Introduction

Soil salinization represents one of the most significant constraints for crop production globally. It is estimated that currently more than 20% of all cultivated land is already affected by high salt content in the soil, increasing abiotic stress for crops and, consequently, decreasing the agricultural productivity below the genetic potential of the elite crop varieties employed. Considering the current climate change scenario, a temperature increase of 2–5 °C, combined with reduced annual rainfall, and increased vapor pressure deficit and evaporation are anticipated (IPCC 2021). These projected changes will have a particularly strong impact on large parts of semi-arid and arid regions worldwide. Notably in these regions, the scarcity of water necessitates the use of marginal to poor quality water for irrigation, which, in turn, will accelerate soil salinization and exacerbate its detrimental impact on plant growth and food production (Munns and Tester 2008). According to Jamil et al. (2011), more than 50% of arable land could be affected by soil salinity by 2050.

The root system is the primary organ exposed to salinity in the soil and functions as a key component in the detection of salt (Galvan-Ampudia et al. 2013). Roots respond to salinity by inhibiting their growth, which is achieved through a substantial decrease in cell expansion growth in the root elongation zone (West et al. 2004). Additionally, adaptations of roots to increased salt content in soil involve alterations in the developmental program of the meristematic zone at the root apex (Julkowska et al. 2017). Consequently, salt stress leads to a reduction in the size of the root meristem, which ultimately results in a shift in the relationship between the growth of the lateral roots and the primary root (Duan et al. 2013). In response to the perception of abiotic stresses, including salt stress, roots redirect their growth and adjust their root length, branching, and root hair length and density appropriately and dynamically to respond to the stress condition (Zou et al. 2022).

Root hairs exhibit particular sensitivity to salt and can therefore serve as indicators for monitoring salt stress. Root hair plasticity demonstrates a gradual and relatively rapid response to varying salt concentrations, affecting both its density and elongation (Robin et al. 2016; Wang et al. 2008). The development of root hairs is predominantly regulated by the homeobox-leucin zipper protein (HD-ZIP) transcription factor GLABRA2 (GL2) (Rerie et al. 1994). It negatively regulates root hair growth by controlling the expression of ROOT HAIR DEFECTIVE 6 (RHD6) and RHD6-LIKE1 (RSL1), two primary regulators of root hair initiation (Lin et al. 2015). GL2 undergoes transcriptional regulation in response to abiotic stresses, including osmotic and salt stress (Wang et al. 2020a; Wang and Li 2008). The growth-inhibiting effect of salt is hypothesized to involve the accumulation of abscisic acid (ABA). Elevated ABA levels subsequently trigger ABA-INSENSITIVE 1 (ABI1)-dependent stabilization of DELLA proteins, which function as negative regulators of gibberellin (GA) signaling (Achard et al. 2008). Suppression of GA production in the corresponding GA biosynthesis mutants has previously provided evidence for a promoting effect of low GA contents on root hair elongation (Jiang et al. 2007). However, in the context of root hair growth regulation, the role of ABA remains controversial. An additional study emphasized its negative regulatory effect by upregulating OBF-BINDING PROTEIN 4 (OBP4), which acts as a negative regulator of RSL2, a key transcription factor (TF) that controls root hair elongation (Rymen et al. 2017).

An alternative and agriculturally sustainable solution that simultaneously has the potential to produce resilient ecosystems under the described scenario could be the utilization of plant-interacting microbes that can enhance productivity, as well as the tolerance of host plants to various types of biotic and abiotic stresses, including drought and salt stress. It is becoming increasingly evident that symbiotic microorganisms are also capable of positively modulating the physiological, biochemical, and morphological adaptations of plants that respond to prevailing environmental signals (Dupont et al. 2015; Wani et al. 2015). The spectrum of benefits for host plants includes growth promotion, higher yields, and enhanced resistance to numerous biotic and abiotic stresses, including nutrient limitation, drought, salinity, and altered temperatures (Pérez-Alonso et al. 2020; Scholz et al. 2023).

The genus Fusarium is one of the most abundant fungal genera, comprising approximately 70 species that are characterized by an exceptional divergence in terms of their genetics and ability to proliferate on a broad range of substrates. Along with the genera Aspergillus and Penicillium, members of the Fusarium genus are among the filamentous ascomycete fungi with the broadest distribution. They are characterized by a particularly rich secondary metabolism, which has already attracted considerable interest (Singh et al. 2021; Villavicencio et al. 2021; Yadav and Meena 2021). Many Fusarium species are well-known fungal pathogens, responsible for substantial economic losses in the agricultural and food industry, including Fusarium head blight, which is one of the most significant diseases that affect cereal grains (McMullen et al. 1997). However, there is also mounting evidence that, in addition to these pathogenic strains, there are several Fusarium species that are mutualists, intimately interacting with their host plants and providing fitness benefits to them, especially under environmental stress conditions. A recent study provided the first evidence for the beneficial effect of the Fusarium sp. strain K-23 on tolerance to salt stress in multiple tomato ecotypes (Pallavi and Nataraja 2022). Another strain of Fusarium incarnatum was also reported to improve tolerance to salt stress of a salt-sensitive rice variety (Ayesha et al. 2022). In this study, we utilize the model plant A. thaliana to investigate how Fusarium sp. strain K-23 influences tolerance to salt stress of the plant from a transcriptional and mechanistic perspective.

