Gene 547 (2014) 145–151

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SpMYB overexpression in tobacco plants leads to altered abiotic and biotic stress responses Jing-bin Li, Yu-shi Luan ⁎, Ya-li Yin School of Life science and Biotechnology, Dalian University of Technology, Dalian 116024, China

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Article history: Received 16 December 2013 Received in revised form 16 May 2014 Accepted 23 June 2014 Available online 24 June 2014 Keywords: Tomato MYB transcription factor Abiotic and biotic stress Tobacco

a b s t r a c t The MYB transcription factors are involved in various plant biochemistry and physiology processes and play a central role in plant defense response. In the present study, a full-length cDNA sequence of a MYB gene, designated as SpMYB, was isolated from tomato. SpMYB encodes the R2R3-type protein consisting of 328 amino acids. The expression level of SpMYB was strongly induced by fungal pathogens. Transgenic tobacco plants overexpressing SpMYB had an enhanced salt and drought stress tolerance compared with wild-type plants, and showed significantly improved resistance to Alternaria alternate. Further analysis revealed that transgenic tobaccos exhibited less accumulation of malondialdehyde (MDA) and more accumulation of superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) after inoculation with A. alternate. Meanwhile, changes in some photosynthetic parameters, such as photosynthetic rate (Pn), transpiration rate (Tr) and intercellular CO2 concentration (Ci) were also found in the transgenic tobaccos. Furthermore, transgenic tobaccos constitutively accumulated higher levels of pathogenesis-related (PR) gene transcripts, such as PR1 and PR2. The results suggested that the tomato SpMYB transcription factor plays an important role in responses to abiotic and biotic stress. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Plants are constantly threatened by various abiotic (high salinity or drought) and biotic (pathogen infection) stress. To survive these challenges, plants have evolved complex transcriptional regulation mechanisms to efficiently adapt to these stresses (Su et al., 2014). These complex transcriptional regulation processes are mainly achieved by transcription factors which having the ability of activating the expression of the stress-related genes and synthesis of diverse functional proteins leading to various physiological and biochemical responses (Chen and Zhu, 2004; Riechmann et al., 2000). A typical higher plant transcription factor usually contains a DNA-binding domain, a transcription regulation domain, an oligomerization site and a nuclear localization domain. Transcription factors interact with cis-elements and regulate the expression of target genes through these domains (Zhang et al., 2012a;Zhang et al., 2012b). On the basis of similarities in the DNA-

Abbreviations: CaMV 35S, cauliflower mosaic virus 35S; cDNA, complementary deoxyribonucleic acid; Ci, intercellular CO2 concentration; EST, expression sequence tag; MDA, malondialdehyde; mM, millimolar; PAL, phenylalanine ammonia-lyase; PCR, polymerase chain reaction; PEG, polyethyleneglycol; Pn, photosynthetic rate; POD, peroxidase; PR, pathogenesis-related; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, reverse transcriptase polymerase chain reaction; SOD, superoxide dismutase; Tr, transpiration rate; WT, wild type. ⁎ Corresponding author at: No. 2 Linggong Road, Ganjingzi District, Dalian City 116024, Liaoning Province, China. E-mail address: [email protected] (Y. Luan).

http://dx.doi.org/10.1016/j.gene.2014.06.049 0378-1119/© 2014 Elsevier B.V. All rights reserved.

