Plant-fungal Interactions Research Articles

Natural and Nature-Inspired Catechol Siderophores: A Promising Strategy for Rice Blast Management.

The rice-blast fungus Pyricularia oryzae poses a significant threat to rice production worldwide. Ferroptosis, an iron-dependent form of regulated cell death, has recently been reported to be involved in P. oryzae pathogenicity during plant-fungal interactions. Ferroptosis regulates the developmental cell death of conidia necessary for appressorium maturation. In this study, we have established that a series of benzamides containing a chelating catechol moiety suppresses the formation/maturation of appressoria, which are essential for host infection by the rice blast fungus. Moreover, for the most active compounds we have shown that their activity can be at least partially reversed by adding exogenous Fe3+. These results highlight the close association between iron availability and appressorium maturation, opening new avenues for the development of targeted strategies for P. oryzae management.

Modulation of fungal phosphate homeostasis by the plant hormone strigolactone

Inter-kingdom communication through small molecules is essential to the coexistence of organisms in an ecosystem. In soil communities, the plant root is a nexus of interactions for a remarkable number of fungi and is a source of small-molecule plant hormones that shape fungal compositions. Although hormone signaling pathways are established in plants, how fungi perceive and respond to molecules is unclear because many plant-associated fungi are recalcitrant to experimentation. Here, we develop an approach using the model fungus, Saccharomyces cerevisiae, to elucidate mechanisms of fungal response to plant hormones. Two plant hormones, strigolactone and methyl jasmonate, produce unique transcript profiles in yeast, affecting phosphate and sugar metabolism, respectively. Genetic analysis in combination with structural studies suggests that SLs require the high-affinity transporter Pho84 to modulate phosphate homeostasis. The ability to study small-molecule plant hormones in a tractable genetic system should have utility in understanding fungal-plant interactions.

RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

BackgroundSmall RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions.ResultsC. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs.ConclusionsWe provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

The role of Aspergillus flavus in modulating the physiological adjustments of sunflower to elevated CO2 and temperature

Exploring the interactions between plants and foliar endophytic fungi under varying climatic conditions is crucial for harnessing endophytes that enhance plant resilience to environmental stressors. This study examines the role of a specific strain of Aspergillus flavus as an endophyte in sunflowers under standard and altered CO2 and temperature regimes. We assessed the impact of this fungus on physiological traits such as chlorophyll and flavonoid content, gas exchange, and Chlorophyll a fluorescence using open-top chambers simulating ambient (∼420 µmol mol−1) and elevated (∼880 µmol mol−1) CO2 levels, along with a temperature increase of 3°C. The findings indicate that A. flavus promotes nitrogen assimilation and chlorophyll production under ambient conditions, potentially enhancing sunflower growth and photosynthetic performance. However, under elevated temperatures (eT), inoculation with A. flavus resulted in decreased biomass and reduced Photosystem II efficiency. Elevated CO2 (eCO2) conditions also led to unexpected negative effects, with reductions in foliar nitrogen, leaf area, and light capture efficiency, culminating in diminished biomass. When both elevated CO2 and temperature conditions were combined (eCO2eT), the interaction further impaired Photosystem II efficiency, suggesting exacerbated physiological stress. These results demonstrate that environmental modifications can transform A. flavus from a beneficial endophyte to a potential pathogen, highlighting the dual nature of plant-fungal interactions. This study underscores the complexity of these relationships under changing climatic conditions and suggests a cautious approach to the agricultural use of endophytes to ensure plant health and productivity.

Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

Comparative transcriptome profiling of resistant and susceptible groundnut (Arachis hypogaea) genotypes in response to stem rot infection caused by Sclerotium rolfsii

AbstractThis study aimed to explore transcriptomic distinctions between resistant (CS‐319) and susceptible (JAL‐42) groundnut (Arachis hypogaea) genotypes exposed to Sclerotium rolfsii infection across different developmental stages. Employing a de novo assembly‐based approach, we analysed the transcriptomic response in these groundnut plants under control and infected conditions at 24, 72 and 120 hours post‐inoculation (hpi). Our RNA‐Seq data yielded a total of 133,900,261 reads, revealing 7796 differentially expressed genes (DEGs). We constructed a gene regulatory network with 59 hub genes, identified 6783 transcription factors and uncovered 88,424 putative markers, including 17,236 simple‐sequence repeats (SSRs), 10,099 single‐nucleotide polymorphisms (SNPs) and 78,332 indels. Notably, the majority of DEGs were upregulated at 24 hpi in the resistant genotype, encompassing diverse functional categories such as pathogenesis‐related genes, defence‐related (R) genes, genes involved in plant–fungus interactions, oxidation–reduction‐related genes, transport, metabolism and proteolysis genes, along with transcription factors (FAR1, B3, GATA, NAC, WRKY, MYBC1 and bHLH), secondary metabolic pathway‐related genes and photosynthesis‐related genes. The up‐regulation of WRKY transcripts, associated with the activation of the jasmonic acid defence signalling pathway, potentially induced systemic acquired resistance (SAR). Conversely, these DEGs exhibited down‐regulation in the susceptible genotype. Furthermore, a total of 17,236 expressed sequence tag (EST)‐SSRs were identified from the unigenes, holding significant potential for advancing plant breeding through marker‐assisted methods, facilitating quantitative trait locus (QTL) mapping and evaluating genetic diversity among genotypes. This study's approach contributes to a more profound understanding of the molecular‐level defence mechanisms involved in the interaction between groundnuts and S. rolfsii.

New insights into decoding the lifestyle of endophytic Fusarium lateritium Fl617 via comparing genomes

Fungal-plant interactions have persisted for 460 million years, and almost all terrestrial plants on Earth have endophytic fungi. However, the mechanism of symbiosis between endophytic fungi and host plants has been inconclusive. In this dissertation, we used a strain of endophytic Fusarium lateritium (Fl617), which was found in the previous stage to promote disease resistance in tomato, and selected the pathogenic Fusarium oxysporum Fo4287 and endophytic Fusarium oxysporum Fo47, which are in the same host and the closest relatives of Fl617, to carry out a comparative genomics analysis of the three systems and to provide a new perspective for the elucidation of the special lifestyle of the fungal endophytes. We found that endophytic F. lateritium has a smaller genome, fewer clusters and genes associated with pathogenicity, and fewer plant cell wall degrading enzymes (PCWDEs). There were also relatively fewer secondary metabolisms and typical Fusarium spp. toxins, and a lack of the key Fusarium spp. pathogenicity factor, secreted in xylem (SIX), but the endophytic fungi may be more sophisticated in their regulation of the colonization process. It is hypothesized that the endophytic fungi may have maintained their symbiosis with plants due to the relatively homogeneous microenvironment in plants for a long period of time, considering only plant interactions and discarding the relevant pathogenicity factors, and that their endophytic evolutionary tendency may tend to be genome streamlining and to enhance the fineness of the regulation of plant interactions, thus maintaining their symbiotic status with plants.

Unravelling transcriptional responses of the willow to Fusarium kuroshium infection

Fusarium kuroshium and the ambrosia beetle Euwallacea kuroshio attack several tree species, including the arroyo willow (Salix lasiolepis). Despite the importance of this pest, there is no molecular information about this plant-fungus interaction. Using an in vitro pathosystem of S. lasiolepis leaves – F. kurosihum and the RNA-seq approach, this study analyzed for the first time the transcriptome of both organisms at 4-, 8-, and 12-days post-inoculation. De novo assembly was done to generate the willow transcriptome, and a total of 361,2151 unique transcripts (UniGenes) were found. 4,583 UniGenes responded to the infection, and the posterior analysis showed that it contains the molecular elements to develop both pattern- and effector-triggered immunity responses (PTI and ETI, respectively). Significant expression changes of genes involved in an oxidative burst, Ca2+ signaling, and mutually antagonistic interaction between salicylic acid- and jasmonic acid/ethylene-dependent signaling suggest that the willow recognized the fungus and triggered downstream responses. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense. Concerning F. kuroshium, 178 induced genes that show homology with genes involved in the pathogenicity in other ascomycetes fungi plus transport proteins, ribosomal proteins, and uncharacterized proteins with specific domains were identified. Collectively these data provide a basis for future research on the molecular response of this transcendental tree during biotic stress, specifically during Fusarium dieback disease.

