Genome-wide identification of Brassinosteroid insensitive 1-associated receptor kinase 1 genes and expression analysis in response to pathogen infection in cucumber (Cucumis sativus L.)

Abstract

Background

BAK1 (Brassinosteroid insensitive 1-associated receptor kinase 1) plays an important role in disease resistance in plants. However, the function of BAK1 family in cucumber and the decisive genes for disease-resistance remain elusive.

Results

Here, we identified 27 CsBAK1s in cucumber, and classified them into five subgroups based on phylogenetic analysis and gene structure. CsBAK1s in the same subgroup shared the similar motifs, but different gene structures. Cis-elements analysis revealed that CsBAK1s might respond to various stress and growth regulation. Three segmentally duplicated pairwise genes were identified in cucumber. In addition, Ka/Ks analysis indicated that CsBAK1s were under positive selection during evolution. Tissue expression profile showed that most CsBAK1s in Subgroup II and IV showed constitutive expression, members in other subgroups showed tissue-specific expression. To further explore whether CsBAK1s were involved in the resistance to pathogens, the expression patterns of CsBAK1s to five pathogens (gummy stem blight, powdery mildew, downy mildew, grey mildew, and fusarium wilt) reveled that different CsBAK1s had specific roles in different pathogen infections. The expression of CsBAK1-14 was induced/repressed significantly by five pathogens, CsBAK1-14 might play an important role in disease resistance in cucumber.

Conclusions

27 BAK1 genes were identified in cucumber from a full perspective, which have important functions in pathogen infection. Our study provided a theoretical basis to further clarify the function of BAK1s to disease resistance in cucumber.

Introduction

BAK1 (Brassinosteroid insensitive 1-associated receptor kinase 1), also known as SERK3 (Somatic embryogenesis receptor-like kinase 3), involves in plant growth and development via brassinosteroid (BR)-mediated signaling [1,2,3]. BAK1 is a co-receptor of brassinosteroid-insensitive1 (BRI1) that senses and transmits BR signal, and is necessary for BR signal transduction to response to various external environment signals [3, 4]. Moreover, as one of the major components both in pattern recognition receptor (PRR)-triggered immunity (PTI) and effector-triggered immunity (ETI), BAK1 also play a major role in plant innate immunity [5]. As a LRRs-RLK (Leucine-rich repeats receptor-like kinase) and co-receptor for different LRR-type PRRs (Pattern recognition receptors), BAK1 forms a receptor complex with multiple PRRs and involved in multiple PTI signaling pathways to regulate plant immunity [5]. BAK1 interacts with FLS2/EFR to mediate plant innate immune response [6, 7]. BAK1 could be involved in a BR-independent cell-death pathway negatively by interacting with BAK1-interacting receptor-like kinase 2 (BIR2) [8,9,10,11]. In addition, BAK1 participates in nucleotide-binding domain and leucine-rich repeat (NLR)-mediated effective defense responses as a negative regulator and is required for ETI [12]. ETI response often induce hypersensitive response (HR) and cause cell death in plants [13]. BAK1 participates in NLR-mediated effective defense responses as a negative regulator. BAK1 regulates ETI activation as a target for many effectors and acts upstream of certain NLRs to inhibit R gene expression [14]. In turn, NLRs could guard BAK1 and other SERKs, and when BAK1-mediated PTI pathway is countered by pathogen, NLRs are activated and initiate autoimmune response by ETI response.

