- Research article
- Open Access
Comprehensive analysis of multiprotein bridging factor 1 family genes and SlMBF1c negatively regulate the resistance to Botrytis cinerea in tomato
BMC Plant Biology volume 19, Article number: 437 (2019)
Multiprotein bridging factor 1 s (MBF1s) are members of the transcriptional co-activator family that have involved in plant growth, development and stress responses. However, little is known about the Solanum lycopersicum MBF1 (SlMBF1) gene family.
In total, five SlMBF1 genes were identified based on the tomato reference genome, and these genes were mapped to five chromosomes. All of the SlMBF1 proteins were highly conserved, with a typical MBF1 domain and helix-turn-helix_3 domain. In addition, the promoter regions of the SlMBF1 genes have various stress and hormone responsive cis-regulatory elements. Encouragingly, the SlMBF1 genes were expressed with different expression profiles in different tissues and responded to various stress and hormone treatments. The biological function of SlMBF1c was further identified through its overexpression in tomato, and the transgenic tomato lines showed increased susceptibility to Botrytis cinerea (B. cinerea). Additionally, the expression patterns of salicylic acid (SA)-, jasmonic acid (JA)- and ethylene (ET)- mediated defense related genes were altered in the transgenic plants.
Our comprehensive analysis provides valuable information for clarifying the evolutionary relationship of the SlMBF1 members and their expression patterns in different tissues and under different stresses. The overexpression of SlMBF1c decreased the resistance of tomato to B. cinerea through enhancing the gene expression of the SA-mediated signaling pathway and depressing JA/ET-mediated signaling pathways. These results will facilitate future functional studies of the transcriptional co-activator family.
Transcriptional regulation is a key step in the expression of genomic information during complex biological processes in all organisms. Transcriptional co-activators are important components of gene expression that function by interacting with transcription factors and/or other regulatory elements and the basal transcription machinery . Multiprotein bridging factor 1 (MBF1) proteins are members of the transcriptional co-activator family and are highly conserved in eukaryotic organisms. MBF1 mediates the transcriptional activation of downstream genes by bridging regulatory transcription factors and TATA-box-Binding Protein . MBF1 proteins are composed of a N-terminal domain, a conservative helix-turn-helix (HTH) domain and a short C-terminus . The HTH domain is critical to maintain the functional activity of MBF1 .
Several MBF1 genes have been identified in plants and have been shown to participate in plant growth, development and stress response. For example, Arabidopsis thaliana has three MBF1 genes, and the expression levels of these genes have been found to be induced by various types of abiotic and biotic stress [2, 5, 6]. Arabidopsis plants that overexpress Arabidopsis thaliana MBF1a (AtMBF1a) show higher tolerance to salt stress and infection of pathogens, and they display a phenotype of hypersensitivity to Glucose . The overexpression of AtMBF1c could enhance the tolerance to high temperature in Arabidopsis [8, 9]. The ectopic expression of Vitis labrusca x V. vinifera MBF1 in Arabidopsis increased drought tolerance , and the ectopic expression the Triticum aestivum MBF1c improved thermotolerance in rice . However, not all of the MBF1 genes are positive regulators that can enhance tolerance to environmental stress in plants. For example, Capsicum annuum MBF1 -overexpressing Arabidopsis lines have larger leaves but display sensitivity to cold and salt stress .
The tomato is one of the most widely cultivated vegetable crops in the world and a key model plant for the study of gene function . However, the yield of tomato is seriously constrained by phytopathogens such as Botrytis cinerea (B. cinerea). Although the function of SlER24, a MBF1 family member, has been characterized and demonstrated to play an important role in tomato seed germination , the function of these genes except SlER24 were few reported. In our study, in order to explore the gene number of the SlMBF1 family in tomato, a systematic analysis was performed in tomato with the tomato genome database. A total of five SlMBF1 proteins were identified. The phylogenetic results and motif analysis showed that the SlMBF1 family was highly conserved. In addition, an analysis of promoter response elements and the expression profiling of the SlMBF1s revealed marked responses to various hormones and stresses. Moreover, we obtained transgenic lines in the tomato. The overexpression of SlMBF1c reduced the resistance of tomato to B. cinerea, suggesting SlMBF1c functions as a negative regulator in the tomato resistance to B. cinerea. Overall, the present study laid the foundation for the further study of MBF1 genes, and their potentially use for trait improvement in the tomato.
