Bao-Zhu Dong & Li-Yun Guo 

BMC Biotechnology volume 20, Article number: 14 (2020) 

Abstract

Background

Botryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea.

Results

We established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 10⁶ protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 μg per 10⁶ protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red.

Conclusion

The established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.

See https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-020-00608-z

Figure 2:  Verification of Bdo_05381-disrupted B. dothidea HTLW03 transformants by PCR. Transformants were analyzed with four PCR amplifications. The lanes marked with “+” correspond to the correct GD transformants with Bdo_05381 upstream and downstream fragments that were the correct size, an amplicon that was 400 bp longer than that of Bdo_05381, and no open reading frame fragment