Genome-Wide Analysis of the Wall-Associated Kinase (WAK) Genes in Medicago truncatula and Functional Characterization of MtWAK24 in Response to Pathogen Infection

The wall-associated kinases (WAKs) can perceive and transmit extracellular signals as one kind of unique receptor-like kinases (RLKs) involved in the regulation of cell expansion, pathogen resistance and abiotic stress tolerance. To understand their potential roles and screen some key candidates in Medicago truncatula (M. truncatula), genome-wide identification and characterization of MtWAKs were conducted in this study. A total of 54 MtWAK genes were identified and classified into four groups based on their protein domains. They were distributed on all chromosomes, while most of them were clustered on chromosome 1 and 3. The synteny analysis showed that 11 orthologous pairs were identified between M. truncatula and Arabidopsis thaliana (A. thaliana) and 31 pairs between M. truncatula and Glycine max (G. max). The phylogenetic analysis showed that WAK-RLKs were classified into five clades, and they exhibited a species-specific expansion. Most MtWAK-RLKs had similar exon–intron organization and motif distribution. Multiple cis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions of MtWAK-RLKs. In addition, the expression patterns of MtWAK-RLKs varied with different plant tissues, developmental stages and biotic and abiotic stresses. Interestingly, plasm membrane localized MtWAK24 significantly inhibited Phytophthora infection in tobacco. The study provides valuable information for characterizing the molecular functions of MtWAKs in regulation of plant growth, development and stress tolerance in legume plants.


Introduction
Plants perceive and process various signals on cell surface to modulate biological processes through members of the receptor-like kinase family [1]. WAK is a unique class of RLKs that link cell wall to cytoplasm physically, consisting of extracellular epidermal growth factor (EGF) domain and/or galacturonan-binding (GUB) domain, transmembrane domain and intercellular Ser/Thr kinase domain [2]. Based on the organization of the conserved domains, WAKs are divided into four types: the RLK type, which contains both extracellular domain and kinase domain; the receptor-like cytoplasmic kinase (RLCK), which contains kinase domain alone; the receptor-like protein (RLP), which contains only extracellular domain; and the short protein type, which harbors no domain but shows high similarity in the emerging sequence.
WAKs participate in regulation on plant growth and development as well as biotic and abiotic stress tolerance. Down-regulation of AtWAK4 or AtWAK2 expression leads to impaired cell expansion in Arabidopsis [3,4]. OsWAK11 regulates both stem and seed

Chromosomal Location and Expansion Analysis of MtWAKs
Fifty-four MtWAKs were unevenly distributed on eight chromosomes ( Figure 1). Chromosome 1 had 17 MtWAKs, while the other chromosomes had 2 to 9 MtWAKs. Interestingly, 20 MtWAK-RLKs and 7 MtWAK-RLCKs were found in clusters with 10 pairs of tandem duplicated gene pairs, while most MtWAK-RLPs and MtWAK short proteins were separately distributed on 8 chromosomes ( Figure 1, Table S2).
Two pairs of paralogous genes, MtWAK19-MtWAK35 and MtWAK36-MtWAK53, belong to segmental duplications (Table S2). The synteny analysis showed that there were 11 pairs of orthologous genes between M. truncatula and A. thaliana and 31 pairs between M. truncatula and G. max (Figure 2, Table S2).

Phylogeny, Gene Structure, Protein Domain and Motif Analysis of MtWAK-RLK Members
Phylogenetic analysis of WAK-RLKs from M. truncatula, A. thaliana, G. max and O. sativa showed that the WAK-RLKs could be divided into five clades. Clade Ⅰ and Ⅴ contained only OsWAK-RLKs. Clade Ⅱ contained 5 AtWAKs, 14 GmWAK-RLKs, 16 MtWAK-RLKs and 2 OsWAK-RLKs. Clade Ⅲ contained 14 AtWAKL-RLKs, 6 GmWAK-RLKs and 2 MtWAK-RLKs. Clade Ⅳ was comprised of 2 AtWAKL-RLKs, 35 GmWAK-RLKs, 4 Os-WAK-RLKs and 8 MtWAK-RLKs ( Figure 3).   Figure 3).  The gene structure, protein domain and motif of MtWAK-RLKs were further analyzed (Figure 4 and S1). Most members showed similar gene structure, harboring two long exons at both ends and a short one in the middle, except MtWAK3 and MtWAK51 with two exons only and MtWAK4, 24,42,43 and 50 with four exons ( Figure 4B). One or two transmembrane domains were shown in the majority of MtWAK-RLKs except MtWAK3 and MtWAK45. Both Gub_WAK_bind and EGF domains existed in all members of clade Ⅱ and MtWAK36, and there were 51 in clade Ⅳ ( Figure 4C). Moreover, 10 conserved motifs were predicted using MEME program. Motifs 1 to 7 corresponded to the conserved kinase domain in all MtWAK-RLKs ( Figure 4D and S1). A conserved arginine (R) residue was present in front of the motif DxxxxN; thus, all MtWAK-RLKs were classified as RD kinase. In addition, motif 8 was absent in the members of clade Ⅳ.  Figure 4C). Moreover, 10 conserved motifs were predicted using MEME program. Motifs 1 to 7 corresponded to the conserved kinase domain in all MtWAK-RLKs ( Figures 4D and S1). A conserved arginine (R) residue was present in front of the motif DxxxxN; thus, all MtWAK-RLKs were classified as RD kinase. In addition, motif 8 was absent in the members of clade IV.

