The synergistic effect of nitric oxide in Serendipita indica - induced resistance and basal immunity of wheat against powdery mildew

doi:10.21203/rs.3.rs-4619675/v1

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The synergistic effect of nitric oxide in Serendipita indica - induced resistance and basal immunity of wheat against powdery mildew

https://doi.org/10.21203/rs.3.rs-4619675/v1

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Aims：The occurrence of wheat powdery mildew caused by Blumeria graminis f. sp. tritici severely hampers the increase of wheat yield. The utilization of biological control in the management of powdery mildew is highly valuable due to its environmentally friendly and sustainable characteristics. The present study investigates the resistance mechanism against powdery mildew by employing a combination of inorganic small molecule NO and endophytic fungus Serendipita indica (S. indica).

Methods：By spraying nitroprusside spraying (NO donor) on wheat leaves and inoculating of roots with S. indica, the effects of combined application of NO and S. indica on powdery mildew were determined through analysis of physiological and biochemical indexes, transcriptome and metabolome data.

Results：Experimental data have demonstrated that pre-colonization with S. indica, followed by sodium nitroprusside spraying (NO donor), reduce 27.9% disease index caused by Blumeria graminis f. sp. tritici and elevate biomass by 20% in wheat. The synergistic application of both treatments (NO+S. indica) effectively enhancedchlorophyll content in leaves and relative water content in roots, significantly decreased the accumulation of malondialdehyde and electrical conductivity in leaves. Furthermore, this combination promotes antioxidant activity in wheat leaves through their synergistic effect. Transcriptome sequencing revealed that sodium nitroprusside alone did not induce a substantial number of differentially expressed genes; however, co-administration of NO and S. indica had a greater impact on transcriptomic changes. Differential expressed geneswere validated by fluorescence quantitative PCR analysis. Metabolic profiling indicated that co-application of NO and S. indicaled to broader alterations across metabolic pathways. Notably, plant hormones such as gibberellin, auxin, and salicylic acid exhibited significant increases upon combined treatment.

Conclusions：The study presents a novel research concept and tactic for the biological management of powdery mildew in wheat.

Powdery mildew

Biomass increase

NO signal

Serendipita indica

Experimental data have demonstrated that pre-colonization with S. indica, followed by sodium nitroprusside spraying (NO donor), reduced 27.9% disease index caused by Blumeria graminis f. sp. tritici and elevated biomass by 20% in wheat.
On the basis of S. indica promoting the increase of wheat antioxidant oxidase activity, the introduction of NO further improved the process.
The synergistic application of both treatments (NO+S. indica) effectively enhanced chlorophyll content in leaves and relative water content in roots, significantly decreased the accumulation of malondialdehyde and electrical conductivity in leaves.
The MAPK pathway is significantly regulated and 85% of genes are up-regulated under NO+S. indica co-treatment, which plays an important role in mediating wheat resistance to powdery mildew.
The joint action of NO+S. indica further affect the transcriptome and metabolic profile; the addition of appropriate concentrations of NO amplifies signaling cascade and plays a good role in strengthening the plant immune system.

The causal agent of cereal powdery mildew, Blumeria graminis f. sp. tritici (Bgt), belonging to the genus of Blumeria of the Ascomycota, is the most devastating diseases affecting grains production globally (Mehta, 2014; Savary et al., 2019; Kloppe et al, 2023). The occurrence of powdery mildew in wheat can be observed from the seedling stage to the adult stage, with severe infestations reaching up to 50% (Savary et al., 2019; Kloppe et al., 2023), which leads to an estimated yield loss-of approximately 1.07% globally and has emerged as a crucial limiting factor for achieving high yield, superior quality, and efficient production of wheat worldwide (Savary et al., 2019). Control of powdery mildew in wheat has been achieved by fungicide applications (Meyers et al., 2019) and genetic resistance (Cowger et al., 2012; Li et al., 2014; Kang et al., 2020). However, long-term irrational use of chemical pesticides has led to problems such as increased production costs, environmental pollution and food safety (Ratnadass., 2021). On the other hand, it is of great significance to select good wheat varieties with heritable resistance to powdery mildew. Over 100 genes or alleles (Pm genes) that confer race-specific resistance to Bgt have been identified and mapped in domesticated wheat and wild relatives (Wang et al; 2006; McIntosh et al., 2017). However, resistance genes generally materialize as moderately resistant, and fungi are highly variable and can quickly overcome identified resistance genes. The ecological and physiological functions of plant endophytes and their potential biocontrol resources have great application potential in the control of agricultural pests and diseases (Hassani et al., 2018), and have become a worldwide central point of the research.

The endophytic basidiomycete Serendipita indica, belonging to the order Sebacinales, establishes a classical type of mutualistic mycorrhizal symbiosis with a broad spectrum of host plants(Weiss et al., 2004; Qian et al., 2012). S. indica colonization conferred plant disease resistance makes it as a valuable agricultural star. S. indica colonization conferred barley resistance to Cochliobolus sativus (Sahay and Varma, 2006) and induce systemic resistance against Blumeria graminis (Deshmukh and Kogel, 2007). Roylawar et al. (2021) reported that S. indica primes onion response against stemphylium leaf blight disease. Li (2022) evaluated the inhibitory effect of S. indica against Fusarium graminearum and Rhizoctonia cerealis, and the protective mechanism of S. indica to plants (Li et al., 2022). Previous studies proved that jasmonic acid/ethylene (JA/ET) signal pathway was rapidly and effectively activated by S. indica colonization to resist pathogen infection by inducing transcriptome reprogramming to change the expression of JA/ET-related genes (Panda et al. 2019). Additionally, gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA) hormone levels were induced by S. indica colonization, which activate host immunity of microbe-associated molecular model (MAMP) (Jacobs et al., 2011) and self-regulation of mutual-symbiosis (Ding et al., 2008; Waller et al., 2008). S. indica colonization enhances the complete signal transduction cascade, leading to systematic expression of host defense genes (Li et al., 2022). Moreover, studies have also shown that the the production of defensive secondary metabolites in plants, such as artemisinin, abricin, trichroterpene and curcumin are accumulated under S. indica colonization (Prasad et al., 2013; Das et al., 2014; Bajaj et al., 2014; Arora et al., 2016). These studies suggested that S. indica does not directly act on pathogens by producing antibiotics or other compounds, but may indirectly inhibit propagation of pathogens by inducing systemic resistance in plants at different levels.

