The richness and diversity of endophytic fungi in F. cymosum vary across habitats and tissue compartments.
The southwestern region of China, including Sichuan, Yunnan, Guizhou, and Tibet, is renowned as the primary center of origin, distribution, and diversification of buckwheat (genus Fagopyrum) [39]. To assess the diversity and variability of endophytic fungi in wild F. cymosum across six native habitat sites in four regions (Fig. 1A), high-throughput sequencing of the ITS region was conducted. A total of 4,052,907 compelling reads and 4,052,889 high-quality sequences were acquired from 18 F. cymosum tissue samples, leading to the detection of 1,122 operational taxonomic units (OTUs). The sequence lengths varied from 182 to 351 base pairs. The smoothness of the sparse curves suggested that sequencing depth impacted the observed diversity in the samples. As the sequencing depth increased, the curve representing the number of OTUs in the 18 samples leveled off (Supplementary Fig. S1G). This suggests that the sequencing outcomes successfully captured the existing diversity in the sample, and additional increments in sequencing depth did not unveil new OTUs. The Venn diagram of OTUs illustrates that endophytic fungi of F. cymosum from six distinct habitats exhibit 46 shared OTUs, with YL, LZ, HL, NH, KM, and HZ individually harboring 147, 237, 296, 111, 99, and 186 unique OTUs, as depicted in Fig. 1B. Within the various tissue groups, roots, stems, and leaves contain 418, 356, and 267 unique OTUs, respectively, while they share 378 OTUs among them (Fig. 1C).
The species abundance classification plot of the top 10 phyla illustrates that Ascomycota is the predominant phylum among the endophytic fungi of F. cymosum, with Basidiomycota following closely behind (Supplementary Fig. S1A, D). In terms of class, Dothideomycetes, Sordariomycetes, and Agaricomycetes constitute the predominant classes among endophytic fungi, as depicted in Supplementary Fig. S1B, E. Regarding the order level, Capnodiales, Glomerellales, and Pleosporales emerge as the top three abundant orders, as shown in Supplementary Fig. S1C, F. Endophytic fungi of wild F. cymosum from six habitat sites display notable variations in genus-level abundance. For example, Septoria fungi comprise 25.96% of the HL habitat samples, whereas they only comprise 2.01% of the LZ samples. In contrast, Colletotrichum fungi account for 37.99% of LZ but less than 2% in NH samples (Fig. 1D). Distinct variations in genus-level fungi are evident among various tissue compartments. Notably, Ilyonectria is the most abundant genus in roots, accounting for 14.43%, while its presence is less than 4% when considering stems and leaves together. In stems, Vishniacozyma dominates with 16.04%, whereas Septoria prevails in leaves at 28.68% (Fig. 1E).
The alpha diversity of the 18 samples was evaluated utilizing community richness (Chao1 index, Observed species), coverage (Good coverage index), diversity (Shannon index, Simpson index), and evenness (Pielou’s index) metrics, which are outlined in Supplementary Table S6. Among the samples from various tissue compartments (roots, stems, leaves), the stem samples demonstrated the highest richness and diversity of endophytic fungi, along with the most evenly distributed fungal communities, as shown in Fig. 1F, G. HL displayed higher Chao1 and Observed species indices among the six distinct habitat sites, suggesting more extraordinary richness compared to HZ, which had the lowest values. Regarding community diversity, both HL and NH exhibited higher diversity than ZL. The robust fungal sequencing coverage, with Good coverage values surpassing 99.8% across all samples, accurately portrays the fungal communities within the samples. Furthermore, there were no substantial differences in evenness among the samples.
