A methyl esterase 1 (PvMES1) promotes the salicylic acid pathway and enhances Fusarium wilt resistance in common beans

Methyl esterase (MES), PvMES1, contributes to the defense response toward Fusarium wilt in common beans by regulating the salicylic acid (SA) mediated signaling pathway from phenylpropanoid synthesis and sugar metabolism as well as others. Common bean (Phaseolus vulgaris L.) is an important food legume. Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli is one of the most serious soil-borne diseases of common bean found throughout the world and affects the yield and quality of the crop. Few sources of Fusarium wilt resistance exist in legumes and most are of quantitative inheritance. In this study, we have identified a methyl esterase (MES), PvMES1, that contributes to plant defense response by regulating the salicylic acid (SA) mediated signaling pathway in response to Fusarium wilt in common beans. The result showed the role of PvMES1 in regulating SA levels in common bean and thus the SA signaling pathway and defense response mechanism in the plant. Overexpression of the PvMES1 gene enhanced Fusarium wilt resistance; while silencing of the gene caused susceptibility to the diseases. RNA-seq analysis with these transiently modified plants showed that genes related to SA level changes included the following gene ontologies: (a) phenylpropanoid synthesis; (b) sugar metabolism; and (c) interaction between host and pathogen as well as others. These key signal elements activated the defense response pathway in common bean to Fusarium wilt. Collectively, our findings indicate that PvMES1 plays a pivotal role in regulating SA biosynthesis and signaling, and increasing Fusarium wilt resistance in common bean, thus providing novel insight into the practical applications of both SA and MES genes and pathways they contribute to for developing elite crop varieties with enhanced broad-spectrum resistance to this critical disease.


Introduction
Common bean (Phaseolus vulgaris L.) is major source of fiber, minerals, and protein to the diet with food calories and amino acids of legumes being second in importance only to cereals and animal sources (Blair et al. 2012 and. Bean seeds are also nutraceutically important for humans (Suárez-Martínez et al. 2016). Common bean is a species that is plagued by many plant diseases especially those caused by fungal pathogens (Schwartz and Hall 2005). A major fungal disease of common bean is Fusarium wilt, avascular disease of great economic importance globally and in various crops, which in P. vulgaris is caused by Fusarium oxysporum f. sp. phaseoli (Fop). The pathogen has been found and identified in most of the bean-growing regions of the world and causes significant yield losse specially in wet highland environments or intensively cropped lands without adequate rotation (Buruchara and Camacho 2000).
Plants have evolved diverse mechanisms to resist the microorganism infestations. Plants triggers the activation of local defenses at the infection sites that lead to a secondary immune response termed systemic acquired resistance (SAR) in distal tissues (Fu and Dong 2013;Zhou and Zhang 2020). To date, a few molecules have been proposed as mobile signals leading to SAR (Dempsey and Klessig 2012;Klessig et al. 2018). Salicylic acid (SA) is considered as a classical phytohormone with critical roles in both Communicated by Janila Pasupuleti.
1 3 local defense and SAR (Vlot et al. 2009;Ding and Ding 2020). It demonstrated that SA accumulation is required for the establishment of resistance in both local and systemic tissues and exogenous SA was also indicated to play a role as an internal defense signal for plant immunity (Fu and Dong 2013;Varanasi and Talati 2014). SA has also been shown to contribute to shaping the microbial populations associated with the plant roots and lead to enhanced plant fitness in response to threats from pathogens (Lebeis et al. 2015). These have been shown to be the case in Fusarium wilt interactions with common bean as well (Xue et al. 2014). Thus, the roles of SA as a defense hormone have been greatly expanded.
Salicylic acid-binding protein 2 (SABP2) has an esterase activity that mediates hydrolysis of methyl salicylate (MeSA) to SA. It appears to be an essential enzyme for the SAR response in many plant species, working by releasing a long-distance signal required in phloem tissues that results in defense responses in infected plants of Arabidopsis thaliana, tobacco and potato. Transgenic tobacco plants with silenced SABP2 gene that are infected with TMV are compromised in their ability to develop SAR (Kumar and Klessig 2003). By contrast, SA synthesized by SABP2 in tobacco plants is essential for transcomplementing the loss of SAR development in SABP2-silenced plants (Kumar et al. 2006). In Arabidopsis, three orthologs of tobacco SABP2 expressed in SABP2-silenced tobacco plants complement for SAR deficiency (Vlot et al. 2008). The potato plants with suppressed activity of tobacco SABP2 ortholog (StMES1) compromised SAR against the challenge of Phytophthora infestans (Manosalva et al. 2010).
Our goal for this study was to determine the roles of SABP2 and MeSA in the SA mediated resistance pathway and defense mechanism of the common bean-Fusarium wilt pathosystem. Previously, we cloned seven orthologous genes of tobacco NtSABP2 genes in common bean (Xue et al. 2018). However, it was not very clear whether the orthologs of SABP2 proteins in common bean functioned in defense responses. Therefore, in the current study, we analyzed one of the orthologs responsed to Fop infection most dramatically, named Phaseolus vulgaris methyl esterase 1 (PvMES1), to assess its role in the SA mediated resistance signaling pathway against Fusarium wilt. After cloning of the entire gene, our specific objectives were to determine the biological/physiological significance of the PvMES1 gene and transcript encoding product in the disease pathosystem by analyzing (1) in vitro esterase activity of the enzyme; (2) function when overexpressed; (3) results when gene silenced; and (4) transcriptome profiling of differentially expressed genes (DEGs) between control and PvMES1 overexpressed/silenced plants.