Induction of systemic resistance in plants can also be achieved by inorganic small molecules such as nitric oxide (NO). NO is an important signaling molecule in plant cells, participating in regulating growth and developmental processes, disease resistance and defense response (Tada et al., 2008; Sun et al. 2015; Lombardo and Lamattina 2018). NO plays a key role in post-translational modifications of proteins, such as cysteine nitrosation and tyrosine nitrosation and is widely involved in both PAMP-triggered immunity and effector-triggered immunity in plants (Dodds et al 2011). As an important regulatory molecule in plant disease resistance, NO has a complex interaction with key hormones in plant defense such as SA, JA, ET and other signaling molecules such as ROS to regulate plant resistance signal transduction (Hussain et al., 2016). Exogenous NO treatment can induce the expression of pathogenesis related protein (PR1) and phenylalanine ammonia-lyase (PAL1) genes and increase the content of SA in tobacco and Arabidopsis (Wendehenne et al., 2004). However, the internal molecular mechanism of how NO synergistically regulates the expression of a large number of defense-related genes in plant disease resistance is still unclear.

In this study, the internal correlation mechanism of NO in cooperation with S. indica to induce wheat resistance to powdery mildew was revealed through physiological and biochemical tests, combined with whole transcriptome sequencing and metabolome analysis. The molecular interaction network of NO collaborating with S. indica to enhance wheat resistance to powdery mildew was explored based on gene expression regulation and signaling pathway analysis. The findings of this research will provide a theoretical and experimental foundation for further understanding the interaction between plants and microorganisms, as well as for improving crop yield and disease resistance.

Fungal Inocula Preparation and Inoculation

The S. indica isolates were provided by Prof. Karl-Heinz-Kogel of Justus-Liebig University in Germany. S. indica was cultured on a complex medium (CM) plate and incubated at 23 °C until ready for use. The surface of the 21-day CM plate was moistened with sterile distilled water using a spatula to collect chlamydospores spores. The spore suspension was filtered through sterile Miracloth, and the spore concentration was adjusted to 1 × 10⁵/mL using a hematocrit (Nassimi and Taheri, 2017).

The wheat variety Zhongmai 100, provided by Tianjin Academy of Agricultural Sciences, was chosen as the test material. Zhongmai 100 is susceptible to powdery mildew. A total of eight treatments were established (Table 1). Prior to sowing, seeds from the Sere, SNP+Sere, Bgt+Sere, and Bgt+Sere+SNP treatments were soaked in chlamydospores spores for 24 hours. As mentioned earlier, wheat seedlings were grown in a 1:1 (v:v) sand and soil mixture (Li et al., 2022) in each pot. Every pot grows 6 seedlings, and 5 replicates were kept for every treatment. Subsequently, the seedlings in the Sere group, Sere+Bgt group, and Sere+SNP+Bgt group were treated with water for three days. SNP was sprayed on the 7^th day for SNP group, Sere+SNP and Bgt + Sere + SNP group. On the 11^th day after seeding, the uniformly growing seedlings were selected for subsequent Blumeria graminis f. sp. tritici (Bgt) infection.The samples from different treatments were collected at 3, 7 ,and 14 days after powdery mildew infection for physiological, biochemical, and molecular analysis.

Table 1. Respective treatments for different samples

	Mock	Sere	SNP	Sere+SNP	Bgt	Bgt+Sere	Bgt+SNP	Bgt+Sere+SNP
Seeds soaked in chlamydospores of S. indica at 1^st day	-	+	-	+	-	+	-	+
Seedlings inoculated with chlamydospores of S. indica for 3 days	-	+	-	+	-	+	-	+
Seedlings sprayed with SNP at the 7^th day	-	-	+	+	-	-	+	+
Leaves infected by Bgt at 11^th	-	-	-	-	+	+	+	+

Note: “-” stands for no treatment, “+”stands for treatment； mock：seed treated with water; Sere：seed treated with chlamydospores of S. indica; SNP: seed treated with sodium nitroprusside; Bgt: seedling infected with Blumeria graminis f. sp. Tritici.

Plant Disease Evaluation

The severity of disease caused by powdery mildew was evaluated as grades 1, 3, 5, 7, and 9 referenced by ICARDA, 1986. Grade 0 indicated no symptoms of visible spore spots on the wheat leaves; grade 1 indicated spore spots covering ≤5% of the total leaf area; grade 3 indicated spore spots on 6–25% of the total leaf area; grade 5 indicated spore spots on 26–50% of the total leaf area; grade 7 indicated spore spots on 51–75% of the total leaf area and grade 9 indicated extensive spore spots on leaves and stems (≥76%). Following disease assessment, plant height, wet and dry weight of leaves were measured.

Microscopic observation of leaf infected with powdery mildew

After 7 days of infection with powdery mildew causal agent conidia, the treated leaves were selected to observe the the infection degree of powdery mildew. The leaves were fixed with acetic acid: ethanol 1:1 solution for 24 h, and then washed with lactic acid: glycerol: water 1:1:1 solution for 48 h. After that, the leaves were stained with 0.25% Coomassie blue solution for 1 h, washed with distilled water, and then soaked in 50% glycerol. The samples were observed under an Olympus IX83 inverted microscopy.

