Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum−Wheat Interactions

Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum−wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants.

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Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum

Phytopathology ◽

10.1094/phyto-08-17-0268-r ◽

2018 ◽

Vol 108 (4) ◽

pp. 510-520 ◽

Cited By ~ 3

Author(s):

Shunwen Lu ◽

Michael C. Edwards

Keyword(s):

Functional Analysis ◽

Fusarium Graminearum ◽

Expression Patterns ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Transcriptional Analysis ◽

Fungal Genome ◽

Dimensional Structure ◽

Head Blight ◽

Fungal Virulence

The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum–wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

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Early transcriptomic response of the mycoparasite Sphaerodes mycoparasitica to the mycotoxigenic Fusarium graminearum 3-ADON, the cause of Fusarium head blight

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00479-y ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Seon Hwa Kim ◽

Vladimir Vujanovic

Keyword(s):

Fusarium Head Blight ◽

Fusarium Graminearum ◽

Expression Patterns ◽

Transcript Level ◽

Future Research ◽

Glutathione Synthetase ◽

Rna Seq ◽

Head Blight ◽

Transcriptomic Response

AbstractMycoparasites are an assemblage of biotrophic and necrotrophic fungi that occur on plant pathogenic fungal hosts. Biotrophic mycoparasites are often overlooked in transcriptomic-based biocontrol studies. Sphaerodes mycoparasitica (S.m.) is a specific biotrophic mycoparasite of plant pathogenic Fusarium graminearum (F.g.), a devastating Fusarium head blight (FHB) disease in small-grain cereals. To understand the biotrophic mycoparasitism comprehensively, we performed Illumina RNA-Seq transcriptomic study on the fungus–fungus interaction in vitro. The aim is to identify the transcript-level mechanism related to the biotrophic S.m. mycoparasitism, particularly its ability to effectively control the F.g. 3-ADON chemotype. A shift in the transcriptomic profile of the mycoparasite was triggered in response to its interaction with F.g. during recognition (1.5 days) and colonization (3.5 days) steps. RNA-Seq analysis revealed ~ 30% of annotated transcripts with "function unknown". Further, 14 differentially expressed genes functionally linked to the biotrophic mycoparasitism were validated by quantitative real-time PCR (qPCR). The gene expression patterns of the filamentous haemagglutinin/adhesin/attachment factor as well as cell wall-degrading glucanases and chitinases were upregulated by host interaction. Besides, mycoparasitism-associated antioxidant resistance genes encoding ATP-binding cassette (ABC) transporter(s) and glutathione synthetase(s) were upregulated. However, the thioredoxin reductase was downregulated which infers that this antioxidant gene can be used as a resistance marker to assess S.m. antifungal and antimycotoxigenic activities. The interactive transcriptome of S. mycoparasitica provides new insights into specific mycoparasitism and will contribute to future research in controlling FHB. Graphic Abstract

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Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

Scientific Reports ◽

10.1038/s41598-021-82051-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shauna Kehoe ◽

Katarina Jewgenow ◽

Paul R. Johnston ◽

Susan Mbedi ◽

Beate C. Braun

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Ovarian Follicle ◽

Mammalian Species ◽

Expression Patterns ◽

Ovarian Follicles ◽

Domestic Cat ◽

Transcriptional Analysis ◽

Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

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Impact of the biofungicide tetramycin on the development of Fusarium head blight, grain yield and deoxynivalenol accumulation in wheat

World Mycotoxin Journal ◽

10.3920/wmj2019.2494 ◽

2020 ◽

Vol 13 (2) ◽

pp. 235-246

Author(s):

W.Q. Shi ◽

L.B. Xiang ◽

D.Z. Yu ◽

S.J. Gong ◽

L.J. Yang

Keyword(s):

Fusarium Head Blight ◽

Fusarium Graminearum ◽

Spore Germination ◽

Field Experiments ◽

Synergistic Effects ◽

Field Trials ◽

Head Blight ◽

Mycelium Growth ◽

Key Aspects

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease that leads to extensive yield and quality loss in wheat and barley production. Integrated pest management (IPM) is required to control this disease and biofungicides, such as tetramycin, could be a novel addition to IPM strategies. The current study investigated in vitro tetramycin toxicity in Fusarium graminearum and evaluated its effectiveness for the control of Fusarium head blight FHB. Tetramycin was shown to affect three key aspects of Fusarium pathogenicity: spore germination, mycelium growth and deoxynivalenol (DON) production. The in vitro results indicated that tetramycin had strong inhibitory activity on the mycelial growth and spore germination. Field trials indicated that tetramycin treatment resulted in a significant reduction in both the FHB disease index and the level of DON accumulation. The reduced DON content in harvested grain was correlated with the amount of Tri5 mRNA determined by qRT-PCR. Synergistic effects between tetramycin and metconazole, in both the in vitro and field experiments were found. Tetramycin could provide an alternative option to control FHB.