By employing the Bray-Curtis distance algorithm for ANOSIM analysis, clustering was conducted on the endophytic fungal communities of F. cymosum across six distinct habitats. The outcomes revealed notable discrepancies in species composition among sample groups in contrast to within-group variations (R = 0.383, P = 0.01), emphasizing habitat-specific differences in F. cymosum, as illustrated in Supplementary Fig. S1H. Likewise, ANOSIM analysis of distinct plant tissues indicated that the disparities between groups were significantly more pronounced than the differences within groups (R = 0.223, P = 0.001) (Supplementary Fig. S1I). Furthermore, PCoA analysis unveiled variations in endophytic fungal communities within F. cymosum samples, with PCo1 accounting for 13.1% of the variance and PCo2 explaining 10.9% (Fig. 1H, I). Notably, F. cymosum from Linzhi City in Tibet displayed significant distinctions compared to other habitats. KM and NH exhibited a closer relationship, while HZ, HL, and YL showed more dispersion, signifying substantial variations in fungal communities among roots, stems, and leaves. Notably, the fungi in stems and leaves demonstrated more remarkable similarity to those in roots.
Endophytic fungi at the genus level of Fusarium, Colletotrichum, and Ceratobasidium may be involved in drought tolerance of F. cymosum
Endophytic fungi within plants have engaged in extended co-evolution with their hosts, frequently establishing mutualistic associations. The host offers a "habitat" and nourishment to the endophytic fungi, which reciprocate by aiding the host in improved uptake of external nutrients like nitrogen, phosphorus, and other inorganic elements. Furthermore, these endophytic fungi elevate the host's photosynthetic efficiency and fortify its resilience against diverse abiotic and biotic stresses, encompassing drought, salinity, and instances of pest and disease attacks [40, 41]. Thus, our hypothesis suggests the presence of endophytic fungal strains in F. cymosum that stimulate growth and enhance the plant's resistance to external stresses. Each habitat where F. cymosum samples are found exhibits unique ecological climates. We referenced the WordClim database to analyze the climatic characteristics of six native habitat sites, revealing notable variations in precipitation levels (Supplementary Table S7), notably during the driest quarter and the driest month. Notably, Linzhi city in Tibet experiences the lowest annual precipitation, while Huili County in Sichuan records the highest levels. In our quest to identify drought-resistant fungal strains in F. cymosum, we pinpointed 18 common genera of endophytic fungi across 18 sample groups (Fig. 2A). Subsequently, we conducted a Pearson correlation analysis between these shared genera and eight precipitation factors (Supplementary Fig. S2). The findings revealed that the genera Fusarium, Colletotrichum, and Ceratobasidium exhibited negative correlations with precipitation variables, notably annual precipitation, driest month precipitation, and driest quarter precipitation (Fig. 2B). This suggests that these three genera of endophytic fungi are predominantly present in F. cymosum plants from regions with low precipitation levels. In conclusion, it is postulated that these genera may play a role in the drought resistance of F. cymosum.
For the isolation of endophytic fungi belonging to the genera Fusarium, Colletotrichum, and Ceratobasidium from F. cymosum, we utilized the tissue block isolation technique and successfully isolated 59 endophytic fungal strains from the roots, stems, and leaves of F. cymosum. By employing morphological and molecular biological analyses, a total of 37 species were identified (Supplementary Table 8). The growth morphology of the 37 endophytic fungi on plates is illustrated in Supplementary Fig. S3. Through phylogenetic analysis, we identified 13 Fusarium strains (labeled JQ_R1, JQ_R2, JQ_R3, JQ_R5, JQ_R6, JQ_R7, JQ_R8, JQ_R9, JQ_S1, JQ_S2, JQ_S3, JQ_L1, JQ_RS1), 2 Colletotrichum strains (labeled JQ_L4, JQ_L5), and 1 Ceratobasidium strain (labeled JQ_R14) among these 37 isolates (Fig. 2C). The viability of the strains on drought plates was documented to assess their capacity for average growth under drought circumstances, and survival curves were subsequently generated (Supplementary Fig. S4). Specifically, twelve strains exhibited average growth on 8% PEG6000 plates, while eleven strains displayed average growth on 12% PEG6000 plates. Next, the strains' capacity to secrete indole-3-acetic acid (IAA) in vitro was evaluated to determine their potential for promoting host growth (Supplementary Fig. S5). Among them, eight strains exhibited notably high levels of IAA production. Following these findings, three strains demonstrating robust capabilities in drought resistance and growth promotion were identified: JQ_R2 (Fusarium sp.), JQ_R14 (Ceratobasidium sp.), and JQ_L5 (Colletotrichum sp.). The growth profiles of JQ_R2, JQ_R14, and JQ_L5 are depicted in Fig. 2D, E, and F, respectively, while their levels of IAA secretion are detailed in Fig. 2G. The ensuing investigation will examine the colonization of these three fungi within F. cymosum and their impacts on plant growth.
