Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Highlights

• An efficient Agrobacterium-mediated transformation for Sclerotium rolfsii.

• Three reporter genes were efficiently expressed in S. rolfsii.

• The SrGPD promoter without an intron is sufficient to drive transgene expression.

Abstract

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.

Introduction

Sclerotium rolfsii is a soil-borne pathogen that causes disease on hundreds of plants (Punja, 1988; Keinath and DuBose, 2017; Sun et al., 2020). It has a high growth rate and strong secretory system, which are important for its pathogenicity. Besides, the basidiomycete S. rolfsii is also used as a microbial biotechnology platform because it produces some bioactive substances with high value (Ludwig and Haltrich, 2003; Survase et al., 2006; Tan et al., 2019; Zhang et al., 2019). For example, S. rolfsii produces a lectin which strongly binds to breast cancer MDA-MB-468 cells and induces growth inhibitory and antiadhesive effect with fibronectin and collagen, suggesting a potential to be developed as an anticancer drug (Kellens et al., 1989; Hegde et al., 2018). It also produces kinds of exopolysaccharides (EPS) which could be used as zinc supplementation with high antioxidant activities (Dong et al., 2018), or be employed as a promising biosorption for industrial wastewater treatment (Li et al., 2016). More importantly, S. rolfsii produces an industrially exploited EPS scleroglucan, which was used as a multi-purpose compound in many fields, including oil recovery, food industry, cosmetics, and medical applications (Schmid et al., 2011; Viñarta et al., 2013).

However, there is no available genetic transformation method and molecular tools for S. rolfsii, resulting in the stagnation of molecular biological research of this important basidiomycete fungus, including the plant pathogenesis and the key genes and gene regulations of biosynthesis pathway of important metabolic products.

Agrobacterium tumefaciens-mediated transformation (ATMT) is a famous method to transform many types of species, especially plant and fungal cells (Frandsen, 2011). De Groot et al. (1998) applied this method to transform several ascomycete species and a single basidiomycete. Thereafter, more basidiomycete strains were transformed successfully using ATMT, such as Phanerochaete chrysosporium (Sharma et al., 2006), Pleurotus ostreatu (Ding et al., 2011) and Flammulina velutipes (Okamoto et al., 2014).

There are some great advantages of ATMT. Firstly, nearly any fungal cell types could be used as starting material with high efficiencies, such as spores, fruiting bodies, and vegetative mycelia (Michielse et al., 2008; Ding et al., 2011). Protoplast preparation and regeneration, the major difficulties in the PEG-mediated protoplast method, are not required in ATMT. This advantage is of great significance to filamentous basidiomycetes since the preparations of spores, fruiting bodies, and protoplasts are either time-consuming or difficult. Secondly, ATMT typically results in single T-DNA integrations in short co-cultivation time (De Groot et al., 1998; Michielse et al., 2005). Besides, ATMT allows the transfer of large (up to 75 kb) DNA fragments (Takken et al., 2004) and the simultaneous introduction of multiple different T-DNA regions in one transformation experiment (Wang et al., 2010). These advantages are also helpful to further functional genomics study and metabolic engineering of basidiomycetes.

Reporter genes, including fluorescent proteins and catalytic enzymes, are extensively used in gene function and regulation researches. The red fluorescent protein reporter DsRed-Max was a brighter and pure RFP, forming homodimers in the cells (Strack et al., 2008). Another RFP reporter tdTomato was a monomeric RFP with the highest brightness among all known fluorescent proteins (Shaner et al., 2004). One famous catalytic enzyme reporter is GUS, the β-glucuronidase gene of Escherichia coli, is extensively applied in gene activity, gene regulation, and histochemical analyses of plants (Jefferson, 1989; Beltran-Aguilar et al., 2019). GUSPlus is a codon-optimized Staphylococcus gusA gene with a catalase intron to ensure expression in plants but not bacteria, detectable at ten-fold higher sensitivity to E. coli GUS (Broothaerts et al.2005).

In this study, we report the development of an efficient and convenient Agrobacterium-mediated transformation system for the filamentous fungus S. rolfsii, including the transformation process and basic transgene plasmids. With this ATMT method, we successfully transformed the homogenized S. rolfsii mycelial cells with three kinds of TDNA transgene expression cassettes, including three reporter genes (DsRed, tdTomato, and GUS) and the basta resistance gene bar. These reporters were efficiently expressed in the transformed fungal cells and could be detected easily and robustly. This system provides the basic transformation method and genetic tools for the functional genomics research and metabolic engineering of S. rolfsii.