Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus.

Rundong Liu,Jaycee Augusto Paguirigan,Jae-Seoun Hur,Min-Hye Jeong,Wonyong Kim

doi:10.3390/jof7040252

Abstract

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

Highlights

Lichens can be defined as intimate symbioses between fungi and phototrophic partners, such as unicellular green algae and/or cyanobacteria, as the main symbionts
There have been a very few studies focusing on the ecological roles of these lichen secondary metabolites; some studies provide evidence that lichen secondary metabolites have contributed to adapting to harsher habitats [21] and deterring herbivores [22,23]
The sensitivity of the C. macilenta mycobiont against hygromycin was evaluated to determine a minimal concentration of the antibiotics for the selection of transformants having T-DNA insertion

Summary

Introduction

Lichens can be defined as intimate symbioses between fungi and phototrophic partners, such as unicellular green algae and/or cyanobacteria, as the main symbionts. Of all known fungi are thought to have been lichenized and nearly 98% of them belong to the phylum Ascomycota [5,6,7]. As much as their species diversity, lichens produce a variety of secondary metabolites [8] that exhibit antitumor [9,10,11], antimicrobial [12,13,14,15,16,17], anti-inflammatory [18,19,20], and antioxidant activities [13,14,17,19], most of these bioactive compounds are considered to be originated from mycobionts. There have been a very few studies focusing on the ecological roles of these lichen secondary metabolites; some studies provide evidence that lichen secondary metabolites have contributed to adapting to harsher habitats [21] and deterring herbivores [22,23]

Objectives

Methods

Results

Conclusion