JQ_R2, JQ_R14, and JQ_L5 demonstrate growth-promoting and drought-resistant effects upon inhabiting F. cymosum.
The process of fungal colonization from external sources to the interior of plants is influenced by various factors, with the primary influence stemming from the fungi themselves. Different fungal species demonstrate varying efficiency in colonization and differences in preferred colonization sites and modes [42–44]. Despite being isolated from F. cymosum, the colonization efficiency and post-colonization morphology of JQ_R2, JQ_R14, and JQ_L5 remain obscure. To address this gap, we employed the fluorescent dye WGA-AF488 for fungi labeling within the plant, enabling the observation of their colonization efficiency and morphology. The fluorescence staining analysis revealed that all three strains exhibited the capability to colonize the roots of F. cymosum from exogenous sources (Fig. 3A). Among the strains, JQ_R14 displayed the most significant and dense colonization, predominantly through hyphae and sclerotia within root cells. The hyphae of JQ_R14 were thick, densely packed, and featured well-defined septa. Sclerotia were interspersed among the hyphae, forming elliptical structures. Similarly, JQ_R2 also exhibited colonization through hyphae and sclerotia, although its hyphae were thin, sparsely distributed, and lacked septa. The sclerotia predominantly exhibited elongated shapes. In contrast to the two other strains, JQ_L5 colonized exclusively through thin, curved, and extensively branched hyphae. Furthermore, small protrusions, likely representing newly divided short hyphae, were frequently noticeable along the hyphae.
The F. cymosum plants cultivated in the soil to mimic field conditions revealed that under NM circumstances, the inoculation with fungal strains notably enhanced plant growth (Fig. 3B). Mainly, substantial improvements were evident in plant height and central stem thickness, displaying significant increments post-inoculation with JQ_R2, JQ_R14, and JQ_L5 (Fig. 3C). Moreover, there was a significant increase in the number of central stem nodes and leaf size. Specifically under NM conditions, the inoculation with JQ_R2 notably boosted the activities of POD, SOD, and CAT enzymes. Similarly, injection with JQ_R14 resulted in significant enhancements in the activities of POD and SOD, whereas immunization with JQ_L5 markedly elevated the activities of POD and CAT. Moreover, the levels of proline and soluble sugars surged post-fungal inoculation, with JQ_R2 and JQ_L5 amplifying the GSH content, although the PAL content remained unaltered. In DM conditions, the plant phenotype (including height, stem thickness, water content, etc.) exhibited a substantial decline when contrasted with NM conditions, whereas enzyme activities and osmolyte contents showed a marked increase. In DM conditions, inoculated plants exhibited elevated plant height, stem and leaf water content, and main stem node numbers compared to non-inoculated plants. Moreover, there was a significant increase in the activities of POD, SOD, and CAT enzymes, alongside higher osmolyte levels and reduced MDA content (Fig. 3C, Supplementary Fig. S6).
To model abrupt extreme drought conditions, we cultivated F. cymosum seedlings hydroponically and supplemented the nutrient solution with varying concentrations of PEG6000 to replicate severe drought stress. To ascertain the optimal concentration of PEG6000 for drought simulation, we subjected one-week-old F. cymosum seedlings to pre-treatment with PEG6000 concentrations of 0%, 5%, 10%, 15%, and 20%. We monitored leaf curling hourly, documenting our observations through photography and recording. The condition of the seedlings was assessed at two distinct time points: initially, at the commencement of leaf water loss and curling, and subsequently at a stage of severe water loss and curling (Supplementary Fig. S7). The findings revealed that leaf curling was induced by 5% and 10% PEG6000 at 56 and 48 hours, respectively, with severe water loss observed at 80 and 72 hours. Conversely, exposure to 20% PEG6000 resulted in leaf curling within 1 hour, followed by severe water loss leading to near-fatality after 5 hours. On the other hand, with 15% PEG6000, leaf curling manifested swiftly at 2 hours and endured for a significant period, enabling the plants to react and withstand, with severe water loss only manifesting after 56 hours. Consequently, 15% PEG6000 was chosen as the concentration for simulating drought stress.
