After performing de novo transcript assembly of >1 billion RNA-Sequencing reads obtained
from 22 samples of different Norway spruce (Picea abies) tissues that were not surface sterilized,
we found that assembled sequences captured a mix of plant, lichen, and fungal transcripts.
The latter were likely expressed by endophytic and epiphytic symbionts, indicating
that these organisms were present, alive, and metabolically active. Here, we show that these
serendipitously sequenced transcripts need not be considered merely as contamination, as is
common, but that they provide insight into the plant’s phyllosphere. Notably, we could classify
these transcripts as originating predominantly fromDothideomycetes and Leotiomycetes species,
with functional annotation of gene families indicating active growth and metabolism, with
particular regards to glucose intake and processing, as well as gene regulation.
S1 Fig. Samples collected from Norway spruce. For each sample a brief description and sample
ID are shown below a representative image of the associated plant tissue, while the sampling
date is shown above.
S2 Fig. Bioinformatics workflow of RNA data processing. We assembled reads from all samples
into a single assembly (left column), computed Tau scores, GC content, and mapped the
transcripts to the genome as well as to the Uniref90 protein database. For enriching for fungal
transcripts (right column), we applied GC content and expression breadth filters to the reads
and assembly respectively, clustered sequences by similarity, and performed functional annotation
as well as phylogenetic analyses.
S3 Fig. Putative taxonomic characterization of transcripts via protein alignments. Bar plot
showing the number of transcripts by taxonomy (super)kingdoms. Parent summarises taxons
hierarchically higher than the represented (super)kingdoms, NA summarises transcripts with
no sequence similarity in the UniRef90 database. The number of transcripts is indicated at the
top of every bar.
S4 Fig. Taxonomic class and phylum of the fungal transcripts. (a) Number of transcripts per
fungal phylum. The phylum are sorted by abundance top to bottom with Ascomycota
(n = 81,181) and Basidiomycota (n = 4,839) being the most represented; the remaining phyla
varying from n = 11 to n = 2. (b) A graph of the taxonomic hierarchy from species to phylum
of the fungal transcripts, showing the broad species diversity of the largest clusters: Ascomycota
(bottom) and Basidiomycota (top). (c) Similar to (a) for the fungal classes, with the Eurotiomycetes
and Dothideomycetes classes being over-represented among the fungal transcripts. (d)
Similar to (b) for the fungal classes (n = 24).
S5 Fig. Characterisation of transcripts lacking taxonomic assignment by their GMAP alignments
to the P. abies genome. (a) Boxplot of the tau scores for the no taxon transcripts split
based on their GMAP alignments to the P. abies genome. The tau score ranges from 1 for complete
specificity to 0 for equal expression in all samples. The transcripts having a GMAP alignment
in the genome (99% of the GMAP hits cover 80% of the transcripts with at least a 90%
identity) show a wide tau score distribution indicative of the presence of ubiquitously expressed
transcripts as well as that of more tissue-specific transcripts. The transcripts having no GMAP
alignment show a distribution typical of only tissue-specific expression (mean tau score of 0.98). (b) Percentage GC density distribution of the no taxon transcripts split based on their
GMAP alignments to the P. abies genome. Transcripts having a GMAP alignment to the
genome present a GC distribution typical of the P. abies transcripts. The transcripts without a
GMAP alignment show a distribution enriched for higher percentage GC, similar to that of
fungi. The shoulder observed under the peak of transcripts with GMAP alignments may indicate
transcripts where the assembly contained gaps or created chimeras. (c) Scatterplot of log2
FPKM expression values vs. the percentage GC content for the transcripts with a GMAP alignment.
Colouring indicates density, which is shaded from yellow (high) to blue (low). The
expression of transcripts with a GMAP alignment resembles that of the Embryophita phylum.
(d) Scatterplot of log2 FPKM expression values vs. the percentage GC content for transcripts
with a GMAP alignment. Colouring as in (c). The expression of transcripts with no GMAP
alignment resembled that of the fungal kingdom.
S6 Fig. Phylogeny built on four nuclear genes. Shown are maximum-likelihood phylogenies
based on fungal nucleotide sequences assembled from the spruce samples in context of known
sequences, with highest sequence similarity to: (a) phosphoenolpyruvate carboxykinase; (b)
NADP-dependent medium chain alcohol dehydrogenase; (c) beta lactamase; and (d) unspecific
lipid transporter. Only branch with support values > 0.9 are shown. While clusters with more
representative sequences yield better branch support (a, b), placement of clusters with fewer
sequences is less certain (c, d). However, in all cases, at least one sequence is grouped with
Dothideomycetes, and for (a,b) with Leotiomycetes.
S1 Table. Sample IDs, description, and ENA submission IDs. Correspondence between the
sample IDs as described in Nystedt et al., (2013), this manuscript and the ENA are shown in
columns one to three. The fourth column contains a succinct description of the samples, refer
to Nystedt et al., (2013) for full details.