Materials and growth conditions
Common bean genotypes BRB130 (Fusarium wilt susceptible) and CAAS260205 (Fusarium wilt resistant) were obtained from the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences (CAAS) in Beijing, China (Xue et al. 2015). Ten-day-old greenhouse grown plants were used for the varying treatments at the fully-expanded, 'cotyledonary' leaf, seedling, growth stage. Treatments included (1) control with no applications; (2) infection by an aggressive F. oxysporum f. sp. phaseoli isolate, FOP-DM01 (Fop) at a density of 5.0 × 10 6 colony forming units, cfu g −1 at 12 d after inoculation with the bean pod mottle virus (BPMV)-derived vectors or leaf extracts challenged by vectors; or (3) spraying of the leaves for five consecutive days with 2 mM salicylic acid (SA) diluted in Tween 20 (0.02% v/v) using previous described methods (Xue et al. 2014). The three plants for each treatment and each time point were fertilized every week from then on. The plants were grown in a greenhouse maintained at approximately 22-28 °C. All treatments were grown in the same greenhouse with normal daylight with supplemental lighting for a total of 14 days. Root tissues were harvested at 0, 24, 48, 72, 96 and 120 h post inoculation (hpi) in the case of Fop treatment and with an additional 6 h collection for SA treatment.

RNA extraction and quantitative real-time PCR analysis
Total RNAs were extracted from root samples of the treatments described above using TRIzol reagent followed by DNAse treatment (Tiangen, China). These were quantified by Nanodrop 2000 spectrophotometer and qualified by running in a gel electrophoresis unit. A total of 200 ng of RNA was then used to synthesize cDNA and subsequently used as a template for PCR and quantitative realtime PCR (qRT-PCR) analysis using a reverse transcription kit (Tiangen) according to manufacturer's protocol. Expression analysis was performed using a qTOWER 2.2 Real-time PCR system (Analytikjena, Germany) with three biological replicates and the actin gene as an internal control to standardize the data (Chen et al. 2009). Threshold values (CT) were used to quantify relative gene expression followed by the comparative 2 −ΔΔCT method (Livak and Schmittgen 2001). Primers used in this study are listed in Table S2.

PvMES1 gene cloning and bioinformatic analysis
Gene-specific primers (MES-F1/MES-R1) were used to amplify the full-length gene both from a cDNA source and for the full-length genomic (gDNA) copy of the PvMES gene from common bean, P. vulgaris. The cDNA and gDNA amplified fragments were cloned into a pMD18-T vector and sequenced. The PCR products were sequenced with an ABI3730xl DNA analyzer using standard techniques and consumables by the Sanger method. Sequence homology and gene identity was determined using BLAST software (Altschul et al. 1990) and the nucleotide database at NCBI (www. ncbi. nlm. nih. gov). Other MES protein sequences from different plants were downloaded from Genbank and compared with the PvMES1 sequence. Bioinformatic analysis of PvMES1 gene included the following aspects: number of amino acids (NA), molecular weight (MW), and isoelectric points (pI) predicted using the protein/peptide characterization tool ProtParam provided on the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (http:// www. expasy. org/ tools/ protp aram. html). The signal peptide and subcellular localization were predicted by iPSORT (http:// ipsort. hgc. jp/), and WoLF PSORT (https:// www. gensc ript. com/ wolf-psort. html) programs, respectively. The three-dimensional structure of the protein was constructed using the Swiss-model Workspace Package (http:// swiss model. expasy. org/) and confirmed with PyMol software (http:// www. pymol. org).

Protein expression and activity assay
The PvMES1 gene was also cloned into an expression vector, pET28A, and this construct was used to transform E.coli BL21(DE3) for protein expression. The resulting 6 × His-tagged recombinant protein was purified using Ni Sepharose TM6 Fast flow columns (GE Healthcare) following the manufacturer's instructions. The purified protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL). MeSA esterase activity was measured by a two-step radiochemical esterase assay using 0.5 μg of purified PvMES1 transcripts encoding product incubated with MeSA as the substrate (Forouhar et al. 2005).

SA quantification
To evaluate MeSA conversion to SA, all root samples were extracted and quantified, then analyzed automatically on a Wakopak LC 2010A reversed-phase HPLC with C18 analytical column (PTH 250 × 4.6 mm) from Wakosil (Wako Inc., Osaka, Japan) using our previously described method (Xue et al. 2014).

Fungal quantification
For quantification of bean root pathogen colonization, total genomic DNA was extracted directly from 500 mg of fresh plant root tissues using a Tiangen Plant Genomic DNA kit (Tiangen Inc., Beijing, China) as described by the manufacturer. The primer pair QFopA/QFopB reported by Xue et al. (2014) was used for amplification and quantification of pathogen DNA, and the reaction conditions were adjusted experimentally to optimize the PCR protocol.

Measurement of pathogen defense-related factors
A series of pathogen induced pathways were measured based on activity of key enzymes: Phenylalanineammonialyase (PAL) activity was assayed following an established method (Assis et al. 2001), with some modifications; Peroxidase (POD) activity was assayed according to a previous method as well (Do et al. 2003); Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were assayed following the method . Total hydrogen peroxide (H 2 O 2 ) content was measured according to Sagisaka (1976), with a few modifications: namely, absorbance of the supernatant was measured at 480 nm after 12,000 rpm spin of the reaction mixture in an E5804 centrifuge (Eppendorf, Germany).

Carbohydrate, phenolics and lignin analysis
Samples of approximately 0.5 g of roots each were subject to extraction for 30 min with 10 ml of an ethanol/watermixture (80:20, v/v) used as a high temperature (80℃) extraction solvent. The solution was filtered and evaporated to dryness under nitrogen, with an aliquot of the filtrate redissolved in 0.5 ml of 0.1 mM ethylenediaminetetraacetic acid calcium disodiumsalt (CaEDTA) solution and filtered through a 0.45 μm Milipore filter before being used for carbohydrate analysis. Glucose, fructose and sucrose were estimated enzymatically as described (Morkunas et al. 2005), and starch was measured by the method (Fredeen et al. 1989). Total phenolic and lignin content was measured as previously described (Velioglu et al. 1998;Syros et al. 2004).