Relative Water Content Measurement

The relative water content (RWC) is a measure of the water present in a plant's leaves and can be calculated using Weatherley's (1950) formula: RWC = (FW - DW)/(TW - DW) × 100. Fresh weight (FW) of the first leaf segment of wheat in each treatment and control plants was determined after infection with Bgt at 3, 7, and 14 days. Subsequently, the leaf sample was immersed in distilled water at room temperature for 24 hours to obtain the turgid weight (TW), while the dry weight (DW) was recorded after drying the leaf in a 70℃ oven for 48 hours.

Relative Conductivity

To determine the relative conductivity, please refer to the study by Yamei M et al., 2017. In brief, 0.1g of wheat leaves were washed three times in deionized water and then placed in 15ml of deionized water at room temperature for 12 hours. The initial solution conductivity (C1) was measured using a DDS-11A conductivity meter. Subsequently, the sample was boiled for 15 minutes to eliminate all cells and completely release the electrolytes. After cooling the sample to room temperature, the final solution conductivity (C2) was measured. The relative conductivity was calculated using the formula (C1/C2)×100%.

Malondialdehyde content

The content of malondialdehyde (MDA) was determined by using 10% trichloroacetic acid (TCA) homogenized wheat leaves (200 mg). The homogenate is then centrifuged at 12000 rpm at 4°C for 10 minutes. Subsequently, 1.5 ml of the obtained supernatant was mixed with 0.6% thiobarbituric acid (TBA) in equal volume. The absorbance of the samples at 532 nm, 450 nm and 600 nm was measured by UV-VIS spectrophotometer. The determination method of MDA was slightly modified according to Allagulova C et al (2023)

Chlorophyll Content

Quantification of chlorophyll a and total chlorophyll: .Fresh leaf tissue (0.5 g) were weighed and immersed in 25 ml of 95% anhydrous ethanol. The leaves were extracted in darkness at room temperature for 3 days until compiete decolorization of the leaves. The absorbance was measured at 665 nm and 649 nm. The chlorophyll content was calculated according to the following formula described in Gao (2006) .

Antioxidant Analysis

The activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) were calculated using the methods of Chance and Maehly(1955) and Aebi(1984). Antioxidant enzyme activities were expressed as µmol min^-1 mg^-1protein.

Quantitative Real Time Polymerase Chain reaction (qRT-PCR) analysis

The total RNA (1µg) of the control and each treatment was synthesized using M-MLV reverse transcriptase (Takara, Japan) according to the supplier's protocol, respectively. After treatment with DNase I (Sigma, Germany), cDNA was used as a qRT-PCR template. The expression levels of each gene were detected by quantitative RT-PCR. Quantitative RT-PCR was detected using the SYBR Green fluorescence method described above (Livak et al., 2001). In short, the qPCR experiment was performed on the Light Cycler96 Fast real-time PCR system (Roche). The reaction solution contains a 2×Ultra SYBR mixture of 10µL, 100 ng cDNA template, and 10µM forward and reverse primers. TaActin (NW_024468909.1), as a normalized gene, was replicated at least 3 times in all experiments. The amplification procedure was as follows :95℃, 5 min for the first initial activation, 95℃ for 30 s, 55℃ for 35 s, 72℃ for 35 s, 72℃ for 2 min for 30 cycles. At the end of each cycle, the melting curves were measured separately to ensure amplification of a single PCR product.

Determination of carbohydrates and hormone

In order to extract soluble sugars, 0.5 g fresh root samples were cut and ground into homogenates and quantified sucrose was tested using coupled optics at 620 nm according to the guidance of Wang (2006). The solution was resolved by HPLC analysis using a reverse-phase column (Thermo Acclaim 120 C18, particle size 5μm, length 250 mm, internal diameter 4.6 mm; Thermo) as described by Huang et al (2020). The contents of plant auxin (IAA), gibberellin (GA3), abscisic acid (ABA) and salicylic acid (SA) were determined by high performance liquid chromatography referenced by specific methods (Wang et al., 2020).

Metabolic Analysis

At jointing stage, leaves were harvested from each treated plant and metabolomic analysis was performed with 6 replicates. Leaf samples from each group were chopped and extracted with a mixture of 1mL methanol and water (1:1,v/v). The extracted metabolites (5 μL) were crushed and injected into Waters ACQUITY UPLC BEH C18 column (5 μm, 150 × 4.6 mm; Water crops). Subsequently, the Waters UPLC I-Class/Xevo G2 XS-Quadrupole time-of-Flight system (Waters Corp., Milford, MA, USA) was used at a flow rate of 0.3 mL/min and a temperature of 4°C. Liquid phase elution was carried out with 0.1% formic acid water and 0.1% formic acid acetonitrile as mobile phase. The detection condition of mass spectrum is set as follows: capillary voltage: 0.5 kV; Cone voltage :35 v; Extractor voltage: 4.0 V; Source temperature :11℃; Dissolution temperature :550℃. Positive mode electrospray ionization mass spectrometry was used for the analysis. Metabolic data collection and analysis were performed using MassLynx NT4.1 software and the QuanLynx program (Waters Corp.). The relative concentrations (g/DW) of different metabolites in the leaf tissue of each bioreplicate are expressed as comma-separated values (.csv). Significantly regulated metabolites between the two groups' leaves were identified based on fold changes(Log2FC >2 or Log2FC <0.5, p<0.05) and false discovery rates. Pathway analysis was performed utilizing KEGG database (http://www.genome.ad.jp/kegg/pathway.html accessed on July27th ,2020), aiming to better elucidate the functions of altered metabolites (p<0 .05 significant),with cluster Profile package being employed (Yu et al., 2012).