Identification and chromosomal location of SlMBF1 genes in the tomato
To identify the putative MBF1 genes in the tomato genome, we used the three Arabidopsis MBF1 protein sequences and the conserved MBF1 and HTH_3 domains as queries to search the tomato genome database using the BlastP program (Additional file 3: Table S3). A total of five putative SlMBF1 proteins were obtained with default parameters. Then, the existence of the conserved MBF1 and HTH_3 domains was confirmed by SMART and CD-Search. As described by Sanchez-Ballesta et al. , the four SlMBF1 genes were named SlMBF1a to c and SlER24, and the newly identified SlMBF1 gene was named SlMBF1d.
The molecular property analysis revealed that these SlMBF1 proteins display similar lengths (139 amino acid for SlMBF1a, SlMBF1b, SlMBF1d, and 146 amino acid for SlER24). The predicted molecular weights of the five SlMBF1 proteins ranged from 15.272 (SlMBF1b) to 16.033 (SlER24) Dalton (Da). The predicted pI values ranged from 9.95 (SlMBF1a and SlMBF1d) to 10.11 (SlER24). The gene IDs and genomic positions were summarized for these SlMBF1 proteins (Additional file 1: Table S1). By analyzing the genomic location information obtained from tomato genome database, these five SlMBF1 genes were mapped on tomato chromosome 1, 7, 9, 10, 12, respectively (Fig. 1.a).
Phylogenetic analysis, gene structure and conserved motifs of the SlMBF1 genes
The full sequences of the five SlMBF1, three AtMBF1, and two OsMBF1 proteins were used to perform protein sequence alignment and phylogenetic analysis (Fig. 1b). These MBF1 proteins were defined as members of the other corresponding plants MBF1 subgroups . Among these two subgroups, subgroup I is composed of four SlMBF1, one OsMBF1 and two AtMBF1 proteins, and subgroup II composed of one OsMBF1, one SlMBF1 and one AtMBF1 proteins. Due to evolutionary differences between these three species, subgroup I could be further divided into two groups, subgroup I-A and subgroup I-B. Among them, subgroup I-A included only four tomato SlMBF1 proteins and subgroup I-B included one OsMBF1 and two AtMBF1 proteins.
The gene structure analysis of the MBF1 family genes from the tomato, Arabidopsis and rice were conducted and the results are consistent with the phylogenetic tree analysis. As shown in Fig. 1b, the number of exons in the SlMBF1, AtMBF1 and OsMBF1 genes ranges from one to five exons. We found that the two subgroups, subgroup II and subgroups I-B, have similar intron-exon structures (Fig. 1b). The three members, OsMBF1c, SlER24 and AtMBF1c, in subgroup II contain one exon, and the members, OsMBF1a, AtMBF1a and AtMBF1b, in subgroups I-B four exons. However, in subgroups I-A, SlMBF1b and SlMBF1c contain four exons, while SlMBF1a five exons and SlMBF1d one exon (Fig. 1b).
The motif analysis of the MBF1 proteins was conducted and four distinct motifs were identified (Fig. 1b; c and Additional file 2: Table S2). Motif 2 and 3, which are MBF1 domains, and motif 1, which is an HTH_3 domain, were identified in all MBF1 proteins. Interestingly, motif 4 was only identified in the SlMBF1a and OsMBF1c proteins. Therefore, the similar motif distribution of the MBF1 proteins in these three model plants may promote to the prediction of the functions of MBF1s.
Potential cis-elements in the promoters of SlMBF1 genes
Previous studies have shown that many MBF1 genes play regulatory roles in developmental processes and tolerance to environmental stresses in plants. To predict the putative functions of the SlMBF1 genes, the 2.0-kb promoter regions of the SlMBF1 genes were isolated for the analysis of the potential cis-elements using the Plant-CARE database (Fig. 2), and many elements related to stress responsiveness and plant hormones were predicted. As shown in Fig. 2, the promoters of SlMBF1 genes contain many stress elements: drought response element, low temperature response element and defense and stress response element. Moreover, hormone responsive elements including abscisic acid (ABA) response element, gibberellin (GA) response element, jasmonate acid (MeJA) response element, salicylic acid (SA) response element and auxin response element were also discovered in the SlMBF1 promoters. These results suggest that the five SlMBF1 genes may play important roles in the response to several hormones and various stresses.
