Polyethylene glycol–mediated genetic transformation and fluorescent labelling of Colletotrichum sublineola for studying sorghum anthracnose pathogenicity

Anthracnose, a disease primarily caused by the fungus Colletotrichum sublineola , affects sorghum production in China. However, the infection mechanisms and complex host interactions of C. sublineola remain to be elucidated. Thus, this study aimed to establish and optimize a protoplast transformation system of C. sublineola so as to lay a foundation for further research on the infection mechanisms and complex host interactions of C. sublineola . A genetic transformation system was established and optimized for C. sublineola strain HX‐5‐1 and used to introduce fluorescent reporter genes, namely, green fluorescent protein ( GFP ) and mCherry . Results showed that the growth of HX‐5‐1 was completely inhibited on a regenerating medium containing 300 μg mL −1 hygromycin B, indicating that hygromycin B acted as a transformant‐selective marker. HX‐5‐1 produced the highest yield of protoplasts when the mycelia were cultured for 60 h and dissolved in a 0.1% lysozyme solution (0.7 M NaCl) for 3 h. Subsequently, a 10 6 mL −1 protoplast solution transformed using 5 μg of plasmid DNA exhibited the highest transformation efficiency. Laser confocal microscopy showed that GFP and mCherry fluorescent signals were successfully expressed in the mycelia and spores of HX‐5‐1, indicating that they were stably inherited in the transformants and did not affect colony morphology, growth rate, or pathogenicity. In conclusion, we successfully established a genetic transformation system for C. sublineola and employed it to obtain stable fluorescent protein expression. This study may serve as a foundation for understanding the infection process of C. sublineola .