Genetic analysis revealed some alterations, mainly a triploidy of chr7, a whole chr6 homozygosis, and an SNP in the <em>FLO8</em> gene (located in the chr6), resulting in a truncated protein version. A wild type <em>FLO8</em> gene complemented filamentation and bile salt sensitivity but showed an intermediate fitness phenotype in colonization. Alterations in bile salt sensitivity were also evident in <em>bmt</em> mutants, defective in β-mannosylation, and transcriptional targets of Flo8, suggesting a link between the fungal cell wall and mammalian gut colonization via the Flo8 transcriptional regulator.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103939"},"PeriodicalIF":2.4,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142565144","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-30DOI: 10.1016/j.fgb.2024.103938
Chibuike Ibe, Carolina H. Pohl
Candida albicans is an important human pathogenic yeast, that can become resistant to commonly used antifungal agents, such as azoles. One mechanism of drug resistance is efflux via ATP binding cassette transporters, such as Cdr1. Several studies have investigated the structural organization, binding mechanisms, function and regulation of Cdr1. This review summarizes the findings on the structure and function of Cdr1 and highlights important aspects to consider in future research relating to multidrug ABC transporters.
白色念珠菌是一种重要的人类致病酵母菌，可对常用的抗真菌剂（如唑类）产生耐药性。耐药性的一个机制是通过 ATP 结合盒转运体（如 Cdr1）外流。一些研究对 Cdr1 的结构组织、结合机制、功能和调控进行了调查。本综述总结了有关 Cdr1 结构和功能的研究结果，并强调了今后有关多药 ABC 转运体研究中需要考虑的重要方面。
{"title":"Update on the structure and function of Candida albicans drug efflux protein, Cdr1","authors":"Chibuike Ibe, Carolina H. Pohl","doi":"10.1016/j.fgb.2024.103938","DOIUrl":"10.1016/j.fgb.2024.103938","url":null,"abstract":"<div><div><em>Candida albicans</em> is an important human pathogenic yeast, that can become resistant to commonly used antifungal agents, such as azoles. One mechanism of drug resistance is efflux via ATP binding cassette transporters, such as Cdr1. Several studies have investigated the structural organization, binding mechanisms, function and regulation of Cdr1. This review summarizes the findings on the structure and function of Cdr1 and highlights important aspects to consider in future research relating to multidrug ABC transporters.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103938"},"PeriodicalIF":2.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142565148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-11DOI: 10.1016/j.fgb.2024.103937
Laura Vilanova , Maria Caballol , Ke Zhang , Åke Olson , Irene Barnes , Michael J. Wingfield , Jonàs Oliva
Diplodia sapinea is the causal agent of Diplodia shoot blight, an emerging disease affecting pine forests worldwide. The range expansion of this pathogen in northern Europe has been suggested to be partially facilitated by recent warmer conditions. Although D. sapinea has been studied extensively, critical aspects of its infection biology and population structure remain unexplored. In this study, we developed nine simple sequence repeat (SSR) markers mined from D. sapinea genomes to assess the genetic diversity at higher resolution. Isolates from northern Spain, an area formerly regarded as having low genetic diversity and samples from a Californian population that was formerly regarded as clonal, were analysed in the study. In Spain, the nine SSR markers identified 56 genotypes in 285 samples. Isolates from symptomatic shoots, cones and asymptomatic tissues collected from different stands, suggested admixture between local populations. The same genotype tended to dominate within a single cone, and the same genotypes were usually found in both symptomatic and asymptomatic shoot tissues. The nine new SSR markers developed in this study revealed a high level of genetic diversity in both the northern Spanish and northern Californian populations than previously anticipated. Analyses using these nine SSR markers should contribute to a better understanding of the epidemiology, evolution and origin of D. sapinea, a pathogen that is gaining prominence in many parts of the world.
