# Facultative symbiosis with a saprotrophic soil fungus promotes potassium uptake in American sweetgum trees

Long Peng, Xiaoliang Shan, Yuzhan Yang, Yuchen Wang, Irina Druzhinina, Xueyu Pan, Wei Jin, Xinghua He, Xinyu Wang, Xiaoguo Zhang, et al.

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## To cite this version:

Long Peng, Xiaoliang Shan, Yuzhan Yang, Yuchen Wang, Irina Druzhinina, et al. Facultative symbiosis with a saprotrophic soil fungus promotes potassium uptake in American sweetgum trees. Plant, Cell and Environment, 2021, 44 (8), pp.2793-2809. 10.1111/pce.14053. hal-04000987

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Submitted on 22 Feb 2023

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ORIGINAL ARTICLE

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WILEY

# Facultative symbiosis with a saprotrophic soil fungus promotes potassium uptake in American sweetgum trees

Long Peng<fnref n="1" />,<fnref n="2" /> ID | Xiaoliang Shan<fnref n="1" />,<fnref n="2" /> ID | Yuzhan Yang<fnref n="2" /> ID | Yuchen Wang<fnref n="1" />,<fnref n="2" /> ID |

Irina S. Druzhinina<fnref n="3" /> ID | Xueyu Pan<fnref n="2" /> | Wei Jin<fnref n="2" /> | Xinghua He<fnref n="2" /> ID |

Xinyu Wang<fnref n="2" /> | Xiaoguo Zhang<fnref n="2" /> | Francis M. Martin<fnref n="4" />,<fnref n="5" /> ID | Zhilin Yuan<fnref n="1" />,<fnref n="2" /> ID

**Correspondence**

Francis M. Martin, INRA, UMR 1136 INRA-University of Lorraine, Interactions Arbres/Microorganismes, Laboratory of Excellence ARBRE, INRA-Nancy, 54280, Champenoux, France.

Email: francis.martin@inra.fr

Zhilin Yuan, State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 10091, China.

Email: yuanzl@caf.ac.cn

**Funding information**

Fundamental Research Funds for the Central Non-profit Research of Chinese Academy of Forestry, Grant/Award Number: CAFYBB2020QB002; Martin's lab is funded by the Laboratory of Excellence ARBRE, Grant/Award Number: ANR-11-LABX-0002_ARBRE; National Natural Science Foundation of China, Grant/Award Numbers: 31722014, 31901290

**Abstract**

Several species of soil free-living saprotrophs can sometimes establish biotrophic symbiosis with plants, but the basic biology of this association remains largely unknown. Here, we investigate the symbiotic interaction between a common soil saprotroph, *Clitopilus hobsonii* (Agaricomycetes), and the American sweetgum (*Liquidambar styraciflua*). The colonized root cortical cells were found to contain numerous microsclerotia-like structures. Fungal colonization led to increased plant growth and facilitated potassium uptake, particularly under potassium limitation (0.05 mM K⁺). The expression of plant genes related to potassium uptake was not altered by the symbiosis, but colonized roots contained the transcripts of three fungal genes with homology to K⁺ transporters (ACU and HAK) and channel (SKC). Heterologously expressed *ChACU* and *ChSKC* restored the growth of a yeast K⁺-uptake-defective mutant. Upregulation of *ChACU* transcript under low K⁺ conditions (0 and 0.05 mM K⁺) compared to control (5 mM K⁺) was demonstrated in planta and in vitro. Colonized plants displayed a larger accumulation of soluble sugars under 0.05 mM K⁺ than non-colonized plants. The present study suggests reciprocal benefits of this novel tree-fungus symbiosis under potassium limitation mainly through an exchange of additional carbon and potassium between both partners.

**KEYWORDS**

endophytes, mycorrhizal fungi, RNA-seq, root-fungus symbiosis, soil saprotrophs, soluble sugars

# 1 | INTRODUCTION

The interaction with diverse symbiotic microbes is pivotal for improving the fitness of most land plants (Almario et al., 2017). In terrestrial

ecosystems, these symbiotic associations can range anywhere between mutually beneficial, such as mycorrhizal mutualism, with no apparent benefit or detriment to each other (i.e., commensalism), or harmful to one but beneficial to other (i.e., parasitism) (Plett &

<footnote n="1">¹State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, China</footnote>

<footnote n="2">²Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, China</footnote>

<footnote n="3">³Fungal Genomics Group, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China</footnote>

<footnote n="4">⁴INRA, UMR 1136 INRA-University of Lorraine, Interactions Arbres/Microorganismes, Laboratory of Excellence ARBRE, INRA-Nancy, Champenoux, France</footnote>

<footnote n="5">⁵Beijing Advanced Innovation Centre for Tree Breeding by Molecular Design, Institute of Microbiology, Beijing Forestry University, Beijing, China</footnote>
Martin, 2018). In particular, the contribution of several types of mycorrhizal interactions to plant nutrient acquisition and stress tolerance responses is well recognized (Genre, Lanfranco, Perotto, & Bonfante, 2020; Martin, Kohler, Murat, Veneault-Fourrey, & Hibbett, 2016; Qin, Druzhinina, Pan, & Yuan, 2016; Strullu-Derrien, Selosse, Kenrick, & Martin, 2018). In the past few years, metabarcoding surveys revealed a high diversity of root-associated endophytic fungi, which can confer a suite of beneficial effects upon plants (Terhonen, Blumenstein, Kovalchuk, & Asiegbu, 2019). Thus, there is an emerging consensus that both mycorrhizal and endophytic fungi constitute an important component of the root mycobiome (Peay, Kennedy, & Talbot, 2016).

More interestingly, besides mycorrhizal and endophytic fungi interacting mutualistically with plant roots (Gibert, Tozer, & Westoby, 2019), mounting evidence suggests that a wide range of soil free-living saprotrophic fungi—spanning the lower to higher fungi— can also frequently establish biotrophic interactions with plants (Bonito et al., 2016; Kariman, Barker, & Tibbett, 2018; Selosse, Schneider-Maunoury, & Martos, 2018; Terhonen et al., 2019; Thoen et al., 2020; Zuccaro, Lahrmann, & Langen, 2014). These beneficial associations can take place in three main ways. First, some saprotrophic fungi could act as facultative mycorrhizal partners along the so-called saprotrophy-mycorrhizal continuum. For instance, some achlorophyllous orchids (Selosse et al., 2010) or ericaceous plant (e.g., Vaccinium corymbosum. L) (Grelet et al., 2017; Vohnik et al., 2012) recruit saprotrophic Mycena or other hitherto unknown fungal associates for developing orchid mycorrhizas and ericoid mycorrhizas. Similarly, some basidiomycetous wood-decayers can dif- ferentiate ectomycorrhiza-like structures in roots from Picea abies and Pinus sylvestris plantlets (Baldrian & Kohout, 2017; Smith, Finlay, Ste- nlid, Vasaitis, & Menkis, 2017; Vasiliauskas, Menkis, Finlay, & Stenlid, 2007). Secondly, common soil saprotrophs may enter into the endophytic phase with plant roots, but without forming typical mycor- rhizal structures, thus referred to here as the saprotrophy- endophytism continuum. For instance, Mortierella elongata (Mortierellomycotina) is a common species of soil fungi that is also recognized as a plant growth-promoting endophyte (Becker, 2017; Liao et al., 2019). Thirdly, several soil saprotrophs could switch to end- ophytism and then adopt a mycorrhizal habitat. The Morchella (Pezizales, Ascomycota) (Baynes, Newcombe, Dixon, Castlebury, & O'Donnell, 2012; Hobbie, Weber, & Trappe, 2001) and sebacinaceous fungi (Agaricomycetes, Basidiomycota) (Weiß, Waller, Zuccaro, & Selosse, 2016) occurring in forest ecosystems are perfect examples of this category. The “waiting room” hypothesis was proposed to explain the transition from endophytic to mycorrhizal habitats (Strullu-Derrien et al., 2018; Vanegas-León et al., 2019). Together, these three catego- ries of interactions exemplify novel types of plant-fungal mutualisms, mainly arising from the ecological plasticity of soil saprotrophs (Selosse et al., 2018; Zhou, Li, Huang, & Dai, 2018). Although the ubiquity and ecological significance of dual niches occupied by a diverse array of saprotrophic fungi are known, detailed information on their mode of action is missing and little is known on physiological and molecular mechanisms driving the nutrient exchanges taking place

in these beneficial associations. Recent work reinforced the assump-
tion that these fungi may benefit their partners via distinct mecha-
nisms (Kariman, Barker, Jost, Finnegan, & Tibbett, 2014).

