# Abscisic acid supports colonization of Eucalyptus grandis roots by the mutualistic ectomycorrhizal fungus Pisolithus microcarpus

Richard Hill, Johanna Wong-Bajracharya, Sidra Anwar, Donovin Coles, Mei Wang, Anna Lipzen, Vivian Ng, Igor Grigoriev, Francis Martin, Ian Anderson, et al.

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Richard Hill, Johanna Wong-Bajracharya, Sidra Anwar, Donovin Coles, Mei Wang, et al. Abscisic acid supports colonization of Eucalyptus grandis roots by the mutualistic ectomycorrhizal fungus Pisolithus microcarpus. New Phytologist, 2022, 233 (2), pp.966-982. 10.1111/nph.17825. hal-04003919

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Abscisic acid supports colonization of *Eucalyptus grandis* roots by the mutualistic ectomycorrhizal fungus *Pisolithus microcarpus*

Richard A. Hill<fnref n="1" /> ID, Johanna Wong-Bajracharya<fnref n="1" />,² ID, Sidra Anwar<fnref n="1" /> ID, Donovin Coles<fnref n="1" /> ID, Mei Wang³ ID, Anna Lipzen³, Vivian Ng³ ID, Igor V. Grigoriev³,⁴ ID, Francis Martin⁵ ID, Ian C. Anderson<fnref n="1" /> ID, Christopher I. Cazzonelli<fnref n="1" /> ID, Thomas Jeffries⁶, Krista L. Plett<fnref n="1" />,² ID and Jonathan M. Plett<fnref n="1" /> ID

Author for correspondence:
Jonathan M. Plett
Email: j.plett@westernsydney.edu.au

Received: 26 August 2021
Accepted: 20 October 2021

New Phytologist (2022) 233: 966-982

doi: 10.1111/nph.17825

**Key words:** carotenoid, ectomycorrhizal fungus, hormone, immunity, plant-microbe interactions, symbiosis.

Summary

* The pathways regulated in ectomycorrhizal (EcM) plant hosts during the establishment of symbiosis are not as well understood when compared to the functional stages of this mutualistic interaction. Our study used the EcM host *Eucalyptus grandis* to elucidate symbiosis-regulated pathways across the three phases of this interaction.

* Using a combination of RNA sequencing and metabolomics we studied both stage-specific and core responses of *E. grandis* during colonization by *Pisolithus microcarpus*. Using exogenous manipulation of the abscisic acid (ABA), we studied the role of this pathway during symbiosis establishment.

* Despite the mutualistic nature of this symbiosis, a large number of disease signalling TIR-NBS-LRR genes were induced. The transcriptional regulation in *E. grandis* was found to be dynamic across colonization with a small core of genes consistently regulated at all stages. Genes associated to the carotenoid/ABA pathway were found within this core and ABA concentrations increased during fungal integration into the root. Supplementation of ABA led to improved accommodation of *P. microcarpus* into *E. grandis* roots.

* The carotenoid pathway is a core response of an EcM host to its symbiont and highlights the need to understand the role of the stress hormone ABA in controlling host-EcM fungal interactions.

Introduction

*Eucalyptus grandis*, a major forestry crop accounting for > 20 million hectares of timber plantations globally, is known for fast growth and adaptability (Myburg *et al.*, 2014; Wingfield *et al.*, 2015; Vivas *et al.*, 2019). Supporting its growth and establishment, *E. grandis* enters into mutually beneficial symbioses with mycorrhizal fungi including the ectomycorrhizal (EcM) fungus *Pisolithus microcarpus* (Duplessis *et al.*, 2005; Plett *et al.*, 2015a). Mycorrhizal fungi, as a broad class, engage in mutualistic associations with *c*. 93% of all plant species, exchanging mineral nutrients for photosynthetically fixed carbon from the plant. EcM fungi, meanwhile, associate with *c*. 2% of plant species, but their hosts typically make up the dominant flora in most of the forested biomes of the world (Tedersoo *et al.*, 2010; Brundrett & Tedersoo, 2018). As opposed to arbuscular mycorrhizal (AM) fungi, which have specialized fungal hyphae that can penetrate plant cell walls, EcM fungi grow only in the apoplastic space of the root and form a structure called a 'Hartig net' that is the site

of the mutualistic exchange of nutrients with the plant (Brundrett & Tedersoo, 2018; Becquer et al., 2019). Given the invasive nature by which EcM fungi colonize their hosts, a number of host transcriptional and metabolic pathways are impacted by these associations, although the pathways that are altered during colonization vary depending on which plant/fungal species are being studied (Larsen et al., 2015; Plett et al., 2015a; Liao et al., 2016; Martin et al., 2016; Bouffaud et al., 2020).

Regardless of type, all mycorrhizal fungi follow the same three general stages of root colonization: an initial stage of pre-symbiosis prior to physical interaction during which signals are exchanged between the two organisms, a stage of physical integration during which the fungus aggregates on the root surface and then begins to grow into the root followed by a final stage of functional symbiosis during which the fungus is established within the root and nutrients begin to be exchanged between the two partners (Hogekamp & Küster, 2013; Plett et al., 2015b). The progression through each of these stages is thought to involve both a distinct set of differentially regulated genes that

<footnote n="1">¹Hawkesbury Institute for the Environment, Western Sydney University, Richmond, NSW 2753, Australia; ²Elizabeth Macarthur Agricultural Institute, New South Wales Department of Primary Industries, Menangle, NSW 2568, Australia; ³US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; ⁴Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA; ⁵INRAE, UMR Interactions Arbres/Microorganismes, Laboratory of Excellence ARBRE, INRAE GrandEst-Nancy, Université de Lorraine, 54280 Champenoux, France; ⁶School of Science, Western Sydney University, Richmond, NSW 2753, Australia</footnote>
support the stage-specific metabolic and physical changes taking
place, as well as persistent set of genes whose change in expression
are required to enable the symbiotic interaction (Hogekamp &
Küster, 2013; Garcia et al., 2015; Larsen et al., 2015; Plett et al.,
2015b).

During the pre-symbiotic stage, EcM fungi secrete a cocktail
of signalling molecules, including proteins and other soluble
and volatile metabolites (Menotta et al., 2004a,b; Splivallo et al.,
2009; Garcia et al., 2015; Cope et al., 2019; Wong et al., 2019;
Rush et al., 2020; Plett et al., 2021a). Upon perception of these
signals, physical and metabolic changes occur within the plant,
underpinned by a change in gene expression (Sebastiana et al.,
2009; da Silva Coelho et al., 2010; Li et al., 2014; Larsen et al.,
2015; Wong et al., 2019). Metabolites induced within the host
during the early stages of colonization include flavonoids, fatty
acids, terpenoids, and carotenoids (Wong et al., 2019). Genes
involved in the plant immune system are typically repressed
(Seddas et al., 2009; Garcia et al., 2015; Wong et al., 2019).
Lateral root formation is also stimulated by EcM fungal signals
independent of contact (Felten et al., 2009; Splivallo et al.,
2009; Ditengou et al., 2015). Contact and integration between
the two organisms trigger further metabolic and physical
changes in the host. During this stage of the interaction, plant
transcriptomic changes linked to pathways involved with
immune and stress responses have been reported, as have
increases in gene expression of signal receptors (Sebastiana et al.,
2009; Balestrini & Bonfante, 2014; Larsen et al., 2015; Plett
et al., 2015b; Liao et al., 2016). In the final stage of the interac-
tion, during which the fungus becomes fully established within
the host as determined by the presence of a Hartig net, plant
nutrient transporter genes are induced and nutrient fluxes
between the partner organisms can be measured (Duplessis
et al., 2005; Willmann et al., 2014; Garcia et al., 2015; Hortal
et al., 2017; Basso et al., 2019; Plett et al., 2020; Plett et al.,
2021b).

Many phytohormones are important to the development of
EcM interactions, with previous research highlighting how the
sensitivity to, and regulation of, plant hormones such as jasmonic
acid, ethylene, and auxin affect colonization (Plett et al., 2014;
Basso et al., 2019). In addition to these classic phytohormones,
only a few studies in EcM fungi have explored the role of abscisic
acid (ABA) in host interactions (Luo et al., 2009; Calvo-Polanco
et al., 2019; Lorente et al., 2021) despite the finding of its crucial
role in the establishment of AM fungal symbioses (Herrera-
Medina et al., 2007; Charpentier et al., 2014; Lou et al., 2021).
ABA is synthesized from carotenoids and functions to coordinate
the plant's response to abiotic stress as well as promote pathogen
defence responses (Finkelstein, 2013; Jia et al., 2017; Fiorilli
et al., 2019). Carotenoids can also be cleaved into other apoc-
arotenoid metabolites important for plant-microbe interactions,
such as strigolactones and blumenin which have been found to be
significant to establishment of AM symbiosis (Maier et al., 1995;
Akiyama et al., 2005; Besserer et al., 2006; Stauder et al., 2018).
Carotenoids can also serve to reduce damage from stress-induced
oxidation in some plant-mycorrhizal interactions (Alvarez et al.,
2009; Hamilton & Bauerle, 2012).