Diplodia sapinea 是 Diplodia 枝枯病的病原体，这种新出现的病害影响着世界各地的松树林。这种病原体在北欧的分布范围扩大，部分原因是近期气候变暖。尽管已经对 D. sapinea 进行了广泛研究，但其感染生物学和种群结构的关键方面仍未得到探索。在这项研究中，我们开发了九个简单序列重复（SSR）标记，这些标记是从沙皮氏菌基因组中挖掘出来的，用于评估更高分辨率的遗传多样性。研究分析了来自西班牙北部的分离株（该地区以前被认为遗传多样性较低）和来自加利福尼亚的样本（该样本以前被认为具有克隆性）。在西班牙的 285 个样本中，9 个 SSR 标记确定了 56 个基因型。从不同林分采集的有症状嫩枝、球果和无症状组织的分离物表明，当地种群之间存在混杂。同一基因型往往在单个球果中占主导地位，而在有症状和无症状的嫩枝组织中通常都能发现相同的基因型。本研究开发的九个新的 SSR 标记显示，西班牙北部和加利福尼亚北部种群的遗传多样性比以前预期的要高。利用这九个 SSR 标记进行分析，有助于更好地了解 D. sapinea 的流行病学、演变和起源。
{"title":"New simple sequence repeat markers reveal undetected diversity in Spanish and Californian Diplodia sapinea populations","authors":"Laura Vilanova , Maria Caballol , Ke Zhang , Åke Olson , Irene Barnes , Michael J. Wingfield , Jonàs Oliva","doi":"10.1016/j.fgb.2024.103937","DOIUrl":"10.1016/j.fgb.2024.103937","url":null,"abstract":"<div><div><em>Diplodia sapinea</em> is the causal agent of Diplodia shoot blight, an emerging disease affecting pine forests worldwide. The range expansion of this pathogen in northern Europe has been suggested to be partially facilitated by recent warmer conditions. Although <em>D. sapinea</em> has been studied extensively, critical aspects of its infection biology and population structure remain unexplored. In this study, we developed nine simple sequence repeat (SSR) markers mined from <em>D. sapinea</em> genomes to assess the genetic diversity at higher resolution. Isolates from northern Spain, an area formerly regarded as having low genetic diversity and samples from a Californian population that was formerly regarded as clonal, were analysed in the study. In Spain, the nine SSR markers identified 56 genotypes in 285 samples. Isolates from symptomatic shoots, cones and asymptomatic tissues collected from different stands, suggested admixture between local populations. The same genotype tended to dominate within a single cone, and the same genotypes were usually found in both symptomatic and asymptomatic shoot tissues. The nine new SSR markers developed in this study revealed a high level of genetic diversity in both the northern Spanish and northern Californian populations than previously anticipated. Analyses using these nine SSR markers should contribute to a better understanding of the epidemiology, evolution and origin of <em>D. sapinea</em>, a pathogen that is gaining prominence in many parts of the world.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103937"},"PeriodicalIF":2.4,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142481368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-04DOI: 10.1016/j.fgb.2024.103936
Hayase Kojima , Moriyuki Kawauchi , Yuitsu Otsuka , Kim Schiphof , Kenya Tsuji , Akira Yoshimi , Chihiro Tanaka , Shigekazu Yano , Takehito Nakazawa , Yoichi Honda
The clade A APSES family transcription factors (Mbp1, Swi4, and Swi6) contribute to cell wall synthesis regulation in fungi. Herein, evolutionary relationships among these proteins were clarified by phylogenetic analysis using various ascomycetes and basidiomycetes, and then the detailed function of Mbp1 in cell wall synthesis regulation was analyzed in Pleurotus ostreatus. Our phylogenetic analysis revealed that Mbp1 and Swi6 are widely conserved among various fungi, whereas Swi4 is a protein specific for Saccharomycotina. In P. ostreatus, two putative clade A APSES family transcription factors, protein ID 83192 and 134090, were found and identified as Mbp1 and Swi6, respectively. The mbp1 gene was then disrupted through homologous recombination using P. ostreatus 20b strain (Δku80) as a host to obtain mbp1 disruption strains (Δmbp1). Disruption of mbp1 significantly decreased the growth rate and shortened aerial hyphae, suggesting that Mbp1 is involved in mycelial growth, especially aerial hyphal growth. Furthermore, thinner cell walls, decreased relative percentage of β-glucan, and downregulation of all β-glucan synthase genes were observed in Δmbp1 strains. Therefore, Mbp1 plays an essential role in β-glucan synthesis regulation in P. ostreatus. Disruption of mbp1 also impacted the expression profiles of chitin synthase genes, septum formation, and sensitivity to a chitin synthesis inhibitor, suggesting that Mbp1 also regulates chitin synthesis. In conclusion, Mbp1 is responsible for normal mycelial growth and regulates β-glucan and chitin synthesis in P. ostreatus. To the best of our knowledge, this is the first report on the detailed function of Mbp1 in cell wall synthesis regulation in fungi.