Analysis of Cis-Acting Elements in the Promoter Region of MtWAK-RLKs
To understand the potential function of MtWAK-RLKs, the putative cis-acting elements were analyzed using PlantCARE software. Abundant phytohormone and stress responsive elements were observed in the promoter regions ( Figure 5). Twenty-four MtWAK-RLKs possessed a larger number of ABA-responsive elements, ethylene-responsive elements (ERE) and MeJA responsive elements (CGTCA-motif, TGACG-motif). Twelve MtWAK-RLKs had salicylic-acid-responsive elements (TCA-element), while nine MtWAK-RLKs had auxin-responsive elements (TGA-element). Most MtWAK-RLKs had anaerobic induction elements (ARE) and stress-responsive elements (STRE) in the promoter region ( Figure 5). A series of growth and development related cis-elements, such as meristem-expression element (CAT-box) and CCGTCC motif were observed in MtWAK-RLKs. The results suggested that MtWAK-RLKs might be involved in phytohormone regulation and stress response in M. truncatula.

Analysis of Cis-Acting Elements in the Promoter Region of MtWAK-RLKs
To understand the potential function of MtWAK-RLKs, the putative cis-acting elements were analyzed using PlantCARE software. Abundant phytohormone and stress responsive elements were observed in the promoter regions ( Figure 5). Twenty-four MtWAK-RLKs possessed a larger number of ABA-responsive elements, ethylene-responsive elements (ERE) and MeJA responsive elements (CGTCA-motif, TGACG-motif). Twelve MtWAK-RLKs had salicylic-acid-responsive elements (TCA-element), while nine MtWAK-RLKs had auxinresponsive elements (TGA-element). Most MtWAK-RLKs had anaerobic induction elements (ARE) and stress-responsive elements (STRE) in the promoter region ( Figure 5). A series of growth and development related cis-elements, such as meristem-expression element (CAT-box) and CCGTCC motif were observed in MtWAK-RLKs. The results suggested that MtWAK-RLKs might be involved in phytohormone regulation and stress response in M. truncatula.

Expression Analysis of MtWAK-RLKs across Different Tissues and Developmental Stages
To obtain insights into their temporal and spatial expression patterns, 12 MtWAK-RLKs having corresponding probesets in the gene expression database were prioritized for analysis (Table S3). MtWAK1, 4, 7, 8 and 18 were almost equally expressed in all tissues ( Figure 6A). MtWAK10, 24 and 50 were exclusively and highly expressed in roots. MtWAK36 and 45 were highly expressed in roots, whereas MtWAK3 was highly expressed in stem. From base to apex of stem, MtWAK24 and MtWAK53 expression was increased gradually, while MtWAK3 exhibited the opposite trend with the lowest expression level in the top internode ( Figure 6B). Interestingly, MtWAK24 and 53 were significantly up-regulated along with seed development ( Figure 6C). In addition, most MtWAK-RLKs were down-regulated during nodulation, except that MtWAK1 was up-regulated greatly at 6 dpi and 20 dpi ( Figure 6D). AuxRR-core and TGAelement, auxin-responsive elements; ERE, ethylene-responsive element; CGTCA and TGACG motif, MeJA-responsive elements; SARE and TCA-element, salicylic-acid-responsive elements; GARE-motif and P-box, GA-responsive elements; ARE and GC-motif, anaerobic induction elements; TC-rich repeats and STRE, stress-responsive elements; MBS, drought-induced element; DRE-core, dehydration-responsive element; LTR, low-temperature-responsive element; WRE3 and WUN-motif, wound-responsive element; CAT-box, meristem expression element; CCGTCC motif and NON-box, meristem-specific activation elements; circadian, circadian control element; GCN4_motif, endosperm-expression element; HD-Zip 1, palisade mesophyll cell-expression element; MBS, flavonoid biosynthetic gene regulation element; O2-site, zein metabolism regulation element.