In this study we sought to further our understanding of the
*E. grandis* genomic pathways that are significantly regulated
during the colonization process of the EcM fungus *P. microcar-
pus*. Using transcriptional profiling across the major stages of
EcM root colonization, we present significantly regulated host
signalling pathways associated with specific interaction phases.
The carotenoid biosynthetic pathway and ABA signalling pro-
cesses were highly enriched with significantly altered gene
expression throughout this interaction, thereby representing a
core response of *E. grandis* to *P. microcarpus* colonization. We
quantified ABA during the fungal integration steps pathway
and found that the synthesis of ABA is significantly regulated
by the presence of *P. microcarpus*. Exogenous application of
ABA altered Hartig net development of *P. microcarpus*, suggest-
ing that ABA may play a more important role in EcM symbio-
sis than previously found.

Materials and Methods

Plant and fungal growth conditions and mycorrhizal root
tip formation

*Eucalyptus grandis* seeds (Seed lot 20974) were obtained from the Commonwealth Scientific and Industrial Research Organization tree seed centre (CSIRO, Clayton, Australia). *Pisolithus microcarpus* isolate SI-14 was isolated from a single fruiting body from Sussex Inlet (36°50 041"S, 150°59 086"E), New South Wales, Australia (scientific license number S13146). *Eucalyptus grandis* seeds were sterilized in 30% hydrogen peroxide (H₂O₂, v/v) for 10 min and then washed with sterile deionized water five times prior to germination on 1% (w/v) water agar and incubated in a controlled environment growth cabinet for one month (25°C; 16 h light cycle). Following this, seedlings were transferred into half-strength modified Melin-Norkrans (MMN) media (pH 5.5; 1 g l⁻¹ glucose; 1% agar) placed between two cellophane membranes to prevent growth into the media (Kleerview Covers by Fowlers Vacola Manufacturing Co. Ltd, Melbourne, VIC, Australia) and grown for another month (22–30°C night/day temperature; 16 h light cycle). Two weeks before contact with the plants, *P. microcarpus* SI-14 was propagated onto cellophane covered half-strength MMN plates and grown in the dark at 25°C. All Petri dishes used were Sarstedt 92 × 16 mm polystyrene vented plates.

Once the *E. grandis* seedlings were two months old and the
fungal cultures were 2 wk old, the plants were separated into one
of three treatment categories: (1) ‘axenic controls’ consisted of
seedlings transferred onto new half-strength MMN medium
without any fungal inoculum; (2) ‘pre-symbiosis seedlings’ were
transferred onto new half-strength MMN medium in indirect
contact with fungal mycelium for 24 h by separating the two
organisms by a permeable cellophane membrane; (3) ‘physical
contact’ seedlings were transferred onto new half-strength MMN
medium and then placed into direct contact with the active grow-
ing edge of a fungal colony and then samples were harvested at
24 h, 48 h, 1 wk, and 2 wk post-contact. These plates were then
closed using micropore tape to allow for gas exchange with the
external environment.
RNA extraction, sequencing, and analysis

The plant root tips of each group were harvested and immedi-
ately frozen in liquid nitrogen. For each timepoint, three biologi-
cal replicates were harvested and extracted. Root samples were
then stored in a -80°C freezer until RNA extraction. The RNA
from each of the samples was then extracted using the ISOLATE
II Plant miRNA kit (Bioline, Sydney, Australia) as per the manu-
facturer's instructions. Following extraction, the RNA samples
were sequenced at the Joint Genome Institute (JGI). Plate-based
RNA sample preparation was performed using the Perkin Elmer
Sciclone NGS robotic liquid handling system, utilizing Illu-
mina's TruSeq Stranded mRNA HT sample prep kit utilizing
poly-A selection of messenger RNA (mRNA) following the pro-
tocol outlined by Illumina in their user guide for TruSeq
Stranded mRNA workflow: https://support.illumina.com/
sequencing/sequencing_kits/truseq-stranded-mrna-workflow.
html with the following conditions: total RNA starting material
was 100 ng per sample and 10 cycles of PCR was used for library
amplification.

The prepared RNA-sequencing (RNA-Seq) libraries were then quantified using KAPA Biosystem's next-generation sequencing library qPCR kit, run on a Roche LightCycler 480 real-time PCR instrument. Quantified RNA-Seq libraries were then multiplexed with other libraries. This pool of libraries was then prepared for sequencing on the Illumina HiSeq sequencing platform, utilizing a TruSeq paired-end cluster kit (v4) and Illumina's cBot instrument to generate a clustered flow cell for sequencing. Sequencing of the flow cell was then performed on the Illumina HiSeq2500 sequencer using HiSeq TruSeq SBS sequencing kits (v4), following a 2 × 150 indexed run recipe. The JGI QC pipeline was then used to filter and trim the raw RNA-Seq reads. These raw RNA-Seq reads were then evaluated for artefact sequence by kmer matching (kmer = 25), allowing one mismatch and detected artefact, trimmed from the 3' end of the reads using BBDUK (https://sourceforge.net/projects/bbmap/). Following this, RNA spike-in reads, PhiX reads and reads containing any Ns were removed. Quality trimming was then performed using the PHRED trimming method, set at Q6. Following trimming, reads under the minimum length threshold of 25 bases were removed unless one-third of the original read length of those reads were longer. The filtered reads from each RNA-Seq library were then all aligned with the E. grandis genome, identifying each gene sequence and their existing characterization (Myburg et al., 2014). This characterization includes their gene ontology (GO), KEGG (Kyoto Encyclopedia of Genes and Genomes) identifier (ID) and closest Arabidopsis thaliana homologue. Only primary hits assigned to the reverse strand were included in the raw gene counts (-s 2 -p —primary options). The gene count data was used to identify significantly differentially expressed genes (DEGs) using DESeq2 v1.30.0 in RSTUDIO v4.0.3 (Love et al., 2014). Relative log2-fold change was calculated through pairwise comparison of each timepoint to axenic plant-only controls using DESeq2. These values were then sorted by significance whereby a DEG was deemed to have a log2-fold change of greater than 1 and a P-value (adjusted for false-discovery; Padj) of ≤ 0.05. Normalized, rlog

transformed count data was used to perform principal component
analysis (PCA) using the MIXOMICS package v.6.14.0 in RSTUDIO.
The Pearson statistical method was used to calculate the relative
similarity dendrogram, using all 6687 genes to measure the rela-
tive similarity of each timepoint based on their differential gene
expression profiles. Heat-map figures were produced using MOR-
PHEUS (https://software.broadinstitute.org/morpheus).

The RNA-Seq expression patterns were validated using quanti-
tative polymerase chain reaction (qPCR). Briefly, RNA from
these root tips was used to synthesize complementary DNA
(cDNA) using the Tetro cDNA synthesis kit (Bioline) according
to manufacturer's instructions using only the oligo dT₁₈ primer
and a standardized quantity of RNA. The qPCR reaction utilized
SensiFAST SYBR no-ROX Q-PCR kit (Bioline) following man-
ufacturers' instructions. The expression levels of two plant con-
trol genes (Eucgr.C00350 and Eucgr.K02046) were used to
normalize the results and the log₂-fold change was calculated for
12 genes across all timepoints (Supporting Information
Table S1). These expression values were plotted against the log₂-
fold change of each gene using a bivariate scatter plot. The signif-
icance of the correlation was determined.

Gene enrichment and KEGG analyses

Gene enrichment analyses were used to identify which genomic
pathways were significantly overrepresented within the data.
These were performed using two different programs to improve
the robustness of the analyses. PLANTREGMAP (Tian et al., 2020;
http://plantregmap.gao-lab.org) was used with E. grandis gene
IDs for GO enrichment while gPROFILER utilized the orthologous
A. thaliana gene ID as a complementary enrichment tool and
were both performed at *P* < 0.05 (Raudvere et al., 2019). In order
to further investigate genes and metabolic pathways regulated
during the different colonization timepoints considered, we uti-
lized the KEGG mapper tool (https://www.genome.jp/kegg/;
accessed 8 March 2020) to associate the E. grandis DEGs to
known metabolic pathways using the KEGG orthology (KO)
numbers associated with each gene within the data set (Kanehisa
& Goto, 2000; Kanehisa et al., 2017, 2019). Heat maps were
generated and overlaid onto the carotenoid metabolic pathways
of interest to show the relative differential gene expression over
time using the MORPHEUS online tool. The carotenoid pathway
utilized in this article was based on Sandmann (2021).