Materials and Methods

Plant and Fungus Material and Corresponding Growth Conditions

In this study, wild-type Arabidopsis thaliana (Col-0, stock N1092) and several previously described mutant lines were used, including the NAM/ATAF/CUC (NAC) TF mutants jub1-1 and JUB1-OX (Wu et al. 2012), the root hair deficient double mutant rsl2 rsl4 (Datta et al. 2015), and the reporter line RGAp::GFP-RGA (Silverstone et al. 2001). After stratification (2 days, 4 °C), plants were grown under sterile conditions on solidified 0.5 × MS medium supplemented with 1% (w/v) sucrose (Murashige and Skoog 1962). The plants were grown in growth chambers under strictly controlled environmental conditions (16 h light, 8 h darkness, constant temperature of 22 °C, 100–105 µmol photons m−2 s−1 photosynthetically active radiation). The fungus Fusarium sp. strain K-23, which has been collected from the Himalayas, in proximity to Kargil in the Jammu and Kashmïr region (34°34′22’’N 76°7′57’’E), was obtained from the fungal collection maintained in the School of Ecology and Conservation Laboratory, Department of Crop Physiology, University of Agricultural Sciences (UAS-B), Gandhi Krishi Vignana Kendra (GKVK), Bengaluru. The fungus was grown in darkness at a constant temperature of 28 °C on solidified potato dextrose agar (PDA) medium. The fungus was refreshed weekly.

Plant–Fungus Co-Cultures and the Determination of Growth Promotion

To investigate differences in Fusarium sp. K-23-triggered growth promotion in the different genotypes of Arabidopsis, surface-sterilized seeds were plated and stratified as described above. The plates were transferred to a growth chamber where the seedlings were grown vertically for seven days. Subsequently, four seedlings were transferred to fresh square Petri dishes containing solidified Plant Nutrition Medium (PNM) (Johnson et al. 2013). The roots of each seedling were then inoculated with 15 µl of a solution containing 1 × 104 spores ml−1. The PNM plates were then transferred to a growth chamber and kept at 22–24 °C, 16/8 h photoperiod, 100 µmol photons m−2 s−1 light intensity for another seven days. Finally, the plants were photographed for further analysis of the root system before they were dissected in root and shoot tissue to determine the fresh and dry weight of the plants (n = 24).

Quantitative Analysis of the Root System Architecture

The root system of the control and fungus-infected Arabidopsis seedlings was captured with a digital camera at a fixed distance (29 cm). Using GIMP v2.10.36 (https://www.gimp.org/, accessed on 15 December 2023), the images were cropped to a height of 14 cm, keeping only the part comprising the root system. The images were then converted to black and white. The root network traits of the plants were analyzed using the RhizoVision Explorer v2.0.3 software (Seethepalli et al. 2021; Seethepalli and York 2021). For comparative analysis, the total length and branching frequency of the network were used as readout. Primary root length was measured using ImageJ2 v2.14.0/1.54f software (Rueden et al. 2017). Regarding the biological variability of the root system of the plant, at least 24 individual plants per genotype and growth condition were analyzed.

Quantitative Analysis of the Root Hair Phenotype

For the analysis of root hair length in the different mock and fungus-treated Arabidopsis genotypes, around 100 fully elongated root hairs were measured in 10 individual plants seven days after infection. Images were captured using a Leica MZ10F stereo microscope equipped with a DFC 400C CCD camera. The length of the root hairs has been determined using ImageJ2 v2.14.0/1.54f (Rueden et al. 2017).

Trypan Blue Staining

For the analysis of root colonization, 10 root samples from control and infected plants were used. After thoroughly washing the root samples with deionized water, they were cut into 1 cm long pieces and incubated overnight in 10 N KOH. The explants were then rinsed 5 times with sterile H2O, before being incubated for 5 min in 0.1 N HCl. The samples were then incubated in a 0.05% lactophenol–trypan blue solution (w/v), before partially decolorizing with chloral hydrate for ten minutes following a previously published protocol (Keogh et al. 1980). The samples were washed once with 100% ethanol and three times with sterile H2O and stored in 60% glycerol (v/v) prior use. Examination of the samples mounted on glass slides was done using a Zeiss Axiophot microscope equipped with a Euromex DC 20000i CMOS camera.

Re-Isolation and Genetic Characterization of the Fungus

To further assess the colonization of Arabidopsis roots with K-23, root tissue from infected and control plants was cut into 1 cm long segments, which were surface sterilized with 70% (v/v) ethanol for 2 min, followed by a washing step with 5% (v/v) NaOCl for 5 min, and three rinses with sterile distilled water. The explants were then placed in Petri dishes with solidified PDA medium and incubated for 5–7 days. The genomic DNA of the fungal mycelium was isolated following the previously reported cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987). Then, the ITS region of the fungus was amplified by polymerase chain reaction (PCR) using the primers ITS1 forward (5′-TCCGTAGGTGAACCTGCC-3′) and ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′) (Gardes and Bruns 1993). The resulting 600-bp DNA fragment was purified and sequenced by the Stab Vida Genetics service (Caparica, PT). Fungus identity was confirmed by performing a BLASTn search against the ITS region database of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/, accessed on 15 November 2023) using the obtained sequence as query. Based on the maximum homology and percent similarity, the identity was assigned in accordance with previously reported criteria (Higgins et al. 2007).

Confocal Laser Scanning Microscopy

Differences in GA signaling in mock- and K-23-infected roots were analyzed at three and seven dpi, respectively, using a Leica SP8 microscope with the Leica Application Suite (Las AF Lite) X software and the plant hormone signaling reporter line RGAp::GFP-RGA. The green fluorescent protein (GFP) was excited at 488 nm using an Argon multiline laser and detected using a 494/596 nm broadband filter.