Statistical analysis

In this study, all data were expressed as means ±SE and represented at least three independent biological experiments.Two-way analysis of variance (ANOVA) with Duncan’s multiple range test method was used to analyze the differences of significance.

Combination of S. indica and NO enhanced plant disease resistance to powdery mildew

The anti-powdery mildew effect of S. indica combined with NO was tested in the pot and field experimental. Four different treatments including Bgt, SNP+Bgt, Sere+Bgt, SNP+Sere+Bgt were applied. Wheat roots were pre-colonized with S. indica, following by sodium nitroprusside spraying (NO donor) with 200μM, twice a week for three weeks. The effect of powdery mildew infection on wheat leaves was photographed, as shown in Figure 1A-B. The results showed that the combination of S. indica and NO could reduce the disease effect caused by powdery mildew compared with any single treatment, namely S. indica alone, or NO alone. According to the statistics of disease index (Figure 1C), the disease index was reduced by 17.6 % and 8.8% when S. indica or NO was applied, respectively; whereas NO combined with S. indica could further induced the disease resistance to Blumeria graminis by 27.9%. These data implied that S. indica has advantages in mediating plant disease resistance, and the combined application of NO and S. indica can further enhance plant disease resistance to powdery mildew. The immunohistochemical staining revealed a significant reduction in the number of powdery fungi in the plant leaves following treatment with S. indica and NO, compared to plants treated solely with S. indica or NO (Figure 1D). In contrast to the treated group, control leaves exhibited a high abundance of Blumeria graminis mycelium, which agrees well with the findings from physiological phenotype (Figure 1A-B)

Combination of S. indica and NO promote wheat growth under pathogens infection

The growth status of plants under different treatment conditions was statistically analyzed at 14 and 28 days post-Bg(Blumeria graminis)-infection (dpi). The results demonstrated infection with powdery mildew stunted plant development, whereas colonization by S. indica significantly enhanced plant height at 14 and 28 days post-infection; in NO treatment, the wheat height was promoted at 14 and 28 days, and the effect was not significant for dry weight at 28 days. The combined administration of S. indica and NO effectively mitigated the dwarfing symptoms induced by the pathogen, surpassing the individual effects of S. indica or NO at 14 and 28 days (Figure 2A). In addition, the above-ground dry weight of different treated plants was also statistically analyzed. The results demonstrated that powdery mildew infection resulted in an approximate 15% reduction in yield of the above-ground portion of the plant. In contrast, the Sere+Bgt and SNP+Bgt groups exhibited respective decreases in production by 4.4% and 10.4% after 14-day of powdery mildew infection. Surprisingly, 20 % dry weight (biomass) of above ground was increased in Sere+SNP+Bgt treatment compared to the powdery mildew infection (Figure 2B) .

Relative conductivity, malondialdehyde content, relative water content, and chlorophyll content Determination

In addition, relative conductivity, malondialdehyde content, relative water content, and chlorophyll content by measuring wheat crowns of plant leaves, were evaluated among various treatments, inducing Mock, Sere, SNP, Bgt, Sere+Bgt, SNP+Bgt, Sere+SNP+Bgt (Figures 3A-D). As shown from Figure 3A, the relative conductivity of wheat plants inoculated with powdery mildew was always increased compared with the control at 3 dpi,7 dpi and 14 dpi, respectively, indicating that the infection of powdery mildew caused plant cell damage. Compared with the control group, the experimental group treated with S. indica or NO showed little difference in relative conductivity. The relative conductivity of wheat pre-inoculated with S. indica spores (Sere+Bgt) was lower than that of wheat only inoculated with powdery mildew (Bgt), suggesting that wheat treated with S. indica could increase wheat resistance to powdery mildew infection. But he effect of NO alone was not as good as that of S. indica spores alone. However, the combination of S. indica spores and NO can significantly reduce the relative conductivity, which indicates that the synergistic effect of S. indica spores and NO can more effectively improve the resistance of wheat to powdery mildew.

As shown in Figure 3B, the relative water content of wheat leaves treated with S. indica or NO was higher than that of mock samples at 3 dpi, 7 dpi and 14 pi. And the relative water content of wheat leaves pre-treated with S. indica spores or NO was higher than that of only pathogens infection samples at 7 and 14 days after powdery mildew infection. However, the relative water content of wheat leaves treated both by S. indica spores and NO together was higher than the two treated separately, which indicated that the collaboration of S. indica spores and NO could better improve the survival ability of wheat and promote its resistance to biotic stress.

Malondialdehyde (MDA) is one of the most important products of lipid membrane peroxidation in plants under stress conditions. Its accumulation may change the structure and function of the membrane, damage the membrane system, increase membrane permeability, electrolyte extravasation, and change the relative electrical conductivity in leaves, which can reflect the degree of lipid peroxidation of the membrane and is an important indicator of plant stress resistance. As shown from Figure 3C, the content of MDA of wheat plants inoculated with powdery mildew was always increased compared with the control at 3 dpi,7 dpi and 14 dpi, respectively. Compared with the control group, the experimental group treated with S. indica or NO did not show obvious differences in MDA content. The MDA content of wheat pre-inoculated with S. indica spores (Sere+Bgt) was lower than that of wheat inoculated with powdery mildew alone. But the effect of NO alone (NO+Bgt) was not as good as that of S. indica spores alone. However, the combination of S. indica spores and NO (Sere+NO+Bgt) can significantly reduce the MDA content, which indicates that the synergistic effect of S. indica spores and NO can more effectively improve the resistance of wheat to powdery mildew.

