Volume 234, Issue 6 p. 1910-1913
Letters
Free Access

Transcriptional acclimation and spatial differentiation characterize drought response by the ectomycorrhizal fungus Suillus pungens

Sonya R. Erlandson

Sonya R. Erlandson

Department of Biology, Stanford University, Stanford, CA, 94305 USA

Department of Agronomy, Horticulture and Plant Science, South Dakota State University, Brookings, SD, 57007 USA

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Rogerio Margis

Rogerio Margis

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Allegre, 90040-060 Brazil

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Andrea Ramirez

Andrea Ramirez

Department of Agronomy, Horticulture and Plant Science, South Dakota State University, Brookings, SD, 57007 USA

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Nhu Nguyen

Nhu Nguyen

Department of Tropical Plants and Soil Sciences, University of Hawai'i at Manoa, Honolulu, HI, 96822 USA

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Lotus A. Lofgren

Lotus A. Lofgren

Department of Microbiology and Plant Pathology, University of California, Riverside, CA, 92507 USA

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Hui-Ling Liao

Hui-Ling Liao

North Florida Research and Education Center, University of Florida, Quincy, FL, 32351 USA

Soil and Water Sciences Department, University of Florida, Gainesville, FL, 32611 USA

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Rytas Vilgalys

Rytas Vilgalys

Department of Biology, Duke University, Durham, NC, 27708 USA

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Peter G. Kennedy

Peter G. Kennedy

Department of Plant Biology, University of Minnesota, St Paul, MN, 55108 USA

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Kabir G. Peay

Corresponding Author

Kabir G. Peay

Department of Biology, Stanford University, Stanford, CA, 94305 USA

Woods Center for the Environment, Stanford University, Stanford, CA, 94305 USA

Author for correspondence: email [email protected]

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First published: 19 October 2021
Citations: 3

Increasing temperature and decreasing precipitation has led to more frequent and extreme drought events in many regions throughout the world. In the western United States, multi-year drought events have led to widespread plant mortality and extreme wildfires (Asner et al., 2016; Pickrell & Pennisi, 2020). Communities of ectomycorrhizal fungi (EMF) – root symbionts which play a critical role in forest health – are also thought to be threatened by these climatic changes (Fernandez et al., 2017; Steidinger et al., 2019). However, altered soil moisture conditions have complex direct and indirect effects on both fungi and ecosystem processes, such as nutrient availability (Schimel, 2018), making it difficult to elucidate the primary drivers of community composition based on field observations or experiments (Pena & Polle, 2014). As a result, efforts to identify the genes or traits involved in response to drought events are critical for accurate prediction of future EMF composition and function (Allison & Treseder, 2008; Romero-Olivares et al., 2019). Despite this fact, we are not aware of any studies that have used gene expression analyses to measure the response of individual EMF to drought events or other climatic stressors.

In this study, we used RNA-sequencing (RNA-Seq) to measure transcriptional changes in the fungus Suillus pungens (SP) exposed to experimental drought. We grew SP in symbiosis with Pinus muricata (PM) in a growth chamber where we manipulated both long-term (chronic) and short-term (acute) soil moisture and sequenced messenger RNA (mRNA) extracted from both ectomycorrhizal roots and extraradical mycelium (ERM). Our primary aims were to: (1) demonstrate the utility of RNA-Seq to quantify response to global change stressors for EMF; (2) identify biological functions involved in drought response by comparing gene expression across moisture treatments and in different organs of EMF; and (3) estimate the potential for fungal acclimation by comparing gene expression between chronic and acute drought stress treatments.