Preparation of gene silencing and overexpression constructs, infectious clones from transcription
An in planta gene silencing vector was prepared using a 501-bp DNA fragment amplified from the PvMES1 cDNA clone using sequence-specific primers (MESvigs-F/R) with the BamHI and ClaI restriction sites. Simultaneously, a PvMES1 overexpression vector for the same BPMV system was made using the open reading frame (ORF) fragment amplified from the PvMES1 cDNA clone by the primer pair OEMES-F/R with the same restriction enzymes. The amplified PCR products were gel purified and digested with BamHI and ClaI and subcloned into pG7R2V previously digested with the same restriction enzymes resulting in silencing and overexpression vectors, pG7R2V-501 and pG7R2V-PvMES1, respectively. The empty vector plasmid pG7R2V was digested by MscI and then set aside to use as a control in both inoculations. All vectors were further verified by Sanger sequencing.
In vitro transcription was conducted with the plasmid constructs above using RiboMAX™ Large Scale RNA Production Systems Kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Yield and integrity of the transcripts were analyzed by electrophoresis on a 1.0% agarose gel.

BPMV-derived vectors used for inoculation
The pG7R2V-501 and pG7R2V-PvMES1 RNAs were separately expressed and used as RNA2 transcripts in combination with RNA1 from the infectious BPMV viral strain Kentucky-Hopkins (K-Hop). These combinations were used for inoculation of common bean seedlings planted in a greenhouse in Shenyang, Liaoning province at Liaoning Academy of Agricultural Sciences (LAAS). Inoculation was done on the primary leaves by rubbing with a sponge/carborundum. However, specially for gene overexpression analysis, leaf extracts prepared from plants infected with empty vector and pG7R2V-PvMES1 vector by the method above were used to treat the primary leaves of ten-days-old plants as control and treatment (Díaz Camino et al. 2011). The wild type plants were also used as controls of both analysis.

Transcriptome sequencing
RNA-seq experiments for transcriptome sequencing were designed with the infected plants described above, consisting of infection of ten-day-old seedlings in all cases. Treatments consisted of seedlings infected with pG7R2V-PvMES1(overexpression) labeled by O-PvMES1 and with pG7R2V-501 (gene silencing) labeled by S-PvMES1. The seedlings treated with leaf extracts from plants infected with empty vector were used as the control of O-PvMES1 named by E-BPMV1, and plants treated with the empty pG7R2V vector were used as the control of S-PvMES1 named by E-BPMV2. The wild type plants were also used as controls for both treatments. Root tissues were collected at 12d (0 d post inoculation with Fop) and 15 d (3 d post inoculation with Fop) after inoculation by the BPMV-derived vectors. Each treatment and control was designed with three biological replicates (8 seedlings each). The libraries were made with indices for identification, then sequenced using an IlluminaHiSeq 2500 at BioMarker Technologies.

Bioinformatics data analysis
The statistical analysis of the RNA-seq data was performed by means of two-way ANOVA for the factors of genotype (empty BPMV control vs. PvMES1 overexpression vs. PvMES1 silencing) and pathogen infection (non-infected control vs. 5.0 × 10 6 cfu g −1 Fop inoculation). In detail, this involved cleaning reads and mapping them to the genome after examining sequence quality and removing low-quality sequence using Tophat v. 2.0.1. We compared our sequences to those of the common bean genome (Phaseolus vulgaris L, 2n = 22) from Phytozome v12.1 (https:// phyto zome. jgi. doe. gov/ pz/ portal. html). The transcriptome assemblies were performed for each library by Cufflinks (Trapnellet al. 2012). To estimate the read count of each gene in each library, the mapping results were filtered to maintain only uniquely mapped reads before being piped into Cuffdiff (http:// cole-trapn elllab. github. io/ cuffl inks/). The fragments per kilobase of exon per millionfragments mapped (FPKM) value was calculated using the internal script of the count table based on Cuffdiff's output. DESeq was used to detect DEGs according to Anders and Huber (2010). The thresholds to identify DEGs and the corrected P values from this method accounted for multiple tests by using the key factor, which was based on a false discovery rate (FDR) < 0.01 and fold change > 2 or < 0.5. The assembled sequences were compared with the non-redundant database (NR, Deng et al. 2006) from NCBI, the Swiss-Prot database (Apweiler et al. 2004), the GO database (Ashburner et al. 2000), the Clusters of Orthologous Group database (COG, Tatusov et al. 2000) and the Kyoto Encyclopedia of Genes and Genomes database (KEGG, Kanehisa et al. 2004). Fold changes in the expression levels between samples were used as the criteria in the screening process.

Cloning and characterization of the PvMES1 gene
Previous study indicated seven SABP2's putative orthologs were found and designated as PvMES1-PvMES7 in common bean, all with strong sequence homology to NtSABP2 (Table S1). The members of the PvMES gene family can be divided into three subfamilies according to the genetic relationship with the NtSABP2 ( Figure S1). The expression level of seven PvMES genes in susceptible varieties BRB130 and resistant varieties CAAS260205 infected by Fop was detected by qRT-PCR (Xue et al. 2018). The results

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showed that the expression of PvMES1 was significantly upregulated at 3 dpi, with most significant increase of 5.6 folds in CAAS260205. Thus PvMES1 gene was selected for this study to continue all our work. The genetic locus for PvMES1 was found at Phvul.003G248200.1 located from bp position 48,561,200 to 48,564,505 on Chromosome 3, with 59.2% sequence identity with NtSABP2 (Table S1).
The PvMES1 gene was found to contain three exons and two introns (Fig. 1a). The original cDNA clone indicated that the mature mRNA sequence contained a single ORF of 834 bp encoding a 277-aa extracellularly secreted protein with a calculated molecular mass of 30.99 kDa and a putative 22 amino acid signal peptide highlighted in red (Fig. 1b). The protein also contained a catalytic triad (Ser102, His227, and Asp255) that formed part of the active site pocket (Fig. 1c, highlighted in orange and red), also highly conserved among other NtSABP2 orthologs from Arabidopsis (AtMESs, Vlot et al. 2008) and potato (StMES1, Manosalva et al. 2010). Finally, PvMES1 contained 14 of the 15 aa identified in NtSABP2 that interact with SA ( Fig. 1b and c, highlighted in yellow and orange), suggested that these two proteins have similar biological functions and biochemical properties.
A full-length gene with a sequence length of 973 bp was cloned from the common bean genotype CAAS260205 infected by Fop using RT-PCR method and sequenced ( Figure S2). Comparison of the 973 bp sequence with non-redundant (nr) sequences in the GenBank nucleotide database indicated that the cDNA fragment isolated was a member of SABP2 gene family, named and deposited in the database as PvMES1 (Genbank accession no. MT471345). The 3265 bp full-length genomic (gDNA) fragment was also obtained which contained a 3182 bp gene sequence ( Figure  S2).