Sugar Transporter HmSWEET8 Cooperates with HmSTP1 to Enhance Powdery Mildew Susceptibility in Heracleum moellendorffii Hance.

The powdery mildew caused by Eeysiphe heraclei is a serious concern in Heracleum moellendorffii Hance. Therefore, exploring the mechanisms underlying sugar efflux from host cells to the fungus during the plant-fungus interaction showed great significance. The study successfully cloned HmSWEET8 and HmSTP1 genes based on RNA-seq technology. The complementation assays in yeast EBY.VW4000 found HmSWEET8 and HmSTP1 transporting hexose. Over-expressing or silencing HmSWEET8 in H. moellendorffii leaves increased or decreased powdery mildew susceptibility by changing glucose concentration in infective sites. Meanwhile, over-expressing HmSTP1 in H. moellendorffii leaves also increased powdery mildew susceptibility by elevating the glucose content of infective areas. Additionally, HmSTP1 expression was up-regulated obviously in HmSWEET8 over-expressed plants and inhibited significantly in HmSWEET8 silenced plants. Co-expressing HmSWEET8 and HmSTP1 genes significantly increased powdery mildew susceptibility compared with over-expressed HmSWEET8 or HmSTP1 plants alone. The results demonstrated that HmSTP1 may assist with HmSWEET8 to promote E. heraclei infection. Consequently, the infection caused by E. heraclei resulted in the activation of HmSWEET8, leading to an increased transfer of glucose to the apoplasmic spaces at the sites of infection, then, HmSTP1 facilitated the transport of glucose into host cells, promoting powdery mildew infection.

Botrytis cinerea causes different plant responses in grape (Vitis vinifera) berries during noble and grey rot: diverse metabolism versus simple defence.

The complexity of the interaction between the necrotrophic pathogen Botrytis cinerea and grape berries (Vitis vinifera spp.) can result in the formation of either the preferred noble rot (NR) or the loss-making grey rot (GR), depending on the prevailing climatic conditions. In this study, we focus on the functional gene set of V. vinifera by performing multidimensional scaling followed by differential expression and enrichment analyses. The aim of this study is to identify the differences in gene expression between grape berries in the phases of grey rot, noble rot, and developing rot (DR, in its early stages) phases. The grapevine transcriptome at the NR phase was found to exhibit significant differences from that at the DR and GR stages, which displayed strong similarities. Similarly, several plant defence-related pathways, including plant-pathogen interactions as hypersensitive plant responses were found to be enriched. The results of the analyses identified a potential plant stress response pathway (SGT1 activated hypersensitive response) that was found to be upregulated in the GR berry but downregulated in the NR berry. The study revealed a decrease in defence-related in V. vinifera genes during the NR stages, with a high degree of variability in functions, particularly in enriched pathways. This indicates that the plant is not actively defending itself against Botrytis cinerea, which is otherwise present on its surface with high biomass. This discrepancy underscores the notion that during the NR phase, the grapevine and the pathogenic fungi interact in a state of equilibrium. Conversely the initial stages of botrytis infection manifest as a virulent fungus-plant interaction, irrespective of whether the outcome is grey or noble rot.

Fungal–Plant Interactions: Latest Advances and Prospects

During the evolution of higher plants, many microorganisms evolve alongside them, creating a synergistic plant–microbe world [...]

The Contribution of Trichoderma viride and Metallothioneins in Enhancing the Seed Quality of Avena sativa L. in Cd-Contaminated Soil.

