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

Abstract

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.

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

“…This genetic modification is neutral with regard to all tested phenotypic properties assayed at the optimal growth temperature for this fungus. These observations and conclusions are similar to those reported for ku70/ku80 mutations in many other filamentous fungi including the previous Dku80 strains in P. digitatum (Xu et al 2014).…”

“…After succeeding in stablishing protocols for the isolation, selection and regeneration of protoplasts with our P. digitatum strains, we were unable to demonstrate protoplast transformation (data not shown). This negative result is consistent with the reported difficulty of transforming P. digitatum protoplasts (Xu et al 2014). As an alternative, we tried to select spontaneous FRT recombination events that would excise the nat1 marker.…”

“…In our study, the P. digitatum Dku70 strains appeared as the wild type at the optimal temperature with very similar growth, conidia production, colouration or pigment content. Both A. alternata and P. digitatum Dku70 strains were not affected in their pathogenicity to citrus, as is the case of the P. digitatum Dku80 (Xu et al 2014). Interestingly, the A. alternata ku70 mutation had unexpectedly no effect on homologous integration and therefore did not enhance gene disruption efficiency.…”

“…For example, in our two recent publications the highest HR frequency was 0.6 % in the case of the protein Oemannosyltransferase Pmt2 mutant (Harries et al 2015). A recent work has demonstrated that the deletion of ku80 increased gene targeting efficiency in P. digitatum and did not affect growth, sporulation, tolerance to salt, or virulence (Xu et al 2014). In this study we explored a similar approach by mutating the alternative ku70 gene in P. digitatum, and discovered unexpected results that must be taken into account for the future use of ku70/80 mutants in filamentous fungi.…”

CRISPR/Cas9‐based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi

“…This method has been more successful in yeast than in filamentous fungi due to lower sequence homology requirements in yeast [30, 31]. However, the HR frequency in filamentous fungi can now be improved by replacing ku70 and ku80 , which are responsible for nonhomologous end joining (NHEJ) [32–34].…”