Expression pattern of the SlMBF1 genes in different tissues
To understand the potential function of the tomato SlMBF1 genes, the expression pattern of these five SlMBF1 genes were examined using qRT-PCR in different tomato organs, including the root, stem, leaf, flower and ripe fruit. As shown in Fig. 3, all of the SlMBF1 genes were detected in these five tissues. The expression of SlMBF1a, SlMBF1b and SlMBF1c were at relatively high levels in most tissues, but SlMBF1d was expressed at relatively lower levels in all tissues. SlER24 was expressed at relatively lower levels in root, stem and leaves but at relatively high levels in fruit and flower.
Expression pattern of SlMBF1 genes under different stress and different plant hormone conditions
To explore whether these five SlMBF1 genes respond to biotic and abiotic stresses in tomato, we examined the expression pattern of the SlMBF1 genes under different stress conditions, including salt, drought, low temperature, B. cinerea and wounding using qRT-PCR (Fig. 4). As expected, most of the SlMBF1 genes responded to different stress treatments. For example, SlMBF1c was induced during the late stage of all stress treatments (Fig. 4). The expression level of SlER24 was upregulated during the late stage of the salt and low temperature conditions (Fig. 4a, c). The expression level of SlMBF1a was initially downregulated then upregulated and then downregulated at the late stage under drought and B. cinerea conditions (Fig. 4b, d). Moreover, SlMBF1b displayed the same expression trend with SlMBF1a under drought conditions (Fig. 4c).
To further study how these five SlMBF1 genes respond to plant hormones in the tomato, we also examined the expression pattern of the SlMBF1 genes under different hormone treatments, including 1-amino cyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), methyl jasmonate acid (MeJA), abscisic acid (ABA), and brassinosteroids (BR) using qRT-PCR (Fig. 5). As shown in Fig. 5, most of the SlMBF1 genes responded to different hormones. For example, the expression level of SlMBF1a and SlMBF1c was initially upregulated then downregulated at late stage under ACC and MeJA conditions. In contrast, the expression level of SlMBF1a was initially induced then repressed at the late stage under ACC and MeJA conditions. Some of the SlMBF1 genes were also induced under the SA, ABA and BR conditions (Fig. 5c, d, e).
The susceptibility of SlMBF1c overexpressing lines to B. cinerea
To investigate the function of SlMBF1c in the defense response to B. cinerea, we generated 35S::SlMBF1c transgenic tomato plants (OE) by the Agrobacterium-mediated method. Using kanamycin as selection marker and genomic PCR detection, two independent and homozygous T3 transgenic lines were selected for further assays. These two OE lines display significantly higher expression levels of SlMBF1c than the WT plants (Fig. 6). Then, we examined the response of the leaves from 5-week-old OE and WT seedlings to B. cinerea infection in Petri dishes, using the method previously described by Du et al., 2017 . As shown in Fig. 7a and b, after infection with B. cinerea, the OE leaves showed significantly larger necrotic lesions compared with WT. Moreover, we also conducted the whole plant inoculation experiments. Similarly, the OE plants displayed a sensitive phenotype, compared with WT, after infection with B. cinerea (Fig. 7c, d and e). In addition, the expression level of B. cinerea Actin was significantly increased in OE plants compared with WT (Fig. 7 f). Taken all together, these results demonstrated that tomato SlMBF1c is a negative regulator in the response to B. cinerea infection.
