Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors.

Rosellinia necatrix is a fungus that infects a wide range of host plants and ruins a variety of commercially important crops. DNA fragments can be introduced into R. necatrix using conventional protoplast-PEG transformation and genome-integrating vectors; however, transformation efficiency with this strategy is quite low. Therefore, to establish a more effective transformation system for the studies of R. necatrix, an autonomously replicating vector was constructed using AMA1 sequences derived from Aspergillus nidulans, which is distantly related to R. necatrix. Use of this vector with AMA1 sequences increased transformation efficiency in R. necatrix, and the vector was maintained as a plasmid in the transformants. Transient and multivariate functional analyses in R. necatrix were performed using co-transformation of multiple pAMA-H vectors, which each carried either an expression cassette for eGFP, mOrange2, or a geneticin resistance gene. Furthermore, fluorescent proteins expressed from the autonomously replicating vectors were dispersed throughout fungal colonies even though the vectors themselves were restricted to the center of each colony. This intriguing phenomenon indicated that gene products could move from the center to the margin in a colony of the filamentous fungi via a cell-to-cell transport system.