binding domain, a range of transcription factors have been found to be induced by the stresses (Yang et al., 2014). The MYB proteins belong to a large family of transcription factors and known to possess diverse roles in plant development and response to environmental stress (Du et al., 2012). Based on the numbers of adjacent imperfect repeats (51–53 amino acids) in their DNA binding domains, MYB proteins in plants are classified into four subfamilies: MYB-related type has a single MYB repeat, R2R3-MYB has two repeats, 3R-MYB (R1R2R3-MYB) has three consecutive repeats and 4R-MYB has four repeats (Dubos et al., 2010). The R2R3-type MYB subfamily constitutes the largest number of MYB proteins in plants (Cai et al., 2011). Currently, an increasing number of plant R2R3-MYB transcription factors have been identified and characterized in numerous plant species, such as Arabidopsis thaliana (126 R2R3-MYB) (Chen et al., 2006), Oryza sativa (109 R2R3-MYB) (Chen et al., 2006), Populus trichocarpa (192 R2R3-MYB) (Wilkins et al., 2009), Glycine max (244 R2R3-MYB) (Du et al., 2012) and Arachis hypogaea (9 R2R3-MYB) (Chen et al., 2014). The functions of various R2R3-MYB transcription factors have been gradually clarified using genetics and molecular biology methods (Katiyar et al., 2012; Liu et al., 2011). Increasing evidence indicated that numerous plant R2R3-MYB genes are involved in responses to diverse abiotic stresses (Al-Attala et al., 2014). For instance, transgenic Arabidopsis overexpression AtMYB15 (Ding et al., 2009), AtMYB44 (Jung et al., 2008) and OsMYB4 (Vannini et al., 2006), TaMYB2A (Mao et al., 2011) and TaMYB33 (Qin et al., 2012) exhibit significantly enhanced tolerance to salt and drought stress.

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Plant pathogenesis-related (PR) genes are marker genes for plant pathogenesis, and play primary roles in disease resistance response, which have been used routinely for the defense status of plants with positive antimicrobial activity (Zhang et al., 2010). Overexpression of some R2R3-MYB transcription factors activates the expression of some PR genes, and the resulting increase in resistance to bacterial, fungal or virus pathogens has been well demonstrated (Seo and Park, 2010; Vailleau et al., 2002). For example, overexpression in Arabidopsis and tobacco of AtMYB30 increased resistance against different bacterial pathogens and a virulent biotrophic fungal pathogen, and the expression of PR genes was altered in the transgenic plants (Raffaele et al., 2006; Vailleau et al., 2002). Likewise, overexpression of the AtMYB96 in transgenic Arabidopsis exhibited an enhanced disease resistance, and a subset of PR genes was up-regulated (Seo and Park, 2010). Furthermore, overexpression of OsMYB4 effectively improved resistance to tobacco necrosis virus, Pseudomonas syringae and Botrytis cinerea in transgenic Arabidopsis plants, and OsMYB4 also induced the expression of PR gene (Vannini et al., 2006). Tomato plants overexpressing OsMYB4 acquired a higher resistance to tomato mosaic virus (Vannini et al., 2007). However, only a few abiotic stress-inducible R2R3-MYB genes from tomato have currently been isolated, and their functions have been studied (Abuqamar et al., 2009; Zhang et al., 2011). The functions of most tomato R2R3-MYB genes are unknown thus far. In the present study, we report the isolation of a pathogen-inducible R2R3-MYB gene, designated as SpMYB. To investigate the roles of SpMYB in the plant stress response, we functionally characterized this gene. The detailed characterization of SpMYB overexpression in tobacco suggested that it is involved in the responses of abiotic and biotic stress. We also performed a comparison of physiological and biochemical changes and the expression of PR genes in the wild-type (WT) and transgenic tobacco plants after inoculation with Alternaria alternate. From this comparison, we identified a pathogen-inducible R2R3-MYB from tomato, which provides more clues for exploring the resistant mechanism and identify an excellent potential candidate gene for plant genetic engineering. 2. Materials and methods 2.1. Plant materials and growth conditions Tomato (Solanum pimpinellifolium L3708) seeds were sown into pots filled with soil and placed in a greenhouse (16 h light/8 h dark period at 25 ± 3 °C). For tissue-specific expression analysis, the root, stem and leaf at the five-leaf stage were collected separately for RNA isolation and used for tissue specific expression analysis. Tobacco seeds (Nictiana tabacum L. cv 89) were surface-sterilized and germinated on MS medium (16 h light/8 h dark period at 25 °C). Four week old sterile WT tobacco plants were used for transformation. 2.2. Pathogen and inoculation Phytophthora infestans was grown on oatmeal medium and maintained in darkness at 20 °C. Collection of sporangia and induction of zoospore production from P. infestans was performed according to previously described methods (Xiang and Judelson, 2010). Subsequently, 2 ml aliquots of P. infestans zoospores (1 × 105 zoospores/ml) was applied to plant leaves with a hand held sprayer until run off. The inoculated plants were kept in the dark at high humidity for 24 h, and then moved to a growth room at 23 °C. Samples were harvested at 0, 6, 12, 24, 48 and 72 h post-inoculation and stored at −80 °C for RNA extraction. 2.3. Isolation and sequence analysis of the SpMYB gene The full-length cDNA of SpMYB was cloned from tomato by homologous cloning and RT-PCR methods (Li et al., 2012). Degenerate PCR

