MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

Kito, Hideki; Fujikawa, Takashi; Moriwaki, Akihiro; Tomono, Ayami; Izawa, Masako; Kamakura, Takashi; Ohashi, Miho; Sato, Hiroyoshi; Abe, Keietsu; Nishimura, Mika

doi:10.1016/j.fgb.2008.09.005

Cited by 33 publications

(32 citation statements)

References 38 publications

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“…These fungi include Neurospora crassa (Ninomiya et al 2004), Kluyveromyces lactis (Kooistra et al 2004) Aspergillus nidulans (Nayak et al 2006), A. fumigatus (da Silva Ferreira et al 2006;Krappmann et al 2006), A. oryzae (Takahashi et al 2006), A. sojae (Takahashi et al 2006), Sordaria macrospore (Pöggeler and Kück 2006), A. niger (Meyer et al 2007), Botrytis cinerea (Choquer et al 2008), Candida glabrata (Ueno et al 2007), Magnaporthe grisea (Villalba et al 2008), A. parasiticus (Chang 2008), Trichophyton mentagrophytes (Yamada et al 2009) and Hypocrea jecorina (Guangtao et al 2009). Another component of NHEJ, a DNA ligase IV, has been also inactivated in some filamentous fungi, such as N. crassa (Ishibashi et al 2006), A. oryzae (Mizutani et al 2008), M. grisea (Kito et al 2008) and Pichia ciferrii (Schorsch et al 2009). Compared to these, C. neoformans appears to be a rather laborious organism for gene targeting.…”

Section: Introductionmentioning

confidence: 99%

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

Virtudazo

Toh‐e

et al. 2010

Mycoscience

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The efficiency of gene targeting by integration through homologous recombination (homologous integration, HI) in the human pathogen Cryptococcus neoformans remains unsatisfactory. In order to achieve a much more efficient gene targeting system in C. neoformans, a new double knockout strain in genes involved in the nonhomologous end joining (NHEJ) pathway was constructed. HI frequency was elevated by as much as approximately fivefold in the single or double knockout strains in NHEJ genes, and the frequency depended on the gene targeted. None of the NHEJ gene knockouts showed significant differences in regular growth, sensitivity to DNA-damaging drugs or UV, and virulence compared to the wild-type control, suggesting that the NHEJ pathway does not play a significant role in these biological stresses in C. neoformans. It was also suggested that the genes analyzed in this study are components of a single NHEJ pathway, as the mutants (including the double mutant) displayed the same phenotypes.

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Section: Introductionmentioning

confidence: 99%

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

Virtudazo

Toh‐e

et al. 2010

Mycoscience

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“…Recently, it has been demonstrated that a Ku-independent NHEJ pathway exists in N. crassa and M. grisea (Ishibashi et al 2006;Kito et al 2008). A ChKU70-independent NHEJ pathway may also exist in C. higginsianum.…”

Section: Discussionmentioning

confidence: 99%

“…Similar results have been obtained in A. oryzae (Mizutani et al 2008). On the other hand, MgLig4, a homologue of Mus-53, is not strictly required for NHEJ in the plant pathogenic fungus M. grisea (Kito et al 2008). In order to obtain a recipient strain that is even more suitable for targeted gene mutagenesis, it may be worth investigating HR in a mutant for the Mus-53 homologue in C. higginsianum.…”

Section: Discussionmentioning

confidence: 99%

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

et al. 2010

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The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.

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“…These results suggest that the DNA ligase IV coding genes in C. neoformans and N. crassa contribute differently to NHEJ. In addition, MgLig4 of Magnaporthe grisea has been shown to be involved in NHEJ only in part; the HI efficiency of the knockout strain was relatively higher than that of the wild-type strain, and the event was gene dependent (Kito et al 2008). It is considered that the mechanism of doublestrand break repair, which influences HI efficiency, may be widely different even within the fungal kingdom.…”

Section: Discussionmentioning

confidence: 99%

Deletion of CnLIG4 DNA ligase gene in the fungal pathogen Cryptococcus neoformans elevates homologous recombination efficiency

Shimizu

Virtudazo

et al. 2010

Mycoscience

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In eukaryotes from yeasts to human, DNA double-strand breaks are repaired by nonhomologous endjoining (NHEJ) or homologous integration (HI). In the human pathogenic yeast Cryptococcus neoformans, gene manipulation by HI does not occur frequently because ectopic integration by NHEJ is predominant, and it has been necessary to screen 30-100 transformants per experiment to obtain transformants with the desired genotypes. To overcome this problem, we constructed a strain in which one of the NHEJ-related genes, CnLIG4, was deleted. CnLIG4 encodes a homologue of the human DNA ligase IV involved in the last step of DNA repair by NHEJ. Gene targeting in the URA5 locus of a URA5-lacking strain TAD1 with URA5 gene fragments having 1-kb flanking sequences achieved 80% HI efficiency, which is higher than that of the wild-type control (50%). Growth phenotypes and virulence were not attenuated by deletion of the CnLIG4 gene. Such results suggest that the CnLIG4 knockout strain created in this study provides an additional alternative for the molecular genetics study of C. neoformans.

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MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

Cited by 33 publications

References 38 publications

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

Deletion of CnLIG4 DNA ligase gene in the fungal pathogen Cryptococcus neoformans elevates homologous recombination efficiency

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