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

• Disruption of ku70 enhanced homologous recombination frequency in Penicillium digitatum.
• The mutation of ku70 did not affect P. digitatum phenotypes at optimal temperature.
• Δku70 strains showed defects in growth and conidia production at high temperatures.
• Fungal Δku70 recipient strains should be used with caution.

The dominant mechanism to repair double-stranded DNA breaks in filamentous fungi is the non-homologous end joining (NHEJ) pathway, and not the homologous recombination (HR) pathway that operates in the mutation of genes by replacement of target DNA for selection cassettes. The key to improve HR frequency is the inactivation of the NHEJ pathway by eliminating components of its Ku70/80 heterodimeric complex. We have obtained ku70 mutants of Penicillium digitatum, the main citrus postharvest pathogen. The increased efficiency of HR in Δku70 strains was demonstrated by the generation of mutants in two different chitin synthase genes (PdchsII and PdchsV). P. digitatum Δku70 strains showed no differences from the parental strain in vegetative growth, asexual development or virulence to citrus fruit, when experiments were conducted at the optimal temperature of 24 °C. However, growth of Δku70 strains at temperatures higher than 24 °C demonstrated a detrimental effect in axenic growth and conidia production. These observations are in agreement with previous studies describing differences between ku70 mutants and their parental strains in some fungal species, and must be taken into account for future applications of the Δku approach to increase HR efficiency in fungi.