Pollution of arable land with heavy metals is a worldwide problem. Cadmium (Cd) is a toxic metal that poses a severe threat to humans' and animals' health and lives. Plants can easily absorb Cd from the soil, and plant-based food is the main means of exposure to this hazardous element for humans and animals. Phytoremediation is a promising plant-based approach to removing heavy metals from the soil, and plant growth-promoting micro-organisms such as the fungi Trichoderma can enhance the ability of plants to accumulate metals. Inoculation of Avena sativa L. (oat) with Trichoderma viride enhances germination and seedling growth in the presence of Cd and, in this study, the growth of 6-month-old oat plants in Cd-contaminated soil was not increased by inoculation with T. viride, but a 1.7-fold increase in yield was observed. The content of Cd in oat shoots depended on the Cd content in the soil. Still, it was unaffected by the inoculation with T. viride. A. sativa metallothioneins (AsMTs) participate in plant-fungi interaction, however, their role in this study depended on MT type and Cd concentration. The inoculation of A. sativa with T. viride could be a promising approach to obtaining a high yield in Cd-contaminated soil without increasing the Cd content in the plant.

Establishment of an Infection System for Gentian (Gentiana spp.) Sclerotial Flower Blight Disease.

Gentians (Gentiana spp.) as floriculture crops are constantly exposed to several fungal and viral pathogens in the field. Among the fungal diseases afflicting gentian production, gentian sclerotial flower blight caused by Ciborinia gentianae incurs economic losses, as it affects flowers before and after harvest. Currently, preventive measures for this disease are limited, and no resistant cultivars have been reported. This is partly because of the lack of a reliable infection system that could promote research on this plant-fungus interaction. In this study, Gentiana plant tissue culture material was inoculated with C. gentianae culture filtrate. We successfully demonstrated non-ascospore-mediated infection of C. gentianae. Inoculation of individual hyphal structures present in the culture filtrate suggested that sclerotial primordia are the main agents of this infection. Interestingly, our results indicated that primary infection of C. gentianae occurs in petals rather than leaves, which enables systemic infection and therefore mirrors the fungus's infection strategy observed in the field. Moreover, we showed that (i) non-ascospore hyphal structures can also cause disease in flowers grown in the field, and (ii) ascosporic infection can also be observed using the in vitro system, opening possibilities for both practical and basic research aimed to combat gentian sclerotial flower blight disease.

Pinibarreniales, a new order of Sordariomycetes from pine barrens ecosystem

ABSTRACT Pinibarrenia chlamydospora, sp. nov. isolated from the roots of highbush blueberry in the New Jersey Pine Barrens, is described and illustrated. Based on multigene phylogenetic analysis, as well as morphological and ecological characteristics, Pinibarreniales and Pinibarreniaceae are established to accommodate this novel lineage in Sordariomycetidae, Sordariomycetes. Pinibarreniales, Tracyllalales, and Vermiculariopsiellales are proposed to be included in the subclass Sordariomycetidae. Pinibarreniales likely have a wide distribution and forms association with Ericaceae plants that live in acidic and oligotrophic environments because its DNA barcode matches with environmental sequences from other independent ecological studies. The plant-fungal interaction experiment revealed negative impacts on Arabidopsis, indicating its pathogenicity. This uncovered new fungal lineage will contribute to a better understanding of the diversity and systematics of Sordariomycetes.

Endophytic Trichoderma: Potential and Prospects for Plant Health Management.

The fungus Trichoderma is widely regarded as the most common fungal biocontrol agent for plant health management. More than 25 Trichoderma species have been extensively studied and have demonstrated significant potential in inhibiting not only phytopathogen growth but also insect pest infestations. In addition to their use as biopesticides, there is increasing evidence that several Trichoderma species can function as fungal endophytes by colonizing the tissues of specific plants. This colonization enhances a plant's growth and improves its tolerance to abiotic and biotic stresses. In recent decades, there has been a proliferation of literature on the role of Trichoderma endophytes in crop protection. Although the mechanisms underlying plant-fungal endophyte interactions are not yet fully understood, several studies have suggested their potential application in agriculture, particularly in the mitigation of plant pests and diseases. This review focuses on the diversity of Trichoderma endophytic strains and their potential use in controlling specific diseases and pests of crop plants. Trichoderma endophytes are considered a potential solution to reduce production costs and environmental impact by decreasing reliance on agrochemicals.

