The foliar nematode,
The foliar nematode survives overwinter in soil, dormant buds, and abscised leaves, where its desiccation tolerance allows it to endure freezing temperatures and low relative humidity (Jagdale and Grewal, 2006). Like a number of other nematode species,
We have previously documented the remarkable anhydrobiotic behavior of
Total RNA was extracted from approximately 5,000 nematodes harvested from each biological replicate using the PureLink RNA mini Kit (Life Technologies, Austin, TX) following the manufacturer’s instructions. Total RNA was treated with RNase-Free DNase (Qiagen, Germantown, MD) to remove any contaminating DNA. RNA quality and integrity were verified on an Agilent RNA 6000 Nano LabChip using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Six RNA samples were sent to the Clemson University Genomics Institute (Clemson, SC) for strand-specific, paired-end 125-bp library preparation with the Illumina TruSeq stranded mRNA library kit, followed by sequencing on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA). Raw sequence data were uploaded to the NCBI Sequence Read Archive under accession number SRP148503.
Read quality was assessed with FastQC (
The longest isoform per gene was extracted using a utility script bundled with Trinity v2.6.6 (get_longest_isoform_seq_per_trinity_gene.pl). A fasta file of these transcripts is presented as Supplementary File 1,
Trimmed reads from each sample were aligned back to the assembled transcriptome using Bowtie2 (Langmead and Salzberg, 2012), and transcript abundances in each sample were estimated using RSEM (Li and Dewey, 2011). Reads from all splice forms of a given gene were pooled for downstream analysis. Differential gene expression analysis was performed using edgeR, including only those genes that had counts-per-million above 0.5 in least three samples (Robinson et al., 2010; Chen et al., 2016). Genes whose expression differed significantly between desiccated and control samples were identified using the exact test model in edgeR (Robinson et al. 2010; false discovery rate = 0.05). R package pheatmap was used to run hierarchical clustering with “complete” method for a subset of differentially expressed genes (Kolde and Kolde, 2018). Expression data for all genes are presented in Supplementary File 3,
Gene set enrichment analysis (GSEA v.2.1.0) was performed to identify pre-defined gene sets that showed significant, concordant differences in expression between control and desiccated samples (Mootha et al., 2003; Subramanian et al., 2005). While edgeR identifies individual genes with large, significant fold-changes, GSEA identifies gene sets whose members show concordant, but potentially smaller, changes in expression. A custom GSEA database of 5,988 gene sets, each containing between 5 and 1,500 genes, was created from GO terms and enzyme code annotations of the assembled transcripts. Gene sets whose expression was enriched or depleted in desiccated nematodes were identified using a false discovery rate of 0.05 (Supplementary File 4,
Illumina sequencing of RNA samples from desiccated and control nematodes generated 325 million reads with a mean length of 125 bp and an average GC content of 42%. After filtering and trimming, reads from all samples were combined for
Summary statistics for
Basic sequence statistics | Number |
---|---|
Total raw reads | 324,895,970 |
Mean read length (bp) | 125 |
Raw read GC content | 42% |
Mean read PHRED score after filtering and trimming | 36 |
Number of genes | 48,541 |
Number of isoforms | 147,621 |
Assembly N50 (of all isoforms) | 1293 bp |
Ex90N50 | 1470 bp |
Mean length of all isoforms | 882 bp |
Top BLASTx-hit species |
|
Percent of gene with at least one BLASTx hit (E ≤ 1.0-3) | 35% |
Percent of gene with at least one GO annotation | 23% |
In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes (Fig. 2A). Of the 100 annotated genes with the largest positive fold-changes (box, Fig. 2A), more than one third encoded putative detoxification-related proteins (Fig. 2D). These included numerous Phase I and II detoxification enzymes: CYPs, SDRs, UGTs, and GSTs. Also among the top hundred upregulated genes were a
Across the transcriptome as a whole, large percentages of CYPs (45%), SDRs (32%), UGTs (55%), NHRs (30%), and MRP/PGPs (32%) were upregulated in response to desiccation (Fig. 2B). Only the GSTs did not respond strongly as a group: 28 GST genes were assembled: 5 (18%) were significantly upregulated and three were downregulated.
Results of gene set enrichment analysis were consistent with wholesale induction of detoxification-related genes. Of the top 15 gene sets enriched during desiccation, 5 were dominated by detoxification-related genes. The majority of leading-edge genes in each set (i.e. those that contributed to the enrichment signal) were again CYPs, UGTs, NHRs, and MRP/PGPs. Gene sets associated with other detoxification-related GO terms were also significantly enriched (Fig. 2C): xenobiotic transport (GO:0042908), xenobiotic transporting ATPase (GO:0008559), steroid hormone receptor (GO:0003707), and glucuronosyltransferase (GO:0015020). The leading-edge genes of the former two sets were made up entirely of MRP/PGP genes. The leading-edge genes for GO:0003707 were entirely NHRs, while those of GO:0015020 were primarily UGTs.
CYPs and SDRs are canonical enzymes of Phase I detoxification that add reactive functional groups to a wide variety of endogenous and exogenous compounds. Both are large gene families: 86 CYPs and 68 SDRs have been documented in
Functionalized substrates are further modified by the Phase II reactions catalyzed by UGTs and GSTs. These reactions typically involve the addition of side groups that increase the substrate’s solubility in preparation for excretion. Like CYPs, UGTs act on numerous small lipophilic compounds: xenobiotics, endogenous waste metabolites, steroids, and fatty acids. GSTs participate in the modification and detoxification of substrates by multiple mechanisms, including the addition glutathione to an electrophilic substrate and the direct binding of toxic substrates. These are also expensive reactions: each UGT glucuronidation reaction requires one molecule of glucose, while each GST transferase reaction uses one molecule of reduced glutathione (Gems and McElwee, 2005; Lindblom and Dodd, 2006). Following enzymatic modification by Phase I and II enzymes, toxins and waste metabolites are excreted from the cell by membrane transporters, mainly members of the ATP-binding cassette (ABC) family of efflux pumps (Lindblom and Dodd 2006). Among this large protein family, MRP and PGP transporters have been most extensively characterized for their role in detoxification (Choi, 2005; Hoffmann and Partridge, 2015; Harder, 2016).
