Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae

Transcription

1 Research Article Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae Chun-Lin Yang 1,2,3, Shan Liang 1,2, Hai-Yun Wang 1,2, Li-Bo Han 1,2, Fu-Xin Wang 1,2, Huan-Qing Cheng 1,2,3, Xiao-Min Wu 1,2, Zhan-Liang Qu 1,2, Jia-He Wu 1,2, * and Gui-Xian Xia 1,2, * 1 Institute of Microbiology, Chinese Academy of Sciences, Beijing , China 2 State Key Laboratory of Plant Genomics, Beijing , China 3 University of Chinese Academy of Sciences, Beijing , China *Correspondence: Gui-Xian Xia (xiagx@im.ac.cn), Jia-He Wu (wujiahe@im.ac.cn) ABSTRACT In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants. Key words: major latex protein, ethylene responsive factor, interaction, disease tolerance, cotton Yang C.-L., Liang S., Wang H.-Y., Han L.-B., Wang F.-X., Cheng H.-Q., Wu X.-M., Qu Z.-L., Wu J.-H., and Xia G.-X. (2015). Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae. Mol. Plant. 8, INTRODUCTION Verticillium wilt of cotton is a highly destructive vascular disease that is mainly caused by the soil-borne fungus Verticillium dahliae. Although no germplasm of upland cotton is immune to Verticillium wilt, traditional breeding has produced cultivars with high tolerance to the disease. Previously, efforts have been made to investigate the molecular basis of the disease tolerance in cotton. Global analyses have identified sets of V. dahliae-responsive genes/proteins, including components in jasmonic acid (JA), ethylene (ET), and salicylic acid (SA)-mediated signaling pathways, and lignin biosynthesis (Wang et al., 2011; Xu et al., 2011; Gao et al., 2013a). Several individual genes, such as GhHb1, GhNDR1, GhMKK2, GbVe1, and GhBAK1, were shown to be functionally related to the disease protection (Qu et al., 2006; Gao et al., 2011b; Zhang et al., 2012a; Gao et al., 2013b). These studies have taken important steps toward understanding the complex innate defense mechanisms against V. dahliae infection in cotton. The Bet v 1 family consists of a large group of proteins and could be divided into at least four subclasses, including the major latex proteins (MLPs), pathogenesis-related 10 (PR10) proteins, cytokinin-specific binding proteins (CSBPs), and norcoclaurine synthases (Radauer and Breiteneder, 2007). A common structural feature of these proteins is the formation of a hydrophobic cavity that forms the ligand-binding site for hormones and secondary metabolites (Radauer et al., 2008). Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS. Molecular Plant 8, , March 2015 ª The Author

2 Among the Bet v 1 proteins, members of the PR10 subfamily have been better studied and the major roles of PR10 proteins in response to biotic and abiotic stresses have been documented (Choi et al., 2012; Fernandes et al., 2013). MLP proteins have been identified in various plants such as Arabidopsis, opium poppy, and peach (Radauer et al., 2008). It has been reported that the function of MLPs is related to fruit and flower development in peach (Ruperti et al., 2002) and fruit ripening in kiwifruit (Chruszcz et al., 2013). Meanwhile, a number of studies suggested that the function of MLP genes might be required for pathogen protection. For instance, expression of Arabidopsis MLP3 and MLP28 (AT1G70830) genes was responsive to fungal pathogen Alternaria or Plasmodiophora brassicae (Schenk et al., 2000; Siemens et al., 2006). The melon MLPs were detected in the stem phloem sap of cucumber mosaic virusinfected plants (Malter and Wolf, 2011). In cotton plants, induction of MLP genes has been repeatedly observed following V. dahliae challenge (Qu et al., 2005; Chen and Dai, 2010; Wang et al., 2011; Zhang et al., 2012b). Although these results implied the participation of MLPs in pathogen defense in higher plants, the molecular basis of such a function of MLPs is currently unclear. The ET responsive factors (ERFs) belong to the superfamily of APETALA2 (AP2)/ERF transcription factors. ERFs can bind to the AGCCGCC element (GCC box) located in the promoter region of many plant genes to activate or repress the target gene transcription (Ohme-Takagi and Shinshi, 1995; Fujimoto