Transformation of the nematode-trapping fungusArthrobotrys oligospora

Abstract

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.

Random mutagenesis analysis and identification of a novel C2H2-type transcription factor from the nematode-trapping fungus Arthrobotrys oligospora

“…S1), recognized by enzymes XbaI, KpnI, SmaI, XmaI, SacI, SalI and HindIII. The protoplasts of A. oligospora were prepared according to methods described previously 24, 27 , and 1 × 10 7 −10 8 protoplasts/mL was used for transformation. The sensitivity of A. oligospora against hygromycin B (Hyg, 200 µg/mL) was used to screen the transformants.…”

“…The pMD-hph was digested using restriction enzyme XbaI or SmaI for further use. Protoplasts of A. oligospora were prepared and transformed according to methods described previously 24, 27 . Transformants (mutants) were selected initially on PDASS containing 200 µg/mL Hyg and confirmed further by PCR analysis using primers Hph-1F and Hph-1R (Supplementary Table S2), and the genetic stability of transformants was detected according to methods described previously 24, 27 .…”

“…REMI technique was initially established in Saccharomyces cerevisiae 23 and has been widely used in diverse fungi. In 1999, the hygromycin-B phosphotransferase gene ( hph ) was transformed to A. oligospora using REMI method, and 7 out of 13 transformants were single copy integrations, whereas the remaining were multiple and more complex integrations 24 . Subsequently, the encoding gene of serine protease PII 6 was transformed into A. oligospora by REMI method, and mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes than the wild type strain 9 .…”

The Autophagy-Related Gene Aolatg4 Regulates Hyphal Growth, Sporulation, Autophagosome Formation, and Pathogenicity in Arthrobotrys oligospora

“…Deletion of the Aolatg4 gene was performed using homologous recombination, as described previously (Tunlid et al, 1999;Chen et al, 2013;Zhen et al, 2019). The upstream (2,150 bp) and downstream (2,098 bp) sequences corresponding to the Aolatg4 ORF (5' and 3' flanking regions) in A. oligospora were amplified using paired primers ( Supplementary Table S1).…”

“…Finally, three PCR fragments and a linearized pRS426 vector were co-transformed into yeast strain FY834 via electroporation (Bernhards and Pöggeler, 2011;Park et al, 2011). The complete fragment for gene disruption was amplified from the recombinant plasmid pRS426-ATG4-hph using primers AolAtg4-5f and AolAtg4-3r ( Supplementary Table S1), then it was transformed into A. oligospora using protoplast transformation method (Tunlid et al, 1999;Liu et al, 2020). Hygromycin-resistant transformants were selected on PDAS medium containing 200 mg/ml hygromycin B (Amresco, Solon, United States; Zhen et al, 2019;Liu et al, 2020).…”