Mycelium

Protoplastation were performed as described by Nielsen et al 2006 [31 (link)]. For transformation using either pyrG or argB as genetic marker, 10⁷ protoplasts and ~3 μg of digested DNA and 150 μl PCT solution were incubated for 10 min at room temperature, followed by adding of 250 μl ATB plating on 1 M sucrose based TM with selection. All TM plates were incubated at 30°C, except for A. nidulans transformation at 37°C. For transformation utilizing hygromycin selection, 10⁷ protoplasts and ~3 μg of digested DNA were incubated at 15 min on ice, then 1 mL PCT was added and the mix incubated for 15 min at room temperature. 100 μg/ml hygromycin B (Invivogene, USA) was added to 15 ml molten 1 M sorbitol based TM (TMsh; ~45°C), and immediately poured into an empty 9 cm petri dish. After 24 h incubation at 30°C, an overlay of 15 ml TMsh was added. All candidate transformants were streak purified prior to verification.
All strains were verified by tissue-PCR analysis using mycelium as the source of DNA. For the specific PCR protocol, see S2 Protocol, and for primers refer to S1 Table. Primers for gene-deletion analysis were designed to bind up- and downstream outside the region eliminated by the gene-targeting substrate. This setup detects wild-type sequences present in transformants that were for false positive and heterokaryons as well as for gene-deletion strains derived after marker elimination by direct repeat recombination. Transformants where a deleted gene was replaced by a marker gene was validated in two PCR reactions using a primer pair where one primer binds outside the upstream targeting region and the other binds inside the marker gene; and a pair where one of the primes bind inside the marker gene and the other binds outside of the downstream targeting region. For the experimental design and details of protocol, see S2 Protocol. Wild-type gDNA, as well as wild-type mycelium, was always included as controls to benchmark tissue-PCR efficiency for wild-type reaction success. All primers are listed in S1 Table.

The mycelial mass was
quantified using the standard curve method as described by Alias et
al.^{16 (link)} Briefly, 5 g of homogenized fermented
substrate was collected at 0, 3, 6, 13, 20, 27, and 34 days after
inoculation. Samples were ground to fine powder, and the genomic DNA
was extracted from each sample. After spectrophotometric quantification,
the DNA was submitted to real-time PCR amplification with the TCal
primer pair.^{20 (link)} The experiment was performed
in duplicate.

Immature C. sinensis specimens were purchased from local markets from the Hualong (located at 36°13’N, 102°19’E) and Yushu areas (located at 33°01’N, 96°48’E) of Qinghai Province of China (3800–4600 m’ altitude) in mid-May and characterized by a plump caterpillar body and very short stroma (1.0–2.0 cm) [19 , 27 ]. Mature C. sinensis specimens were collected in mid-June and characterized by a plump caterpillar body and long stroma (>5.0 cm) and by the formation of an expanded fertile portion close to the stromal tip, which was densely covered with ascocarps (Fig 1). Governmental permission was not required for C. sinensis purchases in local markets, and the collections of C. sinensis specimens from sales by local farmers fall under the governmental regulations for traditional Chinese herbal products.
The specimens were washed thoroughly on site in running water with gentle brushing, soaked in 0.1% mercuric chloride for 10 min for surface sterilization and washed 3 times with sterile water. The thoroughly cleaned specimens were immediately frozen in liquid nitrogen on site and kept frozen during transportation to the laboratory and during storage prior to further processing [19 , 27 ].
Some of the mature C. sinensis specimens were harvested along with the outer mycelial cortices and soil surrounding the caterpillar body and replanted in paper cups in soil obtained from C. sinensis production areas (Fig 1A) and were cultivated in our laboratory (altitude 2,200 m) in Xining City, Qinghai Province of China [67 , 68 ]. Because of the phototropism of natural C. sinensis, we kept the windows fully open, allowing sufficient sunshine and a natural plateau breeze blowing over the cultivated specimens in the paper cups. The room temperature was maintained naturally, fluctuating with the lowest temperature at 18–19°C during the night and the highest temperature at 22–23°C in the early afternoon. The humidity of our laboratory was maintained by spraying of water using an atomizer twice a day in the morning and evening.
Fully ejected ascospores of C. sinensis were collected using double layers of autoclaved weighing paper (Fig 1B). During massive ascospore ejection, numerous ascospores adhered to the outer surface of asci, as shown in Fig 1C after removing the upper layer of autoclaved weighing papers for collection of the fully ejected ascospores, and failed to be brushed away using an autoclaved brush; hence, these ascospores were instead gently scratched off using a disinfected inoculation shovel or ring and referred to as semiejected ascospores.
The 2 types of ascospores were cleaned by 2 washes with 10% and 20% sucrose solutions and 10-min centrifugation at 1,000 rpm (desktop centrifuge, Eppendorf, Germany); the supernatant was discarded after each centrifugation. The pellets (ascospores) were subsequently washed with 50% sucrose solution and centrifuged for 30 min, and the ascospores that floated on the liquid were collected [67 ]. The fully and semiejected ascospores were stored in a -80°C freezer prior to further processing.