Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

Villalba, François; Collemare, Jérôme; Landraud, Patricia; Lambou, Karine; Brozek, Viviane; Cirer, Bénédicte; Morin, Damien; Bruel, Christophe; Beffa, Roland; Lebrun, Marc

doi:10.1016/j.fgb.2007.06.006

Cited by 136 publications

(126 citation statements)

References 34 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…All the wild-type and mutant strains used in this study (Table 1) were cultured on oatmeal agar or CM plates as described (Talbot et al, 1993;Park et al, 2006;Villalba et al, 2008). Mycelia harvested from 2-d-old 53YEG (0.5% yeast extract and 1% glucose) cultures shaken at 150 rpm were used for isolation of genomic DNA and protoplasts (Sweigard et al, 1998;Zhao et al, 2005).…”

Section: Culture Conditions and Genetic Manipulationsmentioning

confidence: 99%

“…Primers used to amplify the flanking sequences of HOS2, SNT1, SET3, HST1, and HOS4 are listed in Supplemental Table 1 online. The ligation-PCR products (Zhao et al, 2004) were transformed into protoplasts of Guy11, 70-15, or Ku80 (Villalba et al, 2008) to generate the hos2, snt1, set3, hst1, and hos4 deletion mutants (Table 1). Putative knockout mutants were identified by PCR screens and confirmed by DNA gel blot analysis.…”

Section: Generation Of the Gene Replacement Mutantsmentioning

confidence: 99%

See 1 more Smart Citation

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

et al. 2010

Self Cite

View full text Add to dashboard Cite

Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin b-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

show abstract

Section: Culture Conditions and Genetic Manipulationsmentioning

confidence: 99%

Section: Generation Of the Gene Replacement Mutantsmentioning

confidence: 99%

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Homologous recombination (HR) of exogenous DNA into the genome occurs easily in the yeast species, but it is not a ready mechanism for filamentous fungi since the non-homologous end-joining (NHEJ) pathway seems to be dominant over the HR pathway for repairing of DNA double-strand breaks (DSBs) in filamentous fungi (Ishibashi et al, 2006;Meyer et al, 2007;Maruyama and Kitamoto, 2008;Villalba et al, 2008). The DNA-dependent protein kinase complexes as well as a Ku70-Ku80 heterodimer are the central components of NHEJ (Haber, 2000;Jones et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum

Zhu

Wang

et al. 2014

J. Zhejiang Univ. Sci. B

View full text Add to dashboard Cite

Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation (ATMT), but the frequencies are often very low. In the present study, we replaced the Ku80 homolog (a gene of the non-homologous end-joining (NHEJ) pathway) with the hygromycin resistance cassette (hph) by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in Ku80-deficient strain (∆PdKu80) of P. digitatum. However, using ∆PdKu80 as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous integration and the created ∆PdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum.

show abstract

“…In fungi, traditional methods of gene functional analysis involving single gene replacement, protein localization studies and phenotype screening are already being replaced by high-throughput methodologies. For example, rapid targeted gene replacement of single genes can now utilize double-jointed PCR combined with recipient strains lacking components of the non-homologous DNA endjoining pathway, Ku70 and Ku80 (Kershaw & Talbot, 2009; Fungal physiology -a future perspective Villalba et al, 2008), thereby simultaneously eliminating time-consuming disruption vector construction -with its associated and cumbersome cloning and ligation stepsand the need to screen large numbers of transformants to identify progeny carrying homologous replacements at the loci of interest. Beyond single-gene analysis, RNA interference-mediated gene silencing technologies are being developed for the rapid functional analysis of many genes: for instance the silencing of all genes involved in a particular pathway, or the silencing of all members of a multigene family (Nguyen et al, 2008).…”

Section: Phenotype Microarray Development For Highthroughput Gene Funmentioning

confidence: 99%

Fungal physiology – a future perspective

Wilson

Talbot

2009

Microbiology

View full text Add to dashboard Cite

The study of fungal physiology is set to change dramatically in the next few years as highly scalable technologies are deployed allowing accurate measurement and identification of metabolites, proteins and transcripts within cells. The advent of next-generation DNA-sequencing technologies will also provide genome sequence information from large numbers of industrially relevant and pathogenic fungal species, and allow comparative genome analysis between strains and populations of fungi. When coupled with advances in gene functional analysis, proteinprotein interaction studies, live cell imaging and mathematical modelling, this promises a stepchange in our understanding of how fungal cells operate as integrated dynamic living systems.

show abstract

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

Cited by 136 publications

References 34 publications

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum

Fungal physiology – a future perspective

Contact Info

Product

Resources

About