Phenotypically, under 0% PEG6000 conditions, the inoculation with fungal strains led to increased leaf emergence in F. cymosum seedlings, particularly noticeable in plants treated with JQ_R2. Following three days of exposure to 15% PEG6000, non-inoculated (NI) plants displayed severe drought symptoms characterized by near-complete leaf curling. Fungi-inoculated plants with JQ_R14 and JQ_L5 exhibited reduced drought symptoms, with the curling confined to older leaves, while most new leaves remained unaffected. In contrast, plants treated with JQ_R2 displayed minimal leaf chlorosis and virtually no leaf curling, sustaining regular growth patterns (Fig. 3D). Subsequently, a range of enzyme activities and substance contents were assessed in the hydroponically grown seedlings (Fig. 3E, Supplementary Fig. S7). Without PEG6000 (0% conditions), the injection with JQ_R2 markedly raised the levels of PAL, soluble sugars, and GSH. Conversely, at 15% PEG6000 conditions, notable alterations included a significant surge in POD enzyme activity and a marked reduction in MDA content post-fungal vaccination, particularly with JQ_R2. These patterns aligned with the trends observed in F. cymosum plants cultivated in soil.
A combined transcriptome and metabolome analysis revealed that folate metabolism might be involved in alleviating drought stress in F. cymosum by JQ_R2.
Given the superior drought tolerance exhibited by F. cymosum inoculated with JQ_R2 in prior experiments, we concentrated on evaluating the efficacy of the JQ_R2 strain in subsequent validations. To meticulously investigate the morphology of JQ_R2 hyphae colonizing the roots of F. cymosum, scanning electron microscopy was employed for a detailed examination of the hyphal presence on the root surface (Fig. 4A). In the absence of fungal inoculation, the root surface displayed solely disorganized root hairs and surface grooves of differing depths. Conversely, upon inoculation with the JQ_R2 strain, at low magnification (×300), many hyphae were detected intertwined with root hairs, with some hyphae tightly adhering to the grooves on the surface. The hyphae presented as smooth, elongated rods, with specific hyphal tips swelling to create spherical sporangia structures that released spores upon rupture. When examined at high magnification (×2000), the attachment of hyphae to the root surface, sporangial structure, and the morphology of certain tiny spores were distinctly observable.
To elucidate the molecular mechanisms through which JQ_R2 mitigates drought stress in F. cymosum, we examined the transcriptome data from F. cymosum leaves. Following quality filtering, a cumulative data of 84.32 Gb was sourced from 12 leaf samples of F. cymosum. Each sample provided over 6 GB of data, reflecting a Q20 rate of ≥ 99.385% and a Q30 rate of ≥ 97.826% (Supplementary Table S9). The quality-approved clean data reads were aligned to the reference genome utilizing the HISAT2 software, followed by an alignment assessment through the Qualimap RNA-seq pipeline. The comprehensive mapping rate across the 12 samples exceeded 89.4%, signifying the successful alignment of most sequencing reads to the reference genome. Furthermore, the exonic mapping rate for all samples exceeded 70%, signifying robust data quality and specificity. Moreover, 5'-3' bias values surpassing 1 indicated that the RNA samples were intact and not degraded (Supplementary Table S10). In summary, the transcriptome data are deemed precise and dependable, making them suitable for further analyses. The figure in Fig. 4B illustrates the differentially expressed genes (DEGs) count in the four comparison groups, based on |log2 fold change| ≥ 1 and p ≤ 0.05. The comparison between CK and DCK revealed the most significant number of DEGs, with 185 genes upregulated and 138 downregulated. Conversely, the CK vs. R2 comparison exhibited the lowest count of DEGs, with 30 genes upregulated and 48 downregulated. A Venn diagram, depicted in Fig. 4C, displayed the distribution of DEGs across the four comparison groups, highlighting that the CK vs. DCK group possessed the highest number of unique DEGs (250), followed by the R2 vs. DR2 group with 206 exclusive DEGs. Only five common DEGs were identified between the two experimental groups, suggesting that the injection with JQ_R2 significantly influenced the drought response of F. cymosum plants. Notably, no DEGs were shared between the experimental groups subjected to standard and drought conditions. The number of DEGs observed under drought conditions with fungal inoculation exceeded those identified under normal conditions, indicating a more pronounced impact of JQ_R2 on F. cymosum plants under