primers (forward primer: 5′-GGNAARTCNTGYMGNYTNMGNTGG-3′ and reverse primer: 5′-CCARTGRTTYTTNAYNKCRTTRTC-3′) were designed based on the conserved residues of the R2R3-MYB domains as previously described (Rabinowicz et al., 1999; Romero et al., 1998) and used to amplify a partial sequence of a putative R2R3-MYB gene from cDNA of tomato leaves after inoculation with P. infestans. The PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced. One of the ESTs was used as the query sequence to search the tomato EST database using the BLASTN program. According to the assembled contig sequence, specific primers (forward primer: 5′-TCCGCCTCCCACCACCGTCTAAT-3′ and reverse primer: 5′-AGGGGAGGGTTGGGGGAGGAAGA-3′) were designed to obtain the full length of SpMYB. The cloned cDNA has significant similarity to the Micro-Tom, database named Solanum lycopersicum cDNA, clone: LEFL1098BH02, HTC in leaf (GenBank Accession No. AK325588) (Aoki et al., 2010). A conserved R2R3-MYB domain was found via searching Conserved Domains Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/ cdd/cdd.shtml). Sequence analysis was performed using ProtParam (http://web.expasy.org/protparam/). Similar sequences from other species were obtained by using the BLASTP program. Multiple sequence alignments were analyzed using ClustalX 1.8 and GeneDoc 2.6 software. 2.4. Quantitative real-time PCR analysis To investigate the expression patterns of SpMYB in different organs and in response to P. infestans infection, qRT-PCR was completed with the following primer (forward primer: 5′-TTCCTTTGATGGCAGCGATT ACGC-3′ and reverse primer: 5′-AACAAGCGTCTGCAACAACTCGTC-3′) on a Rotor-Gene 3000 PCR instrument (Corbett Research, Australia) using the SYBR Premix Ex Taq™ kit (TaKaRa, Dalian, China). The thermal cycles used were as follow: an initial denaturation of 95 °C for 3 min, and 40 cycles of 5 s at 95 °C and 20 s at 60 °C. The tomato β-actin gene (forward primer: 5′-TGTGTTGGACTCTGGTGATGGTGT-3′ and reverse primer: 5′-ATCCAAACGAAGAATGGCATGCGG-3′) was used as internal controls. Relative expression levels were calculated by the 2 − ΔΔCT method (Livak and Schmittgen, 2001). Data acquisition and analysis were performed by using Rotor-gene 6 software. All reactions were performed in triplicate. 2.5. Vector construction and transgenic plant generation The full-length coding sequence of SpMYB amplified using the forward primer (5′-CGGGATCCATGGCAGCGATTACGCA-3′) with a BamHI restriction site (underlined) and reverse primer (5′-CGAGCTCTTACTC AATCTTGCTGAT-3′) with a SacI restriction site (underlined) was inserted into the same sites behind the cauliflower mosaic virus (CaMV) 35S promoter in the pBI121 vector. The ligated construct (pBI121-SpMYB) was transformed into Agrobacterium tumefaciens (strain EHA105) by a freeze–thaw method and transformed into tobacco using the leaf disk method (Horsch et al., 1985). The putative transgenic tobacco plants selected with kanamycin (50 mg/L) were further identified by PCR and RT-PCR analysis and then were chosen for further experiments. 2.6. Tolerances of transgenic tobacco plants to salt and drought stress The 7-day-old aseptic tube seedlings of transgenic and WT tobacco plants were transferred to the half-strength MS medium supplemented with 200 mM NaCl or 2% polyethylene glycol. To make a deep analysis, four-week-old transgenic lines and WT plants were transferred to the plastic pots filled with soil and then subjected to salt and drought stress treatments, respectively. For the salt stress treatment, plants were irrigated with 200 mM NaCl for 2 weeks. For drought stress treatment, plants were withheld from watering for 2 weeks and were then rewatered.