NHRs are a very large class of transcription factors (over 280 in
The broad induction of detoxification-related genes was the single most striking pattern to emerge from the desiccation-related transcriptome. This result is intriguing, as it parallels the broad induction of detoxification-related gene expression that has been reported in
This raises the question of what, exactly, is being detoxified in stress tolerant and/or long-lived organisms. Anhydrobitic
Desiccation can cause protein misfolding, damage, and aggregation (Tapia et al., 2015). Indeed, such damaged proteins may be important substrates for the detoxication enzymes highlighted above, such as CYP, UGT, SDR, and NHR. Multiple genes encoding molecular chaperones and components of the unfolded protein response were induced by desiccation in
Of the 98 heat shock protein genes assembled from
Desiccated
Another means by which dehydrating organisms may prevent catastrophic damage to proteins and membranes is through the production of strongly hydrophilic, IDPs. These proteins have been documented in anhydrobiotic species from multiple kingdoms of life. Often classified as late embryogenesis abundant (LEA) proteins, IDPs are thought to stabilize proteins, membranes, and organelles during desiccation (Hand et al., 2011). Recently, novel desiccation-induced IDPs with no homology to other known proteins were identified in tardigrades and shown to mediate desiccation tolerance (Boothby et al., 2017).
A subset of strongly upregulated
Characteristics of 14 putative intrinsically disordered proteins whose expression was significantly upregulated under desiccation in
Gene ID | Predicted length (aa) | Mean FPKM desiccated | Mean FPKM control | Fold-change | Adjusted |
% disordered residues | GRAVY hydropathy value | Blastx hits to LEA database |
---|---|---|---|---|---|---|---|---|
DN15064_c0_g1 | 175 | 398.8 | 0.4 | 1020.9 | 3.15E−64 | 88.1 | −1.836 | − |
DN14203_c3_g3 | 119 | 45.4 | 0.5 | 88.7 | 1.92E−15 | 74.8 | −0.977 | − |
DN10042_c0_g2 | 128 | 366.8 | 5.0 | 72.9 | 3.61E−28 | 82.8 | −0.108 |
|
DN10455_c1_g1 | 191 | 928.2 | 14.0 | 64.1 | 9.30E−99 | 90.6 | −0.503 |
|
DN9957_c2_g1 | 335 | 7.9 | 0.2 | 41.1 | 1.66E−17 | 94.6 | −1.104 |
|
DN10923_c0_g1 | 171 | 17.0 | 0.7 | 25.5 | 1.23E−12 | 81.9 | −1.607 |
|
DN9863_c0_g1 | 250 | 21.7 | 0.9 | 23.4 | 1.70E−43 | 85.2 | −0.951 |
|
DN9710_c0_g1 | 167 | 28.1 | 1.3 | 20.6 | 4.50E−07 | 93.5 | −1.185 | − |
DN12711_c2_g2 | 131 | 92.8 | 6.4 | 13.8 | 4.35E−12 | 77.1 | −0.204 |
|
DN11488_c0_g1 | 192 | 16.40 | 1.30 | 11.50 | 2.52E−03 | 86.5 | −1.167 | − |
DN12459_c0_g4 | 172 | 173.6 | 14.4 | 11.5 | 7.31E−17 | 98.3 | −0.793 |
|
DN15965_c0_g1 | 140 | 294.5 | 25.7 | 10.7 | 2.28E−10 | 72.9 | −0.450 |
|
DN12325_c1_g1 | 96 | 15.5 | 1.9 | 8.1 | 1.38E−17 | 75.0 | −0.672 | − |
DN9469_c2_g1 | 133 | 46.8 | 9.7 | 4.7 | 1.44E−14 | 100.0 | −1.520 |
|
It appeared that many aspects of the desiccation response and network of genes are coordinated by a transcriptional factor
It is important to note that nematodes in our experiment were sampled at only one time point, 24 hr after relatively rapid and severe dehydration. While the desiccated worms were fully viable and capable of recovery within 30 min of rehydration (Fu et al., 2012), their transcriptomes likely reflected both the remnants of transcriptional programs induced early in desiccation and those active in later stages of desiccation. In the future, a more detailed time course of the transcriptional changes that accompany desiccation and rehydration could clarify the order in which specific gene expression changes and signaling events occur.
Anhydrobiosis is more than just a biological curiosity: the conservation of its basic biochemical mechanisms across multiple kingdoms of life – from bacteria to plants to arthropods – suggests that the ability to tolerate significant dehydration is both ancient and conserved. Anhydrobiotic physiology exhibits numerous connections with the basic biology of longevity and aging. A better understanding of anhydrobiosis therefore has genuine implications for human health and lifespan, some of which are already being explored in clinical settings (Crowe, 2015). From an agricultural standpoint, induction of anhydrobiotic stasis offers a means of stabilizing and delivering living amendments such as entomopathogenic nematodes and fungi as biocontrol, while interruption or elimination of anhydrobiosis could disrupt the life cycle of damaging pests such as cyst nematodes and