Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation

Abstract

Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7days at 19°C and 24°C, respectively. Transformation efficiency was 0.75-1.5% for M. furfur and 0.6-7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.

Functional genomics of lipid metabolism in the oleaginous yeastRhodosporidium toruloides

“…Most low-lipid strains appeared morphologically similar to wild type with smaller lipid bodies (Figure 7 – figure supplement 1). However, a BSCL2 -likeΔ (seipin) mutant showed an even larger variation in droplet size than wild type, consistent with observations in S. cerevisiae mutants for the homolog SEI1/FLD1 (107-110) and likely reflective of a conserved function in lipid droplet formation and efficient delivery of lipid biosynthetic proteins to the growing lipid droplet (111, 112). Autophagy mutants ( ATG2 Δ) had the most uniformly small lipid droplets in elongated cells with enlarged vacuoles.…”

Identification of Malassezia furfur Secreted Aspartyl Protease 1 (MfSAP1) and Its Role in Extracellular Matrix Degradation

“…As M. furfur is perhaps the most easily cultivated Malassezia species, it is frequently isolated from human skin in culture studies even though it is much less prevalent than M. globosa and M. restricta (Wu et al, 2015;Leong et al, 2019). Importantly, M. furfur CBS 14141 is one of only three strains of Malassezia that are currently amenable to genetic manipulation (Ianiri et al, 2016;Celis et al, 2017) and understanding the functions of secretory proteases in this strain opens up future opportunities for genetic approaches for further analysis.…”

Expression of aMalasseziacodon optimized mCherry fluorescent protein in a bicistronic vector

“…Transformations were carried out as previously described (Ianiri et al 2016;Celis et al 2017), and 113 randomly selected NAT-resistant colonies were analyzed by PCR to detect the presence of NAT and 114 mCherry ORFs. Predicted amplicons of 576 bp and 708 bp for NAT and mCherry respectively, were 115 observed in 4 out of 23 transformants but absent in wild type (Figure 2A).…”