Expression Analysis of MtWAK-RLKs across Different Tissues and Developmental Stages
To obtain insights into their temporal and spatial expression patterns, 12 MtWAK-RLKs having corresponding probesets in the gene expression database were prioritized for analysis (Table S3). MtWAK1, 4, 7, 8 and 18 were almost equally expressed in all tissues ( Figure  6A). MtWAK10, 24 and 50 were exclusively and highly expressed in roots. MtWAK36 and 45 were highly expressed in roots, whereas MtWAK3 was highly expressed in stem. From base to apex of stem, MtWAK24 and MtWAK53 expression was increased gradually, while MtWAK3 exhibited the opposite trend with the lowest expression level in the top internode ( Figure 6B). Interestingly, MtWAK24 and 53 were significantly up-regulated along with seed development ( Figure 6C). In addition, most MtWAK-RLKs were down-regulated during nodulation, except that MtWAK1 was up-regulated greatly at 6 dpi and 20 dpi ( Figure  6D). The numbers and the depth of red represent the frequency of the elements. ABRE, ABA-responsive element; AuxRR-core and TGAelement, auxin-responsive elements; ERE, ethylene-responsive element; CGTCA and TGACG motif, MeJA-responsive elements; SARE and TCA-element, salicylic-acid-responsive elements; GARE-motif and P-box, GA-responsive elements; ARE and GC-motif, anaerobic induction elements; TC-rich repeats and STRE, stress-responsive elements; MBS, drought-induced element; DRE-core, dehydrationresponsive element; LTR, low-temperature-responsive element; WRE3 and WUN-motif, woundresponsive element; CAT-box, meristem expression element; CCGTCC motif and NON-box, meristemspecific activation elements; circadian, circadian control element; GCN4_motif, endosperm-expression element; HD-Zip 1, palisade mesophyll cell-expression element; MBS, flavonoid biosynthetic gene regulation element; O2-site, zein metabolism regulation element.

Expression Analysis of MtWAK-RLKs in Response to Biotic and Abiotic Stresses
To understand the expression of MtWAK-RLKs in response to pathogens, datasets of 'Cell suspension_Yeast elicitor', 'Root_Macrophomina infected' and 'Root Tip_A17_Ralstonia' from the MtGEA were used for analysis (Table S3)

Expression Analysis of MtWAK-RLKs in Response to Biotic and Abiotic Stresses
To understand the expression of MtWAK-RLKs in response to pathogens, datasets of 'Cell suspension_Yeast elicitor', 'Root_Macrophomina infected' and 'Root Tip_A17_Ralstonia' from the MtGEA were used for analysis (Table S3)

Gene Expression Validation of MtWAK-RLKs by qRT-PCR
To verify the expression profiles obtained from microarray data, transcripts of five MtWAK-RLKs (MtWAK1, 3, 10, 24, and 53) in different tissues, including root, stem, leaf, flower, pod and seed, were detected using qRT-PCR. The data showed the expression patterns in different tissues were in consistence with the microarray data. For example, MtWAK10, 24 and 53 were highly expressed in roots ( Figure 9A). The response of transcript levels in five MtWAK-RLKs (MtWAK3, 8, 10, 21, and 24) were also detected using qRT-PCR. MtWAK3, 8 and 21 were up-regulated after cold treatment, while MtWAK10 was down-regulated ( Figure 9B), which was consistent with the microarray data.

Gene Expression Validation of MtWAK-RLKs by qRT-PCR
To verify the expression profiles obtained from microarray data, transcripts of five MtWAK-RLKs (MtWAK1, 3, 10, 24, and 53) in different tissues, including root, stem, leaf, flower, pod and seed, were detected using qRT-PCR. The data showed the expression patterns in different tissues were in consistence with the microarray data. For example, MtWAK10, 24 and 53 were highly expressed in roots ( Figure 9A). The response of transcript levels in five MtWAK-RLKs (MtWAK3, 8, 10, 21, and 24) were also detected using qRT-PCR. MtWAK3, 8 and 21 were up-regulated after cold treatment, while MtWAK10 was down-regulated ( Figure 9B), which was consistent with the microarray data.