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PCR-RFLP for detection of Fusarium graminearum genotypes with resistance to phenamacril

Plant Disease ◽

10.1094/pdis-06-20-1156-re ◽

2020 ◽

Author(s):

Jiao-Sheng Li ◽

Luo-Yu Wu ◽

Hui Zhang ◽

Xiu-Shi Song ◽

Jian-Xin Wang ◽

...

Keyword(s):

Fusarium Graminearum ◽

Conventional Method ◽

Resistance Mutations ◽

Head Blight ◽

Fusarium Asiaticum ◽

Pcr Rflp ◽

Pcr Products ◽

Resistant Strains ◽

Codon Mutation

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and Fusarium asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in vitro lab experiments. In this study, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217, and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L) or 1649 bp (for mutation of E420K) in myosin-5 gene were amplified respectively by appropriate primer pairs. Restriction enzyme KpnⅠ, TasⅠ or DraⅠ was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L and E420K, respectively. KpnⅠ digested the 841 bp PCR products of phenamacri-resistant strains with codon mutation A135T into two fragments of 256 bp and 585 bp. In contrast, KpnⅠ did not digest the PCR products of sensitive strains. TasⅠ digested the 802 bp PCR products of phenamacril-strains with codon mutation S217L into three fragments of 461 bp, 287bp and 54 bp. In contrast, TasⅠ digestion of the 802 bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515bp and 287bp. DraⅠ digested the 1649 bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 bp and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three resistant mutation genotypes of F. graminearum to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

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Chemotype and aggressiveness evaluation of Fusarium graminearum and Fusarium culmorum isolates from wheat fields in Wisconsin

Plant Disease ◽

10.1094/pdis-06-20-1376-re ◽

2021 ◽

Author(s):

Brian Mueller ◽

Carol Groves ◽

Damon L. Smith

Keyword(s):

Growth Rate ◽

Fusarium Head Blight ◽

Fusarium Graminearum ◽

Fusarium Culmorum ◽

Head Blight ◽

Growing Seasons ◽

Progress Curve ◽

Sporulation Capacity ◽

Derivatives Of

Fusarium graminearum commonly causes Fusarium head blight (FHB) on wheat, barley, rice, and oats. Fusarium graminearum produces nivalenol and deoxynivalenol (DON) and forms derivatives of DON based on its acetylation sites. The fungus is profiled into chemotypes based on DON derivative chemotypes (3 acetyldeoxynivalenol (3ADON) chemotype; 15 acetyldeoxynivalenol (15ADON) chemotype) and/or the nivalenol (NIV) chemotype. The current study assessed the Fusarium population found on wheat and the chemotype profile of the isolates collected from 2016 and 2017 in Wisconsin. Fusarium graminearum was isolated from all locations sampled in both 2016 and 2017. Fusarium culmorum was isolated only from Door County in 2016. Over both growing seasons, 91% of isolates were identified as the 15ADON chemotype while 9% of isolates were identified as the 3ADON chemotype. Aggressiveness was quantified by area under disease progress curve (AUDPC). The isolates with the highest AUDPC values were from the highest wheat producing cropping districts in the state. Deoxynivalenol production in grain and sporulation and growth rate in vitro were compared to aggressiveness in the greenhouse. Our results showed that 3ADON isolates in Wisconsin were among the highest in sporulation capacity, growth rate, and DON production in grain. However, there were no significant differences in aggressiveness between the 3ADON and 15ADON isolates. The results of this research detail the baseline frequency and distribution of 3ADON and 15ADON chemotypes observed in Wisconsin. Chemotype distributions within populations of F. graminearum in Wisconsin should continue to be monitored in the future.

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Characterization of QTL and eQTL controlling early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan 1 x Nyubai doubled haploid wheat population

BMC Plant Biology ◽

10.1186/s12870-019-2149-4 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

François Fauteux ◽

Yunli Wang ◽

Hélène Rocheleau ◽

Ziying Liu ◽

Youlian Pan ◽

...