SlMBF1c regulates the expression of defense-related genes
To explore the signaling pathways, we analyzed and compared the changes in the relative expression of SA signaling-related genes Nonexpressed Pathogenesis-Related 1 (SlNPR1) and Pathogenesis-Related genes (SlPR1a, SlPR1b and SlPR2b), JA signaling-related genes Coronatin Insensitive 1 (SlCOI1), Myelocytomatosis Oncogene 2 (SlMYC2), Proteinase Inhibitor I (SlPI I) and Leucine Aminopeptidase A1 (SlLapA1), and ET signaling-related genes Ethylene Response Factor 1 (SlERF1), Ethylene Receptor (SlNR), ACC Synthase 6 (SlACS6) and Allene Oxide Synthase 2 (SlAOS2) before and after infection with B. cinerea using qRT-PCR. As shown in Fig. 8, before infection, the transcript levels of SlNPR1, SlPR1a, SlPR2b, SlCOI1, SlPI I and SlACS6 display no significantly difference between the two OE lines and WT. However, the transcript levels of SlPR1b, SlERF1, SlNR, SlAOS2 were increased slightly and the transcript levels of SlLapA1 were decreased slightly in the OE lines. After infection with B. cinerea, the transcriptional levels of SA signaling-related genes (SlNPR1, SlPR1a, SlPR1b and SlPR2b) were elevated significantly in the two OE lines compared with WT (Fig. 8a). However, after infection with B. cinerea, the expression levels of the JA signaling-related gene (SlCOI1, SlMYC2, SlPII and SlLapA1) and the ET signaling-related genes (SlERF1, SlNR, SlACS6 and SlAOS2) were significantly decreased in the two OE lines compared with WT (Fig. 8b and c). These results indicated that the overexpression of SlMBF1c in the tomato could repress the JA/ET-mediated signaling pathways upon infection with B. cinerea.
With the genomes of more species completely sequenced, many regulatory gene families such as the MYB , bHLH  and WRKY  transcription factor families, have been identified. In addition to these transcription factor families, there are also transcriptional co-activator families such as MBF1s. Studies of MBF1 genes have mainly focused on the regulation of plant growth, development and stress responses in Arabidopsis [2, 7, 8]. Although in the year 2007, Sanchez-Ballesta et al. identified four MBF1 genes in the tomato and analyzed their structures, tissue-specific expression and response to ethylene treatment during fruit development , the tomato genome sequence completed in 2012 provides more information for the identification of this gene family . Here, five tomato MBF1 genes were identified and confirmed based on the completed tomato genome (Fig. 1a). Meanwhile, the more precise and comprehensive bioinformatics analysis (including the chromosomal location, phylogenetic analysis, gene structure, conserved motifs and cis-elements in the promoters) were performed. Notably, we found five exons in the gene structure of SlMBF1a, but Sanchez-Ballesta et al. only found four exons. Comprehensive expression levels of these genes in different tissues, responses to different stresses (salt, drought, low temperature, B. cinerea and wounding) and different plant hormone conditions (ACC, MeJA, SA, ABA and BR) were also detected (Figs. 4 and 5). More importantly, we identified the biological function of SlMBF1c which negatively regulate the tomato resistance to B. cinerea (Figs. 6,7 and 8).
In this study, five MBF1 genes were distributed on five chromosomes of tomato, respectively (Fig. 1a). Compared with three MBF1s in Arabidopsis  and two MBF1s in rice, the number of MBF1s was greater in the tomato, which means an expansion of MBF1s in tomato. A phylogenetic analysis divided these 10 MBF1 proteins into two main branches (Fig. 1b), the same as in previous description . One branch contained subgroup I-A and B, and the other contained subgroup II (Fig. 1b). This result revealed that there are two different evolutionary directions for these MBF1 proteins in tomato, Arabidopsis and rice. Importantly, subgroup I-A only includes four MBF1 proteins but did not include any Arabidopsis or rice MBF1 proteins (Fig. 1b), which means that this subgroup was lost in Arabidopsis and rice and was acquired in tomato after divergence from the last common ancestor. Moreover, the gene structure analysis showed similar intron-exon structures in subgroup I-B and subgroup II but not in subgroup I-A (Fig. 1B), suggesting that the evolutionary dynamics of intron insertion and loss occurred in subgroup I-A of the tomato MBF1 genes. Previous studies have shown that the yeast mbf1 mutant was fully/partially rescued by the MBF1 genes from human, silkworm and Arabidopsis [2, 21], which revealed that the functions of the MBF1 genes are highly conserved. In this study, the motif analysis showed that these MBF1 proteins share similar pattern of motif composition and that all of them have MBF1 and HTH_3 domains (Fig. 1b), which means that the function of MBF1 proteins among tomato, Arabidopsis and rice might be similar and conserved.