Plasma Membrane Localized MtWAK24 Inhibited Phytophthora Infection in Tobacco
To explore the functions of some candidate MtWAKs in modulating plant immunity, we performed P. parasitica infection experiments in N. benthamiana. The lesion diameter, as well as relative biomass of P. parasitica, in leaves expressing MtWAK24, but not MtWAK36, was significantly decreased compared to the control leaves expressing empty vector, indicat-

Plasma Membrane Localized MtWAK24 Inhibited Phytophthora Infection in Tobacco
To explore the functions of some candidate MtWAKs in modulating plant immunity, we performed P. parasitica infection experiments in N. benthamiana. The lesion diameter, as well as relative biomass of P. parasitica, in leaves expressing MtWAK24, but not MtWAK36, was significantly decreased compared to the control leaves expressing empty vector, indicating that MtWAK24 could inhibit the pathogen infection by P. parasitica ( Figure 10A,B). stress using qRT-PCR. (A) Different tissues. (B) Cold stress. The error bars were obtained from three measurements. nd indicates not detected. Significant differences are indicated as different lowercase letters (P ≤ 0.05, by one-way ANOVA).

Plasma Membrane Localized MtWAK24 Inhibited Phytophthora Infection in Tobacco
To explore the functions of some candidate MtWAKs in modulating plant immunity, we performed P. parasitica infection experiments in N. benthamiana. The lesion diameter, as well as relative biomass of P. parasitica, in leaves expressing MtWAK24, but not MtWAK36, was significantly decreased compared to the control leaves expressing empty vector, indicating that MtWAK24 could inhibit the pathogen infection by P. parasitica ( Figure 10A,B). Subcellular localization of MtWAK24 was further analyzed. The data showed that GFP protein was located in the cytoplasm and nucleus, while MtWAK24 protein was located in plasma membrane because the fluorescence was overlapped with that of AtAKT1, the plasma membrane marker protein ( Figure 10C).  N. benthamiana leaves individually expressing 35S::MtWAK36, 35S::MtWAK24 or 35S::GFP were inoculated with P. parasitica mycelial plugs at 24 h after Agrobacterium infiltration. At 48 hpi, infected leaves were stained using Evans blue staining for lesion determination (A). Relative biomass of P. parasitica was determined by qPCR of P. parasitica genome DNA normalized to tobacco genome DNA (B). The results are indicated with means ± SE, n = 6. Asterisks indicate significant differences (* p < 0.05; Student's t test). The subcellular localization of MtWAK24-GFP proteins and a free GFP protein in tobacco (C). P. M-marker: a plasma membrane localization protein AtAKT1. Bars = 20 µm. Subcellular localization of MtWAK24 was further analyzed. The data showed that GFP protein was located in the cytoplasm and nucleus, while MtWAK24 protein was located in plasma membrane because the fluorescence was overlapped with that of AtAKT1, the plasma membrane marker protein ( Figure 10C).
Multiple cis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions of MtWAK-RLKs, indicating their potential roles in these processes. The GhWAKs/WAKLs having the above cis-elements in the promoter were responsive to multiple phytohormones and abiotic stresses [12]. Transcript levels of five TaWAKs in wheat (Triticum aestivum) were altered by treatments with GA, BR, IAA, JA and ABA [29]. Gene function is associated with its tissue specific expression pattern. Mt-WAK10, 24 and 50 showed a root-specific expression with extremely low expression in other organs. MtWAK36 and MtWAK45 were also mainly expressed in roots, whereas MtWAK3 and MtWAK53 were highly expressed in stem and petiole, respectively. MtWAK1, 4, 7, 8 and 18 were evenly expressed in each organ, but the transcript levels were lower than that of other genes ( Figure 6A, Table S3). Diverse tissue expression patterns of MtWAK-RLKs implied that they might function broadly in plant tissues.
Plant cell expansion and elongation depend on turgor maintenance and cell wall modification, which is associated with the rigidity and elasticity of the cell wall [30]. WAK genes can monitor pectin and participate in both turgor maintenance and cell wall modification [31]. AtWAK4 and AtWAK2 positively regulate cell expansion [3,4], HvWAK1 positively regulates root growth [32], while OsWAK11 regulates both stem and seed elongation [5]. Transcript levels of MtWAK24 and MtWAK53 were increased gradually from bottom to top internodes and along with seed growth, while expressions of MtWAK3 and MtWAK45 increased, followed by a decrease from internode 1 to internode 8 and along with seed development ( Figure 6B,C, Table S3), indicating that they are probably involved in the regulation of cell elongation.
Cell wall modification is involved in symbiosis between rhizobia and legume plants. Passing of an infection thread from cell to cell requires local cell wall degradation [33]. Modifications in the localization of high-and low-methylated homogalacturonans were detected in nodules [34]. The majority of MtWAK-RLKs were greatly down-regulated in the process of nodulation, while MtWAK1 transcript was increased dramatically, implying that MtWAKs are probably associated with nodulation in leguminous plants ( Figure 6D, Table S3).
WAK genes are involved in metal, salt, drought and cold tolerance, although the mechanisms remain unknown. OsWAK112 negatively regulates plant salt tolerance, possibly via direct binding with OsSAMS1/2/3 [17]. The Slwak1 null mutant exhibited disturbed osmotic homeostasis and source-sink balance under long term salinity and thereby reduced fruit yield [46]. The CpGRP1-CpWAK1 complex regulates dehydration-induced morphological changes in Craterostigma plantagineum [47]. Twelve MtWAK-RLK genes were quickly responsive to drought, salt and cold (Figure 8). Ten MtWAK-RLKs transcripts were induced after 2 h of drought ( Figure 8A). Seven and four MtWAK-RLKs were up-regulated or down-regulated by cold stress, respectively ( Figure 8B), and three and four MtWAK-RLKs were up-regulated or down-regulated by salt stress, respectively ( Figure 8C). The results indicate that MtWAK expression is probably involved in abiotic stress resistance.