Clitopilus (Entolomataceae, Agaricales, Basidiomycota) is consid-
ered as a common saprotrophic genus (Raj & Manimohan, 2018), and
several members in the Entolomataceae family can form
ectomycorrhizae with a wide range of tree species (Agerer &
Waller, 1993). Although field investigations suggested that Clitopilus
orcella and C. subvilis are putative ectomycorrhizal fungi (ECM associ-
ated with Quercus petraea (Southworth, 2013) and Q. robur
(Howe, 1964; Keizer & Arnolds, 1994), as well as other trees in the
Fagaceae family (Molina, Massicotte, & Trappe, 1992), the ability of
Clitopilus spp. to form ectomycorrhizas has not yet been confirmed
through in vitro ectomycorrhizal synthesis. Hence, this genus is still
considered saprotrophic (Rinaldi, Comandini, & Kuyper, 2008).

In our previous study, an isolate of *Clitopilus hobsonii*, has been isolated from ectomycorrhizal root tips of several oak tree species (*Quercus* spp.) (Jin, Peng, Zhang, Sun, & Yuan, 2019; Jin, Wang, Sun, & Yuan, 2020), while this fungus had been originally found in the leaf litter layer of *Quercus* (Orton, 1960). We failed to establish an ectomycorrhizal interaction between *C. hobsonii* and *Quercus* spp. in vitro, but we documented its beneficial effects on *Quercus* growth (Jin et al., 2019). Nitrogen utilization patterns suggested that amino acids and bovine serum albumin are the preferred forms of nitrogen for sustaining *C. hobsonii* growth (unpublished data), likely as a proxy for saprotrophic fungal-mediated organic nitrogen mineralization (Wu, 2011). Based on this information, we suggested that *C. hobsonii* has a dual ecological lifestyle, that is, litter saprotroph and beneficial root endophyte.

We further investigated whether *C. hobsonii* could establish mutualistic interactions with other tree species. Our results showed that *C. hobsonii* had a broad host range and was able to colonize roots of the phylogenetically- distant tree lineages, American sweetgum (*Liquidambar* *styraciflua*) and poplars (*Populus* sp.). The former tree is well known for its commercial importance for the paper industry (Ďurkovič & Lux, 2010). The rapid germination of *L. styraciflua* seeds (usually non-dormant) allowed us to easily produce rooting culture of plantlets on a large scale and develop a binary *L. styraciflua*-C. *hobsonii* gnotobiotic interaction system for studying the molecular and physiological mechanisms underlying this beneficial interaction. Therefore, to begin investigating the nature of the saprotrophy-biotrophy continuum, the *L. styraciflua*-C. *hobsonii* biotrophic interaction model was chosen as an initial focus.

Built on this root-fungal association system, the current study had two objectives: first, to characterize the infection structures differentiated in roots by *C. hobsonii*; second, to explore whether this fungus could assist in plant growth and nutrient uptake. More specifically, if evidence for the latter was found, we aimed to provide more mechanistic insight into the mode of action of nutrient uptake. It is well known that mycorrhizal plants can acquire nutrients via the direct pathway as well as the mycorrhizal way, especially under nutrient-poor conditions (Fellbaum, Mensah, Pfeffer, Kiers, & Bücking, 2012). It is reasonable to assume that similar pathways exist in the L.
*styraciflua-C. hobsonii* biotrophic interactions. If so, this prompts two related questions. First, which pathway will lead to improved nutrient acquisition? Or can both pathways operate simultaneously? Second, whether nutrient limitation will confer augmented fitness to the symbiotic association?

## 2 | MATERIALS AND METHODS

### 2.1 | Generation of the gnotobiotic tree plantlets

In October 2018, mature seeds of *L. styraciflua* were collected from Fuyang (Zhejiang Province, China; 30°05′N, 119°95′E). Seeds were air-dried for 1 week to remove excess water and then excised from the utricles. Next, seeds were surface sterilized with ethanol (75%, vol/vol) for 30 s, and NaClO (2%, vol/vol) for 8 min. Finally, the seeds were immersed in ethanol (90%, vol/vol) for 30 s to remove the NaClO residue and rinsed at least five times with sterile distilled water. The clean seeds were sown in plastic germinated trays (LWH: 56 cm × 38 cm × 12 cm), filled with sterilized wet sand, for at least 3 weeks in the growth chamber under the normal watering regime (average 500 ml per week) and with 12 hr light day⁻¹ at the temperature of 25°C and the relative humidity of 50%. Plantlets that were 5–6 cm in height were selected and their roots were cut off. The excised stem cuttings were surface sterilized with 1% (vol/vol) HgCl₂ for 12 min and washed five times with sterile distilled water, then transplanted into sterile glass tubes containing woody plant rooting medium. The woody plant rooting medium was modified from Woody Plant Medium (WPM) (Lloyd & Mccown, 1980), with sucrose (2%, wt/vol) and 0.3 mg/L of indole-3-butanoic acid (IBA). After 3 weeks, those plantlets with well-developed roots were transplanted into Phytatrays II™ (Sigma Chemical Co., LWH: 11.4 cm × 8.6 cm × 10.2 cm) to carry out the fungus inoculation experiments.

### 2.2 | Fungal strain, culture conditions and identification

*Clitopilus hobsonii* isolate QYL-10 was originally recovered from ectomycorrhizal root tips of *Q. lyrata* (Jin et al., 2019). The isolate was kept as mycelial suspensions, in 20% (vol/vol) glycerol at -80°C, until its use. Vegetative mycelium was routinely grown on potato dextrose agar (PDA) medium for a week at 25°C. Taxonomic affiliation of the isolate was confirmed by multi-locus phylogeny using nuclear 28S rRNA, RNA Polymerase II second-largest subunit (rpb2), and translation elongation factor-1α (tef1) sequences (Morgado, Noordeloos, & Hausknecht, 2016). Maximum likelihood phylogenetic tree reconstruction confirmed that isolate QYL-10 clustered together with several currently known *C. hobsonii* species with moderate support (bootstrap value = 72) in 1000 bootstrap replicates run (Figure S1). The partial 28S, rpb2 and tef1 sequences obtained for *C. hobsonii* have been deposited in GenBank under accession numbers MG211139, MN092372 and MN092373, respectively.

### 2.3 | Experimental design

We first monitored the plantlet growth and fungal colonization of roots by co-culturing the *C. hobsonii* with *L. styraciflua* tree plantlets. Twelve gnotobiotic plantlets were transferred into a sterile Phytatray II™. Given the fast growth rate of the fungus, we used only 1/10 strength Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) as a substrate for the inoculation experiment. Plantlets were kept in a growth chamber under a 12-hr light/12-hr dark photoperiod at 25°C. After 10 days, five mycelial plugs (5 mm in diameter) of actively growing fungus were placed in the middle of each Phytatray II™ (Sigma, St. Louis, USA), while the fungus-free controls were mock-inoculated with sterile PDA plugs. A total of 32 phytatrays (384 plantlets) were used. To measure the plants' growth traits, chlorophyll contents and nutrient concentrations, the plantlets (three replicates, 12 plantlets per replicate per treatment) were harvested one-month post-inoculation. We also sequenced the transcriptome (RNA-seq) of their roots to detect symbiosis-induced transcriptional changes over time. The root samples with or without fungal treatment were collected at two-time points: 14 and 28 days after inoculation (DAI) with three phytatrays per treatment as three biological replicates (i.e., 12 RNA samples in total). In addition, a small portion of the remaining colonized roots was used to observe the fungal colonization pattern.

