| 1. |
|
|
| 2. |
- Broberg, Martin, et al.
(författare)
-
Comparative genomics highlights the importance of drug efflux transporters during evolution of mycoparasitism in Clonostachys subgenus Bionectria (Fungi, Ascomycota, Hypocreales)
- 2021
-
Ingår i: Evolutionary applications. - : Wiley. - 1752-4571. ; 14, s. 476-497
-
Tidskriftsartikel (refereegranskat)abstract
- Various strains of the mycoparasitic fungal speciesClonostachys roseaare used commercially as biological control agents for the control of fungal plant diseases in agricultural crop production. Further improvements of the use and efficacy ofC. roseain biocontrol require a mechanistic understanding of the factors that determines the outcome of the interaction betweenC. roseaand plant pathogenic fungi. Here, we determined the genome sequences of 11Clonostachysstrains, representing five species inClonostachyssubgenusBionectria, and performed a comparative genomic analysis with the aim to identify gene families evolving under selection for gene gains or losses. Several gene families predicted to encode proteins involved in biosynthesis of secondary metabolites, including polyketide synthases, nonribosomal peptide syntethases and cytochrome P450s, evolved under selection for gene gains (p <= .05) in theBionectriasubgenus lineage. This was accompanied with gene copy number increases (p <= .05) in ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters predicted to contribute to drug efflux. MostClonostachysspecies were also characterized by high numbers of auxiliary activity (AA) family 9 lytic polysaccharide monooxygenases, AA3 glucose-methanol-choline oxidoreductases and additional carbohydrate-active enzyme gene families with putative activity (or binding) towards xylan and rhamnose/pectin substrates. Particular features of theC. roseagenome included expansions (p <= .05) of the ABC-B4 multidrug resistance transporters, the ABC-C5 multidrug resistance-related transporters and the 2.A.1.3 drug:H + antiporter-2 MFS drug resistance transporters. The ABC-G1 pleiotropic drug resistance transporter geneabcG6inC. roseawas induced (p <= .009) by exposure to the antifungalFusariummycotoxin zearalenone (1121-fold) and various fungicides. Deletion ofabcG6resulted in mutants with reduced (p < .001) growth rates on media containing the fungicides boscalid, fenhexamid and iprodione. Our results emphasize the role of biosynthesis of, and protection against, secondary metabolites inClonostachyssubgenusBionectria.
|
|
| 3. |
|
|
| 4. |
- Broberg, Martin, et al.
(författare)
-
Out in the Cold: Identification of Genomic Regions Associated With Cold Tolerance in the Biocontrol Fungus Clonostachys rosea Through Genome-Wide Association Mapping
- 2018
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- There is an increasing importance for using biocontrol agents in combating plant diseases sustainably and in the long term. As large scale genomic sequencing becomes economically viable, the impact of single nucleotide polymorphisms (SNPs) on biocontrol-associated phenotypes can be easily studied across entire genomes of fungal populations. Here, we improved a previously reported genome assembly of the biocontrol fungus Clonostachys rosea strain IK726 using the PacBio sequencing platform, which resulted in a total genome size of 70.7 Mbp and 21,246 predicted genes. We further performed whole-genome re-sequencing of 52 additional C. rosea strains isolated globally using Illumina sequencing technology, in order to perform genome-wide association studies in conditions relevant for biocontrol activity. One such condition is the ability to grow at lower temperatures commonly encountered in cryic or frigid soils in temperate regions, as these will be prevalent for protecting growing crops in temperate climates. Growth rates at 10 degrees C on potato dextrose agar of the 53 sequenced strains of C. rosea were measured and ranged between 0.066 and 0.413 mm/day. Performing a genome wide association study, a total of 1,478 SNP markers were significantly associated with the trait and located in 227 scaffolds, within or close to (<1000 bp distance) 265 different genes. The predicted gene products included several chaperone proteins, membrane transporters, lipases, and proteins involved in chitin metabolism with possible roles in cold tolerance. The data reported in this study provides a foundation for future investigations into the genetic basis for cold tolerance in fungi, with important implications for biocontrol.
|
|
| 5. |
- Dubey, Mukesh, et al.