Carotenoid analysis

Using the same conditions as described earlier, we generated *E. grandis* seedlings in pre-symbiotic contact with *P. microcarpus* SI-14 for 48 h or their complementary axenic controls, after which the roots were harvested and immediately frozen in liquid nitrogen and stored at -80°C. Root samples from *c*. 10 individual plants were pooled to obtain a recommended minimum of 150 mg of tissue for each replicate for total carotenoid extraction. A total of four independent replicates were extracted per group. A pre-symbiotic setup was chosen as we could sample root-only tissue, thereby avoiding the quantification of potential
carotenoids that may be also produced by the fungus. Frozen roots were finely ground using a mortar and pestle containing liquid nitrogen and extracted in 500 µl of extraction buffer (acetone : ethyl acetate; 60 : 40 v/v) containing 0.1% BHT (butylated hydroxytoluene). An equal volume of MilliQ water was then added to each tube, gently inverted to avoid forming an emulsion and centrifuged for 5 min at 15 000 g. The upper phase was transferred to a glass vial and pigments separated via high-performance liquid chromatography (HPLC) using a C18 column. Absolute quantification of xanthophyll pigments was performed as previously described in Cazzonelli *et al.* (2020) and Alagoz *et al.* (2020).

## Abscisic acid responsive genes

ABA responsive genes were identified and classed based on *A. thaliana* genomic annotation, and cross-checked with the *E. grandis* annotation where available, as well as previously published literature (Amir Hossain *et al.*, 2010; Kulik *et al.*, 2011; Finkelstein, 2013). Genes involved in ABA metabolism and transport, signalling and/or regulated by ABA in *E. grandis* were also annotated against those identified in *A. thaliana* as previously described by Pornsiriwong *et al.* (2017). These ABA responsive genes were then extracted from our relative gene expression matrices and analysed for significant differential expression as described earlier. The expression of various classes of ABA responsive genes were then visualized in heat map form through the MORPHEUS platform.

## Abscisic acid quantification

*Eucalyptus grandis* plants and fungi were grown in the same way as for the time course analysis focusing on the pre-symbiotic timepoint and the fungal integration timepoints (24 h post-contact through 1 wk). At a given timepoint, roots in contact with fungi, or in indirect contact with fungal colonies for pre-symbiosis, were excised from the surrounding mycelium and pooled to obtain 30–40 mg of tissue per replicate. Four separate replicates for plant tissues were extracted as detailed later. To understand if ABA was being produced in *P. microcarpus* mycelium in close contact to the roots, we also excised from the plates fungal mycelium that had been physically touching the root system. As with the roots, samples were pooled to obtain 30–40 mg of tissue per replicate. Due to the small masses recovered, we obtained three separate replicates for these tissues per timepoint. ABA was extracted according to the method of Hall *et al.* (2019) with modifications. In brief, c. 50 mg ground root tissue per sample was extracted with 70% methanol spiked with 2H-ABA (Olchemim, Olomouc, Czech Republic) to yield a final 2H-ABA concentration of 100 ppb. All samples were incubated in a rotor mixer at 80 rpm for 30 min at 4°C before centrifuging at 16 000 g for 5 min at room temperature. The supernatant was then collected into a clean tube and the samples re-extracted with 70% methanol to reach a final volume of 250 µl. The extracts were analysed by ultra-performance liquid chromatography electrospray ionization tandem mass

spectrometry (UPLC-ESI-MS/MS) using an Acquity UHPLC coupled to a Xevo triple quadrupole mass spectrometer (Waters Corp., Milford, CT, USA). Briefly, 5 µl of extract were injected into a 2.1 mm × 50 mm × 1.7 µm, C18 reverse phase column. ABA identification was based on comparison to fragmentation pattern with an authentic standard. Quantification was based on a standard calibration curve and adjusted for sample recovery as compared to the internal ABA standard. Reported ABA values within the manuscript were either fold-change from ABA concentrations in axenic tissues or as absolute concentrations with standard error.

## The effect of exogenous abscisic acid on colonization

In order to ascertain the effect of exogenous ABA application upon symbiosis, the *E. grandis-P. microcarpus* interaction was repeated as described earlier for the 'physical contact' condition but with the addition of ABA to the media in the interaction plates. The concentrations chosen were 0, 0.1, 0.25, and 0.5 µM of ABA (n = 6). ABA was dissolved in ethanol and added to the medium after autoclaving. Dilutions were done such that an equivalent amount of ethanol was added to all treatments (including 0 µM). To verify that the added hormones were not affecting the pH of the medium, we tested this and found that pH was not affected by the addition of ABA (Fig. S1). After a 2-wk period of colonization, the total number of root tips exhibiting a fungal mantle were counted as opposed to the number of uncolonized roots in contact with the fungus. Normalcy of the colonized and uncolonized root data was assayed using a Shapiro-Wilk test and the significance of the data analysed using a Student's t-test. Roots exhibiting mantle formation were excised from the plants and fixed in 4% paraformaldehyde and stored at 4°C until sectioning. To section, samples were embedded in 6% agarose and 30 µm thick cross-sections were cut using a 7000SMZ-2 Vibrotome (Campden Instrument Ltd, London, UK) and stored in 1 × phosphate-buffered saline (PBS, pH 7.4) at 4°C until staining. The roots were each sectioned in the middle of the colonized root tip to ensure the most comparable results. Roots were rinsed three times with 1 × PBS (pH 7.4) after co-staining with Wheat Germ Agglutinin-Fluorescein (WGA-FITC; 100 µg ml⁻¹) for 15 min and propidium iodide (PI; 20 µg ml⁻¹) for 10 min. Confocal microscopy was carried out on a TCS SP5 confocal laser scanning microscope (Leica, Macquarie Park, NSW, Australia) with an excitation wavelength of 488 nm and detection wavelength of 500–550 nm for WGA-FITC imaging and a 561 DPSS laser with an excitation wavelength of 561 nm and detection wavelength of 580–660 nm for PI staining. Plant/fungal contact area was calculated using measurements of sections of each root imaged. Each image was imported into IMAGEJ and the contact surface area of fungal hyphae to plant cells was measured using the free-hand tool and normalized to the outer circumference of the root. Six biological replicates were measured per condition, with two technical replicates per biological replicate. Hartig net depth of penetration and mantle thickness were also measured using IMAGEJ. The former measurement is defined as the distance from the outer rim of a
given epidermal cell to the deepest point of fungal penetration. An average of five technical replicate measurements for each root section image were taken and then averaged to arrive at a value for each biological replicate. Similarly, mantle thickness was a measurement from the outer rim of epidermal cells to the outer rim of the fungal mantle.

To quantify the relative fungal biomass within each colonized root, and support the results obtained from microscopic analysis, we undertook a qPCR approach. This approach was taken as relative mantle thickness was found to be consistent between treatments suggesting that any difference in fungal biomass would be due to increased fungal presence within the root. Individual colonized root tips from four biological replicates of *E. grandis* treated with either 0.25 µM ABA or control were excised from the root systems ensuring that no excess extra-radical mycelium was taken. cDNA was synthesized as earlier. The expression level of two functional control genes (*Pm_670570* and *Pm_679335*; i.e. known to show consistent expression regardless of conditions) and one plant control gene (*Eucgr. C00350*) were assayed using the SensiFAST SYBR no-ROX Q-PCR kit (Bioline) following the manufacturers' instructions. To quantify the copy number of each gene, we PCR amplified each of the target sequences, purified them using the Wizard SV Gel and PCR Clean-Up system (Promega, Madison, WI, USA) according to the manufacturer's instructions and generated a standard curve with known numbers of copies to relate transcript copy number to Ct in our qPCR cycling. Following this, the average ratio of fungal gene expression : plant gene expression was calculated.