Our preliminary results showed that interaction with the *C. hobsonii* led to an increase in potassium uptake by *L. styraciflua* plantlets, while nitrogen or phosphorus uptake was not altered. Thus, we hypothesized that potassium status likely affected the outcome of *L. styraciflua-C. hobsonii* interaction. To assess the effect of potassium level on the *L. styraciflua-C. hobsonii* interaction, potassium concentration of the initial 1/10 MS medium (2 mM K⁺) was adjusted to normal K⁺ (NK, 5 mM K⁺) and low K⁺ (LK, 0.05 mM K⁺) (Liu, Ren, Chen, Wang, & Wu, 2013), respectively. The latter can be defined as the K⁺-limiting condition for plant growth. Plantlets were grown on these two slightly modified MS media, using the same growing environmental conditions and inoculation protocol as described above. A total of 42 phytatrays (504 plantlets) were used in this experiment. To measure the plant growth, the concentrations of soluble sugar and the expression level of K⁺ nutrient-related genes, the plantlets (three replicates, 12 plantlets per replicate per treatment) were harvested 40 days post-inoculation. Experiments of plant inoculation test and K⁺ nutrient-related gene expression analysis by quantitative real-time PCR (qRT-PCR) were repeated at least two times, yielding similar results, so the result of one representative experiment was shown.

We further surveyed the in vitro expression patterns of three fungal K⁺ nutrition-related genes. In this case, apart from two potassium levels (5 mM, and 0.05 mM), a medium depleted in potassium (0 mM) was also used as a treatment. Briefly, five mycelium-colonized PDA plugs were each placed in a 250-ml flask containing 100-ml of the liquid medium, which were then placed in an incubator without shaking at 25°C for 25 days in the dark. The medium was composed of sucrose (10 g/L), NaH₂PO₄ (0.5 g/L), NH₄Cl (0.5 g/L), MgSO₄ 7H₂O (0.5 g/L) and ferric citrate (5 mg/L). The final K⁺ concentrations were
respectively adjusted to 0.375 g/L (5 mM), 0.745 mg/L (0.05 mM) and 0 mg/L (0 mM), by adding KCl. After 1 month of static culture, the mycelium samples were washed with sterile distilled water at least five times to eliminate adsorbed external K⁺ and harvested by filtration through filter paper.

## 2.4 | Microscopic observation of fungal colonization pattern in roots

To observe the fungal infection structures in planta, colonized roots were stained using trypan blue (Padamsee et al., 2016). Colonized roots were also sectioned for microscopic observations. Roots were fixed in FAA (formaldehyde: acetic acid: ethanol, 10/50/5, vol/vol/vol). The tissues were dehydrated using a mix of ethanol and tertiary butanol series, clarified in xylol, and embedded in paraffin. Ten-micrometre transversal sections were cut with a Thermo Scientific HM 325 rotary microtome, and stained with lactophenol aniline blue (0.01%, wt/vol), with ten sections viewed per root sample. All the squash mounts and sections were viewed at 400 to ×1,000 magnification under a light microscope (Axio Scope A1) equipped with an AxioCam MRc5 digital camera and ZEN2 software (Carl Zeiss, Jena, Germany).

## 2.5 | Phenotypic and physiological measurements

To determine total root length, root surface area, root volume, number of root tips of intact roots were rinsed with distilled water and scanned (Epson Expression 11000XL, Epson, CA, USA) with an image analysis software (WinRHIZO Pro version, Regent Instruments, Quebec, Canada). To measure leaf chlorophyll content, 0.2-g fresh leaves were immersed overnight in a 25-ml chlorophyll extraction solution (95%, vol/vol, ethanol). Total chlorophyll content was determined as described by Wellburn (1994), by using the GENESYS 20 spectrophotometer (Thermo Scientific, USA) to measure absorbance at 650 nm and 665 nm.

To determine the nitrogen (N), phosphorus (P) and potassium (K) concentrations of plants, plantlets that were grown on 1/10 MS, NK and LK media were harvested and dried at 65°C for 16 hr to a constant weight. Dried plantlets including roots, stems and leaves were then ground into powder using an oscillating grinder (MM400, Retsch, Haan, Germany). The Kjeldahl digestion procedure was used for total nitrogen determination (Horneck & Miller, 1997), and the P concentration was quantified by the Mo-Sb colourimetric method after digestion with H₂SO₄-H₂O₂ (Bowman, 1988; Sekiya, 1970). An ICP-AES system of iCAP 6,300 (Thermo Fisher Scientific Inc., San Jose, USA) was used for the K⁺ determination. To determine the concentrations of soluble sugar, plantlets grown on NK and LK media were harvested. The total soluble sugar content was quantified by the anthrone sulfuric acid method according to Hansen and Møller (1975).

To measure transient K⁺ flux at the root elongation and mature zones, root segments with 3-cm apices were sampled from the

inoculated and control plantlets (28 DAI) grown under the LK condition. The whole roots were transferred into a Petri dish (35 mm in diameter, Corning Inc., Corning, NY, USA) containing 5 ml of fresh measuring solution (composed of 0.01 mM KCl, 0.1 mM NaCl, 0.1 mM MgCl₂ and 0.1 mM CaCl₂ at pH 5.7) for 10 min. The steady fluxes of K⁺ were recorded, for 5 min, from the meristem (500 µm from the tip), elongation (3 mm from the tip) and mature (15 mm from the tip) root zones. These K⁺ fluxes were measured with non-invasive micro-test technology (NMT100 Series, Younger USA LLC, Amherst, USA) done at the NMT Service Centre (Xuyue Beijing Sci. & Tech. Co., Ltd., Beijing, China) with oxygen microelectrodes (tip diameter: 2–4 µm, XY-DJ-501, Younger, USA) and K⁺ microelectrodes (tip diameter: 2–4 µm, XY-DJ-502, Younger, USA). The K⁺ fluxes were calculated by Fick's law of diffusion, J = −D (dc/dx), where J represents the ion flux, dc/dx is the ion concentration gradient and D is a diffusion coefficient. At least six individuals per treatment were analysed.

## 2.6 | Transcriptome profiling and qRT-PCR

Total RNA from the plantlets' and fungal cultures was extracted using the Trizol reagent (Invitrogen, CA, USA), by following the manufacturer's instructions. The total RNA quantity and purity were analysed with a Bioanalyzer 2,100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA). The RNA was then reverse-transcribed to create the final cDNA library, by following the protocol of the mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA). The average insert size for the paired-end libraries was 300 bp (± 50 bp). We performed the paired-end sequencing on an Illumina Hiseq 4,000 platform (LC Sciences, Hangzhou, China).

