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. 2020 Feb 18;10(3):124. doi: 10.1007/s13205-020-2105-x

Identification of the key genes involved in the regulation of symbiotic pathways induced by Metarhizium anisopliae in peanut (Arachis hypogaea) roots

Feng Wang 1, Xiangqun Nong 1,✉, Kun Hao 1, Ni Cai 1, Guangjun Wang 1, Shaofang Liu 2, Hidayat Ullah 1,3, Zehua Zhang 1,✉
PMCID: PMC7028888  PMID: 32140376

Abstract

We detected and compared the mRNA and protein expression levels of immunity-associated and symbiosis-associated genes in peanut (Arachis hypogaea) roots inoculated with entomopathogenic fungus M. anisopliae or the phytopathogenic fungus Fusarium oxysporum, by RT-qPCR and parallel reaction monitoring (PRM). The selected genes were mainly associated with plant–fungus interactions, signal transduction, regulation of cell death, nitrogen or iron metabolism, nutrient acquisition or transport, and compound synthesis based on previous transcriptome analysis. The results showed that the host basal defense responses were significantly inhibited by both M. anisopliae and F. oxysporum, which suggests that both fungi actively suppress the host immunity for successful colonization and infection. However, only F. oxysporum induced a strong host hypersensitivity, which indicates that the host is strongly resisting F. oxysporum but potentially allowing M. anisopliae. Additionally, the genes (SYMRK, CaM, CCaMK, FRI2, ABCC2, F6H1, SCT, NRT24 and LTP1) related to symbiosis and growth were distinctively observed with an up-regulated expression following M. anisopliae treatment, which implies that the host was actively initiating the establishment of symbiosis with the fungus. This study revealed a synergistic relationship between host immunosuppression and the promotion of symbiosis during interactions with M. anisopliae. It suggested that M. anisopliae benefited plant for symbiotic relationship, in addition to controlling herbivorous insects as an entomopathogen.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-2105-x) contains supplementary material, which is available to authorized users.

Keywords: Symbiosis, Metarhizium anisopliae, Plant immunity, Defense, Gene expression, Arachis hypogaea

Introduction

Peanut (Arachis hypogaea) is one of the four major oil crops worldwide, with 1.67 million tons produced in China in 2015, making it the world’s highest producer. Peanut yields and quality are often impacted by various biological factors. For example, root-gnawing white grubs (Holotrichia spp.) can severely damage the peanut root system and influence quantity and quality (Roberts and Leger 2004; Nong et al. 2011). Hence, chemical, physical and biological control methods have been taken to prevent pests and diseases and maintain yields. One method showing high potential as a biological control agent able to prevent pests is the use of the entomopathogenic fungus Metarhizium anisopliae (Liu et al. 2016; Maniania et al. 2003; Shah and Pell 2003). Furthermore, this fungus has been shown to have another ecological role as a plant rhizosphere and endophytic microorganism, which is beneficial to plant growth (Wyrebek et al. 2011). Haricot beans (Phaseolus vulgaris) and switchgrass (Panicum virgatum) seeds grown in the presence of M. anisopliae were shown to grow faster and had an increased root hair density when compared with controls (Sasan and Bidochka 2012). Additionally, M. anisopliae has increased the dry weight of dicotyledon mung (Vigna radiata) by enhanced growth of shoots (Rekadwad et al. 2016). Moreover, M. anisopliae has been shown to colonize the rhizosphere of the Norway spruce (Picea abies), with a significantly increased population noted over time (Bruck 2005). In bean roots, Metarhizium robertsii was found to endophytically colonize cortical cells, with the mycelium aggregated within the cells and present within the intercellular spaces, and yet no apparent plant damage was noted (Sasan and Bidochka 2012). Additionally, inoculation with M. anisopliae in P. vulgaris, P. virgatum, and L. esculentum resulted in endophytism and growth promotion (Wyrebek et al. 2011; Ownley et al. 2010). As such, it is necessary to gain an understanding of the molecular mechanisms associated with plant responses to beneficial versus pathogenic fungi to aid in effective disease management and ensure higher quality peanuts.