## Results

### Host gene expression shows the greatest variation during the integration stage of *P. microcarpus* colonization

Colonization of the *E. grandis* root system by the mutualistic fungus *P. microcarpus* isolate SI-14 led to 6687 significant plant DEG (*P* < 0.05; Table S2) across five timepoints of colonization, ranging from pre-symbiosis through to a fully colonized mycorrhizal root tip at 2 wk post-contact (Fig. 1a). These data were further verified using qPCR (Fig. S1a). There was a significant correlation between the transcriptional profile predicted by RNA-Seq and gene transcription based on qPCR analysis (R² = 0.31, r = 0.56; F(1,52) = 23.27; *P* < 0.001; β = 0.91; *P* < 0.001). We found that the lowest number of DEGs was observed during the pre-symbiotic phase, when the plant and fungus were separated physically by a permeable membrane, followed by the 2 wk timepoint. This latter timepoint shows a fully developed Hartig net, the development of which has previously been demonstrated to lead to active exchange of nutrients with the host plant in similar *in vitro* conditions (Hortal *et al.*, 2017; Plett *et al.*, 2020). Between these two timepoints are those timepoints concerning the physical integration stage of colonization, covering the timepoints 24 h post-contact through to the 1-wk timepoint, where we observed the majority of DEGs. Additionally, we observed that there were nearly equal numbers of genes that were upregulated or downregulated (Fig. 1a). A comparison of DEGs from each of the three stages of colonization

indicated that a relatively small number of genes were found to be persistently regulated throughout colonization, the majority of which were significantly upregulated (106 that were persistently upregulated throughout colonization, one gene that was upregulated in certain stages and repressed in others, three genes were persistently repressed; Fig. 1b). The 109 persistently regulated gene set made up nearly half of the DEGs during the pre-symbiotic timepoint, whilst also being the third largest grouping in the 2-wk functional symbiosis timepoint.

Hierarchical clustering of the samples based on expression patterns showed clustering of the timepoints during the physical integration stages apart from the pre-symbiotic and functional symbiotic timepoints (Fig. 1c). In order to identify the genes most responsible for driving differences between the timepoints we performed a PCA with our time course data. We found that principal component one (PC1) explained 31.5% of the variation, followed by 14.3% for principal component two (PC2) (Fig. 1d). Therefore, as PC1 presented the same distribution of groupings observed in the dendrogram, we identified those genes having the largest contribution to this PC. These genes most explain the separation of the timepoints during which the fungus is integrating into the root from the pre-symbiotic or the functional (i.e. 2 wk) timepoints. Within this gene set, 13 genes had a percentage contribution of > 0.25 and were significantly differentially regulated across the time course of colonization (Fig. 1e; Table S3). All but one of the 13 influential DEGs were primarily upregulated throughout colonization with the top three genes being an ABA-induced protein phosphatase gene (PP2C; Eucgr.A01486), a *LATE EMBRYOGENESIS ABUNDANT* gene (Eucgr.K01836), and a *Eucalyptus*-specific gene of unknown function (Eucgr.H04961; Fig. 1e). The repressed gene was annotated as a polyketide cyclase/dehydrase and lipid transport superfamily protein (Eucgr.L02071; Fig. 1e). Separation of colonization timepoints along PC2 was largely driven by nutrient and lipid transporters (Table S4).

### *Eucalyptus grandis* root metabolism changes over the course of *P. microcarpus* colonization

The metabolic pathways regulated in *E. grandis* over the course of colonization were identified using annotated GO terms within our datasets (Table 1). During the integration stages, genes associated with the flavonoid and phenylpropanoid biosynthesis pathways were significantly repressed while the latter became positively regulated during functional symbiosis at 2 wk post-contact. Beyond KEGG pathway enrichment, we also considered the enriched biological processes at each of the timepoints and found that genes involved in defence or response to abiotic stress were typically upregulated during the integration stages of colonization (i.e. 24 h and 48 h post-contact timepoints through 1 wk post-contact) after which they were repressed at 2 wk (Table 1). There was also enrichment of genes associated with two main hormone pathways: the ABA response and degradation pathway (GO:0010295; GO:0009737) as well as auxin response and transport (GO:0080161; 35 auxin responsive DEGs, associated and transport related genes).
<figure><img src="image_5.png" /><figcaption>**Fig. 1** Altered transcription of different pathways in *Eucalyptus grandis* linked to each stage of colonization by *Pisolithus microcarpus*. (a) Bar graph showing the number of significant positively and negatively expressed genes at each timepoint (grey bars and black bars, respectively; *P* < 0.05). (b) Venn diagram showing the number of common differentially expressed genes (DEGs) between all five timepoints and all genes unique to individual timepoints. Total number of DEGs within each category are shown. (c) Dendrogram demonstrating the relative similarity of each timepoint based on DEG profiles. (d) Principal component analysis of normalized and r-log transformed transcriptomic data for each replicate across the time course of colonization. (e) Heatmap showing the relative expression of genes most responsible for the separation of the timepoints during *P. microcarpus* colonization of *E. grandis* along the principal component one (PC1) axis with a percentage contribution to PC1 > 0.25.</figcaption></figure>

Finally, we considered the enriched processes associated with the 109 persistently DEGs (Table 2). Genes associated with terpenoid and tetraterpenoid metabolism, lipid metabolism, as well as monocarboxylic acid biosynthesis were found to be abundant.

Based on the consistent enrichment of defence and stress response genes we extracted those genes contributing to the enriched GO defence term, annotated as TIR-NBS-LRR gene family, NB-ARC domain-containing resistance proteins, and dirigent-like protein family. Of the 234 genes across these classes,
<table><caption>**Table 1** Significantly enriched processes associated with root function during the colonization of *Eucalyptus grandis* by *Pisolithus microcarpus*.</caption><thead><tr><th>Timepoint</th><th>Expression relative to axenically grown roots</th><th>Number of significantly regulated genes (P < 0.05)</th><th>Enriched GO terms<br>KEGG (GPROFILER; P < 0.05)</th><th>Enriched GO biological processes<br>(PLANTREGMAP; P < 0.01)</th></tr></thead><tbody><tr><td rowspan="2">Pre-symbiosis (24 h indirect contact)</td><td>Positive</td><td>251</td><td>N/A</td><td>ADP binding<br>Defence response<br>Carotenoid metabolism<br>Phenylpropanoid processes</td></tr><tr><td>Negative</td><td>61</td><td>N/A</td><td>Cellulose synthase<br>Glucosyltransferase<br>ADP binding</td></tr><tr><td rowspan="4">24 h (post-contact)</td><td>Positive</td><td>1670</td><td>MAPK signalling pathway<br>Tyrosine metabolism<br>Carotenoid biosynthesis<br>Biosynthesis of secondary metabolites</td><td>Response to abiotic stress<br>Transcription factor activity<br>Transporters<br>Response to abscisic acid (ABA)<br>Response to hormone<br>Carotenoid biosynthesis</td></tr><tr><td>Negative</td><td>1810</td><td>Metabolic pathways<br>DNA replication<br>Biosynthesis of secondary metabolites<br>Flavonoid biosynthesis<br>Nitrogen metabolism</td><td>Catalytic activity<br>DNA replication<br>Response to abiotic stress</td></tr><tr><td>Positive</td><td>1612</td><td>Protein processing in endoplasmic reticulum<br>Galactose metabolism</td><td>Response to abiotic stress<br>Response to ABA<br>Voltage-gated ion channel activity</td></tr><tr><td>Negative</td><td>1688</td><td>Phenylpropanoid biosynthesis<br>Metabolic pathways<br>Biosynthesis of secondary metabolites<br>Glyoxylate and dicarboxylate metabolism<br>Zeatin biosynthesis</td><td>Response to abiotic stress<br>Oxidoreductase activity<br>Cell wall biosynthesis</td></tr><tr><td>1 wk (post-contact)</td><td>Positive</td><td>1814</td><td>N/A</td><td>ADP binding<br>Ribonucleotide binding<br>Nucleotide binding<br>Response to stress<br>Defence response<br>Signal transduction</td></tr><tr><td rowspan="2">2 wk (post-contact)</td><td>Negative</td><td>1217</td><td>Ribosome</td><td>Plastid</td></tr><tr><td>Positive</td><td>570</td><td>Phenylpropanoid biosynthesis</td><td>Oxidoreductase activity<br>Phenylpropanoid biosynthesis<br>Nutrient reservoir activity<br>Extracellular region and cell membrane</td></tr><tr><td></td><td>Negative</td><td>352</td><td>Porphyrin metabolism<br>Metabolic pathways<br>Biosynthesis of secondary metabolites<br>Carbon fixation</td><td>Oxidoreductase activity<br>Response to abiotic stress<br>Carotenoid biosynthesis<br>Plastid</td></tr></tbody></table>

Selected enriched KEGG pathways (GPROFILER; P < 0.05) and gene ontology (GO) biological processes (PLANTREGMAP; P < 0.05) identified amongst the significantly regulated *E. grandis* genes. N/A indicates that no significantly enriched terms were found in this category.