For data processing, Cutadapt and in-house Perl scripts were used to remove the reads that contained adaptor contamination, low-quality bases and any undetermined bases (Martin, 2011). After removing the adaptors and low-quality data, a total of ~604 million filtered reads were obtained from the 12 samples collected at 14 and 28 DAI, with the number of reads ranging from 41.56 to 56.19 million filtered reads for each sample (Table S2). Then the sequence quality was verified using Fast QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), including the Q30 and GC-content of the clean data. De novo assembly of the transcriptome was performed with Trinity 2.4.0 (Grabherr et al., 2011). The longest transcript in clusters was promoted as the reference "gene" sequence (aka unigene). All assembled unigenes were aligned against the GenBank non-redundant (Nr) protein database (http://www.ncbi.nlm.nih.gov/), Gene ontology (GO) (http://www.geneontology.org), SwissProt (http://www.expasy.ch/sprot/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/), eggNOG (http://eggnogdb.embl.de/) and Pfam (https://pfam.xfam.org/) using DIAMOND (Buchfink, Xie, & Huson, 2015) with a threshold of E value <0.00001. We used the Viridiplantae protein database to identify *L. styraciflua* proteins because the *L. styraciflua* genome sequence was not publicly available at the time of the study. The Salmon method (Patro, Duggal, Love, Irizarry, & Kingsford, 2017) was used to assess the relative expression level of
unigenes by calculating their TPM (Transcripts Per Million) for normal-
ization (Mortazavi, Williams, McCue, Schaeffer, & Wold, 2008). The
K⁺ nutrition-related gene expression levels were visualized in
 heatmaps as log2-transformed TPM values. The heatmaps were con-
structed by the gplot package of R software. The differentially-
expressed unigenes (DEUs) were designated as those with log2 (fold
change) > 1 or log2 (fold change) < -1 that had statistical significance
(i.e., *p* value < 0.05) using the R package "edgeR" (Robinson, McCar-
thy, & Smyth, 2010). Next, GO and KEGG enrichment analyses were
again performed on the DEUs by in-house perl scripts. All unigenes
were deposited in the NCBI and can be accessed in the Short Read
Archive (SRA) under accession number SRR10199935,
SRR10199934, SRR10199933, SRR10199932, SRR10199931,
SRR10199930, SRR10199929, SRR10199928, SRR10199927,
SRR10199926, SRR10199925, SRR10199924.

To validate the DEUs obtained by the RNA-seq, fragments of six
genes relevant to plant and fungal K⁺ nutrition were cloned and sub-
jected to qRT-PCR analysis. The designed primers used for this assay
are listed in Table S1. All the qRT-PCR amplifications were performed
with SYBR Premix Ex Taq™ II (Perfect Real Time; Takara Biotechnol-
ogy Co., Ltd, Dalian, China), according to the manufacturer's instruc-
tions. The amplifications were carried out in 20-µl reaction system
consisting of 10-µl of SYBR Premix Ex Taq™ II, 0.8 µl each of forward
and reverse primers (10 µmol/L), 7.8-µl of nuclease-free water, 0.4-µl
of ROX and 1-µl cDNA (1:10 diluted with nuclease-free water). The
reactions were performed in a QuantStudio 7 Flex Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA) and the ensuing
data analysed using QuantStudio Real-Time PCR Software (Thermo
Fisher Scientific, San Jose, CA, USA). A melting curve was recorded at
the end of each run to detect those primers generating non-specific
PCR products. The genes *tef1* and *tubulin* from *C. hobsonii*, and *CAP*
and *tubA* from *L. styraciflua*, served as the internal standards. Three
independent biological replicates per sample, and three technical repli-
cates per biological replicate, were used.

## 2.7 | Phylogenetic and functional analysis of fungal K⁺ nutrition related genes

To confirm the role of three fungal genes potentially involved in K⁺
nutrition (i.e., *ChACU*, *ChHAK* and *ChSKC*), we cloned their complete
open reading frame (ORF) based on the transcriptomic data from
cDNA library of *C. hobsonii* (data not shown). The primers for amplify-
ing *ChACU*, *ChHAK* and *ChSKC* were listed in Table S1.

The multiple alignments of the predicted amino acid sequences of
ACU, HAK and SKC from several basidiomycetous fungi (Table S4)
were performed by using Multiple Sequence Alignment of DNAMAN
software 6.0. The ACU family is so far exclusive to fungi, while the
HAK transporter and SKC channel are present in both fungi and plants
(Haro & Benito, 2019). Therefore, we constructed the phylogenetic
trees using the neighbour-joining method (MEGA version 6.0) to
reveal the genetic relatedness of the three genes from *C. hobsonii* with
those from other organisms. A bootstrap analysis of 1,000 replicates

was performed. The list of proteins from different species used in this
analysis was provided in Table S5.

To verify if the ChACU, ChHAK and ChSKC contribute directly to K⁺ uptake in the fungus and indirectly benefit host plant potassium acquisition, their full-length coding sequences were introduced into yeast expression vector pYES-2, and the vector was constructed using Trelief Sosoo Cloning Kit (Tsingke Biotech Co., Ltd, Beijing, China), according to the manufacturer's instructions. All primers used for vector construction were detailed in the Table S1. The resulting vectors were transformed into a K⁺-uptake-defective yeast mutant strain R5421 (MATα ura3-52 leu2 trk1Δhis3Δ200his4-15 trk2Δ1::pCK64), in which the two endogenous K⁺ transporter genes (TRK1 and TRK2) were deleted (Gaber, Styles, & Fink, 1988). Transformation of yeast cell was performed using the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol and growth assays were carried out in synthetic K⁺-free medium [yeast nitrogen base without amino acids, ammonium sulphate or potassium (ForMedium™, UK)] supplemented with various amounts of KCl.

## 2.8 | Statistical analysis

The normality and variance homogeneity of all phenotypic and physi-
ological data sets were evaluated by the Shapiro-Wilk's and Levene's
tests, respectively. All data sets were subjected to different statistical
tests depending on the data. Significant differences (the fresh weight,
height, chlorophyll content, N, P, K⁺ concentrations) between inocu-
lated and un-inoculated plantlets grown on 1/10 MS, and significant
differences (K⁺ influx) between inoculated and un-inoculated plantlets
grown under the LK condition were estimated with a Student's t test
at *p* ≤ 0.05 using the GraphPad Prism program version 6.0 (San
Diego, California, USA). Significant differences (the fresh weight,
potassium contents, root analysis, concentrations of soluble sugars,
relative gene expression) between inoculated and un-inoculated plant-
lets respectively grown under the LK or NK condition, and significant
differences (relative gene expression) among mycelium under three
potassium levels (NK, LK, without K⁺) were estimated with a one-way
analysis of variance (ANOVA) was performed using IBM SPSS Statis-
tics 20 software program (SPSS Inc., http://www.spss.com.cn). Each
ANOVA was followed by the Tukey's post hoc test at *p* ≤ 0.05. All
data were expressed as mean values with standard deviations (SD).

# 3 | RESULTS

## 3.1 | Colonization of roots and anatomy of the *L. styraciflua*–*C. hobsonii* association

During the colonization of *L. styraciflua* roots, *C. hobsonii* cannot form ectomycorrhizal structures, such as the intraradicular Hartig net and fungal mantle. Except for cap cells, the meristem, the differentiating and elongating zones of colonized root tips were heavily colonized by microsclerotia-like structures, which were likely formed from
aggregates of spherical hyphal cells (Figure 1a,b,e). The hyphae often
spread into the cortical cells and occasionally the extraradical hyphae
were found at the root surface (Figure 1c,d). Transverse sectioning
revealed that the fungus developed oval-to-spherical vesicle-like
structures occurring intracellularly, in the endodermal cells, close to
the vascular cylinder (Figure 1f-h).

3.2 | C. hobsonii promotes plant growth, root development and K⁺ absorption

Next, we investigated the effects of C. hobsonii colonization on the
growth of L. styraciflua plantlets. We found a conspicuous phenotypic
and physiological changes occurring in this symbiotic association. Spe-
cifically, root colonization by C. hobsonii enhanced the growth of L.
styraciflua plantlets grown on 1/10 MS medium (2 mM K⁺), as shown
by the increased fresh weight, plant height, leaf number, total root
length, total root area and total root volume of, and in almost all cases,
differences between control and inoculated groups were statistically
significant (p = 0.001, 0.072, 0.009, 0.037, 0.028 and 0.051, respec-
tively) (Figure 2a,b and Table 1). In addition, the total chlorophyll,
chlorophyll a and chlorophyll b contents of inoculated plants were
also significantly higher than non-inoculated plants (Table 2).