There has been an ongoing interest in studying the mechanism underlying plant responses to pathogenic verses beneficial fungal interactions. When examining plant responses associated with a pathogen, extracellular and intracellular host receptors that recognize conserved pathogen-associated molecular patterns and more specialized virulence proteins were identified (Abramovitch et al. 2006). The plant innate immunity was also differentiated into basal and resistant (R)-gene-mediated defense responses (Cheng et al. 2016; Meng and Zhang 2013; Wirthmueller et al. 2013). Nevertheless, beneficial fungi can modulate host immunity early on to allow for the establishment of symbiosis by utilizing the common symbiotic pathway that consists of the symbiosis receptor-like protein kinase (SymRK), ion channels (CASTOR and POLLUT), and calcium- and calmodulin-dependent protein kinase (CCMAK) (Cao et al. 2017; Hao et al. 2017; Bonfante and Requena 2011; Parniske 2004). However, how plants distinguish between beneficial verses pathogenic fungi is unclear. In this study, peanut roots were inoculated with M. anisopliae (beneficial) or Fusarium oxysporum (pathogenic) and mRNA, and protein expression was examined for 28 targeted genes mainly associated with plant–fungus interactions, signal transduction, cell death, nitrogen or lipid metabolism, nutrient acquisition or transport, and compound biosynthetic pathways. The aim of this study was to determine how plant temporal cellular responses are similar and yet dissimilar depending on the organismal association.

Materials and methods

Test plants and fungi

The Chinese peanut (A. hypogaea) cultivar Luhua-11 was utilized, with seeds first surface sterilized as previously described with modifications (Miché and Balandreau 2001). Seeds were soaked in a pot with sterile distilled water for 30 min, and then immersed in 4% sodium hypochlorite solution for 2.5 h. The fluid was then decanted and the seeds were washed with sterile distilled water. Next, the seeds were immersed in 15% hydrogen peroxide for 30 min, rinsed three times with sterile distilled water and stored overnight at 4 °C to allow for growth synchronization.

M. anisopliae (M2-2 strain), which functions as a fungal pathogen against white grubs (Holotrichia parallela; Coleoptera: Scarabaeidae) and other scarab species (Nong et al. 2011), was isolated by our laboratory and deposited at the China General Microbiological Culture Collection Centre (No. 4275; Beijing, China). F. oxysporum (F323 strain), a peanut pathogen causing root rot, was provided by Prof. Xiliang Jiang of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing, China). The fungal isolates were grown on potato dextrose agar (PDA) supplemented with 0.5% yeast extract at 27 °C for 2 weeks. The conidia were then collected to generate conidial suspensions.

Plant treatments

The surface-sterilized seeds were sown at one seed per pot into garden pots containing 90 g sterilized vermiculite. Plants were grown in a controlled climate chamber at 25 °C and a light:dark cycle of 14:10. After 7 days, seedlings were at a stage of having two lateral branches. At this time, irrigation was carefully performed to allow 20 mL of conidial suspension (1 × 107 conidia/mL), either M. anisopliae (MA) or F. oxysporum (FO), to reach the peanut roots. The control plants (CK) received 20 mL of sterile water. Every pot was placed on a tray containing approximately 1 cm deep of water. The tray was filled with water with an interval of 2 days to maintain substrate moisture.

Sample collection

At 4 days post-inoculation, peanut root samples were collected by carefully pouring the pots, sweeping aside loose vermiculite, removing the plants and rinsing the roots three times with sterile distilled water. The roots were then cut, snap frozen in liquid nitrogen, and stored at -80 °C until further use.

Detection of gene transcriptional level by RT-qPCR

Total RNA extraction and first-strand cDNA synthesis

Total RNA was extracted from the roots of the three experimental groups using TRIzol® reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions, with the obtained RNA qualified and quantified using a NanoPhotometer® (Implen, München, Germany). First-strand cDNA synthesis was performed using a PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions, with the acquired cDNA quantified and qualified using a NanoPhotometer® (Implen, München, Germany) and stored at − 80 °C.