As can be seen from Figure 3D, the chlorophyll content of wheat leaves decreased after infection with powdery mildew, indicating that the infection of powdery mildew would interfere with the photosynthesis of plant leaves. And there was no significant difference in chlorophyll content between the experimental groups treated with S. indica or NO respectively compared to control group. The chlorophyll content of wheat precolonized with S. indica and infected by powdery mildew later was higher than that of wheat plants only inoculated with powdery mildew, indicating that the treatment of wheat precolonized by S. indica could improve the photosynthesis of infected leaves; whose effect was better than that of only implementation of NO. Moreover, the chlorophyll content in the Sere+NO+Bgt treatment was even higher than that in the Sere+Bgt treatment. The combination of S. indica and NO could significantly increase chlorophyll content thus could be more effective to improve the photosynthetic efficiency of wheat.

Determination of antioxidant enzymes activity

To investigate the effect of various treatments on enzymatic antioxidants, activities of CAT, POD and SOD were assayed in wheat seedlings at 3 dpi, 7 dpi and 14 dpi, respectively (Figures 4A-C). The activity of CAT increased from the 3 dpi to 7dai and then decreased gradually at14 dpi. Compared with the control, both S. indica colonization or NO treatment could increase the CAT activity. The activity of CAT was higher in Sere+Bgt and SNP+Bgt than that Bgt treatment. The combined pretreatment of S. indica and NO could further induce the activity of CAT (Sere+SNP+Bgt) , which was higher than that of either single treatment (Sere+Bgt or SNP+Bgt ) (Figures 4A).

With the continuous infection of powdery mildew, POD activity increased gradually from 3 dpi to 14 dpi. Compared with mock, powdery mildew infection increased POD activity. There was no significant difference in POD activity among the four treatments including SNP, Bgt, Bgt+Sere, Bgt+SNP. However, the combined pretreatment of S. indica and NO could induce the activity of POD (Sere+SNP+Bgt), which was highest than that of either single treatment (Sere+Bgt or SNP+Bgt ) (Figures 4B). SOD activity increased rapidly after 3 days of infection and decreased after 7 days of infection. Among all treatments, SOD activity of wheat plants inoculated only with powdery mildew was the highest. SOD activity of wheat plants treated with S. indica or NO decreased at 3 and 7 days after infection, while SOD activity of wheat plants co-treated with S. indica and NO was strongest at 14 days after powdery mildew infection (Figures 4C).

Profile of different expressed genes analysis in wheat responsive to Serendipita indica and pathogens colonization

The transcriptomes of wheat responsive to various treatments were obtained and sequenced respectively by the BGI Seq500 platform. All the uni-genes were finally obtained by sequence splicing, a redundancy removal based on the sequence clustering software. Functional notation and cluster analysis of unigenes were performed by comparing unigenes to the database. Through data integration analysis, differentially expressed genes (DEGs) of wheat responsive to S. indica and pathogens colonization were analyzed (Table 2). The number of DEGs caused by Bgt was 18796, and the upregulation and downregulation genes were 14097and 4699, respectively. The number of DEGs caused by NO was 1750, and the upregulation and downregulation genes were 825 and 925, respectively. The effect of S. indica on the genome of wheat was to produce 151 up-regulated genes and 507 down-regulated genes, which is the smallest effect on the wheat genome of all treatment groups. Especially, the combination of S. indica and NO produced 3240 DEGs compared to mock in wheat genome, including 2061 upregulation genes and1179 down regulation genes, respectively. Additionally, the DEGs involved in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed (Figures 5A-F). In the Sere vs Mock group, the DEGs were mainly concentrated in the phenylpropanoid biosynthesis, MAPK signaling pathway, plant-pathogen interaction etc.; In the SNP vs Mock group, besides the MAPK signaling pathway, DEGs in the ABC transporters, fatty acid degradation, zeatin biosynthesis and monoterprenoid biosynthesis pathway were included; In the comparison group of Sere_SNP vs Mock, DEGs and the pathways which DEGs enriched in were significantly increased (Figures 5C). Similarly, in the Sere_SNP_Bgt vs Bgt group (Figures 5F), the enrichment pathways of DEGs were significantly increased than those of in SNP_Bgt vs Bgt or Sere_Bgt vs Bgt group, the pathways of DEGs involved in Sere_SNP_Bgt vs Bgt group were increased by appro. 8 times, indicating that the combined treatment of the two (S. indica and NO) could further induce wheat transcriptome rearrangement and promote more DEGs. And those DEGs were mainly enriched in the phenylpropanoid biosynthesis, MAPK signaling pathway, starch, sucrose metabolism and amino sugar / nucleotide sugar metabolism etc.

Table 2. Differentially expressed genes (DEG) in the different comparison group.

Comparison group	DEG number	Up expression of DEG	Down expression of DEG
Sere vs Mock	658	151	507
Snp vs Mock	1750	825	925
Bgt vs Mock	18796	14097	4699
Sere_Snp vs Mock	3240	2061	1179
Snp_Bgt vs Bgt	581	197	384
Sere_Bgt vs Bgt	3580	2756	824
Sere_Snp_Bgt vs Bgt	4560	2978	1582
Sere_Snp_Bgt vs Sere_Bgt	632	321	311
Sere_Snp_Bgt vs Snp_Bgt	5722	3619	2103

QPCR identification of the critical genes in the various treatments

The MAPK signaling pathway is a widely distributed signaling pathway in eukaryotic cells, which has preserved its fundamental structure and function throughout extensive evolution. This pathway plays a crucial role in organism adaptation to the environment by regulating gene expression and cytoplasmic function (Cardinale et al 2002). Transcriptome data showed that the expression patterns of MAPK gene family were induced by NO and S. indica colonization. Forty-six MAPK genes were analyzed, of which 85% showed up-regulated expression patterns (Part shown in table 3). Fluorescent quantitative PCR was used to detect the expression of these genes in different treatments. The results showed that the expression levels of those transcriptions including TraesCSU02G209300, TraesCS4A02G431300, TraesCS1A02G203700 and CS2B02G047300 were in accordance with the RNA-seq. The patterns of gene expression in different treatment groups were different, but the expression levels in the treatment of S. indica colonization group were significantly up-regulated, especially significantly in Sere+SNP+Bgt treatment group at 7 and 14 day after treatment (Figure 6A-D), which is especially true for TraesCS4A02G431300 gene expression. Based on the above results, we fully realized that the addition of NO to S. indica spores further promoted the expression of resistance genes, which was more effective than that of NO alone.