BAK1 belongs to SERK subfamily widely distributed in plants [5], and regulates cell death with SERK4 [14]. In Arabidopsis, 5 SERKs were reported [15], but only one BAK1 gene, AT4G33430, was identified as a specific interactor for BRI1 [16], and has been reported to play a positive role in plant immunity [17]. BAK1 interacted with an atypical LRR-RLK BIR2 to negatively regulate PAMP-triggered immunity [10]. The depletion of BAK1 in Arabidopsis showed significant cell death and ADR1 (Activated disease resistance 1) acted as a sensor for NLRs to trigger ETI [18]. In rice, 2 SERKs were found [19] and the homolog of Arabidopsis thaliana BAK1 gene, OsBAK1, positively regulate resistance to rice blast fungus, Magnaporthe grisea [20] and Magnaporthe oryzae [21], and bacterium pathogen Xanthomonas oryzae pv. oryzae PXO99 [22]. Cotton has two BAK1 orthologs, and GhBAK1 mediates resistance to Verticillium wilt. Silencing GhBAK1 by Virus‐induced gene silencing showed Verticillium wilt susceptibility, induced cell death and ROS production [23]. In Grapevine, heterologous VqSERK3/BAK1 expression in Arabidopsis bak1-4 mutant lines showed increased powdery mildew resistance, reduced cell death, and strengthened stomatal immunity [24]. In wheat, 12 SERKs showed different roles in plant growth and development [25]. 9 SERKs in Medicago truncatula and 17 in Glycine max were identified [26]. However, BAK1 and it’s the closest paralog BAK1-LIKE 1 (BKK1) usually play a redundant role in plant immunity and BR signaling pathways [27,28,29]. These findings suggest that BAK1 plays an important role in plants disease resistance.

Cucumber (Cucumis sativus L.) has an important economic value and is main vegetable produce worldwide. However, various diseases cause severe quality damage and yield decline in cucurbits production, practically gummy stem blight (GSB), powdery mildew (PM), downy mildew (DM), grey mildew (GM), fusarium wilt (FW) and others. Therefore, breeding and development of cultivar with broad-spectrum resistance (BSR) is the most important strategy to cucumber production. However, the function of BAK1 in cucumber for disease-resistance remain elusive. In this study, we identified a total of 27 BAK1 genes in cucumber, and divided them into five subgroups. Phylogenetic, gene structures, motif compositions, synteny, gene duplication and cis-elements were analyzed. In addition, the expression patterns of CsBAK1s between susceptible and resistant lines were analyzed after inoculation with five different pathogens and speculated the potential functions of CsBAK1 gene family for pathogens infection. This study provided a theoretical foundation to further clarify the function of BAK1s to disease resistance in cucumber.

Results

Identification and characterization of cucumber BAK1 gene member

We identified a total of twenty-seven BAK1 genes in cucumber by an HMM model for Pkinase domain (PF00069) and BLASTP (Additional file 2: Table S1). All CsBAK1s were mapped onto seven chromosomes (Fig. 1), and named CsBAK1-1 to CsBAK1-27 based on their position on the Cucumis sativus genome (Chinese Long v3) (http://cucurbitgenomics.org/ftp/genome/cucumber/Chinese_long/). As shown in Fig. 1, twenty-seven CsBAK1s were distributed on all seven chromosomes, of which Chr.1 had four members, Chr.2, 3, and 4 each had two members, Chr.5 and 6 each had three members, and Chr.7 had ten members. The twenty-seven CsBAK1 protein sequence lengths ranged from 992 (CsBAK1-5) to 156 (CsBAK1-27) amino acids (aa), the molecular weights (MVs) ranged from 17,422.90 Da (CsBAK1-27) to 112,297.85 Da (CsBAK1-4), and isoelectric points (pI) varied from 4.81 (CsBAK1-12) to 9.23 (CsBAK1-17) (Additional file 1: Table S1). Subcellular characterization predicted cell membrane localization for ten proteins, nucleus localization for seven proteins, cell wall localization for six proteins, cytoplasm localization for three proteins, and cell membrane/nucleus localization for one protein. 11 of 27 CsBAK1s were characterized as unstable and most of CsBAK1s were hydrophilic proteins. All the data collected suggested a high variability among the CsBAK1s. The present study provided a comprehensive analysis of candidate CsBAK1s.