RT-qPCR analysis

Real-time quantitative PCR (RT-qPCR) was performed to verify the expression of the targeted genes using SYBR® Premix ExTaq™ II (TaKaRa, Dalian, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The 60 s ribosomal protein L7 (RPL7) gene was used as a reference control. The reaction was performed under the following conditions: 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 40 s. Three technical replicates were performed for the targeted samples, with relative expression level determined using the 2−ΔΔCt method. All results are expressed as a mean ± standard error (SE), with p < 0.05 deemed significant following one-way ANOVA using Duncan’s multiple-range test (SPSS version 16.0, SPSS). Target genes were selected based on a previous transcriptome analysis comparing peanut roots inoculated with M. anisopliae or F. oxysporum. Gene-specific primers (Table S1) were designed based on unigene sequences acquired from the NCBI database (RSA Accession Number SRR4294183, SRR4294184, SRR4294185).

Examination of associated protein expression level via parallel reaction monitoring (PRM)

Protein extraction and digestion

Peanut root samples were ground in liquid nitrogen and lysed in lysis buffer [50 mM Tris–HCl (pH 8), 7 M urea, 2 M thiourea, 4% SDS, 1 mM PMSF, and 2 mM EDTA]. After 10 min, 10 mM dithiothreitol (DTT) was added to the lysis solution, followed by centrifugation at 13,000 × g for 20 min at 4 °C. The supernatant was then collected, incubated at − 20 °C for 2 h and centrifuged as before. The supernatant was discarded and the pellet was re-suspended in 800 μL of ice-cold acetone and 10 mM DTT. The suspension was then centrifuged again and the resulting pellet was collected, air dried, and dissolved in 200 μL of lysis buffer. Proteins were quantified using a 2-D Quantification Kit (GE Healthcare). Protein samples were reduced with 10 mM DTT at 56 °C for 30 min, alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark, and then diluted four times with 10 mM TEAB. Equal amounts of protein for each sample were digested with trypsin (enzyme:protein = 1:50) and incubated at 37 °C for 12–16 h. after digestion, peptides were desalted using C18 columns (Phenomenex, USA) and the resultant peptides were dried by vacuum centrifugation.

Proteins quantification

The parallel reaction monitoring (PRM) technology is based on the high-resolution and accurate mass spectrometry (MS) to selectively detect each precursor ions by the quadrupole analyzer, following fragmentation in the collision cell. Finally, the time of flight (TOF) is used to detect all fragments in the secondary mass spectrometry, and a high-resolution secondary mass spectrum is generated to achieve protein quantification by extracting ion information.

Targeted protein quantifications were performed for the selected proteins of interest using PRM on a TripleTOF 5600 + LC–MS/MS system (SCIEX, MA, USA). MS data acquisition was first performed in a data dependent acquisition (DDA) mode to obtain MS/MS spectra for the 30 most abundant precursor ions following each MS1 survey scan. Proteins were identified using the ProteinPilot software, and the results were imported into the Skyline software for MS/MS spectra library building. Peptides were selected based on the ion signals in the spectra library. A list of associated peptides containing m/z values and retention times was exported from Skyline and imported into the Analyst software to perform PRM.

The PRM method was refined by running and evaluating the biological samples until the highest quality method was obtained. Data collection for each sample was performed using the final PRM method on a quadrupole–quadrupole-time-of-flight (QqTOF) mass spectrometer, where each precursor ion is selected in the quadrupole analyzer and then fragmented, with the fragmented ions then quantified in the TOF mass analyzer. Three biological replicates were carried out for each treatment. To eliminate protein carryover, a “blank” was run between adjacent samples to wash the column. Data processing was done in Skyline, and the quantification results were manually inspected for each peptide. Fold-change ratios for each target peptide were statistically calculated to compare protein expressional changes between the different treatment groups. All results are expressed as a mean ± standard error (SE), with P < 0.05 deemed significant following a t test analysis (SPSS version 16.0, SPSS). Raw PRM data can be found in Table S2.

Results

Comparing candidate gene mRNA and protein expression levels

Gene and protein expression levels were examined in peanut roots inoculated with M. anisopliae (MA) or F. oxysporum (FO) using RT-qPCR or PRM. Of the 28 genes that were examined, 22 genes were significantly differentially expressed and 10 proteins were significantly differentially expressed between the two treatment groups. In the PRM experiment, 416 transitions were detected, optimized and found to represent 46 peptides and 25 proteins, to include 2 housekeep proteins (Table 1). However, five genes were found to have undetectable protein levels.

Table 1.