(författare)
-
An ATP-Binding Cassette Pleiotropic Drug Transporter Protein Is Required for Xenobiotic Tolerance and Antagonism in the Fungal Biocontrol Agent Clonostachys rosea
- 2014
-
Ingår i: Molecular plant-microbe interactions. - 0894-0282 .- 1943-7706. ; 27, s. 725-732
-
Tidskriftsartikel (refereegranskat)abstract
- ATP-binding cassette (ABC) transporters mediate active efflux of natural and synthetic toxicants and are considered to be important for drug tolerance in microorganisms. In biological control agents (BCA), ABC transporters can play important roles in antagonism by providing protection against toxins derived from the fungal prey and by mediating the secretion of endogenous toxins. In the present study, we generated deletion and complementation strains of the ABC transporter abcG5 in the fungal BCA Clonostachys rosea to study its role in xenobiotic tolerance and antagonism. Gene expression analysis shows induced expression of abcG5 in the presence of the Fusarium mycotoxin zearalenone (ZEA), secreted metabolites of F. graminearum, and different classes of fungicides. Phenotypic analysis of abcG5 deletion and complementation strains showed that the deletion strains were more sensitive towards F. graminearum culture filtrates, ZEA, and iprodione-and mefenoxam-based fungicides, thus suggesting the involvement of abcG5 in cell protection. The Delta abcG5 strains displayed reduced antagonism towards F. graminearum in a plate confrontation assay. Furthermore, the Delta abcG5 strains failed to protect barley seedlings from F. graminearium foot rot disease. These data show that the abcG5 ABC transporter is important for xenobiotic tolerance and biocontrol traits in C. rosea.
|
|
| 6. |
- Dubey, Mukesh, et al.
(författare)
-
Disruption of the Eng18B ENGase Gene in the Fungal Biocontrol Agent Trichoderm atroviride Affetcs Growth Conidation and Antagonistic Ability
- 2012
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:5
-
Tidskriftsartikel (refereegranskat)abstract
- The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-b-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (ab) 8 barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.
|
|
| 7. |
- Dubey, Mukesh, et al.
(författare)
-
Evolution and functional characterization of pectate lyase PEL12, a member of a highly expanded Clonostachys rosea polysaccharide lyase 1 family
- 2018
-
Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 18
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundPectin is one of the major and most complex plant cell wall components that needs to be overcome by microorganisms as part of their strategies for plant invasion or nutrition. Microbial pectinolytic enzymes therefore play a significant role for plant-associated microorganisms and for the decomposition and recycling of plant organic matter. Recently, comparative studies revealed significant gene copy number expansion of the polysaccharide lyase 1 (PL1) pectin/pectate lyase gene family in the Clonostachys rosea genome, while only low numbers were found in Trichoderma species. Both of these fungal genera are widely known for their ability to parasitize and kill other fungi (mycoparasitism) and certain species are thus used for biocontrol of plant pathogenic fungi.ResultsIn order to understand the role of the high number of pectin degrading enzymes in Clonostachys, we studied diversity and evolution of the PL1 gene family in C. rosea compared with other Sordariomycetes with varying nutritional life styles. Out of 17 members of C. rosea PL1, we could only detect two to be secreted at acidic pH. One of them, the pectate lyase pel12 gene was found to be strongly induced by pectin and, to a lower degree, by polygalacturonic acid. Heterologous expression of the PEL12 in a PL1-free background of T. reesei revealed direct enzymatic involvement of this protein in utilization of pectin at pH5 without a requirement for Ca2+. The mutants showed increased utilization of pectin compounds, but did not increase biocontrol ability in detached leaf assay against the plant pathogen Botrytis cinerea compared to the wild type.ConclusionsIn this study, we aimed to gain insight into diversity and evolution of the PL1 gene family in C. rosea and other Sordariomycete species in relation to their nutritional modes. We show that C. rosea PL1 expansion does not correlate with its mycoparasitic nutritional mode and resembles those of strong plant pathogenic fungi. We further investigated regulation, specificity and function of the C. rosea PEL12 and show that this enzyme is directly involved in degradation of pectin and pectin-related compounds, but not in C. rosea biocontrol.
|
|
| 8. |
- Dubey, Mukesh, et al.
(författare)
-
Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungusTrichoderma atroviridereveals a role in conidiation and antagonism
- 2021
-
Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 296, s. 131-140
-
Tidskriftsartikel (refereegranskat)abstract
- Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin geneagl1in the mycoparasitic fungusTrichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression ofagl1during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungusRhizoctonia solanias the sole carbon source. Expression ofagl1was supressed under iron-limiting condition, whileagl1transcript was not detected duringT.atrovirideinteractions with the prey fungiBotrytis cinereaorR.solani. Phenotypic analysis ofagl1deletion strains (Delta agl1) showed reduced conidiation compared toT.atroviridewild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Delta agl1strains display reduced antagonism towardsB. cinereaandR.solanibased on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungusT.atroviride.
|
|
| 9. |
|
|
| 10. |
- Dubey, Mukesh, et al.
(författare)
-
Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea
- 2014
-
Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 14
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.Results: Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the Delta Hyd3 strain, but not for the Delta Hyd1 strain. Furthermore, enhanced root colonization ability for the Delta Hyd1 Delta Hyd3 strain was found in comparison to WT.Conclusions: These results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.
|
|
