Protoplast Transformation of Filamentous Fungi

Turgeon, B. Gillian; Condon, Bradford; Liu, Jinyuan; Zhang, Ning

doi:10.1007/978-1-60761-611-5_1

Cited by 73 publications

(64 citation statements)

References 16 publications

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“…A homokaryotic strain of Termitomyces was obtained from a heterokaryon in a colony of M. natalensis, using a standard protoplasting procedure (52,53). DNA was extracted from pure culture material, after which 12.5 Gb raw paired reads were generated for five insert libraries with HiSeq 2000, which allowed for de novo assembly of the 83.7-Mbp genome.…”

Section: Methodsmentioning

confidence: 99%

Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Poulsen

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

351

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Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.carbohydrate-active enzymes | eusocial | symbioses | cellulose | lignin

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Section: Methodsmentioning

confidence: 99%

Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Poulsen

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

351

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“…The maize pathogen Cochliobolus heterostrophus (strain C4) was grown for 7 days on complete xylose medium (27) in the same controlled environment as the plants. The second leaf was inoculated with 7 l droplets of 0.02% Tween 20 in deionized water containing 1000 spores.…”

Section: Methodsmentioning

confidence: 99%

“…Knockout Strain Generation: Construction, Transformation, and Screening of Transformants-Transformation techniques followed established methods (26,27). The split-marker method (29,30) was used for the construction of a linear DNA fragment, which upon integration into the genome of the wild type strain (Gv 29 -8), replaces the gene of interest with a hygromycin selectable marker (27).…”

Section: Concentration Of the T Virens Secreted Protein (Sp)mentioning

confidence: 99%

“…The split-marker method (29,30) was used for the construction of a linear DNA fragment, which upon integration into the genome of the wild type strain (Gv 29 -8), replaces the gene of interest with a hygromycin selectable marker (27). The list of primer pairs for construction of each mutant is provided in supplemental Table S2.…”

Section: Concentration Of the T Virens Secreted Protein (Sp)mentioning

confidence: 99%

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Secretome of Trichoderma Interacting With Maize Roots: Role in Induced Systemic Resistance*

Lamdan

Shalaby

Ziv

et al. 2015

Molecular & Cellular Proteomics

104

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Trichoderma virens is a biocontrol agent used in agriculture to antagonize pathogens of crop plants. In addition to direct mycoparasitism of soil-borne fungal pathogens, T. virens interacts with roots. This interaction induces systemic resistance (ISR), which reduces disease in aboveground parts of the plant. In the molecular dialog between fungus and plant leading to ISR, proteins secreted by T. virens provide signals. Only a few such proteins have been characterized previously. To study the secretome, proteins were characterized from hydroponic culture systems with T. virens alone or with maize seedlings, and combined with a bioassay for ISR in maize leaves infected by the pathogen Cochliobolus heterostrophus. The secreted protein fraction from coculture of maize roots and T. virens (Tv؉M) was found to have a higher ISR activity than from T. virens grown alone (Tv). A total of 280 fungal proteins were identified, 66 showing significant differences in abundance between the two conditions: 32 were higher in Tv؉M and 34 were higher in Tv. Among the 34 found in higher abundance in Tv and negatively regulated by roots were 13 SSCPs (small, secreted, cysteine rich proteins), known to be important in the molecular dialog between plants and fungi. The role of four SSCPs in ISR was studied by gene knockout. All four knockout lines showed better ISR activity than WT without affecting colonization of maize roots. Furthermore, the secreted protein fraction from each of the mutant lines showed improved ISR activity compared with WT. These SSCPs, apparently, act as negative effectors reducing the defense levels in the plant and may be important for the fine tuning of ISR by Trichoderma. The down-regulation of SSCPs in interaction with plant roots implies a revision of the current model for the Trichoderma-plant symbiosis and its induction of resistance to pathogens. Molecular & Cellular Proteomics

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“…All four candidate RR genes were deleted (Table 1 and see Table S1 in the supplemental material) using the split-marker approach (see Fig. S1 in the supplemental material) (11) and the transformation protocol described earlier (11,41). Primers for deletion are listed in Table S2 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Histidine Kinase Two-Component Response Regulator Proteins Regulate Reproductive Development, Virulence, and Stress Responses of the Fungal Cereal Pathogens Cochliobolus heterostrophus and Gibberella zeae

et al. 2010

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Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein.Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.

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Protoplast Transformation of Filamentous Fungi

Cited by 73 publications

References 16 publications

Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Secretome of Trichoderma Interacting With Maize Roots: Role in Induced Systemic Resistance*

Histidine Kinase Two-Component Response Regulator Proteins Regulate Reproductive Development, Virulence, and Stress Responses of the Fungal Cereal Pathogens Cochliobolus heterostrophus and Gibberella zeae

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