Table 3. MAPK gene family were induced by NO+S. indica treatment

MAPK Gene name	Log2 (T/CK)	P-value	Number of Amino Acid	Up or down regulation
TraesCS4A02G431300.1	12.14529132	0.00000521	GO:0001071	Up
TraesCS2B02G047300.1	12.14529132	0.00000521	GO:0003700	up
TraesCS1A02G203700.1	10.1998561	0.001196567	GO:0001071	up
TraesCSU02G209300.1	10.1998561	0.001196567	GO:0003700	up
TraesCS5A02G532300.1	9.955511294	0.0000359	GO:0001071	up
TraesCS1A02G203600.1	9.955511294	0.0000359	GO:0003700	up
TraesCS4D02G357300.1	8.979022049	0.0000879	GO:0001071	up
TraesCS4B02G363800.1	8.979022049	0.0000879	GO:0003700	up
TraesCS1D02G207100.1	5.887513129	0.002162047	GO:0001071	up
TraesCS5D02G408200.1	5.887513129	0.002162047	GO:0003700	up
TraesCS1D02G207100.1	5.806224792	0.015540276	GO:0001071	up
TraesCS5D02G408200.1	5.806224792	0.015540276	GO:0003700	up
TraesCS7D02G053700.1	5.685358034	0.014044741	GO:0001071	up
TraesCS4D02G357200.1	5.685358034	0.014044741	GO:0003700	up
TraesCS1D02G207000.2	-3.025924133	0.027368266	GO:0001071	Down
TraesCS2A02G098200.1	-3.025924133	0.027368266	GO:0003700	Down
TraesCSU02G092300.1	-3.10901611	0.027521161	GO:0001071	Down
TraesCS5A02G532200.1	-3.10901611	0.027521161	GO:0003700	Down
TraesCS4B02G363900.1	-4.868783154	0.034462531	GO:0001071	Down
TraesCS4B02G363900.1	-4.868783154	0.034462531	GO:0003700	Down

S. indica combined with NO resist to powdery mildew by regulating plant hormone

Physiological and biochemical data suggest that S. indica combined with NO exhibits anti-powdery mildew effect. Therefore, the effect of S. indica spores combined with NO on wheat metabolism was also explored. The relative metabolites, including the amount of hormones, free sugars and soluble protein in leaves of wheat were identified among the different treatments. The results demonstrated that the concentration of indole-3-acetic acid (IAA) in the treatment group colonized by S. indica was significantly higher than that in the other treatment groups at 3 dpi and 7 dpi. When nitric oxide (NO) was applied alone, the IAA content showed a slight increase compared to the control group, but remained lower than that of plants colonized by S. indica. Notably, the combined application of NO and S. indica resulted in the highest IAA content in plants (Figure 7A). In addition, GA3 content increased in the S. indica colonization wheat at 7 dpi, the GA3 content in SNP was higher than control but lower than in Sere. After Blumeria graminis infection, the GA3 content in Sere+Bgt or SNP+Bgt was increased compared to the Bgt treatment, and this increase was especially obvious in Sere+SNP+Bgt group.The plants colonized by S. indica still maintained a high level of gibberellin on the 14^th day of Blumeria graminis infection. GA3 content decreased in SNP+Bgt treatment at 14 dpi compared to the same treatment at 7 dpi. Whereas GA3 content in Sere+SNP+Bgt was still highest among all the treatments (Figure 7B). The results showed that the salicylic acid (SA) content in the treatment pre-colonized by S. indica was significantly higher than that in the other treatment groups at 3 and 7 dpi. In particular, when NO was added, the SA content in the treatment that pre-colonized by S. indica was further increased (Figure 7C). There was not obvious difference for the ABA content in the 7 treatments including Mock, Sere, SNP, Bgt, Sere+Bgt, SNP+Bgt, Sere+SNP+Bgt at the 3dpi. However, the ABA content was increased in Bgt, Sere+Bgt and Sere+SNP+Bgt treatment at 7 dpi. And this upward trend remained at the 14 dpi, additionally, the ABA content in SNP+Bgt was enhanced, too (Figure 7D).

S. indica combined with NO resist to powdery mildew by regulating primary metabolite

At the other hand, sucrose content was identified in the 7 treatments at 3 dpi, 7 dpi and 14 dpi. On the whole, the sucrose content in S. indica colonized plants was lower than that of control at three different time points including 3 dpi, 7 dpi and 14 dpi. However, the sucrose content was significantly increased in NO treatment no matter the plant was infected by Blumeria graminis or not. And in the Sere+SNP+Bgt treatment, the sucrose content was higher than Sere+Bgt but lower than SNP+Bgt (Figure 8A). Additionally, total soluble protein content was compared among the seven treatments at 3 dai, 7 dai and 14 dai. The soluble protein content in the NO treatment group was higher compared to that in the S. indica colonization group, while the S. indica colonization resulted in lower soluble protein content than the control group. Powdery mildew infection led to the lowest levels of soluble protein content in plants. In the NO+Bgt group, an increase of soluble protein content was observed. Furthermore, a combined administration of NO and S. indica also resulted in an increase of soluble protein content in plants (Figure 8B).