RNA Isolation and RNA Sequencing Analysis

For each condition, 100 mg of root and shoot tissue of seven-day-old mock- and fungus-infected seedlings that were grown in the absence and presence of 50 mM NaCl, respectively, was harvested for total RNA extraction as previously described (Oñate-Sánchez and Vicente-Carbajosa 2008). Total RNA obtained was quantified using a Nanodrop ND-1000® UV/Vis spectrophotometer (ThermoFisher). RNA quality was also checked on a Bioanalyzer 2100 (Agilent) by the Novogene Genomics Service (Cambridge, UK). Subsequently, library construction and sequencing (150-nt paired-end reads) on Illumina NovaSeq™ 6000 platforms were performed by the Novogene Genomics Service, which also provided basic data analysis applying their RNA-seq pipeline. This included data filtering and quality control, as well as sequence alignment using HISAT2 v2.0.5 (Kim et al. 2019), transcript quantification against the A. thaliana reference annotation (TAIR10, Ensembl release 51) with featureCounts v1.5.0-p3 (Liao et al. 2014), and differential gene expression analysis using the DESeq2 v1.20.0 algorithm (Love et al. 2014). For each tissue, treatment, and condition, respectively, three biological replicates were processed. The resulting p values were adjusted for multiple testing using the Benjamini–Hochberg correction (Benjamini and Hochberg 1995). A cut-off value of |log2(fold change)|≥ 1 and a FDR ≤ 0.05 was established to select DEGs.

To analyze general expression profiles, the ClustVis tool (Metsalu and Vilo 2015) was used. Venn diagrams have been generated using the Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 24 February 2024) online tool, to analyze overlapping patterns in differentially expressed genes. For the gene ontology (GO) enrichment analysis, we used either the ShinyGO online tool (Ge et al. 2020), DAVID (Huang et al. 2009), or the Metascape gene annotation and analysis resource (Zhou et al. 2019). In addition, we used a locally installed instance of DIANE v1.0.6 (Cassan et al. 2021) to re-analyze the obtained normalized expression data and run Poisson-based mixture models for expression-based clustering. Functional relationships between DEGs were investigated employing stringApp v2.0.3 (Doncheva et al. 2019) in Cytoscape v3.10.2 (Shannon et al. 2003).

Statistical Analysis

The statistical assessment of the data was performed using the JASP v0.18.3 software (https://jasp-stats.org/, accessed on 24 February 2024). Data were analyzed with one-way anova followed by Tukey–Kramer post hoc test to allow comparisons between all means or with Student's t test when two means were compared. The results were considered significant when the p value < 0.05. The values reported represent means ± standard error (means ± SE).

Results

Characterization of the Plant-Fungus Interaction

Fusarium sp. strain K-23 was obtained from a fungal collection maintained at the University of Agricultural Sciences (UAS-B), Bengaluru. A previous study reported the considerable salt tolerance of strain K-23 (Pallavi and Nataraja 2022). Additionally, the authors provided evidence that the fungus is capable of colonizing the roots and shoots of tomato plants. To validate the identity of the fungus, the salt tolerance experiment was replicated, and the strain was grown axenically on PDA plates with increasing concentrations of NaCl in the medium. Light can exert a significant influence on diverse aspects of fungal physiology, including pigmentation, the circadian clock, and secondary metabolism (Corrochano 2007; Herrera-Estrella and Horwitz 2007). Consequently, the effect of light on the growth of the fungus was also examined. As anticipated, the fungus exhibited considerable salt adaptation, demonstrating growth in high-salt conditions of up to 2 M NaCl in the medium (Fig. 1).

Fig. 1
figure 1

Representative images showing the growth behavior of Fusarium sp. strain K-23 six days after inoculation growing in darkness or under constant light on PDA plates with either none (Control) or indicated amounts of NaCl in the medium

Full size image

Upon comparing fungal growth in darkness and constant light conditions, no significant differences were observed in the phenotype of the fungal mycelium. However, growth in light with salt appeared to be marginally accelerated. Subsequently, genomic DNA was extracted from the fungus, and the internal transcribed spacer (ITS) region was amplified via polymerase chain reaction (PCR). The resulting ITS fragment (Supporting information Data Sheet 1) was sequenced and analyzed using the nucleotide basic local alignment search tool (BLASTn) against the ITS database of the National Center for Biotechnology Information (NCBI). This analysis confirmed the identification of K-23 as a member of the Fusarium incarnatum-equiseti complex from the Incarnatum clade. Considering the high similarity of the ITS sequence and the phenotype of the fungal mycelium on PDA plates, K-23 exhibited close resemblance to previously reported Fusarium irregulare strains (Wang et al. 2019). The subsequent investigation addressed whether the tomato root-colonizing fungus was capable of penetrating the root cortex cells of Arabidopsis or remained on the root surface. Re-isolation experiments from Arabidopsis plants co-cultivated with the fungus yielded no evidence of substantial fungal invasion of the roots (Fig. 2b). This observation was corroborated by lactophenol–trypan blue staining of multiple root sections, which suggested the localization of fungal hyphae external to the root in Arabidopsis (Fig. 2d).

Fig. 2
figure 2

Re-isolation experiments and infection of Arabidopsis roots with Fusarium sp. strain K-23. a Re-isolation control samples. Shown are two plates with surface sterilized root explants from non-infected plants that have been incubated on PDA medium for 6 days. b Similarly treated PDA plates containing surface sterilized root explants from Arabidopsis seedlings that were co-cultivated with K-23 for seven days. c Representative image of a lactophenol–trypan blue stained root segment from a non-infected plant. d Image of a lactophenol–trypan blue stained root segment from a K-23 infected Arabidopsis plant. The arrowhead in the image highlights the fungal hyphae on the outside of the root. Scale bar = 20 µm

Full size image

Extensive hyphal structures were observed on the root surface, from which the fungus can establish an effective interaction with the plant. Given the relatively low colonization rate of the examined Arabidopsis roots, our findings cannot definitively exclude the possibility that the fungus may penetrate the intercellular space of the root to a limited extent. Such growth behavior could potentially enhance the relationship between the interacting organisms.

