Genetic transformation system of Cytospora chrysosperma, the causal agent of poplar canker

Abstract:

[Objective] To explore the molecular pathogenic mechanism of C. chrysosperma, a protoplast preparation and transformation method is established, and the transformation efficiency and the stability of transformants are analyzed. [Methods] In this study, strain CFCC 89981 served as the recipient. The protoplasts were prepared using cell wall degrading enzymes, and transformed by gGFP plasmid mediated by PEG. PCR amplification, Southern blot and fluorescent observation were used to confirm the transformation efficiency and the genetic stability of GFP-tagged transformants. [Results] High-quality protoplasts with excellent regeneration efficiency (63.74%±9.73%) were generated using Driselase and Lysing enzyme digesting fresh mycelium with 1.2 mol/L KCl in pH 5.5 for 4 h and transformed with the gGFP plasmid using PEG. A total of 304 hygromycin B resistant transformants was obtained though added 4 μg DNA. FDA staining results showed that 98% of protoplasts exhibited high activity. The GFP fragments were detectable in the genomes of transformants by both PCR amplification and Southern blot analysis, and the fluorescence detection results also indicated that the GFP gene had been integrated and was stably expressed in the C. chrysosperma genome. Highly intense green fluorescence was observed in single-spore purified transformants. The GFP gene and hph gene were stably expressed after subculturing in PDA plates without hygromycin B resistant. [Conclusion] The high quality and viable protoplast of C. chrysosperma preparation and transformation system are established, it will provide a solid foundation and greatly facilitate the future studies on functional genomics and pathogenic molecular mechanism in C. chrysosperma.