Phenotypic and PvMES1 gene expression differences under Fop infection
To further analyze the biological role of the PvMES1 gene we evaluated its expression in contrasting Fusarium wilt susceptible and resistant genotypes. Notable phenotypic differences between BRB130 and CAAS260205 symptomatology were found when infected with Fop ( Figure S3). We followed this phenotypic characterization with a time course study to evaluate the expression of PvMES1 under Fop infection and SA treatments using qRT-PCR and relative expression compared to controls. We found that the transcription of PvMES1 in the infected but resistant CAAS260205 roots was strongly and rapidly induced by Fop infection especially at 48 and 72 hpi (Fig. 1d). In response to SA treatment, PvMES1 expression was repressed over the period from 24 to 72 hpi in both genotypes and then gradually recovered to the original expression level when CAAS26025 had higher SA induced expression of the gene (Fig. 1e).

PvMES1 displays SA-inhibited MeSA esterase activity
To determine the biochemical properties of PvMES1, its corresponding full-length cDNA was expressed in Escherichia coli, the protein affinity purified, and analyzed for MeSA esterase activity. The results showed that the C-terminal His × 6-tagged PvMES1 transcript encoding product was successfully expressed and found isolated to an inclusion body. Dissolved in an 8 M urea solution, it electrophoresed well and was estimated to have a molecular weight of 30 kDa (Fig. 1f). Purified PvMES1 transcript product readily converted MeSA to SA. Simultaneously, we also tested the transcript product in a SA-binding assay and found that PvMES1 transcript encoding product reproducibly bound SA, and that the esterase activity was dramatically inhibited by SA when an LC 50 reached approximately 6.2 μM (Fig. 1g).

Overexpression and silencing of PvMES1 gene in roots mediated by BPMV-based method
The transcript product mixture of recombinant the pG7R2V constructs (pG7R2V-PvMES1 for overexpression and pG7R2V-501 for silencing, respectively) and pGHopR1 containing RNA1 from BPMV were inoculated as infective virus onto the fully expanded unifoliate leaves of plants which would be later infected by Fop at 12 dpi after inoculation with the virus. The difference in disease phenotype between the wild type (WT) plants, E-BPMV1 controls and PvMES1-overexpressed plants was even more noticeable at 4 weeks post inoculation with Fop (Fig. 2a,upper portion). Similarly, the typical stem browning and leaf yellowing phenotype of PvMES1-silenced plants appeared earlier than WT and its E-BPMV2 control, even at 2 weeks after Fop infection, PvMES1-silenced plants almostly withered away, while Fusarium wilt symptoms just occurred on the control seedlings of WT and E-BPMV2 obviously (Fig. 2a, lower portion). Disease scores were significantly different (P < 0.05) in treatments versus controls (Fig. 2b).
The qRT-PCR results indicated that fungal pathogen DNA provided evidence for the level of Fop infection and was significantly lower (P < 0.05) in roots of PvMES1overexpressed plants compared to control roots at 18 dpi, but higher in the PvMES1-silenced plant roots than the control (Fig. 2c). Gene expression analysis showed levels in the PvMES1-overexpressed plants were significantly higher than control at 6 dpi with the BPMV vector and continued to increase up to a 13.6-fold difference at 18 dpi by BPMV (6 dpi by Fop) (Fig. 2d, upper portion). The PvMES1-silenced plants had lower gene expression every day of measurement during dual fungal and viral infection, which reduced with 0.51-fold difference compared to control at the final time point (Fig. 2d, lower portion). Similar patterns were seen with MeSA esterase activity and SA accumulation. The MeSA esterase activity of PvMES1-overexpressed plants increased dramatically during the infection with BPMV, especially inoculated with Fop pathogen, reaching a maximum of 0.43 nmol/min/μg at 18 dpi with BPMV (Fig. 2e, upper portion), and only 0.024 nmol/min/μg in the silenced plants at the same time point (Fig. 2e, lower portion). Correspondingly, SA accumulation in PvMES1-overexpressed plants constantly increased with BPMV inoculation, especially after infection by Fop, which almost double that of control at 18 dpi with BPMV (Fig. 2f, upper portion). In PvMES1-silenced plants, the SA level significantly decreased sharply declined after 12 dpi with BPMV (Fig. 2f, lower portion). These results strengthened the hypothesis that PvMES1 gene played a vital role in the resistance against Fusarium wilt through the level regulation of the active free SA in common beans.