Fig. 1

Chromosomal location of CsBAK1 genes in cucumber. Blue bars represent the chromosomes. Chromosome numbers are shown at the top of the bar. CsBAK1 genes are labeled at the right of the chromosomes

Phylogenetic analysis and classification of CsBAK1s

In order to thoroughly investigate the BAK1 in plants, 83 BAK1s from 54 different species with 27 CsBAK1s protein sequences were used to conduct a neighbor-joining phylogenetic tree (Additional file 2: Table S2). As shown in Fig. 2, all BAK1s were divided into four groups. Most of CsBAK1 members were classified into Group II and clustered with BAK1s from Oryza sativa and Arabidopsis. Interestingly, only CsBAK1-3 was classified into Group II, and closely related to Cucurbitaceae and other dicotyledonous species, such as Glycine max, Citrus clementina, and Medicago truncatula, etc. Most of CsBAK1s were clustered with other species in Group I other than Cucurbitaceae, which implied a relatively high synteny between Group I across various species, and Group II in Cucurbitaceae might occurred genetic divergence.

Fig. 2

Phylogenetic analysis of BAK1 proteins from different species. BAK1 proteins were firstly aligned using the ClustalW, and the phylogenetic tree was then constructed using iTOL. A total of 1,000 bootstrap replications were applied. Red letters represented CsBAK1s in cucumber

CsBAK1 structure and motif composition

In order to reveal the evolution of cucumber BAK1 family precisely, 27 CsBAK1s were further classified into five subgroups based on conserved amino acid sequence (Fig. 3a). The exon–intron structure of CsBAK1s showed that the number of exons ranged from 2 to 16. The same subgroup didn’t show similar gene structure, and exhibited a diversity of exon/intron structure (Fig. 3b).

Fig. 3

Gene structure and conserved motif analysis of CsBAK1 gene family. a Phylogenetic analysis of CsBAK1s. b Gene structure of CsBAK1s. Green boxes, yellow boxes and gray lines represent untranslated regions, exons and introns respectively. c Motif composition of CsBAK1s. Different motifs are shown in different colors, and their detailed information is provided in Table S4

Motif analysis identified ten conserved motifs of CsBAK1 proteins (Fig. 3c and Additional file 4: Table S4). Eight CsBAK1s in Subgroup II contained one specific Motif 2. CsBAK1 proteins in Subgroup III had longer amino acid sequences, but less motifs than those in the other subgroups. They only contained two motifs, Motif 1 and Motif 9. CsBAK1s in Subgroup IV had the most variable number of motifs. CsBAK1-24 and CsBAK1-17 had one motif (Motif 5), CsBAK1-10 and CsBAK1-27 had two motifs, CsBAK1-23 had three motifs, CsBAK1-2 had four motifs, and CsBAK1-21 had five motifs. All CsBAK1s in Subgroup V had Motif 8, five of six CsBAK1s had Motif 1 and Motif 3, except CsBAK1-14. CsBAK1s in the same subgroup shared the similar motif composition, indicating that members in the same subgroup might have similar biological functions.

Synteny and gene duplication analysis of CsBAK1s

To further invested the evolution and extension of CsBAK1 gene family, genome-wide synteny analysis identified three segmentally duplicated pairwise genes, including CsBAK1-8/CsBAK1-20, CsBAK1-14/CsBAK1-25, and CsBAK1-17/CsBAK1-24 (Fig. 4a). Among these gene pairs, CsBAK1-8/CsBAK1-20 and CsBAK1-17/CsBAK1-24 were clustered together and had similar gene structures. These results indicated that CsBAK1s duplication mainly result from segmental duplication other than tandem duplication. Five comparative syntenic maps of CsBAK1s with five species were used to investigate of syntenic blocks, including two monocots (Oryza sativa and Zea mays) and three dicots (Arabidopsis thaliana, Glycine max, and Medicago truncatula) (Fig. 4b). The orthologous pairs between two monocots (Oryza sativa and Zea mays) were 6 and 3, between three dicots (Arabidopsis thaliana, Glycine max, and Medicago truncatula) were 9, 43, and 18, which showed that dicots may have more syntenic conservation than monocots. In addition, Ka/Ks analysis of the three orthologous gene pairs showed that the values of all three gene pairs were less than 1 (Additional file 5: Table S5), and indicated that CsBAK1s were under positive selection during evolution.