Target proteins examined using parallel reaction monitoring (PRM)

List Protein name Gene name
1 LysM domain receptor-like kinase 3 LYK3
2 Feruloyl CoA ortho-hydroxylase 1 F6H1
3 High-affinity nitrate transporter 2.4 NRT24
4 Spermidine coumaroyl-CoA acyltransferase SCT
5 Calmodulin CaM
6 Symbiosis receptor-like protein kinase SYMRK
7 Programmed cell death protein 4 PCD4
8 Metacaspase-4 MCA4
9 4-coumarate–CoA ligase-like 5 4CLL5
10 Transcription factor MYC4 MYC4
11 Mitogen-activated protein kinase MMK1 MMK1
12 MAP kinase kinase 2 MAP2K2
13 Calcium-dependent protein kinase SK5 CDPK
14 Calcium-binding protein CML49 CML49
15 ABC transporter C family member 2 ABCC2
16 Ferritin-2 FRI2
17 4-coumarate—CoA ligase 1 4CLL1
18 Gibberellin 20 oxidase 1 GAOX1
19 Cell wall integrity protein scw1 SCW1
20 Disease resistance protein RML1A RML1A
21 Probable LRR receptor-like serine/threonine -protein kinase RPK
22 Pto-interacting protein 1 PTI1
23 Transcription factor MYB86 MYB86
24 Alcohol dehydrogenase class-1 ADH1
25 Alcohol dehydrogenase class-3 ADH3
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Gene expression relating to defense and immunity

Plants have developed defense mechanisms and an immune system to resist external aggressions. Of the candidate genes examined herein, 17 are related to pathogen defense. Most of these genes were found to have significantly down-regulated mRNA and protein expression levels in both treatment groups (MA and FO) compared to the control. The genes included one receptor (RML1A), which is associated with resistance; two receptor-like kinase (LYK3 and RPK), which perceives microbial signals and transduces, extracellular stimuli into intracellular response and an innate immune kinase (PTI1). Additionally, other immune system genes that are induced following receptor recognition were also down-regulated to include mitogen-activated protein kinases (MMK1 and MAP2K2), a transcription factor (MYB86) and a cell wall integrity protein (SCW1). Furthermore, 4-coumarate-CoA ligase-like (4CLL5 and 4CLL1) expression, which is associated with jasmonic acid (JA) biosynthesis and the modulation of intercellular and systemic signaling systems in immunity, were down-regulated compared to the control (Fig. 1a). This consistently down-regulated expression indicates that both fungi actively suppressed the host immunity for successful colonization and establishment.

Fig. 1.

Fig. 1

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Temporal expressional analysis of selected candidate genes involved in defense and immunity responses. Gene expression levels (filled bars) were determined via RT-qPCR and protein expression levels (open bars) were determined using PRM. Protein expression levels are shown as fold changes = (normalized protein abundance in treatment group)/ (normalized protein abundance in control group). CK, MA and FO, respectively, refer to check, M. anisopliae and F. oxysporum. All results are expressed as a mean ± standard error (SE), with P < 0.05 deemed significant. The lowercase ‘a’, ‘b’ and ‘c’ indicated statistically significant difference of protein or mRNA level following a t test analysis (SPSS version 16.0, SPSS) or one-way ANOVA using Duncan’s multiple-range test (SPSS version 16.0, SPSS)

In spite of these commonalities, immunosuppressive differences were still noted between the MA and FO groups. A relatively lower immune response was noted in the MA group, thus suggesting that a more harmonious interaction may coordinate the symbiotic development in this case. Of the differential genes between the FO and MA groups, three genes encoding proteins that play key roles in programmed cell death (PCD), PCD4, MCA4 and PDF, were significantly up-regulated in FO relative to MA at the transcriptional level. However, PCD4 and MCA4 protein expression levels were reduced in FO, with PDF protein expression level being undetectable. Furthermore, two transcription factors, MYC4, an activator of the JA-regulated immune response, and WRKY33, a mediator for fungal pathogen resistance, were up-regulated in FO (Fig. 1b). Lastly, two calcium-signaling sensors, a calcium-dependent protein kinase (SK5) and a calcium-binding protein (CML49), were not significantly altered when compared to control group (Fig. 1c).