Growth Promoting Effect of K-23 on Arabidopsis Plants Under Salt Stress Conditions

Despite observed differences in Arabidopsis colonization and previously tested Indian tomato varieties, an analysis of the plant growth-promoting effect of the fungus on Arabidopsis plants under control and moderate salt stress conditions was conducted. Several phenotypic growth parameters were compared, including the weight of the roots and shoots and various traits characterizing the root system architecture, in mock-treated Arabidopsis plants and plants co-cultivated with K-23 in the absence and presence of moderate salt contents. As illustrated in Fig. 3, Arabidopsis roots grown in the presence of the Fusarium sp. strain K-23 exhibited several notable differences in the monitored physiological parameters.

Fig. 3
figure 3

Analysis of the growth-promoting effect of Fusarium sp. strain K-23 on Arabidopsis plant. The radar plots show the effect of the fungus on Arabidopsis growth under control and moderate salt stress conditions on total plant fresh weight (TPL), shoot fresh weight (SW), root fresh weight (RW), lateral root number per plant (#LR), primary root length (PRL), and the length of the total root system (TRL). Compared are mock-infected plants (−F) and plants 7 dpi with the fungus (+ F). The plots show means of the relative values (n = 24), where the particular average value for each tested parameter of Arabidopsis plants grown under control conditions was set to 100% (%Ctrl). Significant differences are highlighted with black bars. Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

The fungus demonstrated no substantial effect on Arabidopsis growth when co-cultivation was performed under control conditions. A significant reduction of approximately 9% (p = 0.03) was observed only for total root system length in fungus-inoculated plants compared to mock-treated plants. All other observed differences were not significant. However, a minor reduction in the primary root length and the fresh weight of the root was noted, while the fresh weight of the shoot was slightly higher. In contrast, the fungus appeared to exert a marked beneficial impact on infected Arabidopsis plants when grown under salt stress conditions. Indicative of increased salt tolerance, Arabidopsis plants co-cultivated with K-23 exhibited an approximately 17% (p = 0.0001) longer total root system, 13% (p = 0.04) higher shoot fresh weight, and 14% (p = 0.0009) higher total plant fresh weight. In summary, these results corroborate the plant growth-promoting effect of Fusarium sp. strain K-23 previously observed in tomato under salt stress conditions (Pallavi and Nataraja 2022).

mRNA Sequencing Reveals an Overlap Between the Responses to K-23 and Salt

To investigate the biological processes affected by the co-cultivation with K-23, a comprehensive transcriptomic approach was employed. RNA extracted from roots grown under control or salt stress conditions in the presence or absence of the fungus was subjected to mRNA sequencing (RNA-seq). Principal Component Analysis (PCA) was conducted to reduce the dimensionality of the multivariate data set obtained. The analysis provided evidence for a group-wise clustering of the individual samples. The first two principal components explained 62.6% of the variance in the data set. Fungus infection and salt treatment demonstrated a clear effect on the variance. Notably, the infection with the fungus shifted the samples toward the two salt-treated sample groups.

Transcript profiling provided a comprehensive overview of differentially expressed genes (DEGs) under the tested conditions and facilitated the analysis of their functional relationships. Utilizing an adjusted p value (false discovery rate (FDR)) of ≤ 0.05 and an arbitrarily selected differential expression value of log2(fold change) ≥|1|, 928 induced and 159 repressed DEGs were identified for the co-cultivation under control conditions (+ F Ctrl versus −F Control). When examining the effect of the fungus under salt stress conditions, a change in the number of DEGs dependent on infection with K-23 was observed, with 593 induced and 309 repressed DEGs (+ F Salt versus −F Salt). The results of the differential expression analysis are summarized in Fig. 4.

Fig. 4
figure 4

Effects on gene expression of wild-type Arabidopsis seedlings under salt treatment and Fusarium sp. strain K-23 infection. a Principal component analysis (PCA) of transcriptome-wide normalized gene expression counts assessing the difference in mock (−F) and K-23 (+ F) infected wild-type Col-0 seedlings under control and salt stress conditions. Each dot/square represents a sample, color and symbols are coded by infection status and stress condition. The x-axis denotes the PC1; y-axis denotes values for PC2. b DEGs statistics for mock and K-23-infected Col‐0 plants in the absence or presence of 50 mM NaCl at 7 dpi. c Venn diagram showing the numbers of DEGs shared between K-23 infected plants (7 dpi) and similarly grown Arabidopsis seedlings that were salt-treated for the same time. d Venn diagram showing the numbers of DEGs in K-23 infected (+ F) versus non-infected Col‐0 plants (−F) that were either grown in the absence (Ctrl) or presence of 50 mM NaCl (Salt) at 7 dpi. Induced (up) and repressed (down) DEGs were separately evaluated. e GO enrichment analysis of the 460 induced DEGs in salt-treated and K-23-infected Col‐0 plants. Each circle in the figure represents a distinct GO term. The circle size indicates the number of genes enriched in the corresponding GO term. The significance of the observed gene enrichment is represented by a color gradient referring to the -log10(FDR) value

