Technology and optimization of preparation and regeneration of protoplasts from rice sheath blight fungus Rhizoctonia solani

Rice sheath blight caused by Rhizoctonia solani Kühn is one of the most important diseases on cultivated rice worldwide. Unlike most other fungal pathogens, R. solani forms heterokaryotic vegetative mycelia with multiple nuclei per hyphal cell and is unable to produce haploid asexual spores under normal conditions. These characteristics of R. solani may make it difficult to perform genetic transformation and functional analysis of genes. Production of large amount of protoplasts from this fungus is a prerequisite for the studies of molecular genetics, such as protoplast fusion and fungal transformation. Previously, some lytic enzymes and conditions for releasing R. solani protoplasts have been tested and optimized and several protocols for the preparation and regeneration of protoplasts from R. solani mycelium have been developed by some researchers. However, the efficiency of R. solani protoplasts releasing by these protocols is sometimes unstable due to different strains of R. solani or experimental conditions. Therefore, it is necessary to develop an efficient method for preparing protoplasts of rice sheath blight fungus.The objectives of the present study were to evaluate various cell wall degradation enzymes and their combinations for releasing protoplasts from R. solani mycelium, and to develop an efficient protocol for yielding protoplasts.Using 0.7 mol/L NaCl as stabilizer solution, seven different cell wall degradation enzymes, including Glucanex, lywallzyme, cellulase-R-10, macerozyme-R-10, snailase, driselase and lysing enzyme, and their combinations were evaluated for releasing protoplasts from R. solani GD-118 mycelium which was harvested from potato dextrose liquid medium cultured at 28℃ for 36 h. The number of released protoplasts was counted by using haemocytometer under microscopy. The optimal concentration of lytic enzymes for the generation of protoplasts was determinated and the conditions to obtain and regenerate protoplasts of the fungus were also optimized.Among the seven tested lytic enzymes, Glucanex was the most suitable enzyme for the digestion of R. solani GD-118 cell wall. The protoplast yield in the treatment with 20 mg/mL Glucanex for 4 h was 23.7×10⁴ cell/mL (e. g. 118.5×10⁴ cells per gram mycelium). Moreover, the results showed that the optimal mixture enzymes of 15 mg/mL Glucanex and 10 mg/mL lywallzyme were effective in releasing protoplasts with the production of 3.09×10⁷ protoplasts from per gram R. solani GD-118 mycelium, and the obtained 58% protoplasts could be regenerated. In addition, the combination had similar effects on digesting cell wall of different strains of rice sheath blight fungus.Taken together, the mixture lytic enzymes of 15 mg/mL Glucanex and 10 mg/mL lywallzyme can effectively digest the cell wall of rice sheath blight fungus and produce abundant protoplasts.