In the first experiment, 2-month-old SP colonized seedlings were subjected to two watering regimes (drought, control) for 10 wk. The amount of water added (4 or 10 ml, biweekly) was chosen to maintain distinct but field-relevant soil moisture levels (Kennedy & Peay, 2007), while mimicking longer-term, climatic trends expected in California, USA, where SP and PM are native. In the second experiment, seedlings from the control treatment were exposed to dry down (i.e. no additional watering) for periods of 5, 7, 9, 11, 14 and 15 d. This treatment was intended to simulate the onset of acute short-term drought events that occur regularly in California. After each experiment, we measured plant biomass, scored SP root colonization, conducted soil chemical analyses, and removed a subset of colonized roots and soil containing ERM for mRNA extraction. Complementary DNA (cDNA) libraries were sequenced on an Illumina HiSeq 4000 2 × 150 bp, with 228 × 106 reads obtained from 15 root and 22 soil samples (Supporting Information Table S1). Fungal transcripts were mapped against the annotated transcriptome of SP strain FC27 (JGI GOLD Project ID Gp0251823) using Kallisto (Bray et al., 2016). Analyses of variance was used to compare plant biomass and SP root colonization and DESeq2 (Love et al., 2014) to compare fungal gene expression across drought and control treatments, respectively. Additional details on the experiment, molecular methods, bioinformatics and statistics can be found in Methods S1.

At a transcriptional level, SP responded very differently to acute and chronic drought. The acute drought treatment caused major changes in gene expression (Fig. 1). After 5–15 d of dry down, 21% of all genes (2069) were downregulated and 20% (1968) upregulated in ERM (adjusted P < 0.1; Fig. 1a; Dataset S1). A sizable but somewhat modified pattern was seen in ectomycorrhizal roots, with 12% of genes downregulated (1209) and 16% upregulated (1599) (adjusted P < 0.1; Fig. 1c; Dataset S2). By contrast, while 10-wk of chronic drought treatment was physiologically stressful, as evidenced by an c. 50% reduction in both pine seedling growth and SP root colonization (Fig. 2a,b), changes in gene expression between chronic drought and control treatments were relatively small (Fig. 2c). In ERM, only 0.01% (1) and 0.14% (14) of genes were either upregulated or downregulated (adjusted P < 0.1) between the drought and control treatments, respectively. In ectomycorrhizal roots the fungal response was even more muted, with only 0.11% (11) and 0% (0) of genes either upregulated or downregulated, respectively (Dataset S3).

Details are in the caption following the image
Comparison of messenger RNA (mRNA) transcriptional changes by Suillus pungens in response to acute drought. Panels compare gene expression for extraradical mycelium (a, b) and ectomycorrhizal roots (c, d) taken from control plants (1 d post watering) and acute drought (5–15 d after last watering). Panels (a) and (c) show expression changes for all genes. Vertical lines demarcate log2 fold change > 2 and horizontal line P < 10e−6. Panels (b) and (d) display the number of genes highly differentially expressed (adjusted P < 0.001) by KOG class. Blue indicates genes downregulated in response to acute drought while red indicates upregulated genes.
Details are in the caption following the image
Physiological and genetic responses to experimental drought. (a) Pine seedling total biomass and (b) seedling percent root colonization by Suillus pungens after a 10-wk experimental chronic drought. For (a) and (b) error bars are one standard error and letters indicate significantly different treatment groups. (c) Patterns of gene expression by S. pungens across all experimental treatments, as illustrated by principal component analysis (PCA). Small points show individual root (circles) or soil samples (triangles) with larger points showing mean and standard error for treatment groups.

Acclimation is defined as the ability of organisms to modify their physiology or behavior to enhance tolerance of stressful conditions within a single lifetime (Somero, 2010). In our experiment, the lack of sustained transcriptional response between acute and chronic drought treatments is consistent with SP being able to acclimate to drought. Metabolomic and proteomic data will be critical to further understand how EMF acclimate to drought stress, as one potential explanation for our results is that genes related to stress tolerance are expressed transiently but metabolites or proteomes have longer lasting effects on EMF physiology. While there are no comparable studies of EMF, similar results have been seen in other symbiotic systems. Corals exposed to chronic heat stress also showed no initial transcriptional differences with controls (Bay & Palumbi, 2015) but were more able to resist acute bleaching events (Palumbi et al., 2014). While we do not yet know whether the transcriptional acclimation we demonstrate in SP can increase plant or fungal resiliency to future drought events, the capacity of mycorrhizal fungi to acclimate to stressful conditions has important consequences for forest resilience in the face of climate change (Pickles et al., 2012) and deserves greater research.