drought stress. We conducted a Gene Ontology (GO) enrichment analysis to ascertain the roles of the identified DEGs, as depicted in Supplementary Fig. S8. The analysis revealed enrichment of GO terms across three principal functional categories: molecular function (MF), cellular component (CC), and biological process (BP). Within the CK vs. DCK group, the DEGs were predominantly enriched in the MF and BP categories, with a notable enrichment identified in transferase activity, specifically in transferring glycosyl groups. The CK vs. R2 comparison group exhibited enrichment across the MF, CC, and BP categories, particularly emphasizing ribosome and cytosolic ribosome in the CC category. Conversely, in the DCK vs. DR2 group, the DEGs were predominantly enriched in the BP category, with only a few in the CC category. Notably, cellular response to acid chemicals was the most enriched GO term within the BP category. In the R2 vs. DR2 comparison group, most DEGs were enriched in the BP category, with a notable enrichment observed in the organic hydroxy compound metabolic process. Furthermore, the KEGG enrichment analysis offered comprehensive insights into the metabolic and signal transduction pathways influenced by the DEGs. Within the CK vs. DCK experimental group, differentially expressed genes (DEGs) were predominantly linked to plant hormone signal transduction and the MAPK signaling pathway. Conversely, in the CK vs. R2 comparison group, DEGs were primarily enriched in the flavone and flavonol biosynthesis and the phenylpropanoid biosynthesis pathways. In the R2 vs. DR2 experimental group, DEGs were predominantly enriched in fatty acid degradation and several amino acid metabolic pathways, including tyrosine, cysteine, and methionine metabolism (Supplementary Fig. S9). In the DCK vs. DR2 comparison group, DEGs were significantly associated with carbon metabolism and the metabolism of tryptophan, glutathione, glycine, and serine (Fig. 4D).
Transcriptomic analysis facilitates the identification of differentially expressed genes, whose upregulation or downregulation can influence the levels of downstream substances, consequently eliciting diverse plant responses. Nevertheless, gene expression regulation and metabolic regulation are intricate processes influenced by multiple layers and factors. It is important to note that alterations in gene expression levels do not always correlate directly with changes in the content of downstream substances. To address this uncertainty and precisely elucidate the pathways involved in regulating drought tolerance by JQ_R2 in F. cymosum, we performed untargeted metabolomic analysis on the leaves of four experimental groups. Compared to the control group, the OPLS-DA analysis unveiled notable biochemical alterations in the leaves of plants treated with the JQ_R2 strain (Supplementary Fig. S10). This suggests that VIP analysis is a valuable tool for metabolite screening. The differential accumulation of metabolites (DAMs) in the four experimental comparison groups was determined using |log2 fold change| > 1 and raw. pval < 0.05, as shown in Fig. 4E. The CK vs. DCK comparison group exhibited the highest count of differentially accumulated metabolites (DAMs), comprising 47 down-regulated and 86 up-regulated metabolites. Subsequently, the CK vs. R2 comparison group identified 93 DAMs, while the DCK vs. DR2 and R2 vs. DR2 experimental groups revealed 70 and 55 DAMs, respectively. A Venn diagram illustrating the differential metabolites across the four experimental groups was presented (Fig. 4F). It revealed that the CK vs. DCK and CK vs. R2 comparison groups shared 33 common DAMs. Conversely, the CK vs. DCK and R2 vs. DR2 groups exhibited only 4 shared DAMs, while the CK vs. DCK group contained 64 unique DAMs, indicative of the substantial influence of JQ_R2 strain inoculation on metabolite secretion in drought-stressed plants. Following this, KEGG enrichment analysis was conducted on the differentially accumulated metabolites (DAMs). In the CK vs. DCK experimental group, DAMs were predominantly enriched in nucleotide metabolism and the synthesis and degradation of flavonoids. Similarly, in the CK vs. R2 comparison group, the metabolites were chiefly enriched in purine, nucleotide, and amino acid metabolism. Moreover, in the R2 vs. DR2 comparison group, the DAMs were primarily enriched in the biosynthesis and metabolism of phenylpropanoids, flavonoids, and amino acids (Supplementary Fig. S11). Finally, in the DCK vs. DR2 comparison group, the DAMs were predominantly enriched in the biosynthesis and metabolism of various amino acids, including cysteine, methionine, arginine, and proline (Fig. 4G).