Transcriptome profiling of PvMES1-regulated genes in Fop infected bean roots
To comprehensively understand the effects of PvMES1 on the resistance to Fusarium wilt in common beans, we performed RNA-seq experiments and compared the transcriptome profiles: (1) PvMES1-overexpressed DEGs versus Fop infected DEGs versus both PvMES1-overexpressed and Fop infected DEGs (Fig. 3a); (2) PvMES1-silenced DEGs versus Fop infected DEGs versus both PvMES1-silenced and Fop infected DEGs (Fig. 4a). The transcriptome sequencing results of WT plants have no significant difference from E-BPMV1 and E-BPMV2 (Data not shown), thus we respectively selected E-BPMV1 and E-BPMV2 as controls of gene overexpression and silencing for the transcriptome analysis. The numbers of total reads, mapped reads, and the percentage of mapped reads in each replicate are shown in Figure S4. Principal component analyses showed that both Fop attack and PvMES1-regulation profoundly influenced the transcriptome results ( Figure S5).
The were also regulated by PvMES1 in the gene overexpression experiment (Fig. 3b). In the interaction, 589 DEGs (173 up and 416 downregulated) were affected by PvMES1 overexpression. Among the upregulated pathogen-induced DEGs, 93.3% (360 of 386) of the genes were upregulated by both pathogen and PvMES1. Almost all the genes downregulated by pathogen (1074 of 1077; 99.7%) were also decreased by PvMES1 (Fig. 3c, d).
In  Fig. 4b; Data S2). Among the PvMES1silenced and pathogen-induced genes, 1557 were regulated by the fungal pathogen, 64.9% of the total; and among these Fig. 1 Characteristic of the cloned PvMES1 gene and the encoded protein from common bean (Phaseolus vulgaris L.). a Gene structure of the common bean ortholog of SABP2, named P. vulgaris (Pv) methyl esterase 1, PvMES1. Boxes indicate exons, and lines indicate introns. The numbers below the bars and lines indicate the s lengths. Start and stop codons indicated at beginning and end of sequence, respectively. b Multiple sequence alignment of PvMES1 with genes from Solanum tuberosum L., potato (StMES1) and from Nicotiana tabacum L., tobacco (NtSABP2), as we as with three Arabidopsis thaliana orthologs (AtMES1, AtMES4 and AtMES9). Identical residues are shaded in black and similar residues in gray. The catalytic triad residues are indicated by arrows, and residues that contact salicylic acid are indic with black triangles. The signal peptide sequence is marked with a red box. Numbers to right indicate amino acid position. c Three-dimensional (3D) model of the PvMES1 protein. The predicted of a catalytic triad that formed part of the active pocket are marked in orange and red. The amino acid positions that interact with SA are marked with yellow and orange. d Expression pattern of the P. vulagris methyl esterase PvMES1 in the roots of Fusarium oxysporum f. sp. phaseoli (Fop) from a susceptible common bean genotype BRB130 and from a resistant genotype CAAS260205 for plants infected with the pathogen at a density of 5.0 × 10 6 colony forming units, cfu mL −1 and sampling at 0, 24, 48, 72, 96 and 120 h post inoculation (hpi). The relative expression values were determined by qRT-PCR from three biological replicates. The error bars indicate ± SD (n = 3). Single and double asterisks indicate a significant difference based on Student's t-tests at P < 0.05 and P < 0.01, respectively. e Expression pattern of PvMES1 in 10 days-old pot-grown plants of the same common bean genotypes, BRB130 and CAAS260205, whose leaves were sprayed with a 2 mM salicylic acid (SA) solution and sampled at 0, 6, 24, 48, 72, 96 and 120 h after treatment. The relative expression values were determined by qRT-PCR from three biological replicates. The error bars indicate ± SD (n = 3). Single and double asterisks indicate a significant difference based on Student's t-tests at P < 0.05 and P < 0.01, respectively. f Purification of the expected 30 kDa (kilo Dalton) PvMES1 recombinant protein fused with 6 × Histidine tag at the C-terminus. Size marker to the right of the PvMES1-His tag lane. g Biochemical characterization of PvMES1 esterase activity from common bean measured using methyl salicylate (MeSA) as a substrate, where inhibition of PvMES1 MeSA esterase activity was performed using SA at five different initial concentrations (0.5, 1.0, 5.0, 10.0 and 50.0 µM) compared to control at 0 µM. The error bars are shown with standard deviations (n = 3). Lines represent a global fit of all data (n = 3) to the equation for competitive inhibition ◂ 1 3 487 were upregulated and 1070 were downregulated. Interestingly, 99.8% (486 of 487) of the genes upregulated by the fungal pathogen were also upregulated by PvMES1. Conversely, 97.8% (1046 of 1070) of the genes that were downregulated by the fungal pathogen were also downregulated by PvMES1 (Fig. 4c,d).
GO analysis indicated that genes regulated by Fop and PvMES1 are enriched in three categories of biological process, cellular component and molecular function (Figure S6). KEGG analysis of the genes in the interaction term indicated that DEGs induced by both affects are enriched in pathways related to phenylpropanoid biosynthesis, starch and sucrose metabolism, plant hormone signal transduction, plant-pathogen interaction confirming that PvMES1 plays a key role in orchestrating the transcription of pathogen-induced genes ( Figure S7).

Phenylpropanoid pathway-related genes upregulation promotes lignin biosynthesis
A subset of genes participating in multiple branches of the phenylpropanoid pathway, including lignins, flavonoids, and cinnamaldehyde, were identified based on RNA-seq analysis (Fig. 5a). All of the enzymes indicated with red boxes were significantly upregulated by the enhanced SA biosynthesis induced by PvMES1 overexpression. When PvMES1 was silenced, the DEGs in red boxes are those downregulated both before and after Fop inoculation. Enzymes such as 4-coumarate-CoA ligase, cinnamoyl-CoA reductase, mannitol dehydrogenase and peroxidase were upregulated in O-PvMES1 but downregulated in S-PvMES1 versus control with or without Fop inoculation shown in a heatmap with log 2 FPKM ( Fig. 5b; Data S3). The result was further verificated by qRT-PCR analysis (Fig. 5c). Pathogen defense-related indices such as PAL, SOD, POD activity, H 2 O 2 contents, total phenolic and lignin contents were significantly higher in the PvMES1-overexpressed, and lower in the PvMES1-silenced plants compared to the controls after 72 h post inoculation with Fop, but MDA contrastly changed ( Figure S8).

