Phleomycin resistance gene as a reliable selection marker for Agrobacterium tumefaciens-mediated transformation of the citrus postharvest pathogen Penicillium digitatum

Abstract

Penicillium digitatum exists in nature as a causative agent of green mould disease in citrus fruits at the postharvest stages. Inspections of the molecular mechanism of the host invasion by P. digitatum usually require suitable selection markers for genetic manipulation. In this study, we recruited the phleomycin resistance gene as a selection marker for the genetic transformation of P. digitatum using Agrobacterium tumefaciens. The results showed that the growth of the wild strain P. digitatum PdVN1 from fungal spores is quite sensitive to phleomycin and is inhibited at a concentration of 50 µg/ml, whereas growth from fungal mycelium is more tolerant and completely suppressed at a concentration of 200 µg/ml. Under optimised conditions, the A. tumefaciens-mediated transformation (ATMT) efficiency of P. digitatum with the phleomycin resistance marker could reach over 1000 transformants per 106 spores. All the tested transformants presented the integration of T-DNA in their genomes and were mitotically stable for the phleomycin resistance. Furthermore, the results also revealed the success for heterologous expression of the DsRed fluorescent gene in the fungus, in which the strong red fluorescent signal could be observed over the whole fungal mycelium and spores. Our work demonstrates for the first time that the phleomycin resistance gene can serve as a reliable selection marker for A. tumefaciens-mediated transformation of the postharvest pathogen P. digitatum. This selection marker can be exploited for T-DNA insertional mutagenesis and for functional investigations of target genes involved in citrus decay by P. digitatum.