The K⁺ concentration (expressed as g/g DW) in the whole inocu-
lated plantlets was enhanced compared to non-inoculated plantlets

(Student's two-sample t test, *p* = 0.079) (Figure 2c). Notably, the interaction resulted *in approx.* Two-fold increase of the plantlet K⁺ content (expressed as mg/plant) compared with un-inoculated controls (Student's two-sample t test, *p* = 0.009) (Figure 2d). By contrast, N and P contents in plantlets were not significantly affected by the fungal colonization (Figure 2d). Even though the N and P concentrations in C. hobsonii treatment were significantly lower than controls (Figure 2c). These changes likely result from the dilution effects of N and P in the higher plant biomass.

3.3 | Transcriptomic analysis of potassium nutrition in the symbiotic association.

To gain further insight into gene expression patterns induced by C. hobsonii colonization, we performed a RNA-seq-based transcriptomic analysis, focusing on potassium uptake genes. De novo assembly of RNA reads from colonized roots produced a total of 26,324 unigenes (Figure S2). A total of 51 DEUs encoding K⁺ transporters/channel proteins were identified according to GO functional annotation (Table S3). Of particular interest are the ACU, HAK and SKC genes having a very high expression in colonized roots at two stages, but are barely expressed in controls, suggesting that these genes likely belong to the fungal partner (see below). The expression levels of most remaining transcripts are not differentially expressed, although a few

<figure><img src="image_4.png" /></figure>

**FIGURE 1** Infection structures established by C. hobsonii in the *L. styraciflua* root tissues. Roots were cleared and stained with trypan blue. The elongating zones (a) and the meristem (b) of the root tip are colonized by hyphae forming microsclerotia-like structures. The hyphae spread in cortical cells and occasionally hyphae extend to the surface of the root (c, d). Bulbous intracellular hyphae of *C. hobsonii* are observed in root cells (e). The transverse section reveals that *C. hobsonii* develops intracellular oval-to-spherical vesicle-like structures in endodermal cells (f-h). Co, cortex endodermal cell; Ep, epidermal cells; Vc, vascular cylinder cell
<figure><img src="image_5.png" /><figcaption>**FIGURE 2** The beneficial effects of *C. hobsonii* on *L. styraciflua* growth. Growth of non-inoculated and inoculated plantlets on 1/10 MS medium (2 mM K⁺) in phytatrays II (a, b); concentration (c) or content (d) of N, P, and K⁺ in *L. styraciflua* plantlets in 1/10 MS medium. Six phytatrays approximately (72 plantlets) were used for measuring the plant growth and nutrient contents; plantlets were harvested one-month post-inoculation. Error bars represent SD of replicates. Statistical analysis of the data is performed using the Student's t test to determine significant differences between non-inoculated and inoculated plantlets. *p* values indicating the significant level are shown [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<table><caption>**TABLE 1** Effects of *C. hobsonii* inoculation on the growth and root development of *L. styraciflua* plantlets</caption><thead><tr><th></th><th>Fresh weight (mg)</th><th>Plant height (cm)</th><th>Primary root length (cm)</th><th>Number of leaves</th><th>Total root length (cm)</th><th>Total root area (cm²)</th><th>Total root volume (cm³)</th></tr></thead><tbody><tr><td>Control</td><td>71.48 ± 11.71</td><td>2.87 ± 0.30</td><td>1.89 ± 0.61</td><td>5.83 ± 0.87</td><td>8.92 ± 3.31</td><td>1.104 ± 0.368</td><td>0.0111 ± 0.0037</td></tr><tr><td>Inoculation</td><td>79.88 ± 13.52</td><td>2.98 ± 0.36</td><td>1.92 ± 0.55</td><td>6.46 ± 0.72</td><td>11.36 ± 4.47</td><td>1.363 ± 0.419</td><td>0.0133 ± 0.0040</td></tr><tr><td>p value</td><td>0.00059</td><td>0.07239</td><td>0.79267</td><td>0.00934</td><td>0.03712</td><td>0.02789</td><td>0.05073</td></tr></tbody></table>

Note: The plantlets were grown in the Phytatrays containing 1/10 MS medium (2 mM K⁺). Value are mean Values are M ± SD of replicates.

of them showed a slight upregulation (Figure 3a). Furthermore, the expression of genes coding for the CBL/CIPK complex regulating K⁺ uptake in plants (Ragel et al., 2015; Wang & Wu, 2017) was also not regulated by the fungal colonization (Figure S3). To validate this RNA-seq profiling, the expression of ACU, HAK and SKC and three additional genes related to plant K⁺ nutrition (KAB, TPK5 and TPKC) were

measured using qRT-PCR. As expected, the expression of KAB, TPK5 and TPKC was not significantly changed after colonization, and ACU, HAK and SKC transcripts were only captured in the root-fungal association (Figure S4).

The expression pattern of ACU, HAK and SKC genes raised the intriguing question as to whether these genes were activated in roots
<table><caption>TABLE 2 Comparison of the chlorophyll contents between the control and inoculation groups</caption><thead><tr><th></th><th>Chl a content<br>(mg/g FW)</th><th>Chl b content<br>(mg/g FW)</th><th>Carotenoid content<br>(mg/g FW)</th><th>Chl content<br>(mg/g FW)</th><th>Car/Chl</th><th>Chla/b</th></tr></thead><tbody><tr><td>Control</td><td>2.16 ± 0.03</td><td>0.868 ± 0.009</td><td>0.4419 ± 0.0120</td><td>3.03 ± 0.04</td><td>0.146 ± 0.003</td><td>2.493 ± 0.009</td></tr><tr><td>Inoculation</td><td>2.25 ± 0.04</td><td>0.897 ± 0.023</td><td>0.4427 ± 0.0096</td><td>3.15 ± 0.06</td><td>0.141 ± 0.002</td><td>2.511 ± 0.032</td></tr><tr><td>p value</td><td>0.0149429</td><td>0.0593509</td><td>0.919831</td><td>0.0206997</td><td>0.0144034</td><td>0.301387</td></tr></tbody></table>

Note: The plantlets were grown in the Phytatrays containing 1/10 MS medium (2 mM K⁺). Values are M ± SD of replicates.

<figure><img src="image_6.png" /><figcaption>FIGURE 3 Gene expression profile of K⁺ absorption and gene function of fungal K⁺ absorption. Heat maps displaying the changes in normalized gene expression of K⁺ transporter and channel at different infection stages (L. styraciflua after 14 days cultivation, L. styraciflua after 28 days cultivation, L. styraciflua 14 DAI, initial colonization, L. styraciflua 28 DAI, colonization completed). On the top bar, the colour scale bar indicates decreased (blue) and increased (red) levels of transcripts. The values given in the legend are relative expression-transformed ratios. The non-inoculated and inoculated plantlets were grown on 1/10 MS medium (2 mM K⁺) as mentioned above. The root samples were collected at 14 and 28 days after inoculation (DAI) from 36 plantlets per treatment with three biological replicates (i.e., 12 RNA samples in total) (a). Functional complementation of ChHAK, ChACU and ChSKC for K⁺ acquisition in the K⁺ uptake-defective yeast strain R5421. Growth status of R5421 cells expressing ChHAK, ChACU and ChSKC, and empty vector (pYES2), on synthetic K⁺-free medium supplemented with 5 mM and 2 mM KCl. Each transformant was precultured in liquid synthetic K⁺-free medium containing 100 mm KCl. The 1:10 serial dilutions of yeast cells were spotted on the synthetic K⁺-free medium and then incubated at 30°C for 10 days (b) [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

when interacting with the fungus or expressed by the fungal partner? We thus conducted a bioinformatics analysis to compare their amino acid sequences' similarities with all other known fungal sequences (Figure S5, S6, S7, S8, and Table S4). Multiple alignments showed that the three genes shared a high degree of protein sequence homology with fungal ACU (Alkali Cation Uptake ATPase, transporter), HAK (High-Affinity K⁺ uptake, transporter), and SKC (Shaker-like K⁺ channel), respectively (Figure S9, S10, and S11), which are widely present in basidiomycetous fungi (Haro & Benito, 2019). Furthermore, the phylogenetic trees suggested that the HAK transporter and SKC channel clustered together with other fungal genes, but were distantly related to those in plants (Figure S12 and Table S5). Both the PCR amplification and Sanger sequencing further suggested that ACU, HAK

and SKC belonged to C. hobsonii but not L. styraciflua (Figure S13). Thus, the role if these three genes, referred to here as ChACU, ChHAK and ChSKC, was further investigated.