APSES家族转录因子（Mbp1、Swi4和Swi6）在真菌细胞壁合成调控中做出了贡献。本文利用各种子囊菌和担子菌进行系统进化分析，阐明了这些蛋白之间的进化关系，然后分析了Mbp1在浮皮真菌细胞壁合成调控中的具体功能。我们的系统进化分析表明，Mbp1和Swi6在各种真菌中广泛保守，而Swi4是酵母菌特有的蛋白质。在奥斯特菌中，我们发现了两个推定的 A 支 APSES 家族转录因子，蛋白 ID 分别为 83192 和 134090，并将其分别鉴定为 Mbp1 和 Swi6。然后以 P. ostreatus 20b 菌株（Δku80）为宿主，通过同源重组破坏 mbp1 基因，获得 mbp1 破坏菌株（Δmbp1）。mbp1的破坏会明显降低菌丝的生长速度并缩短气生菌丝，这表明Mbp1参与了菌丝的生长，尤其是气生菌丝的生长。此外，在Δmbp1菌株中还观察到细胞壁变薄、β-葡聚糖的相对比例降低以及所有β-葡聚糖合成酶基因下调。因此，Mbp1 在奥斯特菌的β-葡聚糖合成调控中起着重要作用。mbp1的破坏也会影响几丁质合成酶基因的表达谱、隔膜的形成以及对几丁质合成抑制剂的敏感性，这表明Mbp1也调控几丁质的合成。总之，Mbp1 在奥斯特菌中负责菌丝的正常生长，并调控β-葡聚糖和几丁质的合成。据我们所知，这是首次报道 Mbp1 在真菌细胞壁合成调控中的详细功能。
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Pub Date : 2024-09-21DOI: 10.1016/j.fgb.2024.103935
Daniel A. Salgado-Bautista , Eduardo Callegari , Meritxell Riquelme
Proteomics offers a powerful tool to identify proteins within diverse microbial organisms, environments, and organelles, including extracellular vesicles (EVs). Fungal EVs are of particular interest due to their roles in cellular development and communication. While several methods exist to isolate EVs from cells, a universally accepted approach for EV protein characterization is lacking. This study investigated in-solution digestion (SD) and in-gel digestion (GD), for characterizing proteins from Neurospora crassa EVs, followed by LC-MS/MS analysis. GD identified three to four-times more proteins than SD while using the same number of unique peptides. Although GD requires a higher amount of starting sample, it offers a more comprehensive protein identification for fungal EVs, potentially preventing the omission of crucial data.
蛋白质组学为鉴定不同微生物有机体、环境和细胞器（包括胞外囊泡 (EVs)）中的蛋白质提供了强大的工具。由于真菌 EVs 在细胞发育和通讯中的作用，它们尤其引人关注。虽然有几种方法可以从细胞中分离出EV，但还缺乏一种普遍接受的EV蛋白质表征方法。本研究对溶液消化（SD）和凝胶消化（GD）进行了研究，以表征神经孢子EVs中的蛋白质，然后进行LC-MS/MS分析。在使用相同数量的独特肽时，GD鉴定出的蛋白质数量是SD的三到四倍。虽然 GD 需要更多的起始样本，但它能更全面地鉴定真菌 EV 的蛋白质，从而避免遗漏关键数据。
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Pub Date : 2024-09-05DOI: 10.1016/j.fgb.2024.103925
Valerie A. van Ingen-Buijs , Anouk C. van Westerhoven , Petros Skiadas , Xander C.L. Zuijdgeest , Sajeet Haridas , Christopher Daum , Kecia Duffy , Jie Guo , Hope Hundley , Kurt LaButti , Anna Lipzen , Jasmyn Pangilinan , Robert Riley , Jie Wang , Mi Yan , Francis Martin , Kerrie Barry , Igor V. Grigoriev , Johannes Z. Groenewald , Pedro W. Crous , Michael F. Seidl
Phyllosticta citricarpa is an important citrus-pathogen and a quarantine organism in the European Union. Its recently described relative, P. paracitricarpa, is very closely related and not listed as a quarantine organism. P. paracitricarpa is very difficult to distinguish from P. citricarpa, since its morphological features overlap and the barcoding gene sequences that were originally used to delimit them as distinct species have a low number of species-specific polymorphisms that have subsequently been shown to overlap between the two clades. Therefore, we performed extensive genomic analyses to determine whether the genetic variation between P. citricarpa and P. paracitricarpa strains should be considered to represent infraspecific variation within P. citricarpa, or whether it is indicative of distinct species. Using a phylogenomic analysis with 3,000 single copy ortholog genes and whole-genome comparisons, we determined that the variation between P. citricarpa and P. paracitricarpa can be considered as infraspecies variation within P. citricarpa. We also determined the level of variation in mitochondrial assemblies of several Phyllosticta species and concluded there are only minimal differences between the assemblies of P. citricarpa and P. paracitricarpa. Thus, using several orthogonal approaches, we here demonstrate that variation within the nuclear and mitochondrial genomes of other Phyllosticta species is larger than variation between genomes obtained from P. citricarpa and P. paracitricarpa strains. Thus, P. citricarpa and P. paracitricarpa should be considered as conspecific.