The expression pattern analysis in different tissues, stresses and plant hormones is usually used to predict the potential functions of genes in plant growth, development and the responses to stresses. Through the expression pattern analysis, we found that all of the SlMBF1s genes were expressed in the five tissues, and most of them had much higher expression in the flower and leaf (Fig. 3). In addition, GA response element was found in the promoter regions of these five SlMBF1 genes and IAA response element also in the promoter regions of SlMBF1a, SlMBF1b and SlER24 (Fig. 2). These results indicated that SlMBF1s might be involved in plant growth and development. Besides the roles in plant growth and development , MBF1 genes also participate in the responses to abiotic and biotic stresses, such as salt, drought, temperature and pathogens [5,6,7,8,9,10,11,12]. Indeed, several stress-related elements (drought, low temperature, ABA, defense and stress, JA and SA response elements) were found in the promoter regions of these SlMBF1s (Fig. 2). In addition, SlMBF1 genes were induced by abiotic and biotic stresses (e.g. salt, drought, cold and B. cinerea) and by stress-related hormones (e.g. ABA, SA, JA and ACC) (Figs. 4 and 5). These results indicated that these SlMBF1 genes were involved in the responses to stresses with the functions similar to the MBF1s from other species [7,8,9,10,11,12].
The tomato is an important economic and vegetable crop. However, B. cinerea seriously limits the yield of tomato . In this study, the expression of SlMBF1c was significantly induced by B. cinerea, wounding and defense-signaling related hormones (Figs. 4 and 5). Additionally, several defense related elements were also found in the promoter of SlMBF1c (Fig. 2). Moreover, overexpressing the AtMBF1a gene in Arabidopsis confers increased resistance under the infection by B. cinerea . In order to clarify the function of SlMBF1c in the defense response, tomato plants overexpressing SlMBF1c were generated. To our surprise, the transgenic lines displayed a sensitive phenotype, as compared with WT, under infection with B. cinerea (Fig. 6). The finding that SlMBF1c regulates the resistance to B. cinerea is distinct from the function of its Arabidopsis homolog. This phenomenon might be due to the evolutionary differences between SlMBF1c and AtMBF1a, because SlMBF1c belonged to subgroup I-A, but AtMBF1a to subgroup I-B in the phylogenetic tree (Fig. 1b).
Previous studies showed that B. cinerea can activate the SA signaling pathway to promote its pathogenicity in plants [23, 24]. Meanwhile, plants can activate the JA/ET-mediated defense responses against B. cinerea infection [23, 25, 26]. However, the SA signaling pathway can antagonize the JA/ET signaling pathways in plants under B. cinerea infection [23, 25]. In our study, under control condition, only SlPR1b in the SA signaling pathway showed slightly higher expression in the OE lines compared with WT, but under B. cinerea infection, all of SlNPR1, SlPR1a, SlPR1b and SlPR2b were significantly up-regulated in OE lines (Fig. 8a). Moreover, the overexpression of SlMBF1c further promote the expression levels of the SA signaling pathway genes, especially SlPR1a and b (Fig. 8a). On the contrary, under control condition, only SlLapA1 in the JA signaling pathway showed slightly lower expression in the OE lines compared with WT; but under B. cinerea infection, all of SlCOI1, SlMYC2, SlPI I and SlLapA1 were significantly down-regulated in OE lines (Fig. 8b). These results suggested that the JA-mediated defense responses in infected OE lines was seriously suppressed by the highly activated the SA signaling pathway under B. cinerea infection (Fig. 8b) . In addition, although under control condition, the ET-mediated defense genes (SlERF1, SlNR, SlACS6 and SlAOS2) showed higher expression in the OE lines compared with WT (Fig. 8c), all of these genes were significantly down-regulated in the infected OE lines, indicating that the ET signaling pathway in the infected OE lines was also greatly suppressed by the highly activated SA signaling (Fig. 8c). Taken together, these results clarified that the SlMBF1c-overexpressing tomato plants displayed a sensitive phenotype due to the strongly activated SA pathway which antagonized the JA/ET-mediated defense responses under B. cinerea infection.