Sugar metabolism pathway modulating defense response to Fusarium wilt
Several branches of amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism pathways showed DEGs regulated by PvMES1 expression changes. Those which were significantly upregulated by SA enhancement were marked with the red boxes while downregulated genes were marked in blue boxes (Fig. 6a, b). The production of sugar molecular such as glucose, fructose, sucrose and their phosphorylated derivatives as defense signalling factors were indicated to be affected by the pathosystem and promoted by the upregulation of relevant enzymes. Cellulose and pectinas the essential components for cell wall reinforcement upon fungal infection were also regulated by PvMES1 expression degree change. Digital transcriptome expression analysis of sugar metabolism DEGs were shown in a heatmap with log 2 FPKM values ( Fig. 6c; Data S4). The qRT-PCR results showed that ten key genes in the sugar metabolism pathway were significantly upregulated by overexpressing PvMES1 and downregulated by silencing PvMES1 (Fig. 6d). We also analyzed sugar content in the roots of controls and treatment plants infected by Fop. Glucose, fructose, sucrose and starch concentration in the roots of beans were increased by PvMES1 overexpression, but declined during PvMES1 silencing compared with each corresponding control after 24 h post inoculation with Fop ( Figure S9).

Expression of hormone signaling-related genes
Selected hormone signaling-related genes encoding receptors and response factors, identified from KEGG pathway analysis, were shown to be affected by PvMES1 levels (Fig. 7a, Data S5). SA response factors TGA and PR-1 were upregulated in Fop-inoculated roots of common bean by PvMES1 overexpression and downregulated by PvMES1 silencing. These results indicated the change of SA level had a closed relationship with PvMES1 expression degree. Meanwhile, JAZ and MYC2 from jasmonic acid (JA) mediated signaling, and AUX1, AUX/IAA from auxin mediated signaling were significantly repressed by PvMES1 overexpression and induced to high expression levels by PvMES1 silencing with or without Fop, whereas, ARF and GH3 were activated. The response factors from other selected plant hormone have no obvious connection with the defense response to Fop mediated by SA in common beans. The expression levels of the selected hormone signaling-related genes were analyzed by qRT-PCR (Fig. 7b). Fig. 2 Effect of PvMES1 gene on resistance to Fusarium wilt (Fusarium oxysporum f. sp. phaseoli, Fop) in common beans. a Disease symptoms in PvMES1-overexpressed (O-PvMES1) at four weeks after inoculation with the Fop pathogen strain compared to the control of the wild type (WT) and the empty bean pod mottle virus transient vectors (E-BPMV1), and silenced (S-PvMES1) plants at two weeks after inoculation with the pathogen also compared to the wild type (WT) and the empty bean pod mottle virus transient vectors (E-BPMV2) used as the corresponding controls. Pot-grown plants were inoculated at a density of 5.0 × 10 6 cfu mL −1 of fungal spores and grown in a greenhouse for over one month. The experiment was independently repeated three times with five plants per replicate. b Statistical analysis of disease scores in the PvMES1-overexpressed and its control plants at four weeks, PvMES1-silenced and its control plants at two weeks post inoculation with Fop. The error bar values represent ± SD (n = 15) from three independent biological replicates with five plants per replicate. Single asterisk about the brackets indicate significant differences at P < 0.05 based on Student's t-test. c Quantitative detection of F.oxysporum f. sp. phaseoli pathogen DNA in picogram (pg) ratio compared to common bean host DNA in nanograms (ng) at two time points (3 and 6 days) after inoculation with

Interaction mechanism regulating the oxidative burst and calcium transporting
To investigate whether the PvMES1 had a role in oxidative burst or calcium transport based Fop resistance in common bean, we evaluated genes from the host-pathogen interaction signaling pathway found in KEGG (Fig. 8a, Data S6). The result showed that a calciumdependent protein kinase (Phvul.001G135300) was upregulated by PvMES1 overexpression, but downregulated in PvMES1-silenced plants after Fop infection. We also did see the upregulations of a pair of respiratory burst oxidases (Phvul.002G293700, Phvul.006G090200) with PvMES1 gene overexpression. In contrast, two cyclic nucleotide-gated ion channels (Phvul.008G036200, Phvul.010G113100), calmodulin (Phvul.001G155400) and calcium-binding protein (Phvul.001G095600) were repressed in O-PvMES1, and activated significantly in S-PvMES1 post inoculation with Fop. The expression level of selected DEGs in the interaction pathway were identified through the heatmap analysis based on log 2 FPKM values (Fig. 8b).

Activation of a PvMES1-mediated defense signaling pathway in common bean confers resistance to Fusarium wilt
To better understand how PvMES1-mediated Fop resistance is regulated, we performed RNA-seq to examine the expression of 17,037 genes in the controls, PvMES1-overexpressed and -silenced plants ( Fig. 9a; Figure S7; Data S7). Further comparisons of the RNA-seq data of O-PvMES1, S-PvMES1 and control plants at 72 hpi (FDR-adjusted P value < 0.01) revealed 1,450 DEGs. Compared to the control E-BPMV1, 228 upregulated and 550 downregulated genes were identified in O-PvMES1, whereas 590 upregulated and 442 downregulated genes were identified in S-PvMES1 compared with control E-BPMV2. Inaddition, 116 genes were upregulated in O-PvMES1, but downregulated in S-PvMES1, and 145 genes were downregulated in O-PvMES1, but upregulated in S-PvMES1 after infection with Fop (Fig. 9b). Therefore, these 261 genes were regulated in an opposite manner in the overexpression and silencing lines of PvMES1. Selected DEGs involved in the signaling transduction, transcription factors, defense metabolism and sugar transportion were  (Fig. 9c-f).