163 were significantly differentially regulated in at least one time-point (Fig. 2). The TIR-NBS-LRR and NB-ARC domain-containing genes were mostly upregulated across the time course while the dirigent-like proteins were nearly all downregulated.

## Genes involved in carotenoid biosynthesis were significantly enriched and differentially regulated throughout colonization

We investigated if there were overrepresented metabolic pathways differentially expressed throughout the whole of the colonization process. For this analysis, we looked for KEGG metabolic

pathway gene enrichment within all 6687 DEGs. Of the overrepresented metabolic pathways related to root biology, we found that carotenoid and zeatin biosynthesis pathways exhibited a mixture of both positive and negative gene expression patterns while the phenylpropanoid biosynthesis pathway, metabolism of glyoxylate and dicarboxylate compounds, and flavonoid biosynthesis were universally repressed (Fig. 3a). Fig. 3(b) shows that overrepresented genes pertaining to cellular components were most commonly associated with the cellular membrane and plastid components.

Given the persistence of different carotenoid genes throughout all our analyses, and the fact that plastid related functions were
<table><caption>**Table 2** Enrichment analysis of biological processes from the 109 significant, persistently differentially expressed genes (DEGs) during *Pisolithus microcarpus* colonization of *Eucalyptus grandis*.</caption><thead><tr><th>Gene ontology (GO) identifier (ID)</th><th>GO term</th><th>Total number of annotated genes in genome</th><th>Number of genes within our DEG dataset</th><th>Number of genes expected in a random sample</th></tr></thead><tbody><tr><td>GO:0006952</td><td>Defence response</td><td>1028</td><td>11</td><td>3.14</td></tr><tr><td>GO:0043531</td><td>ADP binding</td><td>791</td><td>10</td><td>2.57</td></tr><tr><td>GO:0016119</td><td>Carotene metabolic process</td><td>10</td><td>2</td><td>0.03</td></tr><tr><td>GO:0045735</td><td>Nutrient reservoir activity</td><td>113</td><td>4</td><td>0.37</td></tr><tr><td>GO:0030145</td><td>Manganese ion binding</td><td>114</td><td>4</td><td>0.37</td></tr><tr><td>GO:0005576</td><td>Extracellular region</td><td>583</td><td>7</td><td>1.54</td></tr><tr><td>GO:0008610</td><td>Lipid biosynthetic process</td><td>359</td><td>6</td><td>1.1</td></tr><tr><td>GO:0042214</td><td>Terpene metabolic process</td><td>19</td><td>2</td><td>0.06</td></tr><tr><td>GO:0016109</td><td>Tetraterpenoid biosynthetic process</td><td>23</td><td>2</td><td>0.07</td></tr><tr><td>GO:0016117</td><td>Carotenoid biosynthetic process</td><td>23</td><td>2</td><td>0.07</td></tr><tr><td>GO:0044255</td><td>Cellular lipid metabolic process</td><td>469</td><td>6</td><td>1.43</td></tr><tr><td>GO:0006950</td><td>Response to stress</td><td>2325</td><td>15</td><td>7.09</td></tr><tr><td>GO:0072330</td><td>Monocarboxylic acid biosynthetic process</td><td>203</td><td>4</td><td>0.62</td></tr><tr><td>GO:0016108</td><td>Tetraterpenoid metabolic process</td><td>31</td><td>2</td><td>0.09</td></tr><tr><td>GO:0016116</td><td>Carotenoid metabolic process</td><td>31</td><td>2</td><td>0.09</td></tr><tr><td>GO:0006629</td><td>Lipid metabolic process</td><td>700</td><td>7</td><td>2.14</td></tr><tr><td>GO:0016469</td><td>Proton-transporting two-sector ATPase complex</td><td>57</td><td>2</td><td>0.15</td></tr></tbody></table>

Results based on output using the gene enrichment tool PLANTREGMAP (*P* < 0.01; Tian *et al.*, 2020).

also enriched, we further investigated the expression of the carotenoid pathway as a whole. From this analysis, *E. grandis* homologues to the carotenoid biosynthesis enzymes from *Arabidopsis* were identified and their expression mapped onto the carotenoid biosynthesis pathway (Fig. 4a). The first committed step in carotenoid biosynthesis involves the production of phytoene that, via a series of desaturations and isomerizations is converted into lycopene, that then diverges into the production of epsilon- and beta-carotenoids and downstream xanthophylls. This first step is controlled by PHYTOENE SYNTHASE (PSY; Fig. 4a); of the three genes encoded by *E. grandis*, one gene was significantly downregulated (Eucgr.F02914; c. four-fold; Table S5) during the pre-symbiotic phase followed by a steady loss of this repression throughout the physical integration timepoints (i.e. 24 h, 48 h and 1-wk post-inoculation). Commensurate with the earliest timepoint, we found that during pre-symbiosis in between *E. grandis* roots and *P. microcarpus* a reduction in carotenoid concentration in the host tissues was observed (Table 3). Following this stage, genes encoding the following steps of the synthesis of lycopene were generally induced. This was most significantly and strongly seen for *ZETA-CAROTENE DESATURASE* (ZDS), which were significantly regulated across all points of colonization (*P* < 0.05; Fig. 4a; Table S5) and represent the carotenoid annotated genes found within the 109 core gene set of *E. grandis*' response to *P. microcarpus* (Fig. 1b). Following this, the biosynthetic pathway branches into the formation of strigolactones and apocarotenoids, β-carotenoids, and lutein. Genes coding for enzymes towards the lutein (*CYP97A/C*) and strigolactone pathways (*CCD7/8*) were not significantly regulated during the early timepoints of the colonization process (Fig. 4a; Table S5). Transcription of genes encoding enzymes responsible for apocarotenoid synthesis (*CCD1/4*) were much more variable in expression across colonization, but the only gene showing

significant expression (Eucgr.D02555) was repressed > four-fold during fungal integration into the roots (*P* < 0.05). Conversely, we found a significant induction of β-CAROTENE 3-HYDROZYLASE (BCH; Fig. 4a) during the integration stages of fungal colonization as well as the following steps involving *ZEAXANTHIN EPOXIDASE* (ZEP; Fig. 4a; Table S5; *P* < 0.05). This latter observation was coupled with a significant reduction in the expression of *VIOLAXANTHIN DE-EPOXIDASE* (VDE; Eucgr.B02180; Fig. 4a; *P* < 0.05) which encodes the enzyme which reverses the flux in metabolism at this point of the pathway. The step of neoxanthin to xanthoxin governed by 9-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED; Fig. 4a) also exhibited genes that were universally significantly induced by the colonization process (*P* < 0.05). Taken together, the majority of gene expression profiles in this pathway would suggesting a flux towards biosynthesis of ABA in the later stages of this pathway.

### Abscisic acid biosynthesis and responsive genes are altered by symbiosis

In addition to highlighting the carotenoid pathway, enrichment analysis of the biological processes GO terms in Table 1 revealed a large number of genes in the early stages of physical interaction between *P. microcarpus* and *E. grandis* were associated with ABA signalling responses, an apocarotenoid compound and plant hormone. As our transcriptomic results may suggest an increased degradation of the xanthophyll precursors via increased *NCED* expression leading to ABA synthesis during the integration of the fungus into the root apoplastic space, we quantified ABA in roots of *E. grandis* during pre-symbiosis through to the latter stage of fungal integration into the root (i.e. 1 wk). In comparison to axenically grown roots, we found that ABA concentrations
<figure><img src="image_6.png" /><figcaption>**Fig. 2** *Eucalyptus grandis* NBS-LRR genes are induced across colonization by *Pisolithus microcarpus*. Heatmap showing the relative expression of 234 defence related genes within the NB-ARC, NBS-LRR, and dirigent-like classes during *P. microcarpus* colonization of *E. grandis*.</figcaption></figure>

increased > 50× after 24 h of contact with the fungus, an induction that fell to between 9- and 16-fold induction at the later timepoints (Fig. 4b). The increase in ABA concentrations was significant at all timepoints with the exception of pre-symbiosis (Table 4), but was found to be highest at 24 and 48 h post-contact with the fungus. As these tissues are a hybrid between plant and fungus, we also analysed the concentration of ABA in extramatrical mycelium (EMM) adjacent to the root as an estimate of fungal contribution to ABA synthesis. We found that ABA concentrations in comparison to axenically grown fungal cultures mildly increased during pre-symbiosis and then fell to approximately half the original concentration by 48 h post-contact (Fig. 4c), although at no timepoint were the differences

in concentrations found to be significantly different from axenic controls (Table 4). Across the samples taken, plant tissues had up to 400× higher concentrations of ABA than the EMM (Table 4).