Keyword(s):

Fusarium Graminearum ◽

Doubled Haploid ◽

Expression Patterns ◽

Doubled Haploid Population ◽

Post Inoculation ◽

Head Blight ◽

Wheat Genotypes ◽

Wheat Population ◽

Fhb Resistance ◽

Moderately Resistant

Abstract Background Fusarium head blight (FHB) is a major disease of cereal crops, caused by the fungal pathogen Fusarium graminearum and related species. Breeding wheat for FHB resistance contributes to increase yields and grain quality and to reduce the use of fungicides. The identification of genes and markers for FHB resistance in different wheat genotypes has nevertheless proven challenging. Results In this study, early infection by F. graminearum was analyzed in a doubled haploid population derived from the cross of the moderately resistant wheat genotypes Wuhan 1 and Nyubai. Three quantitative trait loci (QTL) were identified: 1AL was associated with lower deoxynivalenol content, and 4BS and 5A were associated with reduced F. graminearum infection at 2 days post inoculation. Early resistance alleles were inherited from Wuhan 1 for QTL 1AL and 4BS and inherited from Nyubai for the 5A QTL. Cis and trans expression QTL (eQTL) were identified using RNA-seq data from infected head samples. Hotspots for trans eQTL were identified in the vicinity of the 1AL and 4BS QTL peaks. Among differentially expressed genes with cis eQTL within the QTL support intervals, nine genes had higher expression associated with FHB early resistance, and four genes had higher expression associated with FHB early susceptibility. Conclusions Our analysis of genotype and gene expression data of wheat infected by F. graminearum identified three QTL associated with FHB early resistance, and linked genes with eQTL and differential expression patterns to those QTL. These findings may have applications in breeding wheat for early resistance to FHB.

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One Small RNA of Fusarium graminearum Targets and Silences CEBiP Gene in Common Wheat

Microorganisms ◽

10.3390/microorganisms7100425 ◽

2019 ◽

Vol 7 (10) ◽

pp. 425 ◽

Cited By ~ 6

Author(s):

Jiao Jian ◽

Xu Liang

Keyword(s):

Fusarium Graminearum ◽

Small Rna ◽

Pathogenic Fungus ◽

Effective Means ◽

Transcriptional Level ◽

Head Blight ◽

Protein Coding ◽

Pathogen Invasion

The pathogenic fungus Fusarium graminearum (F. graminearum), causing Fusarium head blight (FHB) or scab, is one of the most important cereal killers worldwide, exerting great economic and agronomic losses on global grain production. To repress pathogen invasion, plants have evolved a sophisticated innate immunity system for pathogen recognition and defense activation. Simultaneously, pathogens continue to evolve more effective means of invasion to conquer plant resistance systems. In the process of co-evolution of plants and pathogens, several small RNAs (sRNAs) have been proved in regulating plant immune response and plant-microbial interaction. In this study, we report that a F. graminearum sRNA (Fg-sRNA1) can suppress wheat defense response by targeting and silencing a resistance-related gene, which codes a Chitin Elicitor Binding Protein (TaCEBiP). Transcriptional level evidence indicates that Fg-sRNA1 can target TaCEBiP mRNA and trigger silencing of TaCEBiP in vivo, and in Nicotiana benthamiana (N. benthamiana) plants, Western blotting experiments and YFP Fluorescence observation proofs show that Fg-sRNA1 can suppress the accumulation of protein coding by TaCEBiP gene in vitro. F. graminearum PH-1 strain displays a weakening ability to invasion when Barley stripe mosaic virus (BSMV) vector induces effective silencing Fg-sRNA1 in PH-1 infected wheat plants. Taken together, our results suggest that a small RNA from F. graminearum can target and silence the wheat TaCEBiP gene to enhance invasion of F. graminearum.

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Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR

Toxins ◽

10.3390/toxins12090560 ◽

2020 ◽

Vol 12 (9) ◽

pp. 560

Author(s):

Elena Maria Colombo ◽

Andrea Kunova ◽

Claudio Gardana ◽

Cristina Pizzatti ◽

Paolo Simonetti ◽

...

Keyword(s):

Fusarium Graminearum ◽

Plant Pathogens ◽

Fungal Growth ◽

Toxin Production ◽

Fungal Mycelium ◽

Head Blight ◽

Wheat Grains ◽

Streptomyces Spp ◽

Dual Plate

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions.

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