Utilizing the outcomes of the multi-omics analysis, our objective was to elucidate the mechanisms by which JQ_R2 aids F. cymosum plants in mitigating drought stress. In the previous KEGG enrichment analysis, our attention was directed toward the findings of the DCK vs. DR2 comparison group. Given that the DEGs were predominantly enriched in carbon metabolism and amino acid metabolism, and the DAMs were also chiefly enriched in amino acid synthesis and metabolism, our attention was drawn to a metabolite closely associated with both carbon and amino acid metabolism—folate. The folate content of F. cymosum plants exhibited a significant decrease under drought stress; however, subsequent inoculation with the JQ_R2 strain restored the folate levels to normal (Fig. 4H). Folate synthesis and utilization are intricately linked to dihydrofolate reductase (DHFR), an enzyme that converts dihydrofolate to folate. Particularly essential is the role of folate in its active form as tetrahydrofolate, a conversion process facilitated by DHFR. In our transcriptome analysis, we noted a significant decrease in the expression of dihydrofolate reductase-thymidylate synthase (DHFR-TS) in F. cymosum plants under drought stress. Remarkably, the expression levels of DHFR-TS markedly increased following inoculation with JQ_R2, aligning with the observed pattern in folate content (Fig. 4H). Therefore, we postulate that the JQ_R2 strain may assist F. cymosum plants in mitigating drought stress via the folate metabolic pathway.
Additionally, we validated the precision of the transcriptome data using real-time quantitative PCR. The expression levels of DHFR-TS in F. cymosum leaves corresponded with the transcriptome findings, indicating decreased expression post-drought and a substantial increase following JQ_R2 inoculation. Investigating the tissue-specific expression of the DHFR-TS gene, we assessed its levels in roots and stems. Our findings revealed a notable decrease in DHFR-TS expression in roots but an increase in stems under drought conditions. Interestingly, post-inoculation with JQ_R2, the expression levels of DHFR-TS in both roots and stems rose during drought stress. Besides DHFR-TS, other pivotal genes in the folate synthesis pathway, including ADCS, ADCL, GTPCHI, DHNA, and DHFS [45], are crucial in folate synthesis. Consequently, we evaluated the relative expression levels of these five genes (Fig. 4I). A significant trend emerged as the expression levels of these genes substantially increased in root tissues under drought conditions and post-inoculation with JQ_R2. In leaf tissues, apart from a reduction in ADCS expression and the absence of significant change in GTPCHI expression, the expression levels of the remaining genes also notably rose under drought conditions and following fungal inoculation. Nevertheless, the alterations in stem tissues displayed inconsistency. For example, ADCS expression increased post-inoculation under normal conditions but decreased significantly after inoculation under drought conditions. A typical pattern emerged where the expression levels of these folate synthesis-related genes in stem tissues rose under drought conditions without fungal inoculation.