In order to gauge the transcriptional alterations of ABA-associated genes, we profiled the expression of genes known to be either ABA-induced (Fig. 4d) or ABA-repressed (Fig. 4e) across the time course of colonization based on gene classifications by Pornsiriwong *et al*. (2017). We found a total of 72 *E. grandis* genes orthologous to *A. thaliana* genes known to be induced by ABA, 45 of which were found to be significantly differentially regulated in *E. grandis* during colonization (Fig. 4d). This gene set includes the PP2C genes Eucgr.G02960, Eucgr.J02003 and Eucgr.A01486, the latter of which is included in the gene set most significantly separating the integration stages of our transcriptomic time course from pre-symbiosis and functional symbiosis (Fig. 1e). Of the 75 *E. grandis* genes orthologous to *A. thaliana* ABA-repressed genes, 35 were found to be differentially regulated (Fig. 4e).

Exogenous abscisic acid increases Hartig net development of *P. microcarpus*

Owing to the ABA responsive gene analysis and ABA accumulation during fungal integration into the root, we tested the effect of exogenous ABA upon colonization to see if this would alter colonization potential of *P. microcarpus*. In all treatments we found that *E. grandis* was able to form mycorrhizal roots with the formation of Hartig nets (Fig. 5a–d). The absolute number of colonized roots per plant was not significantly impacted (Fig. 5e). Despite a trend towards increased lateral rooting when the seedlings were grown on higher levels of ABA, we did not observe a significant increase in the number of observed lateral roots (P = 0.07). Similarly, mantle formation was unaffected by the ABA treatments (Fig. 5f). Therefore, exogenous ABA does not appear to improve the formation of mantle-encased roots during the colonization of *E. grandis*. Since ABA responsive genes were most frequently induced during the physical integration stage of colonization, and as the concentration of ABA within the roots was highest during the earliest stages of colonization, we reasoned that Hartig net development may have been affected. Based on measurements of Hartig net penetration depth into the root apoplastic space, we found that Hartig net formation was significantly promoted when the plant was treated with exogenous levels of ABA ≥ 0.25 µM (Fig. 5g). We also observed that, in addition to the fungus penetrate deeper into the root system, epidermal plant cells were more elongated and that the fungus appeared to be in greater contact with cortical cells at higher concentrations of ABA (Fig. 5d). Therefore, we measured the difference in fungal contact with root cell between the treatments (i.e. the amount of direct contact between fungal hyphae and plant cells in a transverse cross-section of a colonized root normalized to root circumference). We found that exogenous application of ABA significantly increased the contact area between the fungus and plant cells at all ABA treatments (Fig. 5h). We further wished to verify if increased contact area resulted in a greater fungal biomass within the root tissues. As mantle thickness was not
<table><caption>(a)</caption><thead><tr><th rowspan="2">KEGG</th><th rowspan="2">Term ...</th><th rowspan="2">P<sub>adj</sub></th><th colspan="3">stats</th><th colspan="3">Expression pattern</th></tr><tr><th>-log10(P<sub>adj</sub>)</th><th>0</th><th>&le;16</th><th>Over-expression</th><th>Mixed expression</th><th>Repressed</th></tr></thead><tbody><tr><td>Biosynthesis of secondary metabolites</td><td>KEGG:01110</td><td>1.254 &times; 10<sup>-5</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Metabolic pathways</td><td>KEGG:01100</td><td>1.112 &times; 10<sup>-4</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Carotenoid biosynthesis</td><td>KEGG:00906</td><td>5.289 &times; 10<sup>-4</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Phenylpropanoid biosynthesis</td><td>KEGG:00940</td><td>4.537 &times; 10<sup>-3</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Zeatin biosynthesis</td><td>KEGG:00908</td><td>1.140 &times; 10<sup>-2</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Glyoxylate and dicarboxylate metabolism</td><td>KEGG:00630</td><td>2.088 &times; 10<sup>-2</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Flavonoid biosynthesis</td><td>KEGG:00941</td><td>3.947 &times; 10<sup>-2</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th rowspan="2">GO:CC</th><th rowspan="2">Term ...</th><th rowspan="2">P<sub>adj</sub></th><th colspan="3">stats</th><th colspan="3">Expression pattern</th></tr><tr><th>-log10(P<sub>adj</sub>)</th><th>0</th><th>&le;16</th><th>Over-expression</th><th>Mixed expression</th><th>Repressed</th></tr></thead><tbody><tr><td>Cell periphery</td><td>GO:0071944</td><td>3.429 &times; 10<sup>-25</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell part</td><td>GO:0044464</td><td>4.060 &times; 10<sup>-18</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell</td><td>GO:0005623</td><td>6.972 &times; 10<sup>-18</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plasma membrane</td><td>GO:0005886</td><td>6.536 &times; 10<sup>-17</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Thylakoid</td><td>GO:0009579</td><td>1.458 &times; 10<sup>-14</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plastid thylakoid</td><td>GO:0031976</td><td>6.647 &times; 10<sup>-14</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Apoplast</td><td>GO:0048046</td><td>1.256 &times; 10<sup>-13</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plastid thylakoid membrane</td><td>GO:0055035</td><td>2.080 &times; 10<sup>-13</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plastid part</td><td>GO:0044435</td><td>1.321 &times; 10<sup>-12</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Organelle subcompartment</td><td>GO:0031984</td><td>7.749 &times; 10<sup>-11</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plastid</td><td>GO:0009536</td><td>1.091 &times; 10<sup>-8</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell-cell junction</td><td>GO:0005911</td><td>8.828 &times; 10<sup>-8</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell junction</td><td>GO:0030054</td><td>8.828 &times; 10<sup>-8</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plasmodesma</td><td>GO:0009506</td><td>8.828 &times; 10<sup>-8</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Symplast</td><td>GO:0055044</td><td>9.752 &times; 10<sup>-8</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>External encapsulating structure</td><td>GO:0030312</td><td>1.125 &times; 10<sup>-7</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cell wall</td><td>GO:0005618</td><td>1.542 &times; 10<sup>-7</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Plastid envelope</td><td>GO:0009526</td><td>3.291 &times; 10<sup>-7</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<caption>**Fig. 3** Carotenoid biosynthesis genes are significantly overrepresented within the core transcriptomic response of *Eucalyptus grandis* roots during colonization by *Pisolithus microcarpus*. (a) *Eucalyptus grandis* root-associated metabolic pathways significantly enriched during *P. microcarpus* colonization of *E. grandis* (*P* < 0.05). (b) Gene ontology enrichment analysis of root-associated cellular component pathways in *E. grandis* during *P. microcarpus* colonization (*P* < 0.05).</caption>

significantly affected (Fig. 5f), we undertook a qPCR based approach to quantify the ratio between the transcription of stably expressed fungal genes vs stably expressed plant genes. We found that the addition of ABA led to a significantly higher ratio of functional transcripts vs plant transcripts at the ABA concentration tested (64.4% ± 16% increase), further supporting the results of ABA supporting fungal growth within roots (Fig. 5i). As pH of the medium was unaffected by the addition of ABA (Fig. S1b), and as equivalent amounts of solvent were added to each medium, it is unlikely that the difference in phenotype was due to these factors. Therefore, ABA appears to be involved in the accommodation of *P. microcarpus* into the apoplastic space of *E. grandis* roots.