Discussion
Our major results were to find that SA and the newly characterized gene producing it, PvMES1, played key roles in the interaction between Fop, an important fungal pathogen of the common bean and its host plant. PvMES1 displayed SA-inhibitable MeSA esterase activity in vitro similar to NtSABP2 and StMES1, AtMES1, AtMES4, AtMES9. The effective gene overexpression and silencing methods based on BPMV-derived vector were utilized to identify the biological function of PvMES1 gene. Thus, it proved that MeSA and SA participated in a key pathway for defense signaling in common bean.
SA is a signaling molecule naturally found in plants and shown to be involved in various plant defense-related actions against infection by various pathogens. White and his colleague firstly reported that salicylates as disease resistanceinducing chemicals (White 1979;Antoniw and White 1980). The methylated form of SA (MeSA) can be found in various plants, its role as an alternative inactive storage form of free SA (Shulaevet al. 1997;Kawano et al. 1998). Forouhar et al. (2005 have reported a successful demonstration that SA-binding protein isolated from tobacco, designated as SABP2 (SA-binding protein 2) has strong esterase activity with MeSA as substrate. Therefore, release of free SA from MeSA by SABP2 contributed to rapid SA increase in the c Relative expression levels of four representive genes, 4-coumarate-CoA ligars 1, Cinnamoyl-CoA reductase 1, peroxidase 10 and a probable mannitol dehydrogenase in PvMES1-overexpressed (red bar), PvMES1-silenced (green bar) and control plants for E-BPMV1 (blue bar) and E-BPMV-2 (orange bar) inoculated with or without Fop at 72 h. The values were determined by qRT-PCR from three biological replicates consisting of four plants (n = 12). The errorbars indicate ± SD (n = 3). Single and double asterisks above brackets indicate a significant difference at P < 0.05 and P < 0.01 based on Student's t-test, respectively, for the given comparison systemic tissues of TMV-infected tobacco plants for SAR development (Park et al. 2007a, b). In this study, we describe the identification of PvMES1 from common bean based on homology with SABP2 and demonstrate that it shares similar biochemical properties with its orthologs from tobacco, Arabidopsis and potato. Expression of PvMES1, in the same way was induced by pathogen infection and repressed by SA, the PvMES1 transcript product exhibits esterase activity toward MeSA, and this activity is feedback inhibited by SA (Fig. 1). This suggesed that PvMES1 could perform a similar defense function as SABP2 in the SA-mediated defense pathway in common bean. Furthermore, the overexpression of PvMES1 led to increased Fop resistance and PvMES1-silenced plants enhanced the susceptibility and compromised SAR dramatically (Fig. 2). We combined PvMES1 regulation and pathogen infection assays to reveal Fig. 6 The role of sugar metabolism in common beans challenged by the Fusarium wilt pathogen, Fusarium oxysporum f. sp. phaseoli (Fop). a A partial diagram of amino sugar and nucleotide sugar metabolism constructed based on the KEGG pathway and gene expression data. b A partial diagram of starch and sucrose metabolism constructed based on the KEGG pathway and gene expression data. In both subfigures (a) and (b), the upregulated enzymes by enhanced PvMES1 expression, and downregulated by repressed PvMES1 expression are shown in red, the enzymes following the reverse trend in co-regulation by PvMES1 are shown in blue. c Heatmap from the RNA-seq experiments of the up and downregulated genes from two selected sugar metabolism pathway, numbered as in the KEGG. The average FPKM of each gene is color coded based on log 2 scale in upper right. d Relative expression levels of ten representive genes in PvMES1-overexpressed, PvMES1-silenced and each control plants inoculated without or with Fop at 72 h. The values were determined by qRT-PCR from three biological replicates consisting of four plants (n = 12). The errorbars indicate ± SD (n = 3). Single and double asterisks above bracketing lines indicate a significant difference at P < 0.05 and P < 0.01 based on Student's t-test, respectively, for appropriate comparisons indicated by brackets 1 3 that SA-responsive genes and pathogen induced genes in common beans by RNA-seq, and it indicated that PvMES1 plays a pivotal role in plant resistance to the Fusarium wilt pathogen (Fig. 3,4). Through increasing SA levels, PvMES1 regulates the expression of genes from the pathway of phenylpropanoid biosynthesis, sugar metabolism, SA signaling and interaction between Fop and common bean.
Phenylpropanoids are the important and necessary precursors of numerous structural polymers and defense compounds (such as phytoalexins and lignin) in plants, and thus serve as the core mediators of crosstalk between developmental and defense-related pathways (Alessandra et al. 2010). The genes identified by RNA-seq in this study focused on the main branches of the phenylpropanoid pathway with an emphasis on the synthesis and deposition of lignin, and also leading to the synthesis of flavonoid and cinnamaldehyde (Fig. 5).
Previous studies have demonstrated that the phenylpropanoid pathway leading to lignin synthesis and deposition from the oxidative coupling of three monolignols named p-hydroxyphenyl, guaiacyl and syringyl as a physical barrier is activated because lignification and reinforcement of cell walls are important processes in plants during fungal penetration (Smit and Dubery 1997;Bhuiyan et al. 2009;Naoumkina et al. 2010). Particularly, the lignins has been reported to play critical roles in the responses to F. oxysporum in flax with the enhancement of MeSA level (Boba et al. 2017).
Some of pathogen defense-related indices measured in this study, such as PAL and POD, belonged to enzymes involved in the core and entrypathway of the phenylpropanoid pathway and biosynthesis of lignin (Marjamaa et al. 2009;Huang et al. 2010). In addition, phenylpropanoid pathway allows plants to produce various secondary metabolites in defense response to infection including flavonoids (isoflavonoids in particular) as phytoalexinsin the legume specise (Andersen and Markham 2006;Bednarek and Osbourn 2009;Naoumkina et al. 2010) and the cinnamaldehyde which affected the morphogenesis and growth of the pathogen fungus (Xing et al. 2014). Overall, it indicated that change of phenylpropanoid pathway mediated by PvMES1 will provide new insights into the control of Fusarium wilt and disease resistance development in common beans.