The JQ_R2 strain may aid host plants in resisting drought stress by elevating folate content and boosting DHFR enzyme activity in F. cymosum
To investigate the potential of folate in aiding F. cymosum plants in withstanding drought stress, we conducted an in vitro folate spraying experiment. Following slight adjustments to the protocols described by Khan et al. [46] and Ibrahim et al.[37], we utilized foliar spraying of 150 µM folate for the experimental group (with distilled water spraying as the control) and monitored the growth response of F. cymosum plants pre- and post-drought stress. Our observation revealed that F. cymosum plants treated with folate exhibited a proliferation of young leaves akin to the phenotype seen post-inoculation with the JQ_R2 strain (Fig. 3D). After drought stress, in contrast to the control group showcasing extensive leaf curling and wilting, the folate-treated F. cymosum plants demonstrated wilting primarily at the margins of some mature leaves, while the younger leaves displayed no evident symptoms (Fig. 5A). Upon quantifying the folate content in the leaves (Fig. 5B), our analysis revealed that foliar application of folate elevated the folate levels in the leaves under normal conditions. Following drought stress, a substantial reduction in folate content was noted in the control group, whereas in the folate-sprayed experimental group, a slight non-significant decrease was observed. In contrast, DHFR enzyme activity notably decreased in the experimental group before drought but exhibited a substantial increase post-drought stress (Fig. 5C). Simultaneously, we assessed the soluble sugar content and peroxidase (POD) enzyme activity in the leaves of both the experimental and control groups following drought stress (Fig. 5D). Both parameters exhibited significant increases in the folate-sprayed experimental group, with the POD enzyme activity being notably elevated, resembling the physiological response observed post-inoculation with the JQ_R2 strain mentioned previously (Fig. 3C, E).
In the aforementioned experimental findings, we noted that the DHFR enzyme activity in folate-treated plants decreased before drought stress, whereas the folate content increased. This outcome prompted us to explore the potential correlation between the DHFR-TS gene and folate content. Hence, we conducted transient transformation to enhance the expression of the DHFR-TS gene in F. cymosum leaves and subsequently monitored alterations in leaf phenotype, folate content, and DHFR enzyme activity. The outcomes indicated a notable increase of over a hundredfold in the expression level of the DHFR-TS gene in the leaves post-overexpression (Fig. 5F), with the leaves exhibiting enhanced drought tolerance. Following detachment for 5 hours, the leaves overexpressing DHFR-TS exhibited a markedly reduced water loss rate compared to the control group (Ev group, which expressed an empty vector) (Fig. 5E). Additionally, the DHFR-TS overexpressing leaves displayed elevated folate content and DHFR enzyme activity (Fig. 5G). These findings imply a positive correlation between the DHFR-TS gene and folate content in F. cymosum. Hence, we postulate that the JQ_R2 strain may elevate folate levels in F. cymosum plants by amplifying the expression of the DHFR-TS gene.
Prior studies have demonstrated that endophytic fungi are capable of directly secreting metabolites for the benefit of their hosts [47, 48]. This observation inspired us to investigate the possibility of JQ_R2 secreting folate, utilizing a portion for its requirements while supplying the remainder to F. cymosum to enhance its drought stress resilience. Consequently, we assessed the folate secretion and DHFR enzyme activity of the JQ_R2 strain on standard plates (0% PEG6000) and drought plates (containing 12% PEG6000). Observing the phenotype images of JQ_R2 cultivated on varying media (Fig. 5H), it is evident that the growth rate of JQ_R2 appears unaffected by drought; in fact, it demonstrates accelerated growth on the drought plates. Nevertheless, the strain's overall condition was impacted. The mycelium exhibited a dense and thick white appearance on regular plates, whereas it appeared gray, sparse, and thin on drought plates. Moreover, a notable secretion of folate was observed within the mycelium of JQ_R2. The folate content in the mycelium notably decreased on drought plates, while there was a significant increase in DHFR enzyme activity. This indicates that JQ_R2 can secrete folate under drought conditions to benefit F. cymosum directly and boost the host's DHFR-TS expression, consequently enhancing the plant's folate secretion.