Fig. 4

Duplication and collinearity analysis of BAK1s. a Duplications of BAK1s in cucumber. Segmental duplication genes are connected with red lines. b Synteny analysis of BAK1s between Cucumis sativus and Arabidopsis thaliana, Glycine max, Medicago truncatula, Oryza sativa, and Zea mays

Identification of cis-regulatory elements in the promoters of CsBAK1s

Cis-regulatory elements (CREs) at promoter (2,000 bp regulatory region upstream of the ATG) were used to detect the potential regulatory mechanism of CsBAK1s (Table S6). Phytohormone-responsive (129), abiotic and biotic stress (105), and growth development (6) elements were identified on the promoter regions of CsBAK1s (Fig. 5 and Additional file 6: Table S6). Phytohormone responsive elements mainly included abscisic acid (45), gibberellin (28), auxin (10), salicylic acid (13) and methyl jasmonate (MeJA) (33). Among stress-related elements, defense and stress (16), wound (3), anoxic (68), drought (11) and low temperature (7) were identified. Furthermore, we also detected growth development elements, such as circadian response (6). These results showed that CsBAK1s might be regulated by various cis-regulatory elements in the promoters during stress responses.

Fig. 5

Predicted cis-elements in the promoter regions of CsBAK1s. Different colors represent different cis-elements. The bottom scale bar indicates the length of promoter

Expression patterns in different tissues of CsBAK1s

RNA-seq data of 26 CsBAK1 genes based on the public database (PRJNA80169) was used to analyze tissue-specific expression patterns in different tissues of Chinese Long (Root, stem, leaf, female flower, male flower, ovary, and tendril), except CsBAK1-27 had no data. Four CsBAK1s in Subgroup II (CsBAK1-1, CsBAK1-11, CsBAK1-13, and CsBAK1-26) and two genes in Subgroup IV (CsBAK1-2 and CsBAK1-10) had significantly higher expressions in all seven tissues (Fig. 6 and Additional file 7: Table S7), other members in Subgroup II (except CsBAK1-12) and Subgroup IV (except CsBAK1-21) showed relatively higher expressions in all seven tissues than that in other subgroups, which indicated that these CsBAK1s in Subgroup II and IV might have vital roles in cucumber growth development. CsBAK1s in Subgroup I, Subgroup III, and Subgroup V had relatively higher expressions in some specific tissues. CsBAK1-3 in Subgroup I had relatively higher expressions in leaf, root, ovary and stem, except for female and male flowers. CsBAK1-15 in Subgroup III had higher expressions in ovary and female, CsBAK1-8 and CsBAK1-14 in Subgroup V had relatively higher expressions in root, which suggested that these members had important regulatory functions in specific tissues.

Fig. 6

Expression profiles of CsBAK1s in different tissues. The expression levels of CsBAK1s gene family were displayed based on log₂RPKM values

Expression analysis of CsBAK1s in response to different pathogens

To thoroughly understand the possible functions of CsBAK1s in response to cucumber diseases, five pathogens (GSB, PM, DM, GM, and FW) were used to analyze expression patterns of CsBAK1s. Most of CsBAK1s showed significantly different expression patterns to response to different pathogens infection (Fig. 7 and Additional file 8: Table S8).

Fig. 7

Expression profiles of CsBAK1s after inoculation with different pathogens. GSB: gummy stem blight; PM: powdery mildew; DM: downy mildew; GM: grey mildew; FW: fusarium wilt. LM90: GSB susceptible line; LM99: GSB resistant line; D8: PM susceptible recurrent parent line; SSL508-28: PM resistant segment substitution line; Vlas: DM susceptible line; PI88: DM resistant line. 9930: GM and FW susceptible line; 9110Gt: GM and FW resistant line. Numerical values indicated expression level of log₂RPKM values for GSB, PM, and DM, and the relative expression level for GM and FW by qRT-PCR. Normalized z-score values were used to generate color-coded heatmap. CsBAK1-27 had no expression in any situation