Metabolic profiles of wheat leaf in response to S. indica colonization and NO treatment

To further elucidate the mechanism of disease resistance in response to the colonization of S. indica and NO treatment, non-targeted metabolic analysis was performed on Mock, Sere, SNP, Sere+ SNP, Bgt, Sere+Bgt, SNP+Bgt, Sere+SNP+Bgt treatments leaves at 14 days after pathogen infection. The metabolite profiling of Sere, SNP, Bgt, Sere+Bgt, SNP+Bgt, Sere+SNP+Bgt treatments showed different changes compared to mock including phenylpropanoid, flavonoid, fatty acids, amino acids, indole alkaloid, terpenoids, etc. The enrichment pathways of significantly different metabolites were shown in some comparison groups (Figure 9, not show all). In the Sere vs mock group, a total of 458 metabolites were detected in wheat leaves under Sere treatment. Among them, the levels of 322 metabolites increased and 136 metabolites decreased (Table 4). Different metabolites were mainly enriched in tryptophan metabolis, tropane, piperidine/pyridin alkaloid biosynthesis and phenylpropanoid biosynthesis. In the SNP vs Mock group, a total of 350 metabolites were identified, of which the content of 275 metabolites increased and 75 metabolites decreased (Table 4). Different metabolites were mainly enriched in tropane / piperidine and pyridin alkaloid biosynthesis and biosynthesis of unsaturated fatty acids. In Sere+SNP vs Mock group, totally 1080 metabolites has been identified including 687 upregulated and 393 down regulated. Different metabolites were mainly enriched in flavonoid biosynthesis, isoquinoline alkaloid biosynthesis, and penicillin and cephalosporin biosynthesis. In Sere+Bgt vs Bgt group, a total of 886 metabolites were identified, of which the content of 628 increased and 258 decreased, and the metabolites of unsaturated fatty acids, flavonoid biosynthesis, isoquinoline alkaloid biosynthesis, pencillin and nicotinamide were significantly upregulated. Whereas a total of 450 metabolites were identified in SNP+Bgt vs Bgt group, of which the content of 199 metabolites increased and 251 metabolites decreased. The different metabolites enriched in brassinosteroid biosynthesis, zeatin biosynthesis and glucosinolate biosynthesis pathway were significantly upregulated. In Sere+SNP+Bgt vs Bgt group, a total of 893 metabolites were identified, of which the content of 611 increased and 282 decreased. The different metabolites enriched in valine / leucine / isoleucine biosynthesis, diterpenoid biosynthesis, tropane / piperidine / pyridin alkaloid biosynthesis as well as phenylpropanoid biosynthesis were significantly upregulated. Particularly, we noticed that co-inoculation with S. indica and NO resulted in more metabolite changes in plant leaves than than any single treatment.

Table 4 Metabolic changes in wheat leaves in different comparing group

	Total	Up	Down
Sere vs mock	458	322	136
Snp vs Mock	350	275	75
Bgt vs Mock	6896	3568	3328
Sere_Snp vs Mock	1080	687	393
Sere_Bgt vs Bgt	886	628	258
Snp_Bgt vs Bgt	450	199	251
Sere_Snp_Bgt vs Bgt	893	611	282
Sere_Snp_Bgt vs Sere_Bgt	531	358	173
Sere_Snp_Bgt vs Snp_Bgt	785	532	253

Bread wheat (Triticum aestivum L., 2n = 6x = 42) is one of the most important crops in the world and provides 20% of the total daily calories and protein for humans (Zorb et al., 2018). Powdery mildew (PM) is one of the serious wheat diseases caused by Blumeria graminis f. sp. tritici (Bgt) infection, which has become a major threat to global wheat production. Biological control is an effective and promising method to deal with powdery mildew.The endophytic fungus S. indica colonization in plant interacted with the plants defense signaling and can induce plant immune response to resist pathogen infection (Yue et al. BMC Plant Biology 2023). Plant defense signals against fungal pathogen infection is regulated by at least two primary nodes of the host innate immune system, pathogen associated molecular pattern (PAMP)‐triggered immunity (PTI) which belongs to basal resistance and effector‐triggered immunity (ETI) that can activate robust immune signaling always by inducing hypersensitive response (HR) cell death to prevent pathogen growth and proliferation (Song et al., 2022; Sun et al., 2019).

The induction of disease resistance in plants can also be achieved through the use of various inorganic small molecules, such as nitric oxide (NO) (Wendehenne et al., 2004). The signal molecule NO plays a crucial role as a lipid-soluble free radical signaling molecule in the plant's defense against diseases, exerting its regulatory function through various mechanisms such as post-translational modification and induction of defense gene expression (Dodds et al., 2010). Furthermore, NO can interact with plant hormones, including SA, JA, ET, etc.and other signaling molecules like ROS to regulate the transduction of signals related to plant disease resistance. Exogenous NO treatment induced the expression of PR1 and PAL1 genes and increased SA content in tobacco and Arabidopsis (Wendehenne et al., 2004). In addition, the SA-dependent resistance of NO to tobacco Mosaic virus (TMV) suggests that NO plays a role in both biosynthesis and physiological regulation of SA(Tada et al., 2008; Lindermayr et al., 2010). It was found that NO treatment induced transcriptional reprogramming of Arabidopsis genes which involved in various biological and abiotic stress responses, hormone signaling, and developmental regulation(Hussain et al., 2016).

In view of the role of S. indica and NO played, respectively, in plant disease resistance, we hypothesized whether the combination of plant systemic resistance induced by S. indica and plant-based immunity induction by NO would achieve better disease-resistant effect against powdery mildew in wheat.