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2.7. Pathogen challenge of transgenic plants A. alternate was grown on potato dextrose agar medium for 2–3 weeks at 26 to 28 °C. For pathogen-resistant experiments, transgenic and WT tobacco leaves were sprayed the A. alternata spore suspension (1 × 105 conidia/mL). After inoculation, all plants were incubated in a controlled growth chamber in the dark for 24 h at 28 °C with 90% relative humidity and then transferred at 16 h/8 h light–dark cycle and 28/20 °C day/night. Disease symptoms were measured on the 7 days after inoculation and the variation of lesions were calculated by a six-class disease severity as described previously (Tong et al., 2012). Several leaves were inoculated and measured in each plant. Disease resistance was expressed using a disease index and all the percentage data were arcsine-transformed before statistical analysis.

2.8. Measurement of malondialdehyde (MDA) content, antioxidant enzyme activities and photosynthetic characteristics All enzyme extraction procedures were conducted at 4 °C. After being infected with A. alternate, the leaves of transgenic lines and WT plants were collected to measure the MDA content, superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activity as described previously (Chen et al., 2013). The Portable Photosynthesis System CIRAS-2 was used to measure photosynthesis parameters, such as photosynthetic rate (Pn), transpiration rate (Tr) and intercellular CO2 concentration (Ci).

2.9. Analysis of downstream genes regulated by SpMYB To elucidate the molecular mechanism of SpMYB, we performed RTPCR to analyze the expression of PR genes including PR1 and PR2, in transgenic and WT tobaccos after being infected with A. alternate. The gene-specific primers were as follows: PR1 (forward primer: 5′-CCCA AAATTCTCAACAAG-3′ and reverse primer: 5′-TTAGTATGGACTTTCGCC TCT-3′); PR2 (forward primer: 5′-ATGGCTTTCTTGCAGCTGCCCTTG-3′ and reverse primer: 5′-GAGTCCAAAGTGTTTCTCTGTGATA-3′). The tobacco actin gene (forward: 5′-GTGATGGTGTGAGTCACACT-3′ and reverse: 5′-GGGAGCCAAGGCGGTGAT-3′) was used as the internal reference.

2.10. Statistical analysis All data were statistically analyzed using one-way analysis of variance, and the means were separated by Duncan's Multiple Range Test by the least significant difference method using SPSS 17.0 version software. Different letters indicate significant difference at p b 0.05. 3. Results 3.1. Isolation and sequence analysis of SpMYB The full length cDNA of SpMYB contained 114 bp of 5′ UTR and 124 bp of 3′ UTR. This cDNA contained an open reading frame (ORF) of 987 bp encoding a protein of 328 amino acids. The deduced molecular mass of SpMYB protein was about 35,822.8, and the isoelectric point was approximately 9.14. Multiple alignments showed that SpMYB contained two imperfect tryptophan-rich repeats (R2 and R3) and shares high similarity with R2R3-type MYB transcription factors of other plant species (Fig. 1). The MYB gene family is divided into different types according to the number of repeat(s) in the MYB domain (Dubos et al., 2010). All of the above data strongly indicated that SpMYB is a R2R3-type MYB transcription factor.