LM99 showed significant GSB-resistance than LM90 after inoculation GSB pathogen in seedling at 12 hpi, therefore, transcriptome sequencing was conducted at 0 and 12 hpi. A total of 156.08 Gb Clean Data was obtained. After quality control, high-quality reads (Q30 > 92.40%) were used to align with Cucumber (Chinese Long) v3 Genome, and alignment efficiency was from 87.81% to 95.89%. Most of CsBAK1s were downregulated significantly (Log₂|(-Foldchange)|> 1) in resistant line (LM99) after inoculation GSB, only four CsBAK1s (CsBAK1-12, CsBAK1-14, CsBAK1-21, CsBAK1-22) were upregulated in resistant line. Four CsBAK1s (CsBAK1-5, CsBAK1-12, CsBAK1-14, CsBAK1-15) were up-regulated, and six CsBAK1s (CsBAK1-7, CsBAK1-8, CsBAK1-16, CsBAK1-18, CsBAK1-20, and CsBAK1-21) were down-regulated in susceptible line LM90. Four genes (CsBAK1-8, CsBAK1-16, CsBAK1-18, and CsBAK1-20) were down-regulated both in LM99 and LM90, two CsBAK1s (CsBAK1-12 and CsBAK1-14) were up-regulated both in LM99 and LM90. Ten CsBAK1s (CsBAK1-1, CsBAK1-2, CsBAK1-3, CsBAK1-4, CsBAK1-10, CsBAK1-11, CsBAK1-13, CsBAK1-19, CsBAK1-23, CsBAK1-24, and CsBAK1-26) and one gene (CsBAK1-22) were down- and up-regulated merely in LM99, respectively. CsBAK1-21 showed different expression pattern between two lines (Fig. 7). In order to verify the accuracy of RNA-seq data, the expression pattern of 15 genes were examined by qRT-PCR, which showed that there was significantly correlation with RNA-Seq and qRT-PCR (r² = 0.9093) (Additional file 10: Figure S1; Additional file 9: Table S9).

For PM, nine genes were up-regulated and four CsBAK1s were down-regulated in susceptible line (D8), eight and five CsBAK1s were up- and down-regulated in resistant line (SSL508-28), respectively. In particular, the expression of CsBAK1-9, CsBAK1-11 and CsBAK1-14 showed opposite patterns in two lines. For DM, eleven CsBAK1s were up-regulated and one gene was down-regulated in susceptible line (Vias), fifteen CsBAK1s were up-regulated and two CsBAK1s were down-regulated in resistant line (PI88). In addition, three CsBAK1s in Subgroup V (CsBAK1-8, CsBAK1-9, and CsBAK1-14) showed dramatically up-regulated expression level after inoculation DM both in two lines. For GM, twelve and four CsBAK1s were up- and down-regulated in susceptible line (9930) respectively, nine and four CsBAK1s were up- and down-regulated in resistant line (9110Gt) respectively. For FW, nineteen and two CsBAK1s were up- and down-regulated in susceptible line (9930) respectively, twenty-four up-regulated CsBAK1s and none down-regulated gene were detected in resistant line (9110Gt). Notably, the expression level of four CsBAK1s were significantly up-regulated both in two lines after inoculation FW. These results showed that different CsBAK1s responded to specific pathogens, and differential expression CsBAK1s between resistant and susceptible lines might have decisive roles in disease resistance. Interesting, CsBAK1-14 had significant responses to all pathogens, which was up-regulated after pathogens infection in susceptible and resistant lines, except that down-regulated in resistant line after inoculation PM.

Discussion

There are two innate immunity systems to respond to pathogen infection in plants, PTI and ETI [30]. The coreceptor-like protein kinase BAK1 forms a receptor complex with multiple pattern recognition receptors (PRRs) in multiple PTI signaling pathways [5], but also acts upstream of NLRs to inhibits R gene expression in ETI [14]. The members of BAK1 genes have been identified in Arabidopsis [10, 16,17,18], Oryza sativa [20,21,22], Gossypium [23], and Vitis vinifera [24], and so on. However, the systematic identification of BAK1 gene members in cucumber has not been reported. In this study, we conducted comprehensive genome wide analyses of BAK1s in cucumber, and 27 CsBAK1s were identified in our study (Fig. 1 and Additional file 1: Table S1).