Full size image

The PCA revealed that the transcriptome of Arabidopsis plants inoculated with K-23 exhibits greater similarity to salt-treated samples. Consequently, Venn diagram analysis was conducted to investigate the potential overlap of DEGs between K-23- and salt-treated Arabidopsis plants. More than half of the DEGs (547) identified in K-23 inoculated samples were also differentially expressed under salt stress conditions (Fig. 4c). To further elucidate this observation, we segregated the induced and repressed DEGs from the previously tested conditions and performed an additional Venn diagram analysis. This analysis aimed to identify DEGs exclusively dependent on K-23 inoculation and those commonly induced or repressed by both fungal treatment and salt stress. The resulting gene groups were subsequently subjected to Gene Ontology (GO) analyses for functional classification of the encompassed DEGs (Supporting information Data Sheet 2). Initially, we examined the functional association of the 440 and 100 DEGs exclusively induced and repressed by the fungus, respectively. As anticipated, K-23-induced DEGs were categorized into GO terms related to plant defense responses, including response to other organisms, response to external biotic stress, response to salicylic acid, and immune system processes. Conversely, the 100 repressed DEGs were associated with abiotic stress-related GO terms, encompassing response to heat, low oxygen levels, radiation, and responses to light. Of the 460 and 56 DEGs that showed intersections between fungal infection and salt treatment, the commonly repressed DEGs exhibited an enrichment in GO terms related to circadian regulation of gene expression. This highlighted an underrepresentation of the PHYTOCHROME RAPIDLY REGULATED 2 (PAR2) gene and the two MYB-like transcription factor (TF) genes REVEILLE 1 (RVE1) and RVE8, among others. RVE1 is known to regulate auxin biosynthesis in a circadian manner (Rawat et al. 2009). Consequently, we examined the expression of a manually curated list of 142 genes related to auxin metabolism, transport, and signaling for alterations in their expression in the unfiltered gene list, to include genes that were below the established threshold value of log2(fold change) ≥|1|. As presented in Table 1, the targeted search for auxin-related genes revealed 12 DEGs that exhibited significant alterations in their gene expression.

Table 1 Differential expression of auxin-related genes in K-23 infected Arabidopsis seedlings
Full size table

Although RVE1 repression suggested potentially decreased auxin contents, the analysis did not provide evidence for general repression of auxin biosynthesis-related genes. With the exception of the SHY2 gene, which exhibited negative regulation, all other identified genes demonstrated a significant induction of their expression.

The functional classification of the 460 DEGs in the remaining group of genes commonly induced by fungus and salt stress revealed an enrichment in GO terms related to cell wall organization. However, the substantial number of GO term classifications associated with root hair development, including trichoblast differentiation, plant epidermal cell differentiation, and root epidermal cell differentiation, warranted particular attention (Fig. 4e). Notably, 193 of the 1,096 K-23 co-cultivation-dependent DEGs exhibited a relationship with root hair development (Supporting information Data Sheet 3).

Co-Cultivation with K-23 Stimulates root Hair Growth

Considering the induction of a substantial number of genes related to root hair development triggered by the fungus and the essential role of root hairs for plant nutrition and water uptake, we elected to investigate the impact of the fungus on root hair formation. Initially, we examined the length and density of the root hairs under the aforementioned conditions (Fig. 5).

Fig. 5
figure 5

K-23-promoted root hair elongation. a Representative images of root hairs from Arabidopsis control seedlings (Ctrl) grown for 7 days on 0.5 × MS and another 7 days on PNM minimal medium, similarly grown seedlings but with 50 mM NaCl (+ Salt) in the PNM medium, and seedlings infected with K-23 in the absence (+ F) and presence of 50 mM NaCl (+ F, + Salt) at 7 dpi. Scalebar: 400 μm. b Quantification of root hair density and c root hair length. Data are means ± SE (n = 10 roots). One-way ANOVA with a post hoc Tukey–Kramer test was used. Different letters indicate significant differences between means (p ≤ 0.05)

Full size image

As indicated by the transcriptomic analysis, we observed a significant increase in root hair density and, notably, in root hair elongation resulting from fungal treatment. The most significant and discernible difference was evident when plants were cultivated in the presence of the fungus under salt stress conditions. As illustrated in Fig. 5c, salt stress typically induces a pronounced suppression of root hair growth. In the presence of the fungus, this suppression was entirely mitigated. Moreover, the moderate increase in root hair length of plants grown without additional salt in the medium was largely maintained under salt stress conditions. These findings suggest that the fungus promotes root hair growth which, consequently, may contribute to enhanced salt stress tolerance in the host plant.

Induced Root Hair Elongation is Crucial for K-23 Triggered Biomass Promotion Under Salt Conditions

Subsequently, we investigated whether K-23-induced root hair elongation is necessary for enhanced tolerance to salt stress in Arabidopsis. To accomplish this, we initially identified root hair development-specific genes among the 460 up- and 56 down-regulated DEGs that commonly respond to fungus and salt treatments by comparing them with a manually curated list of 1,650 root hair development-associated genes. The analysis yielded 140 candidate genes (27% of the shared DEGs) (Supporting information Data Sheet 3), of which five were TFs. The identified DEGs were utilized to perform a functional association network analysis using the stringApp in Cytoscape. As illustrated in Fig. 6a, we obtained a network with 135 nodes and 1,202 edges.

Fig. 6
figure 6

K-23-induced growth promotion under salt conditions depends on root hair elongation. a Functional network analysis of the identified 140 root hair-specific genes among the differentially expressed genes (DEGs) shared between salt and fungus-responsive genes. b Representative images of root hairs from rsl2 rsl4 mutant plants compared to Arabidopsis control seedlings (Col-0) grown for 7 days on 0.5 × MS and another 7 days on PNM minimal medium (Ctrl), and similarly grown seedlings but with 50 mM NaCl (+ Salt) in the PNM medium, and seedlings infected with K-23 in the absence (+ F) and presence of 50 mM NaCl (+ F, + Salt) at 7 dpi. Scalebar: 400 μm. c Comparison of the impact of K-23 on biomass promotion under salt conditions in control (Col-0) and rsl2 rsl4 seedlings. Data are means ± SE (n = 15 seedlings), where the particular average value for each tested Arabidopsis genotype grown under control conditions was set to 100% (%Ctrl). Student’s t test; ** p < 0.01