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3.2. Expression of SpMYB in different tomato tissues and response to pathogen Expression patterns of SpMYB in different tomato tissues were examined using qRT-PCR. The transcripts were detected in all tested tissues, whereas the expression level was varietal in different tissues. The expression level of SpMYB in the stem was lower than that in the leaf, but much higher than that in the root (Fig. 2A). The expression profile of SpMYB in tomato leaves after inoculation with P. infestans was further determined using qRT-PCR analyses. The results revealed that the relative expression values were significantly different. As shown in Fig. 2B, the transcript of SpMYB was significantly induced and reached the highest peak at 6 h after inoculation and was 2.94-fold higher than that of the control. 3.3. Overexpression of SpMYB in transgenic tobacco confers tolerance to salt and drought stress The SpMYB coding sequence was cloned into pBI121 vector, replacing the GUS reporter gene and yielding pBI121-SpMYB. To further analyze the function of SpMYB, transgenic tobacco plants were generated by the Agrobacterium-mediated method. PCR and RT-PCR result showed that exogenous SpMYB was successfully expressed in tobacco (Fig. 2C and D) and three representative positive transgenic lines (Line 5, Line 6 and Line 8) were selected for the further analysis. To investigate the role of SpMYB under abiotic stress, aseptic seedlings of transgenic and WT tobacco plants were grown in salt or drought conditions. After 45 days, transgenic lines showed more developed root systems and higher fresh biomass compared to the WT plants under salt stress (Fig. 3A and B). This discrepancy was also observed under drought stress (Fig. 3C and D). These results suggest that the overexpression of SpMYB confers an enhanced tolerance to salt and drought stress during early seedling development. Three transgenic lines and WT plants were also transferred to soil and used for salt and drought stress assays. After 2 weeks of treatment with 200 mM NaCl, the growth of the WT plant was completely inhibited and leaf growth was retarded. However, transgenic lines showed stronger development capacity and leaf area increased (Fig. 3E). To examine the role of SpMYB in drought stress tolerance, all plants grown in soil were deprived of water for 2 weeks followed by rewatering for 3 days. After 2 weeks of water deprivation, the WT plants were wilted, while transgenic lines still displayed good growth. After 3 days of water irrigation, the WT plants remained in the wilted stage, whereas transgenic plants recovered with growth and maintained normal developmental (Fig. 3F). Taken together, these results indicate that SpMYB plays an important role in tolerance to salt and drought stress. 3.4. Overexpression of SpMYB in tobacco conferred increased resistance to A. alternate Disease symptoms were measured on the 7 days after inoculation and the degree of resistance was calculated by the disease index. A high disease index value means a low resistance. As show in Fig. 4A, transgenic lines had lower disease index than WT tobacco plants. These results indicated that ectopic expression of SpMYB in tobacco conferred increased resistance to A. alternate infection. Abiotic or biotic stress often causes the accumulation of reactive oxygen species (ROS), which can damage cellular membranes and mediate lipid peroxidation (Tian et al., 2013). MDA is widely recognized as a parameter for lipid peroxidation (RoyChoudhury et al., 2007). SOD, POD and PAL are important antioxidant enzymes in the ROSscavenging system and involved in biosynthesis of antimicrobial compounds during plant responses to various stresses (Wu et al., 2013). After inoculation with A. alternate, the MDA content in transgenic lines was lower than that in the WT plants (Fig. 4B), but SOD, POD and PAL

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Fig. 1. Multiple sequence alignment of SpMYB and MYBs from other plant species, including Nicotiana tabacum, Medicago truncatula, Glycine max and Arabidopsis thaliana. The R2 and R3 MYB regions are indicated by solid line and dotted line, respectively. Solid circles indicate the conserved residues, such as tryptophan (W) and phenylalanine (F). Identical amino acids are shaded black, similar amino acids (at least 75%) are shaded gray and unrelated amino acids are not shaded. Terminal amino acid residues are numbered.

activities in these transgenic lines were higher than that in the WT plants (Fig. 4C–E). These results suggested that reduced MDA content and enhanced antioxidant enzymes activities in the transgenic tobacco lines were likely associated with expression of SpMYB. To assess the effects of pathogen infection on photosynthetic performance, we measured Pn, Tr and Ci in the leaves. The results revealed that the transgenic lines maintained higher Pn (Fig. 4F) and lower Tr and Ci than the WT plants (Figs. 4G and H). PR genes play a vital role in pathogen defenses. It has been well demonstrated that many PR genes are regulated by MYB transcription factors (Seo and Park, 2010). The expression of PR1 and PR2 were analyzed in transgenic plants and compared with WT plants. As shown in Fig. 4I, transcription levels of PR1 and PR2 were higher in three transgenic lines than in the WT plants. The results suggest that SpMYB plays an important role in plant defense response by regulating the expression of various PR genes. 4. Discussion MYB proteins comprise one of the largest transcription factors families in plants, which are classified into four major groups according to the number and position of MYB repeats (Dubos et al., 2010). R2R3MYB is a principal member of the MYB transcription factor superfamily, which has been showed to play an important role in abiotic stress tolerance and disease resistance. For example, AtMYB15 enhances the salt and drought tolerances of transgenic plants (Ding et al., 2009). The overexpression of AtMYB30 increases resistance against different