Discussion

The accommodation of a fungal symbiont within root tissues requires the coordination of a large number of plant metabolic and developmental pathways (Martin *et al.*, 2016). Along with morphological changes, differential host gene expression has been previously linked to the stage of colonization in other model EcM interactions (Duplessis *et al.*, 2005; Le Quéré *et al.*, 2005; Sebastiana *et al.*, 2014; Bouffaud *et al.*, 2020). In this article, we have identified how gene expression changes over time within *E. grandis* roots undergoing *P. microcarpus* colonization. Within our current dataset, the pre-symbiotic and functional stages exhibited the least amount of DEGs as compared to axenic roots, while
each of the timepoints during fungal integration showed a high number of unique DEGs. This is likely due to the fact that at the final timepoint, the fungus is no longer actively invading the root and the plant is largely restricted to nutrient exchange, thus necessitating fewer regulated pathways. The difference between

pre-symbiosis and 24 h post-contact is likely due to the addition of nonsecreted proteins of the fungus (e.g. cell wall proteins) coming into contact with key receptors on the plant host that activate a number of microbe sensitive pathways. Investigating those biological processes most significantly enriched found that

<figure><img src="image_7.png" /></figure>
**Fig. 4** Abscisic acid (ABA) concentration increases and ABA responsive genes are significantly regulated during *Pisolithus microcarpus* colonization of *Eucalyptus grandis*. (a) *Eucalyptus grandis* carotenoid biosynthetic pathway with accompanying heatmaps showing log<sub>2</sub>-fold change in the expression of genes encoding key enzymes in the pathway. The colour and dash style of the arrows link to the same colour/dash style of boxes around each heatmap serve to match the expression of each enzyme-encoding gene to their specific metabolic step. For each enzyme-encoding gene, five timepoints of expression are presented corresponding to pre-symbiosis followed by 24 h, 48 h, 1 wk, and 2 wk post-contact, respectively, from left to right. Pathway map adapted from the KEGG carotenoid biosynthesis reference pathway (Kanehisa & Goto, 2000; Kanehisa *et al.*, 2017, 2019) and all enzymes are labelled with their gene abbreviation as follows: PSY, phytoene synthase; PDS, 15-cis-phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CrtISO, prolycopene isomerase; LCYE, lycopene epsilon-cyclase; LCYB, lycopene beta-cyclase; BCH, β-carotene 3-hydroxylase; CYP97A/C, carotenoid epsilon hydroxylase A/C; CCD1/4/7/8, carotenoid cleavage diosgenase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NXS, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase. Fold change expression values with annotation of significance vs axenically grown *E. grandis* roots can be found in Supporting Information Table S5. (b) Fold change in the concentration of ABA in *E. grandis* roots, as compared to axenically grown roots, during pre-symbiosis or during colonization by *P. microcarpus*; *n* = 4, ppb, parts per billion, ±SE. (c) Fold change in the concentration of ABA in *P. microcarpus* extra-radical mycelium in physical contact with *E. grandis* roots, as compared to axenically grown *P. microcarpus* mycelium, during pre-symbiosis or during colonization by *P. microcarpus*; *n* = 3, ppb, parts per billion, ±SE. (d) Heatmap to show the relative expression of *E. grandis* genes orthologous to *Arabidopsis thaliana* genes known to be positively regulated by ABA during *P. microcarpus* colonization. (e) Heatmap to show the relative expression of *E. grandis* genes orthologous to *A. thaliana* genes known to be negatively regulated by abscisic acid during *P. microcarpus* colonization.

<table><caption>**Table 3** The majority of tested carotenoids in *Eucalyptus grandis* roots are significantly reduced during pre-symbiosis with *Pisolithus microcarpus*.</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="7">Carotenoid content (µg g<sup>-1</sup> fresh weight)</th></tr><tr><th>Neo</th><th>Vio</th><th>Ant</th><th>Lut</th><th>Zea</th><th>β-Car</th><th>Total</th></tr></thead><tbody><tr><td>Control</td><td>1.011 (±0.095)</td><td>1.226 (±0.130)</td><td>0.473 (±0.070)</td><td>1.512 (±0.263)</td><td>0.182 (±0.028)</td><td>0.589 (±0.087)</td><td>4.993 (±0.648)</td></tr><tr><td>Pre-symbiosis</td><td>0.768 (±0.036)</td><td>0.914 (±0.090)</td><td>0.297 (±0.015)</td><td>0.883 (±0.058)</td><td>0.083 (±0.005)</td><td>0.422 (±0.022)</td><td>3.368 (±0.155)</td></tr><tr><td>P-Value</td><td>0.010</td><td>0.065</td><td>0.004</td><td>0.006</td><td>0.001</td><td>0.024</td><td>0.005</td></tr></tbody></table>

Quantified carotenoids after 48 h of pre-symbiotic setup include Neo (neoxanthin), Vio (violaxanthin), Ant (antheraxanthin), Lut (lutein), Zea (zeaxanthin), β-Car (β-carotene); ±SE. Significant differences between control (i.e. axenically grown) roots and pre-symbiotic roots was determined (Student's *t*-test; *n* = 4 biological replicates).

<table><caption>**Table 4** Abscisic acid concentration in *Eucalyptus grandis* roots undergoing colonization by *Pisolithus microcarpus* are significantly induced during fungal integration into the root.</caption><thead><tr><th></th><th>Axenic</th><th>Pre-symbiosis</th><th>24 h</th><th>48 h</th><th>1 wk</th></tr></thead><tbody><tr><td>Root</td><td>0.021 (±0.006)</td><td>0.023 (±0.001)</td><td>1.097 (±0.318)</td><td>0.213 (±0.051)</td><td>0.511 (±0.178)</td></tr><tr><td>P-Value (root)</td><td></td><td>0.75</td><td>5.1 × 10<sup>-4</sup></td><td>2.4 × 10<sup>-4</sup></td><td>2.2 × 10<sup>-3</sup></td></tr><tr><td>EMM</td><td>0.003 (±0.001)</td><td>0.005 (±0.002)</td><td>0.003 (±0.000)</td><td>0.001 (±0.001)</td><td>0.002 (±0.001)</td></tr><tr><td>P-Value (EMM)</td><td></td><td>0.45</td><td>0.72</td><td>0.19</td><td>0.27</td></tr></tbody></table>

Quantification of ABA in roots and in extramatrical mycelium (EMM). Concentrations in parts per billion ±SE. Statistics are based on change from axenic control (Student's *t*-test; *n* = 4 for roots, *n* = 3 for EMM).

defence response genes, linked to various biotic and abiotic stimuli, were activated at different stages in the colonization process, much like previous reports (Duplessis *et al.*, 2005; Le Quéré *et al.*, 2005; Larsen *et al.*, 2015; Plett *et al.*, 2015b; Liao *et al.*, 2016). Most prominent within our defence-related gene dataset were 163 significantly regulated LRR/PR proteins, a class highlighted by Liao *et al.* (2016) to also be differentially regulated in various *Pinus* species in response to colonization by members of the *Suillus* genus. Sebastiana *et al.* (2014) report that these defence-related DEGs might be involved with fungal species recognition. There were, also, a number of dirigent-like defence genes that were downregulated throughout the fungal integration stage; these genes are associated with cell-wall reinforcement and their repression may be required to enable growth of *P. microcarpus* into the root apoplastic space (Sebastiana *et al.*, 2014; Li *et al.*, 2017). In terms of the biosynthesis of metabolic products, using GO, we found that genes involved in the synthesis of

phenylpropanoids, terpenoids, and flavonoids were also enriched within our transcriptional profiling of *E. grandis*. Similar gene classes have been found to be regulated in EcM interaction with *Populus*, *Pinus*, and aspen, although the direction and magnitude of how these pathways are impacted is not consistent (Weiss *et al.*, 1997; Li *et al.*, 2014; Liao *et al.*, 2014; Plett *et al.*, 2015b; Basso *et al.*, 2019). Transcriptomically Weiss *et al.* (1997), and metabolomically Plett *et al.* (2021a), found that phenylpropanoid and flavonoids were increased during the early stages of EcM colonization, compounds that Behr *et al.* (2020) were able to positively correlate with EcM colonization. In our results, however, we found these pathways were downregulated during the physical integration stage of colonization in *E. grandis* before being positively expressed during the functional symbiosis phase. Terpenoid associated genes, meanwhile, identified as being enriched during EcM colonization of host roots both here and by previous research (Behr *et al.*, 2020) are known to be partially responsible
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Exogenous abscisic acid (ABA) enhances Hartig net development. (a) Representative image of a transverse cross-section of an *Eucalyptus grandis* lateral root colonized by *Pisolithus microcarpus* without the addition of ABA (M, mantle; HN, Hartig net; CC, cortical cell), with the addition of 0.1 µM ABA (b), 0.25 µM ABA (c), or 0.5 µM ABA (d); Bar, 10 µm in each image. (e) Percentage of total lateral roots in contact with *P. microcarpus* that were colonized under each concentration of exogenous ABA. No significant difference was found (Student's *t*-test; ±SE; *n* = 6). (f) Average mantle thickness across each treatment. No significant difference was found (Student's *t*-test; ±SE; *n* = 6). (g) Average Hartig net depth across each treatment; asterisk (*), significant difference from 0 µM ABA (Student's *t*-test; ±SE, *n* = 6). (h) Average percentage change in fungal/plant contact area, defined as the surface area of plant cells in primary contact with fungal hyphae normalized to root diameter, between 0 µM ABA and the addition of a given quantity of ABA; asterisk (*), significant difference from 0 mM ABA (*P* < 0.05); ±SE, *n* = 6. (i) Average fungal : plant ratio in excised mycorrhizal root tips. The ratio was calculated based on the transcript copy number of two fungal control genes normalized by the transcript copy number of a plant control gene between with either 0 or 0.25 µM ABA treated root systems; asterisk (*), significant difference from 0 mM ABA (*P* < 0.05); ±SE, *n* = 4.</figcaption></figure>

for differentiation between different stages of the colonization
process (Liao *et al.*, 2014; Basso *et al.*, 2019).