## 3.4 | Functional characterization of ChACU, ChHAK and ChSKC in yeast

To explore the potential roles of ChACU, ChHAK and ChSKC in potassium nutrition, empty pYES-2 vector and pYES-2 vectors containing ChACU, ChHAK or ChSKC were respectively introduced into the auxotrophic yeast mutant strain R5421, which is defective in K⁺ uptake and could not grow under low-K⁺ conditions (Gaber et al., 1988). The
R5421 strain transformed with the control vector (R5421-pYES2) was able to grow at higher (5 mM K⁺) but not at lower (2 mM K⁺) K⁺ levels suggesting its defective phenotype due to the lack of K⁺ transport systems. Yeast cells transformed with the ChACU transporter (R5421-ChACU) were able to grow at 2 mM K⁺, suggesting that the ChACU partially complemented the defect in K⁺ uptake of the R5421 strain by driving K⁺ influx into the cells. Similarly, yeast cells transformed with the vector containing ChSKC and ChHAK were also able to grow at 2 mM K⁺, although ChHAK displayed relatively poor complement activity (Figure 3b).

## 3.5 | Association with C. hobsonii improves the potassium nutrition of L. styraciflua under K⁺-limiting conditions

The aforementioned results supported the idea that the fitness of the C. hobsonii-tree association most likely depended on the external potassium status. We thus assessed whether the fungus could confer more benefits to its host trees under potassium deprivation (Garcia et al., 2014; Wang et al., 2017). To address this hypothesis, we measured the impact of the symbiotic association under two potassium levels: NK (5 mM K⁺) and LK (0.05 mM K⁺). C. hobsonii colonization promoted plantlet growth, root development and K⁺ contents under both NK and LK conditions to a much greater extent than controls (Figure 4a-e). Notably, fungal colonization significantly enhanced the K⁺ concentration of whole plantlets under LK condition compared to the controls, and this remained unchanged under the NK condition (ANOVA, *F* = 5.776, *df* = 11, *p* = 0.021) (Figure 5a). The K⁺ content in inoculated plantlets under LK condition was much higher than in other three treatments, although the difference between colonized plants growing in the NK and LK conditions were not statistically significant (Figure 5b). Furthermore, fungal inoculation also significantly enhanced the K⁺ concentration of the shoots under the LK condition (ANOVA, *F* = 12.695, *df* = 11, *p* = 0.020) (Figure S14). These findings suggest that the beneficial role of the fungus on plant K⁺ acquisition is higher under LK than NK condition.

We further used the non-invasive micro-test technique (NMT) to monitor and compare K⁺ flux profiles in roots between non-inoculated and inoculated plantlets under LK condition. The K⁺ influx rate in meristemic root zones of colonized roots was remarkably higher than that of the control plantlets (Figure 6a). Similar patterns were observed in the root elongation and mature zones (Figure 6b,c). These results suggest that more K⁺ is absorbed by the colonized roots (across the meristematic zone, root elongation and mature zones (Student's two-sample *t* test, all *p* < 0.001) (Figure 6d).

## 3.6 | In vitro and *in planta* expression patterns of the fungal ChACU, ChHAK and ChSKC

Building on the above findings, it appears that the expression patterns of ChACU, ChHAK and ChSKC vary greatly under the LK and NK

conditions. To validate this hypothesis, we first assessed the expression levels of ChACU, ChHAK and ChSKC in planta under the NK and LK conditions. ChACU and ChHAK expression were higher under the LK than the NK condition, while the ChSKC had a stable expression under both conditions (Figure 7a-c).

We further ask whether high-level expression activity of those genes was induced by the LK condition in the absence of the plant partner, we investigated the behaviour of free-living C. hobsonii growing on three K⁺ levels. qRT-PCR showed that the ChACU and ChHAK were significantly upregulated either in the absence of K⁺ or under LK condition compared to the NK condition, while ChSKC was only activated in the absence of K⁺ (Figure 7d-f). Together, in vitro and *in planta* expression of ChACU, ChHAK and ChSKC showed similar patterns under K⁺ limiting condition, these results would suggest that LK condition facilitating K⁺ assimilation in the mycelium of C. hobsonii.

## 3.7 | C. hobsonii increases the soluble sugar content of L. styraciflua under K⁺-limiting conditions

Several studies have shown that photosynthetically-derived carbohydrates maintain root-fungal symbiosis (Kariman et al., 2018; Nehls, 2008). We thus evaluated the impact of fungal colonization on the soluble sugar content in plantlets. The inoculated plants grown under LK condition contained significantly higher contents of soluble carbohydrates compared to the noninoculated plants, while there was only a slight increase under the NK condition (ANOVA, *F* = 9.736, *df* = 15, *p* = 0.002) (Figure 4f).

# 4 | DISCUSSION

## 4.1 | C. hobsonii: A hidden beneficial tree biotrophic fungus

Several fungal lineages exhibit dual lifestyles that transition from saprotrophy to biotrophy (Davis, Franklin, Shaw, & Vilgalys, 2003; Glynou, Nam, Thines, & Maciá-Vicente, 2018; Promputtha et al., 2007; Rogers, 2000). Mounting evidence supports the view that a wide range of soil saprotrophic fungi has the potential to form manyle and Hartig net-like structures within tree roots (Koide, Sharda, Herr, & Malcolm, 2008; Smith et al., 2017; Vasiliauskas et al., 2007). However, our current understanding of the molecular mechanisms governing the biotrophic interactions between soil saprotrophs and their plant partners is still scarce.

In the present study, we established the Liquidambar styraciflua-Clitopilus hobsonii association in vitro to investigate this novel facultative biotrophic interaction. We showed that root colonization by the C. hobsonii led to the formation of microsclerotia-like structures that developed within the root cortical cells (Figure 1). Microsclerotia-like infection structures are common in the dark septate endophytes (DSEs)-root symbiotic associations (Qin et al., 2016). But these structures, as well as hyphae, are often melanized and so cannot be readily
<figure><img src="image_7.png" /><figcaption>**FIGURE 4** The beneficial effects of C. hobsonii on L. styraciflua plantlets growth under the LK (0.05 mM K⁺) conditions. Root total length (a), root surface area (b), root total volume (c), the numbers of root tips (d), fresh weight (e) of L. styraciflua plantlets with or without colonization under the LK and NK conditions. Plantlets were harvested 40 days post inoculation (DPI). Concentrations of soluble sugar in L. styraciflua plantlets (f) with or without colonization under the LK (0.05 mM K⁺) and NK (5 mM K⁺) conditions. Error bars represent SD of replicates. Different lowercase letters indicate significant differences. Similar results to those shown in a-e obtained in two additional experiments are provided in Figure S17 [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

stained with trypan blue. Thus, the *C. hobsonii* isolate *stricto* sensu
cannot be referred to as a DSE fungus. Similar structures have been
reported when the decomposer fungus *Sistotrema brinkmannii*
(Basidiomycota) colonizing fine roots (Potvin, Richter, Jurgensen, &
Dumroese, 2012). The observed intracellular structures suggest that
*C. hobsonii* differentiates a specific interface that may facilitate mutu-
alistic nutrient exchanges. Our findings suggest that the saprotrophic