Phyllosticta citricarpa 是一种重要的柑橘病原体，也是欧盟的一种检疫生物。它最近描述的近亲 P. paracitricarpa 与它关系密切，但未被列为检疫生物。P. paracitricarpa 与 P. citricarpa 很难区分，因为它们的形态特征相互重叠，而且最初用于将它们划分为不同物种的条形码基因序列具有较少的物种特异性多态性，这些多态性随后被证明在两个支系之间重叠。因此，我们进行了广泛的基因组分析，以确定 P. citricarpa 和 P. paracitricarpa 菌株之间的遗传变异是否应被视为 P. citricarpa 内部的种下变异，或者是否表明了不同的物种。通过使用 3,000 个单拷贝直向同源基因和全基因组比较进行系统发生组分析，我们确定了 P. citricarpa 和 P. paracitricarpa 之间的变异可被视为 P. citricarpa 的种下变异。我们还确定了几个 Phyllosticta 物种线粒体组合的变异水平，并得出结论：P. citricarpa 和 P. paracitricarpa 的线粒体组合差异很小。因此，我们在此使用几种正交方法证明，其他 Phyllosticta 物种的核基因组和线粒体基因组内的变异大于从 P. citricarpa 和 P. paracitricarpa 菌株中获得的基因组之间的变异。因此，P. citricarpa 和 P. paracitricarpa 应被视为同种。
{"title":"Phyllosticta paracitricarpa is synonymous with the EU quarantine fungus P. citricarpa based on phylogenomic analyses","authors":"Valerie A. van Ingen-Buijs , Anouk C. van Westerhoven , Petros Skiadas , Xander C.L. Zuijdgeest , Sajeet Haridas , Christopher Daum , Kecia Duffy , Jie Guo , Hope Hundley , Kurt LaButti , Anna Lipzen , Jasmyn Pangilinan , Robert Riley , Jie Wang , Mi Yan , Francis Martin , Kerrie Barry , Igor V. Grigoriev , Johannes Z. Groenewald , Pedro W. Crous , Michael F. Seidl","doi":"10.1016/j.fgb.2024.103925","DOIUrl":"10.1016/j.fgb.2024.103925","url":null,"abstract":"<div><p><em>Phyllosticta citricarpa</em> is an important citrus-pathogen and a quarantine organism in the European Union. Its recently described relative, <em>P. paracitricarpa</em>, is very closely related and not listed as a quarantine organism. <em>P. paracitricarpa</em> is very difficult to distinguish from <em>P. citricarpa</em>, since its morphological features overlap and the barcoding gene sequences that were originally used to delimit them as distinct species have a low number of species-specific polymorphisms that have subsequently been shown to overlap between the two clades. Therefore, we performed extensive genomic analyses to determine whether the genetic variation between <em>P. citricarpa</em> and <em>P. paracitricarpa</em> strains should be considered to represent infraspecific variation within <em>P. citricarpa</em>, or whether it is indicative of distinct species. Using a phylogenomic analysis with 3,000 single copy ortholog genes and whole-genome comparisons, we determined that the variation between <em>P. citricarpa</em> and <em>P. paracitricarpa</em> can be considered as infraspecies variation within <em>P. citricarpa</em>. We also determined the level of variation in mitochondrial assemblies of several <em>Phyllosticta</em> species and concluded there are only minimal differences between the assemblies of <em>P. citricarpa</em> and <em>P. paracitricarpa</em>. Thus, using several orthogonal approaches, we here demonstrate that variation within the nuclear and mitochondrial genomes of other <em>Phyllosticta</em> species is larger than variation between genomes obtained from <em>P. citricarpa</em> and <em>P. paracitricarpa</em> strains. Thus, <em>P. citricarpa</em> and <em>P. paracitricarpa</em> should be considered as conspecific.</p></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"175 ","pages":"Article 103925"},"PeriodicalIF":2.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1087184524000628/pdfft?md5=551c0118874a22b2fde3b8d86173256b&pid=1-s2.0-S1087184524000628-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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