In silico analysis of fungal prion-like proteins for elucidating their role in plant-fungi interactions.

Prion-like proteins (PrLPs) have emerged as beneficial molecules with implications in adaptive responses. These proteins possess a conserved prion-like domain (PrLD) which is an intrinsically disordered region capable of adopting different conformations upon perceiving external stimuli. Owing to changes in protein conformation, functional characteristics of proteins harboring PrLDs get altered thereby, providing a unique mode of protein-based regulation. Since PrLPs are ubiquitous in nature and involved in diverse functions, through this study, we aim to explore the role of such domains in yet another important physiological process viz. plant-microbe interactions to get insights into the mechanisms dictating cross-kingdom interactions. We have evaluated the presence and functions of PrLPs in 18 different plant-associated fungi of agricultural importance to unravel their role in plant-microbe interactions. Of the 241,997 proteins scanned, 3,820 (~ 1.6%) were identified as putative PrLPs with pathogenic fungi showing significantly higher PrLP density than their beneficial counterparts. Further, through GO enrichment analysis, we could predict several PrLPs from pathogenic fungi to be involved in virulence and formation of stress granules. Notably, PrLPs involved in (retro)transposition were observed exclusively in pathogenic fungi. We even analyzed publicly available data for the expression alterations of fungal PrLPs upon their interaction with their respective hosts which revealed perturbation in the levels of some PrLP-encoding genes during interactions with plants. Overall, our work sheds light into the probable role of prion-like candidates in plant-fungi interaction, particularly in context of pathogenesis, paving way for more focused studies for validating their role.

Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Cladosporium psychrotolerans strain T01 enhances plant biomass and also exhibits antifungal activity against pathogens.

An increasing number of microorganisms are being identified to enhance plant growth and inhibit phytopathogens. Some Cladosporium species form beneficial associations with plants, either as endophytes or by colonizing the rhizosphere. Herein, we evaluated the influence of the Cladosporium psychrotolerans (T01 strain) fungus on the in vitro growth of Arabidopsis thaliana plantlets through direct and split interactions. After 9 days post-inoculation with C. psychrotolerans, Arabidopsis plantlets exhibited a notable increase in fresh weight and lateral roots, particularly in split interactions. Chlorophyll content increased in both plant-fungus interaction conditions, whereas the primary root was inhibited during direct interaction. We observed an increase in the GUS signal from the Arabidopsis auxin-inducible DR5:uidA marker in lateral root tips in both contact and split fungal interactions, and primary root tips in a split interaction. Arabidopsis and tomato plants cultivated in soil pots and inoculated with C. psychrotolerans (T01 strain) showed a positive effect on biomass production. GC/MS analysis detected that the T01 strain emitted volatile organic compounds (VOCs), predominantly alcohols and aldehydes. These VOCs displayed potent inhibitory effects, with a 60% inhibition against Botrytis cinerea and a 50% inhibition against C. gloeosporioides. Our study demonstrates that C. psychrotolerans T01 has the potential to enhance biomass production and inhibit pathogens, making it a promising candidate for green technology applications.

The AMSlide for noninvasive time-lapse imaging of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis, the nutritional partnership between AM fungi and most plant species, is globally ubiquitous and of great ecological and agricultural importance. Studying the processes of AM symbiosis is confounded by its highly spatiotemporally dynamic nature. While microscopy methods exist to probe the spatial side of this plant-fungal interaction, the temporal side remains more challenging, as reliable deep-tissue time-lapse imaging requires both symbiotic partners to remain undisturbed over prolonged time periods. Here, we introduce the AMSlide: a noninvasive, high-resolution, live-imaging system optimised for AM symbiosis research. We demonstrate the AMSlide's applications in confocal microscopy of mycorrhizal roots, from whole colonisation zones to subcellular structures, over timeframes from minutes to weeks. The AMSlide's versatility for different microscope set-ups, imaging techniques, and plant and fungal species is also outlined. It is hoped that the AMSlide will be applied in future research to fill in the temporal blanks in our understanding of AM symbiosis, as well as broader root and rhizosphere processes.

Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.