Ectomycorrhizal roots and ERM showed both common and divergent transcriptional responses to acute drought. A core set of 1732 genes had shared differential expression patterns. These suggest an overall reduction in protein synthesis (genes related to ribosomes and translation; Fig. 1b,d) and a modified physical environment, as indicated by consistently reduced expression of hydrophobins, small amphiphilic molecules that coat hyphae, breaking surface tension and facilitating growth across air–water interfaces (Bayry et al., 2012). Ectomycorrhizal roots and ERM also had consistently greater expression of trehalose synthase, a compatible solute involved in drought stress (Welsh, 2000), genes associated with fungal cell wall synthesis, such as 1,3-β-glucan synthase, chitin synthase, and choline kinase, the latter being involved in the production of phosphatidylcholine membrane phospholipids in osmotically stressed plants (Tasseva et al., 2004), and a fungal-specific peroxisome gene (PEX14), which has been shown to be critical to cell wall stability and maintenance of turgor (Li et al., 2017).

While ectomycorrhizal roots had both lower fold changes and fewer significant genes in both of our treatments (Fig. 1c), they also had a higher diversity of highly upregulated KOG classes (adjusted P < 0.001). In general, ERM responses appeared to fine-tune key functional categories, such as inorganic ion transport, carbohydrate, and lipid metabolism (as evidenced by nearly equal numbers of genes upregulated and downregulated), while ectomycorrhizal roots had many more upregulated genes related to energy production, signal transduction, and transport and metabolism of amino acids, lipids, and carbohydrates. Spatial differentiation in gene expression between ectomycorrhizal roots and ERM has been previously documented (Wright et al., 2005; Liao et al., 2014) and our results suggest that roots may provide a greater buffer to the abiotic environment that allows them to function as a resource reservoir and organizing center during stress. As such, ectomycorrhizal roots and ERM should be studied holistically to understand EMF responses to climate change.

With this study, we provide a clear demonstration of how combining transcriptomics with experimental manipulations can be useful for understanding EMF responses to climatic change. We show that drought is a multi-faceted environmental challenge (Schimel, 2018) and requires concerted changes in many functional gene classes. While the large number of genes involved makes simple explanations difficult, we are able to identify important genes with known functions and potential implications for ecosystem function (Treseder & Lennon, 2015). Others, such as atromentin synthase, were highly responsive to our treatments, but require further research to understand their functional role (Tauber & Hintze, 2020). Although our study uses seedlings grown in controlled conditions, we demonstrate a potential for environmental acclimation and functional differentiation across roots and ERM as important components of the EMF response to climatic change. We hope these findings stimulate validation and further exploration using other systems and approaches.

Acknowledgements

Sequencing data was generated on an Illumina HiSeq 4000 that was purchased with funds from National Institutes of Health (NIH) under award number S10OD018220, KGP was supported by NSF CAREER Award DEB #1845544. RM involvement in this project was supported by a CAPES-PRINT fellowship. The Suillus pungens genome was sequenced through the Joint Genome Institute Community Science Program project no. 502931.

    Author contributions

    KGP and SRE designed the study. SRE carried out the experiment and collected plant and molecular data. H-LL helped optimize molecular methods. SRE, KGP, RM, and AR analyzed the data. PGK, RV, H-LL, LAL and NN generated the draft genome for Suillus pungens. SRE and KGP wrote the initial draft of the manuscript, PGK, LAL, RV, H-LL, NN, RM, AR, SRE and KGP collaborated to produce the final draft.

    Data availability

    All sequence files and meta-data are available through the National Center for Biotechnology Information (NCBI) Short Read Archive (PRJNA771376). Seedling biomass and colonization data are available through Dryad (https://doi.org/10.5061/dryad.qnk98sfht).