Fortunately, we found that the combination of the two (NO+S. indica) showed advantages in disease resistance. In fact, the widespread increase in plant yield contributed by S. indica colonization has been demonstrated by plenty of studies (Osborne R et al., 2023; Qiang X, etal., 2012; Johnson et al., 2013), which is consistent with our experimental results (Figure1- 2). At the same time, we noted that the growth promotion effect of NO alone was not significant. However, the combined use of the two (S. indica+NO) showed better growth promoting effect. Especially, the synergistic use of the two (S. indica+NO) showed outstanding advantages in improving wheat resistance to powdery mildew (Figure 2A). PW causal agent conidia stained with Coomassie brilliant blue implied that the colonization of S. indica in roots and NO sprayed on leaves can significantly inhibit the propagation of powdery mildew in wheat leaves (Figure 2C). Data indicated that a 27.9% reduction in disease index in the two co-treated wheat(Figure 2B), suggesting a new strategy for guiding agriculture against powdery mildew.

We were delighted to discover that the combined action of the S. indica andNO showed significant advantages in both reducing the conductivity and malondialdehyde content and increasing the cellular water content and chlorophyll content (Figure 3) which was better than the treatment of S. indica alone. Recent studies have shown that NO plays an important role in the interactions between plant roots and microorganisms and the regulatory effect of NO on plant-microbe relationships is concentration-dependent. An appropriate NO level promotes the formation of plant-microbe symbiosis, and a high concentration of NO enhances plant immunity (Zipfel and Oldroyd, 2017; Kumari et al., 2019; Martínez-Medina et al.,2019a; Martínez-Medina et al.,2019b). The concentration here we used in our experiment is 200 μM, which is proved to be effective to initiate the plant immunity.

Pathogen infection results in excessive ROS accumulation which lead to damaged cells, abnormal proteins and damaged organelles that result in oxidative stress. Plants increase their survival during pathogen infection by enhancing reactive oxygen species (ROS) manipulation mechanisms (Rui et al., 2022). Restoring ROS to normal physiological levels required enhanced antioxidants enzyme activity. S. indica colonization protect plants roots from loss of antioxidant enzymes caused by pathogen infection, which was identified by previous research (Schäfer et al., 2009; Harrach etal., 2013; Li et al., 2022; Narayan et al., 2017 ). Our data indicated that the synergistic effect of S. indica and NO can further improve the enzyme activity of SOD, CAT and POD, which was always higher than that of any single treatment (S. indica or NO) (Figure 4). Studies have shown that S. indica induce changes in the hormone levels of gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA), thereby triggering host immunity in the microbial-associated molecular model (MAMP) and self-regulation of mutually-beneficial symbionts (Deshmukh et al., 2007; Stein et al., 2008). The data we have obtained demonstrate that S. indica and NO possess the capacity to further reprogram plant transcriptional profiles (Figure 5-6) and can significantly elevate plant hormone, such as gibberellin, auxin, and abscisic acid (Figure 7) levels compared to any single treatment. The results emphasize the synergistic impact of S. indica and NO on the induction of systemic resistance.

Transcription analysis showed that 280 DEG induced by S.indica treatment were enriched in plant hormone signaling and MAPK pathways. These data agrees well with the previous studies (Li et al., 2022; Li et al., 2023). The double-layer effect of NO+S. indica combination is more obvious revealed by the transcription profile with more than 3240 DEG (Table 2). Metabolic profile changes were agreed wel with the transcription data including 1080 different metabolites in Sere+SNP vs Mock group (Table 3). The data on physiological and biochemical levels also demonstrated that the combination of NO+S. indica exerted significant effects in enhancing wheat disease resistance index, augmenting cellular water content, and elevating antioxidant activity, surpassing the efficacy of any individual treatment. The improved efficiency of disease resistance indicated that the combination of NO and S. indica did not inhibit plant immune system, but had a signal cascade amplification effect, resulting in a superposition effect on disease resistance against powdery mildew in wheat.

NO+S. indica mediates resistance of wheat to powdery mildew on three levels. First of all, on the basis of S. indica promoting the increase of wheat antioxidant oxidase activity, the introduction of NO further improved the process, showing that it increased the cell water content, reduced the content of oxygen free radicals, reduced the accumulation of malondialdehyde and prevented the increase of electrical conductivity. Second, this oxidative stress response is transmitted into the cell and causes changes in transcriptome expression. The expression of many DEGs caused by the combination of NO+S. indica is a good proof. The MAPK pathway is significantly regulated and 85% of genes are up-regulated, which plays an important role in mediating wheat resistance to powdery mildew. Thirdly, the change of nuclear gene expression caused by the joint action of NO+S. indica will further affect the metabolic profile, which is manifested by the accumulation of many hormones, including the increase of auxin, gibberellin, abolic acid and so on. In fact, S. indica itself can play a role in these three aspects, and the addition of appropriate concentrations of NO amplifies this signaling cascade and plays a good role in strengthening the plant immune system. The mechanism of wheat resistance against powdery mildew was concluded in Figure 10. Our study provides a fundamental theory for further exploration of the potential of NO+ S. indica technical method as biological prevention and control pathway against powdery mildew in wheat.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.

Acknowledgements

The author gratefully acknowledges support from Natural Science Foundation of Hebei Province (Grant No. C2021202005) and S&T Program of Hebei Province (Grant No. 19226505D) as well as National Natural Science Foundation of China (Grant No. 31801948) for financial supports. And the authors would like to thank Professor Karl Heinz Kogel for the gift of the Serendipita indica.

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The synergistic effect of nitric oxide in Serendipita indica - induced resistance and basal immunity of wheat against powdery mildew

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