Full size image

Among the TFs included in the network, RAP2.11 and RSL2 are highly connected nodes, exhibiting 45 and 14 direct connections with other DEGs, respectively. It is noteworthy that 10 of those direct connections are shared between RAP2.11 and RSL2. Root hair elongation is predominantly controlled by the two master regulators RSL2 and RSL4 (Yi et al. 2010), while RAP2.11 appears to be more associated with the regulation of potassium transporters in roots (Kim et al. 2012; Templalexis et al. 2022). Consequently, we focused our attention on RSL2 to examine whether the observed induction of root hair growth is a prerequisite for increased salt tolerance triggered by the fungus in Arabidopsis. Accordingly, we cultivated Arabidopsis wild-type and rsl2 rsl4 double mutant plants under control and salt stress conditions and treated them with K-23 or a mock control. As demonstrated in Fig. 6b, rsl2 rsl4 mutant plants are hairless. Treatment of rsl2 rsl4 seedlings with K-23 under salt stress conditions did not result in increased plant biomass production. In contrast to wild-type plants, the rsl2 rsl4 double mutant exhibited a significant reduction in total plant fresh weight (Fig. 6c). Therefore, it can be concluded that fungus-induced root hair growth is a crucial aspect required to enhance salt tolerance in Arabidopsis plants.

JUNGBRUNNEN 1 is Involved in the Fungus-Triggered Response in the Root

To identify transcriptional regulators contributing to the observed induction of root hair growth, we subjected the RNA-seq data sets to gene expression profile clustering utilizing a Poisson-based Mixture Model approach. This method was employed to identify candidate genes induced by salt and the fungus, with stronger induction observed when plants were co-cultivated with the fungus under salt conditions. After assessing the optimal cluster number fit for our data using the coseq package (Rau et al. 2015), we obtained 9 distinct clusters (Supporting information Data Sheet 4). Figure 7 presents the clusters of interest as determined by the Poisson Mixture estimations. The clusters illustrate the representative distribution of genes in the data set, describing their specific behavior across the tested conditions. Of the 9 clusters, clusters 3 and 5 exhibited the gene expression distribution of interest.

Fig. 7
figure 7

Clustering of RNA-seq data under combinatorial effects. The figure shows the two clusters with profiles of interest generated using DIANE. a Gene expression profiles are given as the normalized expression divided by the mean of the normalized expression across all tested conditions. b Differential expression data for the extracted transcription factors included in clusters 2 and 3, respectively. The expression data originates from the comparative analyses of the  + F Ctrl vs. − F Ctrl and  + F Salt vs. − F Salt conditions. Only genes that show a significant difference in their expression (FDR ≤ 0.05) in at least one of the two conditions are shown. The color is correlated to the value of the differential expression (log2FC). c Root hair length of wild-type Arabidopsis (Col-0), JUB1 knockdown mutants (jub1-1) and overexpression mutants (JUB1-OX) under control and salt stress conditions. JUB1 mutants were additionally treated with K-23 and mock. Data represent means ± SE (n = 10 roots). One-way ANOVA with a post hoc Tukey–Kramer test was used. Different letters indicate significant differences between means (p ≤ 0.05)

Full size image

The 2 clusters contained 199 and 709 genes, respectively. Within these clusters, we identified 9 and 11 TF genes, respectively, when compared to the Arabidopsis Transcription Factor Database (AtTFDB; Yilmaz et al. 2011). Among the identified TFs, we observed a substantial number of WRKY and NAC TFs, including WRKY28, WRKY30, WRKY31, WRKY42, WRKY55, NAC003, NAC004, NAC016, NAC047, NAC053, NAC059, NAC102, and JUNGBRUNNEN 1 (JUB1|NAC042).

In general, the fold changes in differential expression were considerably higher in cluster 3 compared to cluster 5. In cluster 5, the gene expression responses to the fungus were more pronounced without salt, ranging between log2(fold change) values of 1.01–2.81. However, under salt conditions, the induction of gene expression remained below the threshold of log2(fold change) 1, with the exception of WRKY42. In contrast, differential expression levels in cluster 3 ranged between log2(fold change) values of 1.68 and 3.97 after fungal infection in the absence of salt. Five genes in this group demonstrated an additional increase in their differential expression in the presence of 50 mM NaCl (Table 2).

Table 2 Differential expression of genes responding to K-23 infection and salt stress in cluster 4
Full size table

While bHLH041 functions as a repressor of the TF genes PLETHORA 1 (PLT1), PLT2, and WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which are involved in stem cell niche maintenance (Xu et al. 2023), ERF098, WRKY30, and WRKY55 are reported to be involved in abiotic and biotic stress responses (Zhang et al. 2012; Scarpeci et al. 2013; Wang et al. 2020b). JUB1, conversely, has been characterized as a key regulator of longevity in Arabidopsis (Wu et al. 2012). More recently, Shahnejat-Bushehri et al. (2016) reported that JUB1 is also involved in the regulation of the metabolism and signaling of gibberellins and brassinosteroids. JUB1 is a repressor of the gibberellic acid (GA) biosynthesis genes GA3ox1 and GA3ox2, which was confirmed in our data set. However, we did not detect an impact on the expression of the DWF4 gene, which encodes cytochrome P450 monooxygenase 90B1, a key enzyme in brassinosteroid (BR) biosynthesis. In summary, JUB1 exhibits a considerably strong response to K-23 treatment in the absence and presence of salt, with 12- and 36-fold induction, respectively, rendering this TF a particularly interesting candidate. Subsequent analysis of root hair elongation in overexpression (JUB1-OX) and knockdown mutants (jub1-1) of JUB1 provided evidence for the involvement of JUB1 in the maintenance of root hair elongation under salt conditions. As demonstrated in Fig. 7c, the JUB1 mutants responded similarly to wild-type Arabidopsis when the plants were treated with salt. Both JUB1-OX and jub1-1 exhibited a significant reduction in root hair length under salt stress conditions. This observation suggests that JUB1 is likely not directly involved in root hair elongation. However, while the effect of K-23 on root hair elongation in JUB1-OX was also relatively similar to that of wild-type plants, K-23-inoculated jub1-1 mutants demonstrated a significant decrease in root hair length under salt conditions. This finding led to the conclusion that JUB1 induction in K-23-treated plants under salt stress is a requirement to maintain the increased root hair length.