bacterial pathogens and a biotrophic fungal pathogen (Vailleau et al., 2002). TaPIMP1 encodes a R2R3-MYB protein that enhances resistances to abiotic and biotic stress in transgenic tobacco (Liu et al., 2011). Likewise, ectopic expression of the rice OsMYB4 gene in Arabidopsis also increases tolerance to abiotic and biotic stress (Vannini et al., 2006). Some of the R2R3-MYB genes involved in abiotic and biotic stress have been previously isolated and characterized in numerous plant species. However, only a few abiotic stress-inducible R2R3-MYB genes from tomato have been identified and characterized, including SlAIM1 (abscisic acid-induced) (Abuqamar et al., 2009) and SlCMYB1 (cold-induced) (Zhang et al., 2011). Functional study so far mostly focused on growth and metabolism. Considering the multiple functions of R2R3-MYB transcription factor, we conducted research concerning characterization of the pathogen-inducible R2R3-MYB gene from tomato. In this study, a full-length cDNA sequence of a tomato MYB gene, namely SpMYB, induced by pathogen infection, was isolated using homologous cloning and RT-PCR methods. Multiple sequence alignment analysis of SpMYB and other plant MYB proteins indicating that SpMYB is a R2R3-type MYB transcription factor (Fig. 1). We demonstrate that the expression of SpMYB was rapidly and strongly induced by fungal pathogens in tomato, and reached its highest levels at 6 h (Fig. 2B), suggesting that SpMYB may be a positive regulator in the defense response to pathogens at the early stage. To investigate the function of SpMYB in plants, we overexpressed SpMYB in transgenic tobacco under control of a CaMV 35S promoter. Transgenic tobaccos were confirmed by kanamycin selection and then

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Fig. 2. Expression patterns of SpMYB and detection transgenic tobacco. (A) Expression profile of the SpMYB in different tissues of tomato. The expression level of root was set as one. (B) Expression of SpMYB after pathogens infection. The tomato β-actin gene was used as an internal control of gene expression. The expression level of control treatment (0 h) was set as one. The error bars indicate the standard deviation of three biological replicates. Bars with different letters represent significant difference (Duncan's Multiple Range Test, p b 0.05). (C) PCR amplifies the result using the template of DNA from tobacco. Lane 1: ddH2O; Lane 2: WT tobacco; Lane 3: pBI121-SpMYB plasmid; Lanes 4–8: transgenic tobacco plants containing SpMYB. (D) Positive transgenic tobacco clones as detected by RT-PCR. Line 5, Line 6 and Line 8 were transgenic lines. M: DL2000 Marker.

confirmed by PCR analysis (Fig. 2C and D). Overexpression of SpMYB in tobacco remarkably enhanced its tolerance to salt and drought stress. It was observed that transgenic lines showed better growth than WT tobacco when plants were grown in the half-strength MS medium or planted on soils (Fig. 3A–F). These results indicate that SpMYB plays an important role in the tolerance to salt and drought stress. The transgenic plants were further analyzed after inoculation with A. alternate. Each plant was assessed individually and each leaf was

scored for number and size of lesions (Tong et al., 2012). The disease index of transgenic lines was lower than that of WT plants (Fig. 4A), indicating that SpMYB-overexpressing tobacco plants displayed significantly increased resistance to A. alternate. To assess the functional properties of SpMYB, we compared MDA content and SOD, POD and PAL activities of transgenic and WT tobacco plants. MDA is widely recognized as a marker for lipid peroxidation. SOD, POD and PAL are wellknown ROS-scavenging enzymes in the antioxidant defense system

Fig. 3. Salt and drought stress analysis for transgenic tobaccos. The root lengths and fresh biomasses were measured in salt stress conditions (A and B) or drought stress (C and D). Phenotypes of transgenic lines and WT plants under salt (E) or drought stress treatment (F). The error bars indicate the standard deviation of three biological replicates. Different letters indicate significant differences between the transgenic plants and the WT control at values of p b 0.05 with Duncan's Multiple Range Test.