NPR1 is not only a master regulator of SA signaling, but also a key regulator in the antagonism between SA and JA through suppressing the JA signaling gene PI I [23, 27]. Indeed, the expression level of SlNPR1 was increased and SlPI I repressed in the infected OE lines (Fig. 8a-b), suggesting that SlMBF1c could activate SlNPR1 to repress SlPI I in infected OE lines. This result is consistent with the previous study . However, in our study, the expression levels of SlPR1a and SlPR1b were more dramatically increased than SlNPR1 in the infected OE lines, and SlLapA1 more dramatically decreased than SlPI I (Fig. 8a-b). Therefore, it will be interesting to clarify that how SlPR1a and SlPR1b are induced and whether SlPR1a and/or SlPR1b are the new key regulators to suppress SlLapA1 in the antagonism between SA and JA signaling pathways when SlMBF1c is being overexpressed in the tomato under B. cinerea stress condition in the future studies.
In this study, five SlMBF1 genes including SlMBF1d newly-found were identified and confirmed in the tomato genome. The analysis of phylogenetic tree, gene structures and protein motifs revealed that MBF1 proteins are conserved among tomato, Arabidopsis and rice and expanded in the tomato. The cis-elements in the promotors, tissue specific expression pattern and responses to stresses and hormones suggested that the SlMBF1s might participate in plant growth and development and stress responses in the tomato. Finally, transgenic experiments showed that SlMBF1c negatively regulate the tomato resistance to B. cinerea through enhancing SA-signaling genes and repressing the genes in the JA/ET-mediated pathways.
Identification of MBF1 genes in the tomato
To identify the SlMBF1 gene family members from the entire tomato genome, three AtMBF1 proteins were used as query sequences for Blastp searches with an e-value of 10− 10 against the predicted tomato proteins. In addition, the Hidden Markov Model (HMM) profile of MBF1 (PF08523.9) and HTH_3 (PF01381.21) from the Pfam database (http://pfam.janelia.org) were also applied as queries to search the MBF1 genes from the tomato genome database (http://solgenomics.net; ITAG Release 3.20). In order to identify the conserved domains, five candidate genes were further confirmed due to the presence of both the MBF1 (PF08523.9) and HTH_3 (PF01381.21) domains using the Pfam database and SMART database (http://smart.embl-heidelberg.de).
The MBF1 proteins in the representative model plants Arabidopsis and rice were downloaded from The Arabidopsis Information Resource database (https://www.arabidopsis.org) and the Rice Genome Annotation Project Database (http://rice.plantbiology.msu.edu/).
The multiple sequence alignment was constructed by Clustal W (version 1.81, a resident software, European Molecular Biology Laboratory, Heidelberg, Germany.) with default parameters . Full sequences of five SlMBF1, thress AtMBF1 and two OsMBF1 proteins were used to construct the phylogenetic tree using MEGA v7.0 . The Neighbor-Joining method was used with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates; random seed) .
Analysis of physical properties, chromosomal localization, gene structure, conserved motif recognition and response elements in promoter regions
Physical properties such as theoretical protein isoelectric point (pI) and molecular weight of the SlMBF1 proteins were calculated using the ExPASy server’s Compute pI/Mw tool (http://web.expasy.org/compute_pi/) . The information of the chromosomal locations and gene structures were downloaded from the tomato genome database. The conserved motifs were analyzed using MEME database (http://meme-suite.org/) . Additionally, the response elements in the promoter regions were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) . The chromosomal locations were visualized by Mapchart 2.30 software . The gene structures, conserved motifs and response elements in the promoter regions were visualized by GSDS Server 2.0 (http://gsds.cbi.pku.edu.cn/).
Plant materials and growth conditions
Tomato (Solanum lycopersicum L. cv ‘SN1’ ) seedlings were grown in a biotron at Shandong Agricultural University with a 16 h light (28 °C)/8 h dark (22 °C) photoperiod (18.5 μmol m− 2 s− 1). Four-week-old tomato seedlings were used for all types of treatments.