Increased Expression of JUB1 Stabilizes DELLA Repressor Proteins

In addition to the repression of GA biosynthesis, which stabilizes DELLA repressor proteins, JUB1 has also been reported to directly induce the expression of the two DELLA members GAI and RGL2 (Shahnejat-Bushehri et al. 2016). Furthermore, GA has been demonstrated to negatively regulate root hair elongation. The ga20ox2-1 knockout mutant exhibits longer root hairs compared to wild-type plants (Lv et al. 2018). The inhibitory effect of GA on root hair elongation is further substantiated by the finding that the effect of GA appears to correlate with the abundance of DELLA proteins that function as negative regulators of GA signaling (Xu et al. 2014). A study on the effect of phosphorus deficiency on root architecture highlighted that low phosphorus levels reduce the concentration of GA, which results in the accumulation of DELLA and significantly elongated root hairs (Jiang et al. 2007). To investigate the effect of the fungus on GA signaling, we cultivated seedlings of the GA signaling reporter line RGAp::GFP-RGA with and without the fungus (Fig. 8).

Fig. 8
figure 8

Infection of Arabidopsis roots with Fusarium sp. strain K-23 promotes DELLA stability in roots. GFP-fluorescence and bright-field image as well as the overlay of the two images showing lateral root tips of mock- (−F) and K-23-infected (+ F) RGAp::GFP-RGA GA-signaling reporter plants at 3 and 7 dpi. Scale bars = 500 µm

Full size image

Confocal laser scanning microscopy experiments revealed that the fluorescence of the GFP-RGA fusion protein is consistently enhanced in the K-23-inoculated reporter RGAp::GFP-RGA plants compared to the corresponding control plants without infection. At 3 and 7 dpi, fluorescence was more intense in the fungus-infected lateral root tips. This observed stabilization of the GFP-DELLA fusion protein suggests a sustained repression of GA biosynthesis, which is hypothesized to also promote root hair elongation.

Discussion

Roots are essential for plant growth and productivity. They perform fundamental functions, including the uptake of water and nutrients, the anchoring of the plant in the soil, and the maintenance of interactions with microorganisms in the rhizosphere, which have a direct impact on the growth and development of aerial parts of the plant (Paez-Garcia et al. 2015). The root system also plays a central role in plant responses to abiotic stresses (Lynch 1995). Frequently, the root system is the first plant organ to encounter abiotic stresses, such as drought or salinity, which necessitates the integration of its primary tasks of water and nutrient supply with the prevailing biotic and abiotic stress conditions in a timely manner. In this context, root growth and development exhibit considerable plasticity to respond to incoming stress cues appropriately. Potential responses involve variations in the number, extension, placement, and growth direction of individual components of the root system (Giehl et al. 2014).

Root development is regulated by a complex network that involves several plant hormones, including auxin, cytokinin, GA, BRs, ABA, and strigolactones (Pacifici et al. 2015). Auxin plays a crucial role in root development, as it is critical to root meristem development (Roychoudhry and Kepinski 2022), root clock (Perianez-Rodriguez et al. 2021), lateral root formation (Du and Scheres 2018), and root gravitropism (Su et al. 2017). Several studies have already elucidated the significant interplay between auxin- and cytokinin-dependent regulatory processes for the determination of whether meristematic cells divide or undergo differentiation (Dello Ioio et al. 2007; Růžička et al. 2009). The auxin–cytokinin crosstalk-dependent control of meristem size and root development involves the repression of polar auxin transport through the down-regulation of the expression of the auxin transporters PIN1, PIN3, and PIN7 by the auxin signaling repressor SHORT HYPOCOTYL 2 (SHY2) (Dello Ioio et al. 2008). Consequently, the auxin maximum at the root apex is altered, which, in turn, modifies positional information that is crucial for the maintenance of the correct cell division, polarity, and fate (Sabatini et al. 1999).

In this study, we confirmed the high salt tolerance of axenically grown Fusarium sp. strain K-23 and its growth-promoting effect on Arabidopsis seedlings under salt stress conditions. The plant–fungus interaction was observed to occur on the surface of Arabidopsis roots rather than inside the roots. There are instances of fungi that are primarily located on the surface of their host plants' roots, such as Mortierella chlamydospora and M. indohii, which rarely penetrate root cortex cells or grow in the intercellular space within the root (Ansell and Young 1982). This phenomenon could also apply to K-23 interacting with Arabidopsis.