Gene expression relating to symbiotic promotion

Herein, candidate symbiosis-related genes were selected that related to plant–arbuscular mycorrhizal fungi associations, growth and reconstruction. The results identified differential expression between peanut roots inoculated with the beneficial M. anisopliae verses the pathogenic F. oxysporum. A key symbiotic receptor kinase (SYMRK), which may be amongst the earliest to activate symbiotic signaling by perceiving signals emanated from beneficial microbes and transducing those signals through its intracellular kinase domain, was up-regulated in the MA group when compared to the FO group. This specific transduction would promote an intracellular calcium spike. As expected, calmodulin (CMA) was up-regulated in MA when compared to FO in response to changing calcium levels. Additionally, CCMAK, another factor potentially responding to calcium concentration oscillations and previously characterized in rhizobial and mycorrhizal symbiosis was also found to be up-regulated at the transcriptional level in the MA group. However, protein levels were undetected using the PRM method (Fig. 2a). These findings showed that three core genes required for symbiotic establishment induced in the presence of M. anisopliae in peanuts.

Fig. 2.

Fig. 2

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Temporal expressional analysis of selected candidate genes involved in symbiotic promotion. Gene expression levels (filled bars) were determined via RT-qPCR and protein expression levels (open bars) were determined using PRM. Protein expression levels are shown as fold changes = (normalized protein abundance in treatment group)/ (normalized protein abundance in control group). CK, MA and FO, respectively, refer to check, M. anisopliae and F. oxysporum

All six candidate genes relating to growth and reconstitution were markedly up-regulated in the MA group when compared to the FO group. These genes include ferritin-2 (FRI2) and high-affinity nitrate transporter (NRT24), which contribute to iron and nitrogen metabolism; ATP-binding cassette transporter C family member 2 (ABCC2), which transports various molecules across extra- and intracellular membranes; lipid-transfer protein 1 (LTP1), an important component in non-vesicular lipid transport; and feruloyl CoA ortho-hydroxylase 1 (F6H1) and spermidine coumaroyl-CoA acyltransferase (SCT), which are used to synthesize plant secondary metabolites such as anthocyanins, flavonoids, isoflavonoids, coumarins and lignin (Gachon et al. 2004) (Fig. 2b). These factors contribute to plant growth, reconstruction and indicate reciprocal and symbiotic establishment.

Moreover, gibberellin 20 oxidase 1 (GAOX1), which is a key oxidase enzyme in the gibberellin (GA) biosynthesis, was down-regulated in both fungal treatments. In a previous study, examining peas with mutation of GA synthetic genes resulted in a subsequent increase in mycorrhization (Foo et al. 2013). Furthermore, a negative regulator of GA biosynthesis and a DELLA family member, GAIPB, was found to be up-regulated in the MA group. These findings suggest that GAOX1 and GAIPB may promote symbiosis establishment (Fig. 2b) and that genes associated with immunosuppression and growth regulation play an important role in endosymbiotic establishment in peanut root inoculated with M. anisopliae.

Correlation analysis for gene expression and protein abundance

For the 23 candidate genes that were examined, correlation between translation and transcription were assessed using scatter plots, with five genes excluded due to protein levels being undetected. The results showed an overall degree of correlation between the mRNA and protein expression levels, with correlation coefficients of 0.5234 for the MA group and 0.3611 for the FO group (Fig. 3). However, a strong correlation was not noted for PCD4, MCA4 and F6H1 in the FO group.

Fig. 3.

Fig. 3

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Correlation analysis examining candidate genes and protein expression levels. Correlation analyses of peanut roots inoculated with a M. anisopliae or b F. oxysporum