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Fig. 4. Responses of SpMYB transgenic tobaccos to inoculation with A. alternate. (A) Disease index of transgenic lines and WT plants after inoculation. Percentage data were arc-sine transformed before analysis. MDA contents (B), SOD, POD and PAL activities (C, D and E) of transgenic lines and WT plants were measured after infection. Pn, Tr and Ci (F, G and H) were recorded on the leaves of transgenic lines and WT plants. (I) RT-PCR assay of PR1 and PR2 genes expressed in transgenic lines and WT plants. The error bars indicate the standard deviation of three biological replicates. Different letters indicate significant differences between the transgenic plants and the WT control at values of p b 0.05 with Duncan's Multiple Range Test.

resulting in decreased ROS levels and accumulation of antimicrobial compound (Wu et al., 2013). It was reported that R2R3-MYB transcription factors increased resistance to pathogens and exhibited improved tolerances to abiotic stress which may participate in the ROSscavenging pathway and elevate levels of ROS-scavenging enzyme activities (Liu et al., 2011; Qin et al., 2012). In this study, transgenic tobacco lines with increased resistance to pathogens had decreased MDA content (Fig. 4B) and increased SOD, POD and PAL activities (Fig. 4C– E) compared to WT plants. The results suggest that SpMYB confer transgenic tobacco exhibited an increased disease resistance possibly through decreased content of MDA and increased activities of SOD, POD and PAL. As the energy source for plant metabolism, photosynthesis is one of the primary processes to be challenged by pathogen infection in plants (Berger et al., 2004). Analysis of photosynthetic performance in leaves was performed with the aim of discriminating biochemical effects after infection. Transgenic lines seem to be associated with a significant increase in Pn and lower decrease in Tr and Ci (Fig. 4F–H) thereby contributing protection of plants from infection by pathogens. Increasing evidence indicated that R2R3-MYB transcription factors can mediate host resistance to pathogen infections through regulation of PR gene expression (Zhang et al., 2012a; Zhang et al., 2012b; Seo and Park, 2010; Vailleau et al., 2002). Different R2R3-MYB transcription factors induce a diverse subset of PR genes. Consistent with the altered resistance responses to pathogen infection in the transgenic plants, expression of several PR genes were changed in the transgenic plants. We observed that the expressions of PR1 and PR2 were significantly higher in transgenic tobacco plants than in the WT plants (Fig. 4I). Results demonstrate that the SpMYB may confer plant resistance in response to

A. alternata infection through the activation of PR1 and PR2 genes. Nevertheless, the direct downstream genes of SpMYB are unknown, and further studies are needed to identify the downstream target genes of SpMYB to provide a better understanding of its role during biotic stress. 5. Conclusion This study has characterized a tomato R2R3-MYB gene, SpMYB, which exhibited significantly higher expression levels following pathogenic fungal infection. Overexpression of SpMYB in tobacco plants conferred enhanced tolerance to salt and drought stress and increased resistance to A. alternata. We speculate that SpMYB may confer transgenic tobacco an increased disease resistance through the accumulation of defense-related enzymes, modulation of photosynthetic activity and activation of PR genes expression. These findings suggest that SpMYB plays a vital role in modulating responses to abiotic and biotic stress and be a useful candidate gene for improving disease resistance in plants. Conflict of interest statement The authors declare that they have no conflict of interests. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (30972001 and 31272167).

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SpMYB overexpression in tobacco plants leads to altered abiotic and biotic stress responses.

The MYB transcription factors are involved in various plant biochemistry and physiology processes and play a central role in plant defense response. I...
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