Phylogenetic analysis showed that 27 CsBAK1s were classified into two groups (Fig. 2). 26 of 27 CsBAK1s were clustered into Group I, and only CsBAK1-3 was classed into Group II. However, the potential functions of BAK1s in Group II are unclear, and the function of CsBAK1-3 still needs to be clarified by further works. Genome-wide synteny analysis of CsBAK1s showed that the number of BAK1s in G. max was 43, in M. truncatula was 18, in Arabidopsis was 9, in O. sativa was 6, and in Z. mays was 3 (Fig. 4). More BAK1s members in dicots than monocots suggested the more extensive duplication events in dicots. 27 CsBAK1s were distributed into all chromosomes, and Chr.7 carried more CsBAK1s than other chromosomes. A possible explanation was that the same chromosomal segmental duplication caused the clustering of CsBAK1s in Chr.7 [31], and a comprehensive analysis for the whole cucumber genome needs to be conducted to verify this hypothesis. 27 CsBAK1s were further divided into five subgroups, and CsBAK1 genes within the same subgroup showed similar gene structures and motifs (Fig. 3), which speculated that CsBAK1s might be functional differentiation during evolution and duplication and the same subgroup members might have similar functions. The 8 CsBAK1s in Subgroup II were the most homologous to AtBAK1 (ATSERK3) involved in pathogen resistance through regulation of PTI signaling pathway and NLR-dependent cell death [5, 27, 32], which indicated that CsBAK1 members in Subgroup II might involve in BAK1-mediated immune signaling in cucumber.

The interaction between plants and pathogens were mediated by complex mechanisms, and the autoimmunity to pathogen infection in cucumber cannot be regulated by a single gene/pathway, but the synergy of many genes. Cis-elements identified phytohormone responsive elements, stress-related elements, and growth development elements in the promoter regions of CsBAK1s (Fig. 5 and Additional file 7: Table S6), which revealed that CsBAK1s might be regulated by various stress responses and growth regulation. There were a relatively high occurrence of defense and stress responsive elements in the promoters of Subgroup III (4 of 5), indicating that CsBAK1s in Subgroup III were more likely to be involved in the defense response, except for CsBAK1-4. Moreover, the expression patterns after different pathogens infection showed that CsBAK1-25 in Subgroup III almost had no response to four pathogens (GSB, PM, DM, and GM), but had significant response to FW (Fig. 7). This unique expression pattern gave us a potential information that different cis-elements in the promoters of CsBAK1s responded to different pathogen infections.

Tissue expression patterns often reflect the gene functions [33]. Expression profile of CsBAK1s showed that most of members in Subgroup II and IV had constitutively expression in cucumber, and others in Subgroup I, Subgroup III, and Subgroup IV were dominantly expressed in specific tissues, which probably due to functional divergences among different subgroups. Tissue specific expressions of CsBAK1s might have important roles in growth development. CsBAK1 members in different subgroups had preferential expressions to specific tissues, and different members in the same subgroup had similar expression patterns, which probably due to functional divergences among different subgroups. Some members had no expressions or very low expressions, that may due to functional redundancy (Fig. 6 and Additional file 7: Table S7).