Identification of MtWAK Genes
The genome sequences were obtained from M. truncatula genome database (http:// www.medicagogenome.org/, Mt4.0v2). The gene information of WAK family in Arabidopsis and Rice were retrieved from the previous studies. The hidden Markov model (HMM) profiles of the WAKs were down-loaded from the Pfam database (http://pfam.xfam.org/, accessed on 1 August 2020). EGF_CA(PF07645), WAK_assoc (PF14380), GUB_WAK_bind (PF13947) and Pkinase_Tyr (PF07714) were used to identify MtWAKs. Firstly, BLASTP search was performed at M. truncatula genome database with an e-value of 1e-5 using previously reported sequences of AtWAKs and OsWAKs as query. Then, we searched EGF_CA(PF07645), WAK_assoc (PF14380), GUB_WAK_bind (PF13947) and Pkinase_Tyr (PF07714) domain from putative MtWAKs with e-value cut-off at 1.0 by using HMMER v3.1b2 software. The integrity of the four domains was verified by using the online program SMART (http://smart.emblheidelberg.de/, accessed on 1 August 2020) with an e-value < 0.1. Lastly, each candidate gene was assessed for its sequence similarity to other putative MtWAKs. Only genes that fit into one of the four MtWAK types (see Table S1) according to Zhang et al. [18] were defined as MtWAKs. Protein length, molecular weight (MW) and isoelectric point (PI) were predicted by ExPasy program (http://www.expasy. org/tools/, accessed on 10 August 2021).

Chromosomal Location and Synteny Correlation Analysis
The physical position of the MtWAK genes on the chromosome was mapped using TBtools software (Version 1.108). The synteny correlation analysis of WAK genes between the homologs in M. truncatula and A. thaliana or G. max were verified and visualized using TBtools software.

Phylogenetic Analysis and Gene Structure, Motif and Conserved Domain Analysis
Multiple alignments of protein sequences were performed using CLUSTALX software (Version 1.81). The phylogenetic tree was constructed by using MEGAX with the neighborjoining method and 1000 bootstrap replications. The gene structure, conserved domain and conserved motifs were displayed using TBtools software. The conserved domains and conserved motifs of the 26 MtWAK-RLKs were analyzed by SMART program and MEME program (Version 5.4.1) (http://meme-suite.org/tools/meme, accessed on 20 July 2022).

Analysis of cis-Acting Regulatory Elements
The 2000 bp promoter sequences upstream from the initiation codon of MtWAK-RLKs were extracted from the genome of M. truncatula and analyzed using PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 August 2020). However, less than 300 bp promoter sequence of MtWAK10 was available.