Subsequently, we conducted a comprehensive transcriptomics analysis to gain a deeper understanding of the molecular processes triggered by the fungus. Analysis of DEGs that were negatively regulated by both fungal inoculation and 50 mM NaCl treatment indicated the misregulation of 2 MYB-like TFs, RVE1 and RVE8. The RVE clock genes connect the circadian clock to plant growth. A rev3, −4, −5, −6, −8 quintuple mutant was demonstrated to be significantly larger than Arabidopsis wild-type plants, suggesting that RVEs function as negative growth regulators (Gray et al. 2017). RVE1, conversely, was shown to be a positive regulator of hypocotyl growth, as RVE1 overexpression lines were observed to accumulate free auxin (Rawat et al. 2009). Following this observation, we conducted a targeted analysis of auxin-regulated genes that exhibit a common response to K-23 treatment in our RNA-seq data set. Among the monitored genes, 12 were identified with significant alterations in their expression. Notably, 3 of the induced genes are known to be involved in the biosynthesis of l-tryptophan (Radwanski and Last 1995). l-Trp serves as the primary precursor for auxin and several auxin derivatives, including secondary metabolites crucial in plant defense, such as camalexin and indole glucosinolates. The induction of multiple genes involved in the formation of these compounds suggests that the metabolic flux of l-Trp is redirected toward the synthesis of these defense compounds, a phenomenon previously described in the infection of Arabidopsis plants with the root-colonizing fungus Serendipita indica (Pérez-Alonso et al. 2022). Moreover, the induction of numerous genes involved in the conjugation of free, and thus bioactive, indole-3-acetic acid (IAA) to inactive ester and amide conjugates further indicates an efficient regulation of cellular IAA homeostasis in response to fungal infection (González Ortega-Villaizán et al. 2024). Conversely, the analysis revealed a significant repression of SHY2. Loss-of-function shy2 mutants exhibit altered root growth patterns (Tian and Reed 1999), characterized by shorter primary roots compared to wild-type roots, while developing more numerous and elongated lateral roots. From these observations, we infer that the slightly increased number of lateral roots observed is potentially associated with the detected repression of SHY2. However, the alteration in the number of lateral roots was not statistically significant. Consequently, we directed our focus toward the 460 genes identified at the intersection of genes that demonstrated a positive response to both K-23 and salt treatment.

Among the DEGs shared between the fungus and salt treatment, we observed an overrepresentation of the GO term classifications related to root hair development (Fig. 4e). A detailed phenotypic analysis of root hairs in mock and K-23 treated wild-type plants under control and salt stress conditions confirmed a significant induction of root hair elongation in response to the co-cultivation with the fungus (Fig. 5). A subsequent experiment with the hairless rsl2 rsl4 double mutant demonstrated that root hair formation capacity is an essential requirement for the promotion of plant biomass by K-23 under salt stress conditions. Furthermore, our functional network analysis indicated a significant induction of RAP2.11, an AP2/ERF TF that modulates the expression of the high-affinity K+ uptake transporter HAK5 and other components of the low-potassium signal transduction pathway in Arabidopsis (Kim et al. 2012; Templalexis et al. 2022). Consequently, the anticipated increase in potassium uptake could improve the K+/Na+ ratio and, thus, mitigate salt stress (Sun et al. 2015). Our RNA-seq data did not provide evidence for a substantial transcriptional alteration of HAK5 (At4g13420) or KUP1 (At2g30070) in the presence of the fungus. However, we observed a significant induction of the CHX17 (At4g23700) cation transporter gene (log2(FC) = 2, FDR = 7.6E-15) and SOS3 (At5g24270) (log2(FC) = 0.73, FDR = 5.5E-3), which is part of the sodium expulsion pathway. Collectively, these changes in the transcriptome suggest that the K+/Na+ ratio in the root with K-23 could be altered, but further experimental work is necessary to corroborate this hypothesis.

Salt stress results in significant inhibition of root hair growth in Arabidopsis. Upon confirming that the elongation and maintenance of root hairs are critical for the salt tolerance effect promoted by the fungus, we focused on identifying TFs involved in this process. Utilizing a gene expression profile clustering approach, we identified a limited number of TFs whose expression was induced by fungal infection and, to a greater extent, through a combination of fungal and salt treatments. Among the identified molecular targets, we concentrated on JUB1, a NAC TF that has been demonstrated to exert a negative regulatory effect on GA and BR biosynthesis. Both of these plant hormones are known to function as negative regulators of root hair elongation, independently of the canonical regulatory cascade that controls root hair development, including GL2, RHD6, RSL1, RSL2, and RSL4 (Li et al. 2022).

Both the overexpression mutant (JUB1-OX) and the JUB1 knockdown line (jub1-1) exhibited wild-type behavior toward salt in the medium, characterized by a significantly reduced root hair length. However, when examining the impact of K-23 on the jub1-1 mutant line under salt stress, we observed a significant reduction of 30% and 42%, respectively, in root hair length when comparing the + F/ + Salt condition with the control, and seedlings treated only with the fungus (+ F). This finding suggests that a substantial portion of the preservation of root hair growth under salt conditions in response to an inoculation with Fusarium sp. strain K-23 involves JUB1.

The RNA-seq data obtained in our study confirmed the suppression of the GA biosynthesis genes GA3ox1 and GA3ox2. To corroborate this finding and elucidate the impact of K-23 on GA signaling, we investigated the effect of the fungus on the stability of the DELLA protein RGA, utilizing a RGAp::GFP-RGA reporter line. This experiment strongly supported our hypothesis, as the abundance of RGA was significantly increased when the reporter line was inoculated with the fungus. This stabilization of the DELLA protein supports the hypothesis that the fungus reduces GA levels, at least locally, which subsequently results in enhanced root hair growth.

In conclusion, our research provides substantial evidence for a previously undescribed molecular mechanism triggered by the Fusarium sp. strain K-23 that enhanced salt stress tolerance in Arabidopsis plants. The observed beneficial effect is strictly dependent on the plant's capacity to form root hairs, as hairless mutants were demonstrated to lose the growth-promoting effect of the fungus under salt stress conditions. Furthermore, we identified that TF JUB1 is involved in maintaining root hair elongation under salt stress. Finally, we propose that Fusarium sp. strain K-23 may serve as a valuable molecular tool to further elucidate root hair development under various abiotic stress conditions.