Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

Abstract

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Genetic Transformation of the Filamentous Fungus Pseudogymnoascus verrucosus of Antarctic Origin

“…We observed that the incubation of mycelia with lysing enzymes yielded 3.82 × 10 8 ± 0.86 × 10 8 protoplasts after 3 h, enough to perform around 20 transformation experiments. This value was estimated considering 1 × 10 7 protoplasts for each transformation experiment, amount optimal in most protocols of fungal PEG-mediated transformation (Liu and Friesen, 2012; Wang et al, 2018).…”

“…For PEG-mediated transformation of fungi, researchers usually use DNA amounts ranging from 2 to 12 μg (Fincham, 1989; Wang et al, 2018). In our case, we tested three DNA amounts within this range (Table 1).…”

A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone

“…The recombined silencing plasmid was designated as pSilent‐GtCytc 1 , purified, and transferred into the wild‐type strain using the PEG‐mediated transformation method according to Wang et al . (2018). The hairpin RNA silencing plasmids of GtCytb (387 bp) and GgIsp (389 bp) were constructed with the same strategy and verified by PCR with the primer pair PScheck 1F/R (Figure ).…”

“…The same fragment of GtCytc 1 was amplified with the primer pair FGtCytc 1 -F/R, purified, and inserted into the KpnI/SphI site of pSilent-GtCytc 1 -up ( Figure S2b). The recombined silencing plasmid was designated as pSilent-GtCytc 1 , purified, and transferred into the wild-type strain using the PEG-mediated transformation method according to Wang et al (2018). The hairpin RNA silencing plasmids of GtCytb 387 bpand GgIsp (389 bp) were constructed with the same strategy and verified by PCR with the primer pair PScheck 1F/R ( Figure S3).…”

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

“…The currently used transient transformation method is polyethylene glycol (PEG)-mediated transient expression of protoplasts [11]. Effective mesophyll protoplast isolation, purification, and transient transformation systems have been established in many plant species, such as wheat [12], Arabidopsis thaliana [11], and Chinese kale [13]. Studies on plant hypocotyl protoplasts have focused on isolation, purification, and regeneration [14,15,16,17].…”