Discussion

Immunity and symbiosis are both plant adaptation derived following long-term interactions and co-evolution with microbes. However, these adaptations must be able to distinguish between a hostile pathogen and a friendly harmony. Recent study shown that LysM receptor-like kinases (LYKs) are commonly employed to enable molecular pattern recognition of both symbionts and pathogens (Evangelisti et al. 2014). The formation of a ligand-mediated receptor complex drives signal initiation following microbe detection, with a signaling decision then made to orchestrate either a defense or symbiotic response (Hayashi and Parniske 2014). Indeed, the host plant tightly regulates most of these interactions. The plant entering into a symbiotic association can be likened to a pathogenic association, with both requiring an active suppression of the host immunity (Cao et al. 2017; Yang et al. 2010; D'Haeze and Holsters 2004). Therefore, the regulation of the plant’s natural immunity plays a vital role in the recognition, establishment, development and maintenance of symbiosis. In this study, candidate genes involved in host immune and symbiotic responses were selected and examined following induction with the beneficial fungus MA comparing with pathogenic fungus FO (Fig. 4). The results suggest that the host peanut commonly employs LYK3 or/and RPK to distinguish symbiont molecular signals and then determines the appropriate signal transduction to orchestrate a defense and symbiotic response. Basal defenses rely on detection receptors, receptor-like kinases and signaling kinases, as well as transcription factors, to comprise the immune pathway. While similar immune factors were suppressed regardless of the fungi, a stronger degree of suppression was noted following FO inoculation when compared to MA. This finding supports the idea that FO exhibits a stronger pathogenicity, but that even MA must overcome the host immunity to successfully establish symbiosis.

Fig. 4.

Fig. 4

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Schematic representation of key genes and pathways involved in the regulation of symbiosis in peanuts inoculated with M. anisopliae. First, the host plant recognizes conserved fungal-associated molecular patterns and more specialized virulence proteins via extracellular and intracellular receptors, such as RLK, SYMRK and NBS-LRR. The fungus can then manipulate the host signal transduction to induce effector-triggered susceptibility. However, in the presence of F. oxysporum, immunity-associated genes were induced to initiate HR-based cell death. In contrast, M. anisopliae promoted the up-regulation of growth and symbiosis-related genes, thus forming a friendly synergistic relationship with host. Abbreviations: RLK, receptor- like kinase; CNGC, cyclic nucleotide gated channel; Rboh, respiratory burst oxidase homologs; ROS, reactive oxygen species; NBS-LRR, nucleotide binding site- leucine-rich repeat; TFs, transcription factors; and HR, hypersensitive response

To achieve a successful infection, many fungi have evolved so-called effector strategies to circumvent host immune responses. The effectors are delivered by haustoria into the plant cells to suppress basal defenses or manipulate host signaling transduction, such as promoting the degradation of defense components, suppressing protein kinase phosphorylation, and inhibiting defense-related gene expression (Wirthmueller et al. 2013). This approach is utilized to sabotage or reprogram host pathways to facilitate a successful infection and even establish symbiosis. In Cladosporium fulvum, fungal effectors, Avr2 and Avr4, are secreted to inhibit plant cysteine proteases and protect fungal cell walls against chitin degradation via plant chitinases (Stergiopoulos and Wit 2009). However, plants have evolved intracellular receptors that can directly or indirectly perceive the presence of these effector proteins. The recognition of these effectors can result in the activation of a strong resistance, such as HR-mediated cell death, that can limit the spread of biotrophic pathogens (Cui et al. 2015). The metacaspase-1 (MCA1) can positively control HR-mediated cell death in Arabidopsis (Coll et al. 2010). Thus, the host must determine whether a strong defense to a pathogen or a weakened immunity allowing symbiont compatibility and growth is necessary. Herein, a relatively low immune response was seen following MA inoculation, while a significantly heightened response was seen following FO inoculation, thus suggesting that a weakened immune response is necessary for symbiotic development. In the host inoculated with MA, the transcriptional regulation reflects a weakened defense and cooperation on the part of the host to establish symbiosis. However, in the presence of FO, immunity-associated transcription factors, including WRKY33, MYC4 and PDF (plant defense), were induced to repress the pathogen. MYC4, a member of the bHLH transcription factors family, is a positive regulator of JA responses and triggers the activation of JA-induced defense, such as VSP1 (vegetative storage protein 1), PDF, and ERF (ethylene-responsive factor) (Caarls et al. 2015; Ballaré 2014). WRKY33 can bind to the PAD3 promoter and directly activate the expression of camalexin biosynthetic genes (Mao et al. 2012; Qiu et al. 2008). Plant defenses belong to a broad group of cationic peptides that act as the first line of defense against invading pathogens and also play vital roles in normal plant growth and development and sexual reproduction.

