Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum

Gandía, Mónica; Xu, Shaomei; Font, Cristina; Marcos, José F.

doi:10.1016/j.funbio.2015.11.001

Cited by 30 publications

(25 citation statements)

References 36 publications

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“…However, both strains were phenotypically undistinguishable in terms of growth rate and ability to exploit iron from various sources. These observations are in line with other studies in which the NHEJ pathway in several filamentous models was inactivated through the deletion of the ku70 gene (Ninomiya et al, 2004;Nayak et al, 2006;Choquer et al, 2008;Hoff et al, 2010;Li et al, 2010;Qi et al, 2015;Gandıá et al, 2016). The resulting Dku70 strains were not reported to exhibit noticeable phenotypic differences with the WT strains, and they were further used as parent strains to delete genes of interest.…”

Section: Discussionsupporting

confidence: 89%

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence

Govic

Havlı́ček

Capilla

et al. 2020

Front. Cell. Infect. Microbiol.

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Section: Discussionsupporting

confidence: 89%

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence

Govic

Havlı́ček

Capilla

et al. 2020

Front. Cell. Infect. Microbiol.

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“…This report shows that it can also be used to stimulate HDR. Previous approaches for stimulating HDR were based on cNHEJ inhibition or Rad51 recombinase stimulation by genetic or pharmacological manipulation of cells 10 , 27 . The direct fusion strategy reported here is based on targeted activity of a key component of the homologous recombination DNA repair pathway and is potentially less likely to have a global effect on DNA repair.…”

Section: Discussionmentioning

confidence: 99%

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

et al. 2018

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In genome editing with CRISPR–Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9–HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9–HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

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“…The P. digitatum parental strains used were the wild type CECT 20796 (Marcet-Houben et al, 2012) and the transformant PDMG314 (Δ ku70:nat1 ) (Gandía et al, 2016). All P. digitatum strains were routinely cultured on potato dextrose agar (PDA, Difco, Detroit, MI, United States) plates for 7–10 days at 24°C.…”

Section: Methodsmentioning

confidence: 99%

The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum

et al. 2019

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Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new antifungal molecules. Among these are the antifungal peptides and proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the antifungal proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to antifungal proteins in P. digitatum.

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Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum

Cited by 30 publications

References 36 publications

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum

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