BAK1 could participate in PTI pathway by recognizing PRRs as a coreceptor for many PAMPs, but also regulates ETI activation as a target for many effectors [13]. To further detect the response to different pathogens for CsBAK1s, we analyzed expression patterns after GSB, PM, DM, GM, and FW infection. The expression levels of most CsBAK1s were changed after pathogen infection, indicating that CsBAK1 gene family had a non-negligible role in cucumber disease resistance. Notably, the expression patterns of CsBAK1s were discrepant in different pathogens infection, which implied that CsBAK1 gene family has various functions to response for different cucumber diseases. Previous studies showed that BAK1 involved in cell death and innate immunity by independent signaling pathways, and loss function of BAK1 did not lead to cell death [5, 34]. In this study, most of CsBAK1s were down-regulated in the resistant line after GSB infection, and CsBAK1-8 and CsBAK1-14 in Subgroup V had dramatic changes after GSB infection both in susceptible and resistant lines (Fig. 7), but the expression levels were significantly higher in susceptible line than that in resistant line, these two genes might be involved in GSB infection in cucumber. In our study, CsBAK1s were significantly down-regulated in resistant line after GSB infection, and might be involved in GSB resistance negatively. In contrast, most of CsBAK1s were up-regulated by PM, DM, GM, and FW infection. RNA-seq data showed that CsBAK1-14 had significantly opposite expression patterns between resistant and susceptible lines, up-regulated in susceptible line (D8) but down-regulated expression in resistant line (SSL508-28) after PM infection, implying CsBAK1-14 might has a vital role in PM resistant. For DM, we found that four of six genes in Subgroup V were up-regulated significantly to response for DM infection both in two lines, CsBAK1 gene family in Subgroup V might play important roles in DM infection. For GM and FW, most of CsBAK1s were up-regulated in resistant lines, which indicated that CsBAK1s might be involved in GM and FW resistance positively. Notably, CsBAK1-14 could have an important role in several pathogen infections, and the overexpression or gene-editing by CRISPR/Cas9 need to be conducted to further clarify its function. In summary, we speculated that CsBAK1 gene family was an important regulator of disease resistance in cucumber, and different CsBAK1 genes had diverse and specific functions in response to different pathogens with peculiar and complex ways.

Conclusion

In this study, we characterized and analyzed 27 BAK1 genes in cucumber from a full perspective. The expression patterns in different tissues showed that CsBAK1s might be involved in pathogen infection divergently, and play essential roles in cucumber growth development and specific organ development. Different CsBAK1s had specific roles in different pathogen infections, and some specific CsBAK1s could have important function in a particular pathogen infection. However, the biological functions and regulatory pathways of CsBAK1s in cucumber need to be further clarified. This study provided a theoretical basis to further clarify the function of CsBAK1s to different pathogen infection in cucumber.

Materials and methods

Identification of BAK1 family members in cucumber

The whole cucumber sequence was downloaded from the Cucumber (Chinese Long) v3 Genome (http://cucurbitgenomics.org/ftp/genome/cucumber/Chinese_long/). The Hidden Markov Model (HMM) of Protein kinase domain (PF00069) was download from the Pfam protein family database (http:// pfam.xfam.org/) to search BAK1 gene members with E value < 0.01 as criterion. Then, BAK1 amino acid sequences in all plants were obtained using keyword “BAK1” from NCBI (https://www.ncbi.nlm.nih.gov/protein), and a BLASTP-algorithm was conducted to search against cucumber genome. Combined with these two methods, the predicted BAK1 protein sequences were considered as cucumber BAK1 (CsBAK1) (Additional file 2: Table S1).

Sequence analysis and structural characterization

Expasy (https://web.expasy.org/protparam/) and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/#) were used to predict the physicochemical properties and subcellular localization of CsBAK1s, respectively. Gene structures of CsBAK1s were analyzed and visualized by TBtools [35]. MEME (http://meme-suite.org/tools/meme) was used to analyze the conserved motifs of CsBAK1s.

Phylogenetic analysis

The full-length amino acid sequences of CsBAK1 (Additional file 3: Table S2) derived from different plant species were used for phylogenetic analysis. Multiple-sequence alignment was performed using the ClustalW (https://www.genome.jp/tools-bin/clustalw) with the default parameters, and then phylogenetic analysis was conducted using iTOL (https://itol.embl.de/). CsBAK1s were classified into different groups according to the phylogenetic tree in other species and cucumber.

Analysis of Cis-acting element in CsBAK1 genes’ promoters

The 2,000 bp sequences upstream of CsBAK1s were extracted from the cucumber genome database and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify Cis-acting elements and transcription factor binding sites.

Chromosomal locations, gene duplications and selection pressure analysis

The chromosomal locations of CsBAK1s were illustrated with TBtools. Gene duplication analysis of BAK1 was conducted with MCScanX and Circos of Tbtools using the amino acid sequences in Cucumis sativus, Arabidopsis thaliana, Zea mays, Glycine max, and Medicago truncatula. Genomic information was available at Phytozome (https://phytozome-next.jgi.doe.gov/). The Ka/Ks ratio of duplicated CsBAK1 genes were calculated to evaluate the selection pressure using KaKs_calculator2.0 (https://sourceforge.net/projects/kakscalculator2/).