In the presence of MA, members of the common symbiotic pathway, which included SYMRK, CMA, and CCMAK, were up-regulated. These factors are required for both rhizobial and mycorrhizal symbiosis (Parniske 2004; Gobbato 2015). Within this pathway, SYMRK perceives signals emanating from beneficial fungi either directly or indirectly, and transduces the event through its intracellular kinase domain. This response subsequently activates ion channels and leads to a Ca2+-spike that is sensed by CMA. Finally, CCMAK responds directly to the oscillations in calcium concentrations and calcium bound calmodulin levels. Once activated, CCMAK phosphorylates Cyclops/IPD3, which subsequently activates symbiosis-related genes expression, including the transcriptional regulator nodule inception (NIN) (Gutjahr and Parniske 2013; Endre et al. 2002; Kistner and Parniske 2002). Additionally, six growth-related genes (NRT24, FRI2, LTP1, ABCC2, F6H1 and SCT) were also up-regulated following MA inoculation. These genes were enriched to components, energies and important secondary metabolite synthesis pathways. Nitrogen, iron, and lipids are essential components for all plants (Chiapparino et al. 2016; Yin et al. 2007). This occurrence suggests that the host plant may have sensed the symbiotic signal and is returning a favorable response. Moreover, DELLA proteins are negative signaling regulators for GA biosynthesis that act positively on JA signaling by sequestering JAZ repressor. The JAZ proteins can interact with MYC2 transcription factors, which activate JA-responsive genes, thus leading to suppression of JA signaling (Hao et al. 2017; Caarls et al. 2015; Foo et al. 2013). The suppression of GA signaling could cause an increased mycorrhization during arbuscule formation (Yu et al. 2014). These finding suggest that high DELLA protein expression levels repress JA-mediated defenses, thus promoting symbiotic development.

The findings present in this study suggest that the establishment of symbiosis between MA and a host plant involves host recognition of symbiont signaling molecules, followed by a subsequent weakening of the immune response, activation of the common symbiotic pathway and promotion of growth-associated gene expression. The pathways regulating immunity and symbiosis should be side by side instead of in series, and there may be cross-coordination via hormones, such as JA and GA. Given the non-obligate symbiosis observed in the presence of MA and the lack of a symbiotic structure (such as arbuscules or nodules), we propose that the resistance mediators MYC4 and WRKY33, the core symbiotic factors SYMRK and CMA, and the transporters NRT24 and ABCC2, may serve as indicators during the early stage of symbiotic establishment. The endophytic lifestyle of MA, a well-known entomopathogenic fungus, should be one of intermediate transitions. Variety in MA adaptability is not surprising since the fungal strains have an extremely wide distribution within vastly different climates (arctic to true desert) and ecological settings. Many important environmental adaptations made by fungi may be displayed by different MA strains. While the findings presented herein are very interesting, further exploration into the symbiotic mechanism between MA and its host plant are needed. This study showed that the symbiotic relationship in this case began with the induction of immunosuppression in the host, followed by simultaneously reprogramming to promote growth and reconstruction through a complex signal network.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 29 kb) (29.4KB, docx)
Supplementary file2 (XLSX 17 kb) (17.9KB, xlsx)

Acknowledgment

We would like to thank LetPub (www.LetPub.com) for providing linguistic assistance during the preparation of this manuscript.

Author contribution

N.C., X.N., and G.W. Methodology; K.H. and S.L. Formal Analysis; F.W. Data Curation; F.W., and X.N. Writing—Original Draft Preparation, Z.Z., H.U. and X.N. Writing—Review & Editing.

Funding

This work was supported by National Key Research and Development Program of China (2017YFD0201205, 2018YFD0201002), National Natural Science Foundation of China (61661136004) and the STFC Newton Agritech ProgrMAme (ST/N006712/1).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Contributor Information

Feng Wang, Email: wangfeng0925@yeah.net.

Xiangqun Nong, Email: xqnong@sina.com.

Kun Hao, Email: haokun8611@foxmail.com.

Ni Cai, Email: 15027173107@163.com.

Guangjun Wang, Email: wangguangjun@caas.cn.

Shaofang Liu, Email: 15652794633@163.com.

Hidayat Ullah, Email: shabkadar@yahoo.com.

Zehua Zhang, Email: zhangzehua@caas.cn.

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