C. hobsonii is able to develop an endophytic phase, but is not able to
differentiate ectomycorrhizal structures.

To determine whether the facultative biotrophy of C. hobsonii
conveys benefits to plants (Selosse et al., 2018; Smith et al., 2017), we
studied the impact of this endophytic fungus on growth and nutrient
uptake in Sweetgum plantlets. The plant-growth-promoting capacity
was confirmed by our results (Figure 2a,b).
<figure><img src="image_8.png" /></figure>

**FIGURE 5** Potassium limitation promotes K⁺ assimilation in *C. hobsonii*-tree association. Concentrations of K⁺ in *L. styraciflua* plantlets (a) or contents of K⁺ in individual *L. styraciflua* plantlet (b) with or without colonization under the LK and NK conditions; plantlets were harvested 40-day post-inoculation. Error bars represent SD of replicates. Different lowercase letters indicate significant differences. Similar results obtained in two additional experiments are provided in Figure S18 [Colour figure can be viewed at wileyonlinelibrary.com]

<figure><img src="image_9.png" /></figure>

**FIGURE 6** Effect of *C. hobsonii* colonization on the K⁺ fluxes of roots. The steady-state net K⁺ flux was measured from the root meristem (300–600 µm from the tip) (a), the elongation zone (1–3 mm from the tip) (b), the mature zone (10–15 mm from the tip) (c) and the mean rate of K⁺ fluxes in root zones after *C. hobsonii* inoculation under the LK condition (d). The magnitude of steady-state K⁺ fluxes was calculated by data recorded over a 300 s period. A total of approximately 20 plantlets were used for measuring K⁺ fluxes in the root; plantlets were harvested 40-day post-inoculation. Error bars represent SD of replicates. Different lowercase letters indicate significant differences [Colour figure can be viewed at wileyonlinelibrary.com]
<figure><img src="image_10.png" /><figcaption>**FIGURE 7** *In planta* and in vitro measurement of expression patterns of *ChACU*, *ChHAK*, *ChSKC* under the LK (0.05 mM K⁺), NK (5 mM⁺) and without K⁺ (only for in vitro analysis) conditions. Relative expression levels of the *ChACU* (a), *ChHAK* (b), *ChSKC* (c) in the root-fungal association under two K⁺ conditions (NK and LK). A total of approximately 12 phytatrays (144 plantlets) were used for measuring expression of genes; plantlets were harvested 40-day post-inoculation. Relative expression levels of *ChACU* (d), *ChHAK* (e), *ChSKC* (f) in mycelium under three K⁺ condition (NK, LK, and without K⁺). Three independent biological replicates per sample and three technical replicates per biological replicate were conducted. Different lowercase letters indicate significant differences. Similar results obtained in two additional experiments are provided in Figure S19</figcaption></figure>

## 4.2 | Interaction with *C. hobsonii* facilitates Sweetgum K⁺ assimilation.

The role of mycorrhizal and endophytic fungi in facilitating the uptake of minerals by trees and crops has been extensively studied (Chen, Ren, Yang, Peng, & Dai, 2013; Franklin, Näsholm, Högberg, & Högberg, 2014; Hiruma et al., 2016; Meena, Maurya, Verma, & Meena, 2016; Yang et al., 2014). The majority of nutrition uptake studies of plant-fungal symbiotic associations have focused on N and P, while plant K⁺ nutrition has received far less attention (Garcia & Zimmermann, 2014; Liu et al., 2019). Our study showed a striking contribution of *C. hobsonii* to potassium uptake by Sweetgum plantlets. The beneficial effect was strongly expressed under the LK condition (Figures 5 and 6). In contrast, the lack of higher values for N and P under all K⁺ conditions tested (0.05, 2 and 5 mM), suggests that the benefit is not simply due to more vigorous root systems but is specific to K⁺ uptake (Figures 2, 4, and S15). More importantly, this fungus only colonized plant roots, therefore, it is reasonable that an increase in the K⁺ content of the aerial part of the colonized plants would underpin the beneficial role of the fungus in improving plant potassium nutrition (Figures 1, S14, and S16). This inference is very analogous to another study, in which the *Paxillus involutus* (Boletales, Basidiomycota) strain NAU promotes K⁺ influx into *Populus × canescens* roots without the formation of ectomycorrhizal structures (Li et al., 2012).

Soil-dwelling fungi associated with plant roots may improve K⁺ nutrition in plants via three strategies: (a) increasing the availability of K⁺ (Dhillon, Eickhoff, Mullen, & Raun, 2019; Sun, Fu, Finlay, & Lian, 2019); (b) inducing the expression of specific plant genes encoding K⁺ transporters (directly pathway) (Liu et al., 2019); (c) mediating the K⁺ transfer to plants by symbiotic interactions (indirectly pathway) (Guerrero-Galán et al., 2018; Haro & Benito, 2019). In this work, the potassium solubilization activity of *C. hobsonii* has not yet been validated experimentally and merits further investigation. Thus, at this time we are unable to give any conclusive evidence in support of the first claim. Here, we found that higher transcript levels of the fungal transporters could present the most likely explanation for the higher net K⁺ uptake by colonized plants. As discussed below, the fungal K⁺ uptake pathway is active in the transfer of nutrients from fungus to host plant roots (Figure 3a). However, this does not necessarily exclude other explanations. For an example, higher plant potassium transporter activity may also result in increased K⁺ uptake to some extent, despite the fact that neither K⁺ transporters nor channels in plants were significantly up-regulated at early or later stages of the biotrophic interaction (Figure 3a and Figure S3). It also should be noted that we applied both parameters (mg/plant and mg/g DW) to estimate plant K⁺ uptake and accumulation. While the latter is more important for deficiency symptoms, the former is a better reflection of net K⁺ uptake. We argue that the most relevant measurement would be to relate the total K⁺ content per plant to the
actual root surface to assess whether the larger net uptake is simply a consequence of more robust root systems or of a higher number of transporters per surface unit (e.g., due to more K⁺-related transcripts), or higher activity of the existing transporters. This question deserves closer investigation.

## 4.3 | Potassium limitation entails more fitness of the root-fungal association?

In many fungal species, nutrient starvation triggers a general induction of fungal transporters (Becquer et al., 2019; Calabrese et al., 2017). Fungal K⁺ transporter genes are induced in response to low K⁺ conditions and are down-regulated when K⁺ is abundant (Benito, Garciadeblas, Fraile-Escanciano, & Rodríguez-Navarro, 2011; Cabrera, Álvarez, Martín, Siverio, & Ramos, 2012; Liu et al., 2019). Our findings are in line with these observations. Four families of putative K⁺ transport systems, including TRK (derived from the transporter of K⁺), HAK, ACU and PAT proteins (derived from P-type ATPase), and two K⁺ channel families, including TOK (tandem-pore outward-rectifying K⁺) and SKC, have been identified (Benito & González-Guerrero, 2014; Garcia et al., 2014; Garcia, Doidy, Zimmermann, Wipf, & Courty, 2016; Meena et al., 2016), which exist in different combinations across the fungal kingdom (Benito et al., 2011; Haro & Benito, 2019).

