DOI: 10.5061/dryad.n8pk0p301
title: Data from: Symbiont-specific responses to environmental cues in a threesome lichen symbiosis

description: Photosymbiodemes are a special case of lichen symbiosis where one
 lichenized fungus engages in symbiosis with two different photosynthetic
 partners, a cyanobacterium and a green alga, to develop two distinctly
 looking photomorphs. We investigated differential gene expression in
 photosymbiodemes of the lichen Peltigera britannica at different
 temperatures representing mild and putatively stressful conditions and
 compared gene expression of thallus sectors containing cyanobacterial
 photobionts with thallus sectors with both green algal and cyanobacterial
 photobionts. Firstly, because of known ecological differences between
 photomorphs, we investigated symbiont-specific responses in gene
 expression to temperature increases. Secondly, we quantified
 photobiont-mediated differences in fungal gene expression. High
 temperatures expectedly led to an upregulation of genes involved in heat
 shock responses in all organisms in whole transcriptome data. As expected,
 the expression of genes involved in photosynthesis was increased in both
 photobiont types at 15 and 25°C. The green algae exhibited thermal stress
 responses mainly at 25°C, and the fungus and the cyanobacteria already by
 15°C, demonstrating symbiont-specific responses to environmental cues and
 symbiont-specific ecological optima. Furthermore, photobiont-mediated
 differences in fungal gene expression could be identified, with
 upregulation of distinct biological processes in the different morphs,
 showing that interaction with specific symbiosis partners profoundly
 impacts fungal gene expression.
description: RNA was isolated with the
 innuPREP Plant RNA Kit from Analytik Jena. RNA libraries were constructed
 with dual indexing using the TruSeq® Stranded mRNA Library Prep kit
 (Illumina) which included a poly-A selection step. Libraries were
 sequenced on a MiSeq as well as a HiSeq 3000/4000 SR platform (both
 Illumina). The processed paired-end MiSeq data was used
 for <em>de</em> <em>novo</em> transcriptome
 assembly with Trinity software version 2.4.0 (Haas et al., 2013). The
 HiSeq data was pseudoaligned to the <em>de</em>
 <em>novo</em> transcriptome with the RNA-seq quantification
 program kallisto version 0.45.0 (Bray et al., 2016). Differential gene
 expression analysis was conducted with the program DESeq2 version 1.22.2
 (Love et al, 2014). All genes were taxonomically assigned with MEGAN6
 version 6.13.1 (Huson et al., 2007); only ascomycete, chlorophytes and
 cyanobacterial genes were retained for differential gene expression
 analysis. Functional annotation of differentially expressed genes was
 conducted using UniProt BLAST (The UniProt Consortium, 2021). The top-200
 fungal differentially expressed transcripts were also blasted (blastx
 version 2.7.1+, translated nucleotide to protein) (Sayers et al., 2020)
 against our own database consisting of filtered metagenomic sequences of
 <em>Peltigera</em> <em>britannica</em>,
 <em>P</em>. <em>leucophlebia</em> and
 <em>P</em>. <em>collina</em> (unpublished data of
 the authors) using standalone BLAST for Linux Ubuntu (ncbi-blast+
 package). <strong>References</strong>
 Bray, N. L.,
 Pimentel, H., Melsted, P., & Pachter, L. (2016). Near-optimal
 probabilistic RNA-seq quantification. Nature Biotechnology, 34(5),
 525-527. doi:10.1038/nbt.3519 Haas, B. J., Papanicolaou, A., Yassour, M.,
 Grabherr, M., Blood, P. D., Bowden, J., . . . Regev, A. (2013). De novo
 transcript sequence reconstruction from RNA-seq using the Trinity platform
 for reference generation and analysis. Nature Protocols, 8(8), 1494-1512.
 doi:10.1038/nprot.2013.084 Huson, D. H.,
 Auch, A. F., Qi, J., & Schuster, S. C. (2007). MEGAN analysis of metagenomic data. Genome
 Research, 17(3), 377-386. doi:10.1101/gr.5969107 
 Love, M., Huber, W.,
 & Anders, S. (2014). Moderated estimation of fold change and
 dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12), 550.
 doi:10.1186/s13059-014-0550-8 Sayers, E. W., Beck, J., Brister, J.R., Bolton,
 E. E., Canese, K., Comeau, D.C., ... Ostell, J. (2020). Database resources
 of the National Center for Biotechnology Information. Nucleic Acids
 Research, 48(D1), D9-D16. doi:10.1093/nar/gkz899
 The UniProt Consortium.
 (2021). UniProt: the universal protein knowledgebase in 2021. Nucleic
 Acids Research, 49(D1), D480-D489.
 doi:10.1093/nar/gkaa1100
description: The 'TrinityAscomycetePhotomorphGenes.fasta', 'TrinityAscomyceteTemperatureGenes.fasta','TrinityChlorophytesGenes.fasta' and 'TrinityCyanobacteriaGenes.fasta' files contain the transcripts obtained from the <em>de</em> <em>novo</em> transcriptome assembly, comprising only the longest isoforms per 'gene'. The 'TrinityChlorophytesGenes.fasta' file contains only the first 200 chlorophytes transcripts that were identified as significantly differentially expressed with DESeq2. The same is true for the 'TrinityCyanobacteriaGenes.fasta' file which contains the 200 most significantly differentially expressed cyanobacteria genes. Differential gene expression analysis of the chlorophytes and cyanobacteria genes was based on temperature increase (25 °C vs control (4 °C)). The 'TrinityAscomycetePhotomorphGenes.fasta' file contains the top200 photomorph-mediated significantly differentially expressed ascomycete transcripts and the 'TrinityAscomyceteTemperatureGenes.fasta' file contains the top200 temperature-mediated significantly differentially expressed ascomycete transcripts. The 'Pbrit_Seqs_Out.fasta', 'Pleu_Seqs_Out.fasta' and 'Pcoll_Seqs_Out.fasta' files contain those contigs of the unpublished <em>Peltigera</em> genome (<em>Peltigera</em> <em>britannica</em>, <em>P</em>. <em>leucophlebia</em> and <em>P</em>. <em>collina</em>) which were found to be homologous to the top200 photomorph- and temperature-mediated ascomycete transcripts (the gene in question plus 100 bp up- and downstream). The 'norm_counts*' files contain the normalized gene counts for each sample and of each gene identified as differentially expressed by DESeq2. These files were used for a two-way ANOVA to determine statistical significance. <br> The 'Sample_explanations.txt' file gives an overview over the samples and their treatments.