Expression Pattern Analysis
The genome-wide microarray data obtained from MtGEA (https://mtgea.noble.org/ v3/, accessed on 1 August 2020) was used to analyze the expression of MtWAK genes in different tissues and developing stages. The expression data were gene-wise normalized. The clustered heatmap of expression pattern profile on log2 scale was portrayed using TBtools software. Analyses of WAK expression in response to biotic and abiotic stress were conducted on datasets: (i) 'Cell suspension_Yeast elicitor', 'Root_Macrophomina infected' and 'Root Tip_A17_Ralstonia' (microarray data obtained from MtGEA); (ii) powdery mildew Erysiphe pisi treatment [48] RNA-seq data were retrieved from NCBI Database (SRR7589436, SRR7589435, SRR7589438, and SRR7589437); (iii) drought, salt and cold treatment, date from NCBI GEO (dataset accession: GSE136739). The expression abundance was presented by the reads per kilobase per million (Table S3). The relative transcript level after treatments was calculated compared with the untreated control or before treatment (0 h). The clustered heatmap of relative expression pattern on log2 scale was analyzed by the TBtools.

Tissue Samples Collection and Cold and Salt Treatment
M. truncatula plants were grown in a growth chamber at 25 • C with 16 h of light. Root, stem, mature leave, flower, pod and seed were sampled from three-month-old plants.
Six-week-old plants were exposed to low temperature (5 • C) as a cold stress treatment. Leaves were collected at time intervals of 0, 1, 2, 6 and 12 h.

RNA Extraction and qRT-PCR
Total RNA was extracted using the RNAprep pure Plant Kit (Tiangen, Beijing, China). cDNA synthesis was performed with HiScript III RT SuperMix for qPCR (+gDNA wiper) reagent kit with gDNA Eraser (Vazyme, Nanjing, China). qRT-PCR was performed following the instructions of ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The MtActin7 (Medtr3g095530) gene was used as the controls. All primer sequences are shown in Supplementary Table S5. The relative gene expression level was calculated with 2 −∆Ct method.

Subcellular Localization
The full CDSs of MtWAK24 without the terminator were cloned and ligated to pCAMBIA 1305-GFP vector driven by CaMV 35S promoter. Primer sequences for constructions are shown in Supplementary Table S5. Positive Agrobacterium colonies were cultured in LB medium containing 50 µg/mL rifampicin and kanamycin at 28 • C overnight; the cells were harvested by centrifugation at 2500 × g at room temperature for 3-5 min and resuspended in infiltration buffer (10 mM MgCl 2 , 10 mM MES pH 5.6, and 100-200 µM acetosyringone). The cell density was adjusted to OD 600 = 0.2 before the cell suspensions were infiltrated into one-month-old Nicotiana benthamiana (N. benthamiana) leaves. After 48-72 h, fluorescence was observed using confocal laser scanning microscopy (Zeiss LSM800, Jena, Germany).

Phytophthora Infection in N. benthamiana
Vectors expressing selected MtWAK-RLKs under control of CaMV 35S promoter were constructed. The infection experiments were performed as described [49]. Briefly, the infiltrated leaves were inoculated with P. parasitica mycelium at 12 hpi, and leaf lesions were determined using the Evans blue method at 48 hpi. P. parasitica strain was obtained from Yuanchao Wang's lab (Nanjing Agricultural University, Nanjing, China). Relative biomass of P. parasitica was determined by qPCR of P. parasitica genome DNA normalized to tobacco genome DNA at 48 hpi. All primer sequences are shown in Supplementary Table S5.

Conclusions
In summary, a comprehensive genome-wide analysis of WAK family was performed. A total of 54 MtWAKs were identified in M. truncatula, including 26 MtWAK-RLK, 9 MtWAK-RLCK, 10 MtWAK-RLP and 9 short protein type genes. MtRLKs and MtRLCKs were largely tandem duplicated. Most MtWAK-RLKs had similar exon-intron organization and motif distribution. Multiple cis-acting elements responsible for phytohormones, stresses, growth and development were observed in the promoter regions. The expression patterns of MtWAK-RLKs varied in different plant tissues and developmental stages and biotic and abiotic stress conditions. The results suggest that MtWAKs might have multiple functions in M. truncatula. Furthermore, plasma-membrane-localized MtWAK24 significantly inhibited Phytophthora infection in tobacco, indicating its role in pathogen resistance, which is worthy to be investigated in the future.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12091849/s1, Figure S1: The motif of MtWAK-RLKs; Table S1: The predicated information of MtWAK family genes in M. truncatula. Table S2: The synteny analysis of WAK homologs between M. truncatula and A. thaliana or G. max; Table S3: The gene expression profiles of MtWAK-RLKs in different tissues and developmental stages as well as biotic and abiotic stress; Table S4: The number of plant protein kinases and WAKs predicted via iTAK program; Table S5: Primers.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.