Identification of ABC transporter genes of Fusarium graminearum with roles in azole tolerance and/or virulence.
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ABSTRACT:
SUBMITTER: Abou Ammar G
PROVIDER: S-EPMC3823976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA methylation mediates organisms' adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.
| S-EPMC8152758 | biostudies-literature
Project description:Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ~70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.
| S-EPMC3713025 | biostudies-literature
Project description:Sexual reproduction plays a critical role in the infection cycle of Fusarium graminearum because ascospores are the primary inoculum. As a homothallic ascomycete, F. graminearum contains both the MAT1-1 and MAT1-2-1 loci in the genome. To better understand their functions and regulations in sexual reproduction and pathogenesis, in this study we assayed the expression, interactions, and mutant phenotypes of individual MAT locus genes. Whereas the expression of MAT1-1-1 and MAT12-1 rapidly increased after perithecial induction and began to decline after 1 day post-perithecial induction (dpi), the expression of MAT1-1-2 and MAT1-1-3 peaked at 4 dpi. MAT1-1-2 and MAT1-1-3 had a similar expression profile and likely are controlled by a bidirectional promoter. Although none of the MAT locus genes were essential for perithecium formation, all of them were required for ascosporogenesis in self-crosses. In outcrosses, the mat11-1-2 and mat11-1-3 mutants were fertile but the mat1-1-1 and mat1-2-1 mutants displayed male- and female-specific defects, respectively. The mat1-2-1 mutant was reduced in FgSO expression and hyphal fusion. Mat1-1-2 interacted with all other MAT locus transcription factors, suggesting that they may form a protein complex during sexual reproduction. Mat1-1-1 also interacted with FgMcm1, which may play a role in controlling cell identity and sexual development. Interestingly, the mat1-1-1 and mat1-2-1 mutants were reduced in virulence in corn stalk rot assays although none of the MAT locus genes was important for wheat infection. The MAT1-1-1 and MAT1-2-1 genes may play a host-specific role in colonization of corn stalks.
| S-EPMC3691137 | biostudies-literature
Project description:The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.
| S-EPMC6811443 | biostudies-literature
Project description:The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers.
| S-EPMC3843709 | biostudies-literature
Project description:Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized the Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used green fluorescent protein (GFP)-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between the endoplasmic reticulum (ER) and Golgi and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains, which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3-RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearumIMPORTANCEFusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, and limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, deoxynivalenol (DON) biosynthesis, and pathogenicity is therefore important for the development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein, RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum Our findings provide a comprehensive characterization of the golgin family protein RUD3 in plant-pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.
| S-EPMC8104996 | biostudies-literature
Project description:3-isopropylmalate dehydrogenase (IPMD) encoded by LEU2 is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of Fusarium graminearum revealed two paralogous LEU2 genes (designated as FgLEU2A and FgLEU2B) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual FgLEU2A/B gene in F. graminearum assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of FgLEU2A resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas FgLEU2B deletion mutant ΔFgLeu2B-2 was morphologically indistinguishable from the wild type strain PH-1. The growth defects of ΔFgLeu2A-10 could be overcome by exogenous addition of Leu at 0.25 mM. Double deletion of FgLEU2A and FgLEU2B (ΔFgLeu2AB-8) caused a more severe Leu-auxotrophic phenotype as the concentration of Leu exogenously added to medium to rescue the growth defect of ΔFgLeu2AB-8 should be raised to 1.25 mM, indicating a less important but nonnegligible role of FgLeu2B in Leu biosynthesis. Disturb of Leu biosynthesis caused by FgLEU2A deletion leads to slower growth rate, reduced aerial hyphal formation and red pigmentation on PDA plates and completely blocked conidial production and germination. All of the defects above could be overcome by Leu addition or complementation of the full-length FgLEU2A gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking FgLEU2A were dramatically impaired on wheat heads and non-host cherry tomatoes. Additionally, a low level of deoxynivalenol (DON) production of ΔFgLeu2A-10 and ΔFgLeu2AB-8 in wheat kernels was also detected. Taken together, results of this study indicated a crucial role of FgLeu2A and a less important role of FgLeu2B in Leu biosynthesis and fungal infection-related morphogenesis in F. graminearum and FgLeu2A may serve as a potential target for novel antifungal development.
| S-EPMC5106029 | biostudies-literature
Project description:Fusarium graminearum is the predominant causal agent of cereal Fusarium head blight disease (FHB) worldwide. The application of chemical fungicides such as azole antifungals is still the primary method for FHB control. However, to date, our knowledge of transcriptional regulation in the azole resistance of F. graminearum is quite limited. In this study, we identified and functionally characterized a Zn(II)2-Cys6 transcription factor FgAtrR in F. graminearum. We constructed a FgAtrR deletion mutant and found that deletion of FgAtrR resulted in faster radial growth with serious pigmentation defects, significantly reduced conidial production, and an inability to form perithecia. The pathogenicity of the ΔFgAtrR mutant on wheat spikes and corn silks was severely impaired with reduced deoxynivalenol production, while the tolerance to prochloraz and propiconazole of the deletion mutant was also significantly decreased. RNA-seq indicated that many metabolic pathways were affected by the deletion of FgAtrR. Importantly, FgAtrR could regulate the expression of the FgCYP51A and ABC transporters, which are the main contributors to azole resistance. These results demonstrated that FgAtrR played essential roles in asexual and sexual development, DON production, and pathogenicity, and contributed to intrinsic resistance to azole fungicides in F. graminearum. This study will help us improve the understanding of the azole resistance mechanism in F. graminearum.
| S-EPMC8869466 | biostudies-literature
Project description:Candida lusitaniae is an opportunistic pathogen in humans that causes infrequent but difficult-to-treat diseases. Antifungal drugs are used in the clinic to treat C. lusitaniae infections, however, this fungus can rapidly acquire antifungal resistance to all known antifungal drugs (multidrug resistance). C. lusitaniae acquires azole resistance by gain-of-function (GOF) mutations in the transcriptional regulator MRR1. MRR1 controls the expression of a major facilitator transporter (MFS7) that is important for fluconazole resistance. Here, we addressed the role of the ATP Binding Cassette (ABC) transporter CDR1 as additional mediator of azole resistance in C. lusitaniae. CDR1 expression in isolates with GOF MRR1 mutations was higher compared to wild types, which suggests that CDR1 is an additional (direct or indirect) target of MRR1. CDR1 deletion in the azole-resistant isolate P3 (V688G GOF) revealed that MICs of long-tailed azoles, itraconazole and posaconazole, were decreased compared to P3, which is consistent with the role of this ABC transporter in the efflux of these azoles. Fluconazole MIC was only decreased when CDR1 was deleted in the background of an mfs7Δ mutant from P3, which underpins the dominant role of MFS7 in the resistance of the short-tailed azole fluconazole. With R6G efflux readout as Cdr1 efflux capacity, our data showed that R6G efflux was increased in P3 compared to an azole-susceptible wild type parent, and diminished to background levels in mutant strains lacking CDR1. Milbemycin oxim A3, a known inhibitor of fungal ABC transporters, mimicked efflux phenotypes of cdr1Δ mutants. We therefore provided evidence that CDR1 is an additional mediator of azole resistance in C. lusitaniae, and that CDR1 regulation is dependent on MRR1 and associated GOF mutations.
| S-EPMC8467326 | biostudies-literature
Project description:BackgroundIdentifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.ResultsWe used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.ConclusionsThis study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.
| S-EPMC4404607 | biostudies-literature