The tissues expression profile of CsBAK1s

Genome-wide transcriptional data of cucumber were obtained from Cucurbit Expression Atlas (http://cucurbitgenomics.org/rnaseq/cu/3). The expression levels of CsBAK1s in different tissues were obtained from transcriptome data PRJNA80169, including leaf, stem, female flower, male flower, ovary, root, and tendril.

Real-time PCR

Total RNAs of all samples were extracted using the RNA simple Total RNA Kit (TIANGEN, Beijing, China) following instruction and then the quality was determined by 1% agarose gel electrophoresis. For reverse transcription, the first-strand cDNA was synthesized using the TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen, China). For qRT-PCR, gene expressions were examined using cDNA templates by SYBR green super mix and ABI 7900 machine (Bio-Rad, Hercules, CA, USA). Gene-specific primers for the candidate genes were designed using Primer3.0 (https://primer3.ut.ee/). The expression level of CsActin (CsaV3_2G018090) was set as control for normalization and the relative gene expression was calculated followed the 2^−ΔΔCt method [36], and three biological replicates with three technical replicates were used for each gene. All primers used in this study were listed in Additional file 4: Table S3. Heatmap in R package was used to visualize the difference of gene expression.

Expression patterns of CsBAK1s in response to different pathogens

To thoroughly understand the functions of CsBAK1s to major diseases in cucumber, we analyzed the expression patterns of CsBAK1s after inoculation different pathogens, including gummy stem blight (GSB), powdery mildew (PM), and downy mildew (DM), grey mildew (GM), and fusarium wilt (FW).

For GSB, the GSB-resistant cucumber accession ‘PI 183967’ (Cucumis sativus var. hardiwickii (Royle) Alef.) and the GSB-susceptible accession ‘931’ (Cucumis sativus var. sativus), and their 160 recombinant inbred lines were used to analyze GSB resistance. A susceptible RIL (LM90) and a resistant RIL (LM99) with similar genetic backgrounds were selected to GSB pathogen inoculation and further genome-wide transcriptome analysis in this study. LM90 and LM99 were inoculated with Didymella bryoniae. LM90 showed apparent plant diseases at 12 hpi, therefore we chose samples at 0 and 12 hpi to construct RNA-seq library by using Illumina HiSeq 3000. The sequence raw data were submitted to the National Center for Biotechnology Information (NCBI) with accession number of PRJNA1127874.

For PM and DM, the RNA-seq data based on the database was used to clarify the expression patterns of CsBAK1s. To clarify the expression patterns of CsBAK1s to PM, transcriptome data PRJNA321023 was used to analyze the expression patterns of CsBAK1s to PM in SSL508-28 (PM-resistantce) and D8 (PM-susceptibility) at 0 and 48 hpi (http://cucurbitgenomics.org/rnaseq/cu/4). The transcriptome data PRJNA285071 was used to analysis the response to DM in a susceptible (cv. Vlaspik) and resistant (PI 197088) cucumber at 0 and 24 hpi (http://cucurbitgenomics.org/rnaseq/cu/19).

For GM and FW, ‘9110 Gt’ is resistant to GM and FW, and ‘9930’ is susceptible to GM and FW. Therefore, ‘9110 Gt’ and ‘9930’ were used to analyze expression patterns of CsBAK1s for GM and FW. All plants were grown in the greenhouse at Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and were inoculated when the second true leaf was fully opened. Then, all true leaves were collected at 0, 12, 48, and 96 h post inoculation (hpi) and immediately frozen in liquid nitrogen for RNA extraction. Three plants for each sample of three replicates were collected. ‘9930’ (susceptible to GM and FW) showed visible disease symptom quickly at 12 hpi for GM, and at 48 hpi for FW, samples collected at 12 and 48 hpi were used to analyze CsBAK1s expression level for GM and FW by qRT-PCR, respectively.