Different stresses and hormone treatments
For the salt and drought stress treatment assays, tomato plants (4-week-old) were transferred into the 10 L tanks containing half-strength Hoagland nutrient solution and were maintained in this system for one week before supplementation with NaCl (150 mM) and Polyethylene glycol 8000 (20%), as previously described . The tomato plants were transferred to the incubator for cold treatment at 4 °C. The seedling leaves were pressed with hemostatic forceps for the wounding treatment. The inoculation of the tomato plants with B. cinerea (B05.10) was performed as previously described [17, 23, 37], with minor modifications. The seedling leaves were spotted with a single 5-μl droplet of B. cinerea spore suspension (106 spores/ml) for the pathogen treatment. For the hormone treatments, the seedling leaves were sprayed with ACC (100 μM), SA (2 mM), MeJA (100 μM), ABA (100 μM) and BR (200 μM). The leaves from different tomato plants were collected for the qRT-PCR analysis.
RNA isolation and quantitative real-time PCR analysis
The total RNA from tomato leaves was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from one microgram of total RNA using a reverse transcriptase system (Thermo, Beijing, China), according to the manufacturer’s instructions. The reactions were performed using the SYBR Mixture (Juheme) with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). The PCR assays were conducted with the following parameters: 95 °C for 30 s; 40 cycles of 95 °C for 30 s, 60 °C for 15 s, and 72 °C for 15 s. All of the primers that were used in the qRT-PCR analysis are listed in Additional file 4: Table S4, some of which came from the previous studies [24, 38,39,40]. The tomato Actin2 gene was used as the internal control. The results were calculated using the 2−ΔΔCt method . All of the qRT-PCR assays were conducted in three biological replicates and each biological replicate had three technical replicates.
Vector construction and plant transformation
For the construction of the overexpressing SlMBF1c vector, the entire SlMBF1c coding sequence was amplified using the primers SlMBF1c-F: TATCACAAGACTGGGAGC and SlMBF1c-R: GTCGTACTACTAGAGGCA. Then, the amplified products were digested with XbaI and KpnI sites and inserted into the pBI121 vector under the control of the 35S promoter. The 35S: SlMBF1c construct was transferred into the Agrobacterium strain LBA4404 by electroporation, and the Agrobacterium-mediated tomato transformation was performed following the protocols described by Fillatti et al. .
All of the error bars for expression levels, represent the standard deviation (SD) which came from three technical replicates, except that in the phenotypic analysis of OE lines which came from six biological replicates. The analysis of significance level was performed with the Student’s t-test at p < 0.05 using Excel 2010 (Microsoft Cooperation, Washington, NJ, USA).
Availability of data and materials
The data that support the results are included within the article and its additional file. Other relevant materials are available from the corresponding authors on reasonable request.
1-Amino cyclopropane-1-carboxylic acid
Arabidopsis thaliana Multiprotein bridging factor 1
- B. cinerea :
Multiprotein bridging factor 1
Molecular Evolutionary Genetics Analysis
Oryza sativa Multiprotein bridging factor 1
protein isoelectric point
Solanum lycopersicum Multiprotein bridging factor 1
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We thank Professor Jiuhai Zhao, College of Agronomy, Shandong Agricultural University, for providing B. cinerea (B05.10). We also thank Professor Qinghua Shi, College of Horticultural Science and Engineering, Shandong Agricultural University, for providing tomato (Solanum lycopersicum L. cv ‘SN1’) seeds.
This work was supported by fundings from the National Natural Science Foundation of China (31672170 and 31872950), the Natural Science Foundation of Shandong Province (JQ201309), the Shandong “Double Tops” Program (SYL2017YSTD06) and the ‘Taishan Scholar’ Foundation of the People’s Government of Shandong Province (ts20130932). The funds played no role in study design, data analysis, and manuscript preparation.
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Molecular properties of SlMBF1 gene family in tomato.
Ten conserved motifs sequences and the bit score means information content from all MBF1 proteins from tomato, Arabidopsis and rice.
The conserved domains information of five tomato MBF1 protein.
Primers used for qRT-PCR.
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Zhang, X., Xu, Z., Chen, L. et al. Comprehensive analysis of multiprotein bridging factor 1 family genes and SlMBF1c negatively regulate the resistance to Botrytis cinerea in tomato. BMC Plant Biol 19, 437 (2019). https://0-doi-org.brum.beds.ac.uk/10.1186/s12870-019-2029-y
- Expression pattern
- Botrytis cinerea