Terpenoids encompass a wide array of secondary metabolites
including volatile signalling molecules, and carotenoids, and
provide precursors for phytohormone biosynthesis (Huang &

Osbourn, 2019). The comparative lack of information regarding
the roles of carotenoids during EcM colonization, combined with
the prominence of these pathways within our investigation, led
us to focus on understanding their roles during EcM symbiosis.
While these metabolites play a significant role in supporting AM
root colonization (Welling et al., 2016; Felemban et al., 2019), their role in EcM systems has largely been investigated in the shoots of colonized hosts (Sebastiana et al., 2013; Welling et al., 2016). Within roots, AM fungal colonization leads to changes in lutein, lycopene, β-carotene and zeaxanthin (Welling et al., 2016). While we observed reduced carotenoid levels in E. grandis roots during pre-symbiosis, we found that the transcription of genes enzymes leading to the synthesis of lycopene, β-carotene and zeaxanthin were induced while the genes related to lutein synthesis were repressed. Following on from β-carotene, the observed higher expression of ZEAXANTHIN EPOXIDASE and 9-CIS-EPOXYCAROTENOID DIOXYGENASE could reveal a higher biosynthetic flow away from β-carotene and zeaxanthin, the preferred substrates required for the biosynthesis of the apocarotenoids stigolactones and zaxinone that promote AM symbiosis (Felemban et al., 2019; Ablazov et al., 2020). Our results would suggest that the biosynthetic flux of pre-existing intermediates was funnelled instead toward the production of ABA. In future it would be interesting to study if these alterations in carotenoid metabolic flux could further favour EcM colonization at the expense of competing AM fungi in dual host plants.

The plant hormone ABA is another product of the carotenoid pathway that our transcriptomic results would suggest is altered during EcM integration. ABA is known to have influence over various aspects of plant development and cellular metabolism, including mycorrhizal associations. There are a number of possible mechanisms by which ABA may be fostering beneficial plant–microbe interactions. In AM symbioses, ABA in low doses has been shown to support colonization through the signalling PROTEIN PHOSPHATASE 2A while higher levels of ABA negatively impact colonization through an inhibition of mycorrhizal induced host calcium oscillations (Charpentier et al., 2014). ABA also interacts with other plant hormones, and its inhibition of ethylene signalling has been proposed as another pathway by which AM colonization is promoted (Herrera-Medina et al., 2007; Martín-Rodríguez et al., 2011). ABA may also influence colonization of AM fungi through the stimulation of antioxidant pathways (Lou et al., 2021). Reports of the accumulation of, and signalling due to, ABA during EcM symbiosis are conflicting. Some studies found that there was no change to ABA concentration in EcM host roots (Coleman et al., 1990; Calvo-Polanco et al., 2019; Lou et al., 2021), while another, like ours, found significant increases (Luo et al., 2009). Certain reports have not found ABA signalling genes to be induced during EcM symbiosis (Rincón et al., 2005), possibly due to only considering a select number of ABA-responsive genes, while other studies found that an additional stress is required before ABA signalling was affected (Luo et al., 2009). In our system, we found that ABA concentration increased during the initial stages of fungal integration into the root. As a commensurate increase in ABA was not observed in the extraradical mycelium, and as the concentration of ABA in the mycelium was found to be orders of magnitude lower than in the colonized roots, this would suggest that the site of synthesis is in the plant cells. This was supported by the repression of PSY (which is negatively regulated by elevated ABA concentrations; Welsch et al., 2008), by the upregulation of E. grandis genes in

the majority of the biosynthetic pathway previous to ABA pro-
duction, and by the expression of ABA responsive genes in the
host root cell. Of note, we found that the differential expression
of ABA-responsive genes was not uniform: only certain genes
were induced/repressed as would be anticipated by elevated ABA
concentrations. The mechanism by which this selectivity may
occur is unknown, although as *Pisolithus* encodes effector-like
proteins (Kohler *et al.*, 2015; Plett *et al.*, 2020) it may have the
capability of targeting specific components of host hormone sig-
nalling pathways to effect this pattern of differential gene tran-
scription. Our results with exogenous application of ABA also
support the hypothesis that the hormone is involved in the con-
trol of *P. microcarpus* integration into the root during the Hartig
net formation. This is similar to the proposed role of ABA in sup-
porting AM fungal colonization of host roots by Zhang *et al.*
(2019), highlighting a function that may be conserved across the
evolution of mutualistic plants–fungal interactions.

Based on this study, we conclude that the colonization of *E. grandis* by an EcM partner significantly changes the regulation of a large portion of the host gene repertoire, particularly the carotenoid pathway and its derivative ABA metabolic signalling pathway. Future research should be undertaken to understand the triggers, consequences, and regulation of carotenoid derived apocarotenoids, as well as the role of ABA during mutualistic EcM associations in both *in vitro* and more natural soil-based systems. This would give us a more holistic understanding concerning the roles of carotenoids and hormones during both EcM and AM plant–microbe interactions.

Acknowledgements

The authors thank M Mikhael and D Wojtalewicz at the Western Sydney University Mass Spectrometry facilities for assistance with the carotenoid and hormone analyses, and the Western Sydney University Confocal Bio-Imaging Facility for access to its instrumentation. This research was funded by the Australian Research Council for a Discovery grant to ICA, JMP and FM (DP160102684) and the US Department of Energy Joint Genome Institute (CSP1953). This research was also supported by the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01) and the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Programme, the Office of Biological and Environmental Research in the US Department of Energy (DOE) Office of Science. The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US DOE under contract no. DE-AC02-05CH11231.

Author contributions

RAH, JMP, KLP, JW-B, ICA, FM and TJ designed research. MW, AL, VN, IVG performed RNA-sequencing and RAH, JMP, KLP and TJ performed the analysis. RAH, DC, SA, CIC performed the carotenoid and hormone analyses. RAH, KLP, TJ,
CIC and JMP wrote the article. All authors discussed the results and edited the manuscript.

ORCID

Ian C. Anderson https://orcid.org/0000-0002-3507-163X

Sidra Anwar https://orcid.org/0000-0002-0500-1649

Christopher I. Cazzonelli https://orcid.org/0000-0003-3096-3193

Donovin Coles https://orcid.org/0000-0003-3824-1881

Igor V. Grigoriev https://orcid.org/0000-0002-3136-8903

Richard A. Hill https://orcid.org/0000-0002-2678-796X

Francis Martin https://orcid.org/0000-0002-4737-3715

Vivian Ng https://orcid.org/0000-0001-8941-6931

Jonathan M. Plett https://orcid.org/0000-0003-0514-8146

Krista L. Plett https://orcid.org/0000-0001-6422-3754

Mei Wang https://orcid.org/0000-0002-3240-7994

Johanna Wong-Bajracharya https://orcid.org/0000-0001-6119-8645

Data availability

The data that supports the findings of this study are openly avail-
able in the National Centre for Biotechnology Information
(NCBI) Short Read Archive at https://www.ncbi.nlm.nih.gov/
sra, reference numbers SRX5064672-SRX5064689.

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## Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Supporting control experiments to verify RNA-sequencing and influence of abscisic acid on medium pH.

**Table S1** Primers used in the course of this study.

**Table S2** The log₂-fold change and statistical analysis of the 6687 *Eucalyptus grandis* differentially expressed genes (DEGs) during the colonization by *Pisolithus microcarpus*.

**Table S3** List of the *Eucalyptus grandis* genes that most differentiate between colonization stages.

**Table S4** *Eucalyptus grandis* genes whose expression most influences separation of the principal component analysis.

**Table S5** Fold change in gene expression levels of carotenoid-associated biosynthetic enzymes during the colonization of *Eucalyptus grandis* by *Pisolithus microcarpus*.

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.

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