Sugars act as a regulatory factor affecting almost all processes of growth and development in plants (Wind et al. 2010;Stokes et al. 2013). It indicated that sugars constitute the primary substrate providing energy and structural material for defense responses in plants, while they may also act as signal molecules in activating PTI and ETI, interacting with the hormonal signaling network regulating the plant immune system (Bolouri Moghaddam and Van den Eden 2012; Rolland et al. 2002). In most fungal pathogen-plant systems, the high product level of sugar metabolism such as sucrose, glucose, fructose etc. in plant tissues enhances plant resistance (Morkunas and Ratajczak 2014). Gupta and Fusarium oxysporum f.sp ciceri suggesting a vital role of sugars in defense against Fusarium wilt. It has also been reported that sucrose, glucose and fructose can play an important role in resistance to fungal pathogens through stimulation of phenylpropanoid metabolism (Forlani 2010;Morkunas et al. 2011;Gibertia et al. 2012).
Sucrose also functions as a signaling molecule (Wind et al. 2010). Sucrose responsive element is reported to influence the expression of WRKY transcription factor during defense (Sun et al. 2003). Sucrose synthase is also related to cellulose synthesis, it can provide UDP-glucose as an essential substrate for cell wall development (Fujii et al. 2010). As well known, the cellulose and pectin are the essential components made of plant cell wall, which constitutes the first barrier to microbial invasion and processes associated with its reorganization are among the early events occurring in plant defense (Albrecht et al. 2008). Trehalose is also a well-known disaccharide with signaling functions in plant growth and development, as well as defense responses, and its derivativetrehalose 6-phosphate has a dual function as a signal and homeostatic regulator of sucrose levels in plants (Paul et al. 2008;Delatte et al. 2011). It has been shown to partially induce resistance against powdery mildew (Blumeriagraminis f. sp. tritici) in wheat by the activation of phenylalanine ammonia-lyase and peroxidase genes (Reignault et al. 2001;Muchembled et al. 2006). However, sugar production alone is insufficient to control signal transduction. This role is performed by the sugar transporters. It revealed that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection in Arabidopsis (Yamada et al. 2016).
Before our study, not much information existed on the expression of these sugar molecules and transportersin common beans especially as related to disease infection. Our study demonstrated that sugars helped in generating alarm signals and efficiently transmitted them throughout with the aid of sugar transporters and played the key role in the cellulose and pectin synthesis. Therefore, the activation of SA inducible genes in the metabolism and transport of sugar molecules enhanced the defense signaling and the cell wall reinforcement in response to Fop infection in common bean (Figs. 6;9f; Figure S8).
Phytohormones such as abscisic acid, jasmonic acid, ethylene, salicylic acid, auxin, gibberellin, cytokinein, and brassinosteroids appeared to play other critical roles in the complex signalling cascades and were integrated into current models of defence responses (Bari and Jones 2009). Xue et al. (2014) has reported that SA applied to foliar tissue is capable of enhancing the systemic acquired resistance of common bean roots to infection by Fop through the induction of endogenous free SA accumulation. In this study, SA response factors TGA and PR-1 were induced by PvMES1 activation following the endogenous SA accumulation (Fig. 7). Otherwise, the use of mutants and transgenic plants of Arabidopsis indicated that SA was involved in the to Fusarium oxysporum f. sp. phaseoli (Fop) in common beans. The diagram was constructed based on the information of KEGG pathways (Figs. 5,6,7,8 and 9) and shows the roles of Avr, avirulence gene product; Ca 2+ , calcium signaling; CDPK, calcium directed protein kinase; HR, hypersensitive response; PAMP, pathogen associated molecular pattern; PCD, programmed cell death; PM, plasma mem-brane; and ROS, reactive oxygen species. We hypothesize that salicylic acid (SA) is increased by PvMES1 and then activates phenylpropanoid biosynthesis, sugar metabolism, relevant hormone signaling pathways and interaction pathway between fungi and host in response to Fop attack leading to cell wall reinforcement, flavonoid biosynthesis and other host cell defenses against the pathogen sugar dependent activation of these PR protein-coding genes (Thibaud et al. 2004).
For auxin signaling, the previous study indicated that overexpression of GH3.5 conferred enhanced SA accumulation, pathogen resistance, and defense gene expression (Park et al. 2007a, b;Zhang et al. 2007;Westfall et al. 2016). Similar with GH3.5 from subfamily II, mutations in GH3.12 as a member from subfamily III, also reduced SA accumulation and pathogen resistance in Arabidopsis, that indicated GH3.12 likely acts upstream of SA in defense signaling (Lee et al. 2007;Nobuta et al. 2007). However we saw no role for these transcription factors.
To date, a variety of possible receptors and corresponding signaling mechanisms has been proposed for the modes of SA signal transduction in plants. Du et al. (2009) observed that binding of Ca 2+ /CaM to the Arabidopsis transcription factor SR1 repressed expression of EDS1 and suppressed SA accumulation and defense. In addition, Chen et al. (1993) have proposed the involvement of ROS as key signals that relay SA signals in plants. An oxidative burst through F. oxysporum infection led to ROS overproduction in the in vitro cultured embryo axes of yellow lupin, and the processes are much more intensive when treated with exogenous sucrose, the defense signaling molecule shown above (Morkunas et al. 2013). These studies opened the door for SA studies regarding calcium signaling and oxidative bursts. In this study, it indicated that resistance enhancement in common bean to Fop through suppressing calcium-mediated signaling and activating ROS accumulation as mediators by SA induction (Fig. 8).
In summary, PvMES1 plays a pivotal role in regulating the SA inducible resistance to Fusarium wilt in common bean (Fig. 10). The pathway of phenylpropanoid synthesis and sugar metabolism regulated by PvMES1 are demonstrated to be important for the enhancement of defense response. SA and other elements as key signals derived from the phenylpropanoid synthesis, sugar metabolismand interaction activated the resistance pathway based on the signaling transduction, transcription factors, defense metabolism in common bean toward Fusarium wilt (Fig. 9c-e). The current understanding of the interactions between SA signaling and plant pathogen attack mainly comes from studies in Arabidopsis and tomato, and different resistance mechanisms provide an angle to understand the infection process and the defense response of F. oxysporumin the root system of common beans. Collectively, our study provides insights into the roles of SA in regulating defense responses, indicating a central role for PvMES1 in common bean against Fop. The significant practical applications for engineering disease resistance and support to the broad-spectrum resistance in common bean by manipulating SA levels and thereby increasing SAR development remains to be discussed.