As complex multicellular fungi, mushroom-forming fungi (Agaricomycetes) have evolved
numerous cell types. However, how many cell types we can estimate in fungi is still
elusive. Single-cell RNA sequencing (scRNA-seq) is a cutting-edge technology which
has been widely used in animals and plants for exploring cell type diversity at the
finest level, yet to our knowledge has not been applied to complex multicellular fungi.
Here we present an optimized protocol of scRNA-seq in the model mushroom-forming fungus
Coprinopsis cinerea, and demon- strate a diversity of specific cell types in preliminary
experiments. We optimized the enzyme cocktail, homogenization and purification during
the protoplastation of fruiting bodies. Our protocol results in >10⁶ protoplasts/ml
with >80% cell viability in the fruiting body samples after 1.5h digestion, which
has rarely been attempted before. The Pearson-correlation analysis of gene expression
between protoplasted and un-protoplasted samples showed a high correlation (r=0.87),
with most of the differential expressed genes related to the environmental response,
suggesting that protoplastation induced moderate, and controllable gene expression
changes to the cells. Using the 10x Genomics technology, we profiled >7,000 cells
from the mycelium sample and >3,000 cells from fruiting body samples. We clustered
the cells based on their expression profile and identified several distinct clusters.
Compared to bulk RNA-seq data that reveals the cell identify for several of these,
including a distinct subcluster corresponding to asexual spores (oidia) of C. cinerea
was identified in the mycelium sample. Some cluster-specific genes in fruiting body
samples had a high correlation with the tissue-specific expression dataset. Taken
together, our study demonstrates a feasible protocol of scRNA-seq in C. cinerea and
provides a first-generation of cell type landscape of complex multicellular fungi
at single-cell resolution.
