Screening of optimum conditions for isolation and regeneration of protoplasts
Digestion enzyme is a crutial factor controlling the efficiency of protoplast transformation. Three cell wall digestion enzymes (glucanase, lysing enzyme, and snailase) were tested alone or combined for releasing protoplasts from F. oryzae. The results showed that glucanase had a higher yield of protoplasts than the other two enzymes alone (Fig. 1A). Furthermore, the combination of enzymes was more efficient than a single enzyme. The combination of glucanase, lysing enzyme and snailase yielded the highest protoplasts, followed by the combination of glucanase and snailase (Fig. 1A). However, considering the operability and cost, the use of glucanase alone can produce sufficient protoplasts for transformation.
The number of fungal protoplasts increased along with glucanase concentration and reached a maximum at 10 mg mL− 1 (Fig. 1B). The volume of enzyme solution used for digestion also had a great influence on protoplast production. It was found that the protoplast yield increased along with the volume of enzyme digestion solution, reaching the highest yield at 40 mL of glucanase at 10 mg mL− 1 (Fig. 1C).
Along with the increase of incubating time, the number of protoplasts released gradually increased. When incubated for 3 hours, the release of protoplasts reached a peak at 12.6×107 g− 1 (Fig. 1D). Prolonging incubation time did not increase the yield of protoplasts significantly. Prolonged incubation time damaged the plasma membrane and affected the quality of protoplasts (Liu et al. 2010). The yield of protoplasts released from F. oryzae showed no apparent differences when the digestion reactions were incubated at a temperature between 30 ˚C and 32 ˚C. Fewer protoplasts were produced when incubating at temperatures lower than 30 ˚C and higher than 32˚C (Fig. 1E).
The fungal age also affected the release of protoplasts. The cell walls of mycelium is thickened with age, and protoplasts are challenging to be released. In turn, it is easily destroyed, and more protoplasts are released. However, the hyphae in the logarithmic growth stage have stable metabolic activity and strong adaptability. In this work, 4-day-old hyphae of F. oryzae were broken into fragments and re-cultured in CM liquid medium to ensure that the hyphae were young and in logarithmic growth phages. The protoplast yield increased continuously in 1–3 days, keeping stable on the 4th day. The second day is the best appropriate with a high yield (Fig. 1F).
Additionally, the osmotic pressure stabilizers play crucial roles in protoplast isolation and regeneration (Liu et al. 2010). Four different osmotic pressure stabilizers (NaCl, KCl, sucrose, and sorbitol) were tested. The results showed that protoplast yield reached the highest when 0.9 M KCl was used as an osmotic pressure stabilizer (Table 1). Furthermore, the osmotic pressure stabilizer in the regeneration medium plays a fundamental role in the re-growth of protoplasts (Ma et al. 2014). The results also showed that the protoplast regeneration rate reached highest when RgA medium containing 1 M sucrose.
Table 1
Effect of osmotic pressure stabilizers for protoplast formation and regeneration
| Digestion solution | RgA medium |
Osmotic stabilizers | Concentration (mol L− 1) | Protoplast yield (107 g− 1) | Regeneration cells (106 g− 1) | Protoplast regeneration rate (%) | Concentration (mol L− 1) | Protoplast regeneration rate (%) |
KCl | 0.8 | 10.77 ± 0.55b | 6.37 ± 0.42c | 5.91 ± 0.33 | 0.8 | 19.03 ± 0.35f |
0.9 | 12.47 ± 0.75a | 21.80 ± 1.32a | 17.48 ± 0.66 | 1 | 26.93 ± 1.46d |
1 | 9.0 ± 0.26d | 4.73 ± 0.25d | 5.25 ± 0.92 | 1.2 | 16.77 ± 1.10fg |
NaCl | 0.8 | 1.6 ± 0.26fgh | 0.37 ± 0.06f | 2.29 ± 0.16 | 0.8 | 6.43 ± 0.75i |
0.9 | 2.13 ± 0.40f | 0.83 ± 0.06ef | 3.9 ± 0.62 | 1 | 6.73 ± 1.00i |
1 | 1.67 ± 0.32fg | 0.87 ± 0.06ef | 5.2 ± 0.34 | 1.2 | 3.53 ± 0.67j |
Sucrose | 0.4 | 1.97 ± 0.29f | 0.93 ± 0.15ef | 4.74 ± 0.57 | 0.8 | 33.63 ± 2.58c |
0.5 | 9.80 ± 0.89c | 11.03 ± 0.47b | 11.25 ± 0.85 | 1 | 42.2 ± 2.67a |
0.6 | 3.67 ± 0.32e | 5.80 ± 0.2c | 15.81 ± 0.81 | 1.2 | 37.77 ± 1.29b |
Sorbitol | 0.4 | 0.83 ± 0.06h | 0.57 ± 0.06ef | 6.8 ± 0.45 | 0.8 | 15.77 ± 1.56gh |
0.5 | 1.13 ± 0.15gh | 1.23 ± 0.15e | 10.88 ± 0.23 | 1 | 21.93 ± 1.46e |
0.6 | 0.93 ± 0.12gh | 0.53 ± 0.06ef | 5.71 ± 0.42 | 1.2 | 13.3 ± 1.95h |
The bold fonts indicate the best concentration of osmotic pressure stabilizers for protoplasts formation and regeneration, which showed significant differences from the others. Significant differences (One-way anova): lowercase letters means P < 0.05. |
Antibiotic resistance assay and transformation of GFP-vector in F. oryzae
To insert exogenous DNA fragment into F. oryzae, sulfonylurea resistance genes were tested as a selection marker. The minimal inhibitory concentration was determined as the lowest concentration at which no visible hyphal growth was observed. Our analysis showed that when the sulfonylurea concentration reached 300 µg ml− 1, colony growth was completely inhibited (Fig. 2A, B), suggesting that the minimum inhibitory concentration (MIC) value for F. oryzae was 300 µg ml− 1. We thus used this antibiotic concentration for the selection of F. oryzae transformants generated from the protoplast transformation.
Fluorescence expression vector pKD6-GFP was transfected into the protoplasts of F. oryzae. First, the GFP-tagged transformants were grown on DCM medium supplemented with 1M sucrose and 300 µg mL− 1 sulfonylurea for 5–7 days. Then the regenerated transformants were re-cultured on a DCM medium containing 300 µg mL− 1 of sulfonylurea for two generations. Fluorescence observation showed that GFP was strongly expressed in hyphae and conidia from three generations of transformants (Fig. 3). The results also suggested that exogenous promoter SOD1 promoter also has a strong ability to start the gene expression in F. oryzae.
The colonization of GFP-expressing F. oryzae in rice roots
To further clarify the intensity and stability of fluorescence expression, we inoculated the GFP-labeled F. oryzae strain on rice roots. It was found that F. oryzae successfully infected the rice roots, gradually spread from the epidermis to the cortex, and finally reached the endodermis (Fig. 4). And, in the co-culture process of rice and F. oryzae, the fluorescence expression in mycelium was stable and coherent. Furthermore, the intensity of fluorescence expression was vigorous, which facilitated the observation of mycelia in root tissue (Fig. 4).