In the current study, three genes *ChACU*, *ChHAK* and *ChSKC* were identified in *C. hobsonii* by transcript profiling of the symbiotic association. Among them, the ACU ATPases is known to be one of the major actors in the acquisition of soil K⁺ (Benito, Garciadeblas, Schreier, & Rodrigueznavarro, 2004). We found that the expression of *ChACU* either in the free-living fungus (in vitro) or in the root-fungal association (*in planta*) was upregulated by potassium starvation (Figure 7). The HAK belonging to membrane transporter families plays a similar role with the *ChACU* (Benito & González-Guerrero, 2014; Garcia et al., 2016; Guerrero-Galán, Calvo-Polanco, & Zimmermann, 2019). The amino acid sequence of *ChHAK* was homologous to other known HAK transporters (Figure S10 and S12a) and gene expression was also induced by potassium limitation (Figure 7). Unexpectedly, the *ChHAK* appeared to make a marginal contribution to potassium absorption as the yeast mutant expressing *ChHAK* partially restored the growth of the mutant on the medium containing 2 mM K⁺ (Figure 3b). Hence, further functional evidence of *ChHAK* needs to be verified in future studies. As the main K⁺ efflux systems, it has been demonstrated that the SKC and *HcTOK2.2* channels could be actively involved in the transfer of K⁺ from hyphae to host root cortical cells (Garcia et al., 2016; Garcia & Zimmermann, 2014; Haro & Benito, 2019). However, this is not always the case. Some fungal potassium channels also show the strength of inward rectification. For example, the *HcTOK1* from the ectomycorrhizal fungus *Hebeloma cylindrosporum* (Agaricales) is involved in K⁺ influx into the hyphal cells (Guerrero-Galán et al., 2018). Similarly, our data show that the *ChSKC*, although less efficient than *ChACU*, complements the defect in K⁺ uptake of the yeast mutant (Figure 3b). It will be interesting to focus future researches in dissecting the whole K⁺ transportome because

additional types of K⁺ transport systems operating in *C. hobsonii* will be identified thanks to genomic data availability (Peng et al., 2021).

We further reveal that the root-fungal association has evolved to improve the fitness of the fungus from another perspective. It is commonly accepted that the “underground market” between fungus and host plant is situated in the symbiotic interface, where it is well established that mycorrhizal symbionts provide host nutrients in exchange for carbohydrates such as soluble sugars and organic acids (Kariman et al., 2018; Nehls, 2008). In general, the total sugar content was often higher in mycorrhizal plants regardless of the growth conditions (stressed or non-stressed) (Feng, Li, Tian, Tang, & Rengel, 2002; Liu et al., 2019). In this study, particularly under the LK condition, the substantial amounts of soluble sugar accumulated in plants in the presence of the fungus (Figure 4f), where will, in turn, provide more carbohydrates to the fungal partner. The increased chlorophyll contents in leaves of inoculated plants could result in efficient plant photosynthesis (Table 2). Together, these twin lines of evidence suggest that the LK condition would favour more mutualistic interaction between *C. hobsonii* and trees mainly through reciprocal exchange of carbon and potassium to both partners. Such evidence supports the plausibility that trees associated with *C. hobsonii* behave similarly to plants with other mutualistic fungi.

Future research will investigate the key role of the Na⁺/K⁺ ratio in salt tolerance (Guerrero-Galán et al., 2019). We surmise that under low K⁺ conditions, the improved K⁺ acquisition decreases the Na⁺/K⁺ ratio so that *C. hobsonii* would protect its plant partners from salinity stress. *C. hobsonii* inoculation increased the soluble sugar content of plantlets under LK condition most likely to adjust the osmotic potential and to maintain higher turgor pressure, which constitutes an important plant protection mechanism against salinity stress (Garg & Bharti, 2018; Jia et al., 2019; Wang, Sun, & Shi, 2019).

In summary, our study documented morphological and nutritional aspects of the facultative biotrophic interaction between *C. hobsonii* and *L. styraciflua*. We provide several lines of evidence suggesting that the fitness of this symbiosis is controlled by the environmental K⁺ conditions. Although there is no extensive genetic reprogramming of host K⁺ nutrition-related genes during the symbiotic interaction, regulation and fine-tuning of fungal physiology responding to changes of K⁺ concentrations is the most distinctive feature of the beneficial biotrophic partners. An illustrative outlining of the benefits underlying the LK-induced transfer of carbon and potassium in this association is proposed in Figure 8. A recent project of whole-genome sequencing the *C. hobsonii* and comparative genomics of related species (including ectomycorrhizal, saprotrophic and endophytic taxa) within Agaricales is currently underway, and this endeavour will allow us to shed light on the key evolutionary trajectories leading to facultative biotrophy and to identify more candidate genes belonging to putative K⁺ transport systems for functional characterization. Taken together, this work is a new starting point for conducting detailed mechanistic studies on interactions between the tree and sapro-symbiotic fungi. Moving forward, *C. hobsonii* could also underpin the silvicultural management to overcome soil potassium deficiency.
<figure><img src="image_11.png" /><figcaption>**FIGURE 8** A schematic depiction of the facultative biotrophic fungus-induced plant K⁺ acquisition under the K⁺ limitation condition occurred in the *L. styraciflua-C. hobsonii* biotrophic symbiosis. It is proposed that *C. hobsonii* has a dual lifestyle, saprotrophic and biotrophic. During root colonization, the endophytic hyphae establish sclerotia-like structures in cortical cells and promote K⁺ acquisition in *L. styraciflua* roots, especially under K⁺ limitation. The K⁺ transporter, *ChACU*, is hypothesized to increase the uptake of soil potassium under K⁺ depletion. The natural fruiting bodies of the *C. hobsonii* (adapted from https://www.inaturalist.org/taxa/381735-Clitopilus-hobsonii) are presented [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

## ACKNOWLEDGEMENTS

This study was supported by National Natural Science Foundation of China (No. 31722014, 31901290) and Fundamental Research Funds for the Central Non-profit Research of Chinese Academy of Forestry (CAFYBB2020QB002). Martin's lab is funded by the Laboratory of Excellence ARBRE (grant no. ANR-11-LABX-0002_ARBRE). The authors are grateful to Professor Yan Liang for constructive suggestions in this study.

## CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.

## AUTHOR CONTRIBUTIONS

Zhilin Yuan and Long Peng designed the study. Long Peng and Xiaoliang Shan performed the experiments. Wei Jin isolated the fungus. Xinyu Wang constructed the phylogenetic tree. Xueyu Pan established the *C. hobsonii*–*L. styraciflua* in vitro co-culturing system. Zhilin Yuan, Long Peng, Xiaoliang Shan, Yuchen Wang and Xiaoguo Zhang analysed the data. Zhilin Yuan, Long Peng and FMM wrote the manuscript, with the help of Xinghua He, Irina S. Druzhinina and Yuzhan Yang. Xinghua He helped in preparing the figures.

## DATA AVAILABILITY STATEMENT

The RNA-seq data that support the findings of this study are openly available in National Center for Biotechnology Information Search database at https://www.ncbi.nlm.nih.gov/, reference number

SRR10199935, SRR10199934, SRR10199933, SRR10199932, SRR10199931, SRR10199930, SRR10199929, SRR10199928, SRR10199927, SRR10199926, SRR10199925, SRR10199924. Other data that support the findings of this study are available from the corresponding author upon reasonable request.

## ORCID

Long Peng https://orcid.org/0000-0003-1119-4245

Xiaoliang Shan https://orcid.org/0000-0002-7553-8046

Yuzhan Yang https://orcid.org/0000-0002-4206-7495

Yuchen Wang https://orcid.org/0000-0003-1045-712X

Irina S. Druzhinina https://orcid.org/0000-0003-2821-5268

Xinghua He https://orcid.org/0000-0002-9325-6683

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

Zhilin Yuan https://orcid.org/0000-0002-5799-8401

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## SUPPORTING INFORMATION

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

**How to cite this article:** Peng, L., Shan, X., Yang, Y., Wang, Y., Druzhinina, I. S., Pan, X., Jin, W., He, X., Wang, X., Zhang, X., Martin, F. M., & Yuan, Z. (2021). Facultative symbiosis with a saprotrophic soil fungus promotes potassium uptake in American sweetgum trees. *Plant, Cell & Environment*, 44(8), 2793–2809. https://doi.org/10.1111/pce.14053