et al., 2000; Licausi et al., 2013). In addition to their roles in various physiological processes, ERFs are also involved in responses to environmental stresses (Kizis et al., 2001; Singh et al., 2002; Licausi et al., 2013). In Arabidopsis, for example, several ERFs, including ERF1, ERF2, ERF14, ORA59, ERF5, and ERF6 were shown to have important roles in pathogen defense, and changes in the expression levels of some ERFs led to altered disease susceptibility, which was linked with the transcriptional regulation of defense-related genes, such as PDF1.2 and pathogenesis-related genes (PRs) (Brown et al., 2003; Oñate- Sánchez et al., 2007; Pre et al., 2008; Zarei et al., 2011; Moffat et al., 2012; Meng et al., 2013). Previous studies showed that ERF proteins could form stable complexes with other transcriptional regulators, which determined the transcriptional activity and target specificity of ERFs (Song and Galbraith, 2006; Licausi et al., 2013). Although little is known about the partners of the ERF proteins involved in disease protection, two regulatory proteins, including a transcription factor (OBF4) and a mitogenactivated protein kinase (MPK6), have been shown to interact with ERFs to regulate the transcription of defense-related genes in Arabidopsis (Büttner and Singh, 1997; Wang et al., 2013). In this study, we characterized the function and regulatory mechanism of an MLP gene (GhMLP28) from upland cotton (Gossypium hirsutum). We show that expression of GhMLP28 was stimulated by V. dahliae inoculation; inhibition of its expression disrupted the defense ability of plants against pathogen infection, but overexpression of this gene rendered the transgenic plants with increased disease tolerance. More importantly, we found that GhMLP28 interacted with GhERF6 to enhance its transcription factor activity, which facilitated the expression of some GCC-box genes, and the distribution of GhMLP28 proteins in the nucleus was dependent on the presence of GhERF6 in cotton 400 Molecular Plant 8, , March 2015 ª The Author Interaction of MLP28 and ERF6 in Defense Response root cells. Our results provide a critical line of evidence showing the participation of MLP in plant defense response and demonstrate that GhMLP28 acts as a positive regulator of GhERF6. RESULTS Identification of the GhMLP28 Gene and Phylogenetic Analysis Previously, we identified differentially expressed cdnas from a Verticillium wilt-tolerant cotton cultivar by subtractive suppression hybridization (SSH) analysis (Qu et al., 2005). Of these cdnas, a clone whose expression was significantly induced in cotton root after V. dahliae inoculation was further studied in this work. The cdna clone was found to encode an MLP that contains the conserved amino acids of the Bet v 1 family proteins. The full-length cdna was cloned by 5 0 and 3 0 rapid amplification of cdna ends and was found to represent the same gene (Gh-MLP) as that reported by Chen and Dai (2010). Phylogenetic analysis of this cotton MLP and those from other plants was conducted. The result showed that it was closest to the Arabidopsis MLP28 (Supplemental Figure 1), which is considered to be a putative defense-related protein (Siemens et al., 2006). Based on the phylogenetic relationship as well as the fact that multiple MLP genes can be identified in the diploid cotton genome (Li et al., 2014), we renamed this gene GhMLP28. Expression Profile of GhMLP28 Gene The expression pattern of GhMLP28 in various organs of cotton was examined by quantitative real-time PCR (qrt-pcr) analysis. As shown in Figure 1A, the gene was ubiquitously expressed in all organs investigated. In cotton root, where the V. dahliae fungus enters the plant, accumulation of the GhMLP28 transcript was higher than in other organs. The V. dahliae-induced expression of GhMLP28 observed by SSH analysis in our previous study (Qu et al., 2005) was verified by qrt-pcr analysis. Cotton plants were exposed to the fungus for various durations and the changes in GhMLP28 expression were examined. Expression of GhMLP28 was induced 3 days after V. dahliae challenge and reached maximum levels at 4 days post inoculation (Figure 1B). To further investigate the involvement of GhMLP28 in disease responses, gene expression was analyzed after treatment with defense-related signaling molecules, ET, JA, and SA, respectively. As shown in Figure 1C, expression of GhMLP28 was enhanced by exogenous application of ET and JA, but was downregulated following SA treatment. This supports that GhMLP28 is involved in defense against V. dahliae infection in the cotton plant. Knockdown of GhMLP28 Expression Results in Increased Susceptibility of Cotton Plant to V. dahliae Infection Virus-induced gene silencing (VIGS) strategy (Liu et al., 2002; Gao et al., 2011b; Qu et al., 2012; Pang et al., 2013) was used to investigate the cellular function of GhMLP28. The cdna of the GhMLP28 gene was cloned into the virus vector ptrv- RNA2 (ptrv2) (Liu et al., 2002). The Agrobacterium cells harboring this plasmid were inoculated onto the cotyledon of the cotton variety BD18, a Verticillium wilt-tolerant cultivar. The

3 Interaction of MLP28 and ERF6 in Defense Response Molecular Plant Figure 1. Expression Profiles of the GhMLP28 Gene. (A) Expression pattern of the GhMLP28 gene in various organs of cotton plant. Root, stem, and leaf were sampled from the 14-day-old seedlings grown in a greenhouse, and flower was harvested from soil-grown plants on the day of anthesis. (B) Accumulation of GhMLP28 transcripts in cotton roots inoculated with V. dahliae. Total RNAs were extracted from roots of 14-day-old seedlings at 0 5 days after inoculation. (C) Expression of GhMLP28 after treatments with ET, JA, or SA. Total RNAs were extracted from roots of 14-day-old seedlings at 0 6 h after treatment, respectively. Error bars indicate the standard deviation (SD) of three technical replicates within one biological experiment. Three biological repeats were performed. plant transfected with the empty vector ptrv2 was used as a control (TRV:00). The seedlings were grown for 14 days and the top leaves were harvested to confirm the reduced expression of the GhMLP28 gene (Figure 2A). Subsequently, the control and GhMLP28-silenced plants were subjected to V. dahliae challenge. As shown in Figure 2B, GhMLP28-silenced plants grew comparably with the control plants before pathogen inoculation (upper panel); 35 days after inoculation, the control plants displayed obvious disease symptoms, with many wilted leaves arising from the bottom of the plants, however, the GhMLP28-silenced plants were more severely affected than the control plants, and almost all the V. dahliae-inoculated plants had withered (lower panel). The rate of diseased plants and disease index (DI) were calculated; the values for the GhMLP28-silenced plants were higher than the control plants (Figure 2C), indicating that knockdown of the GhMLP28 gene led to increased susceptibility of the plants to V. dahlia infection. Overexpression of GhMLP28 Enhances the Disease Tolerance of Transgenic Tobacco Plants An overexpression strategy was also used to assess the function of the GhMLP28 gene. Due to technical difficulties and the long duration of cotton transformation, the tobacco plant (Nicotiana tabacum) was used in this experiment. More than 20 transgenic lines constitutively overexpressing GhMLP28 were obtained. Three lines with different expression levels of GhMLP28 (Figure 3A) were chosen for further analysis. Wild-type and transgenic plants (line 2) were subjected to V. dahliae challenge by using a modified method of that introduced by Munis et al. (2010). Disease symptoms were examined 10 days post inoculation. The results showed that the necrotic lesions were smaller in the leaves of transgenic plants than those in the leaves of the wild-type (Figure 3B and 3C), suggesting that the spread of necrotic lesions in transgenic plants was inhibited by ectopic overexpression of GhMLP28. The fungus Phytophthora parasitica var. nicotianae, which causes black shank disease in tobacco, was also used to investigate the defense behavior of GhMLP28-transgenic plants. The tobacco plants were challenged by P. parasitica through root inoculation. Ten days after inoculation, control plants wilted, whereas GhMLP28-overexpressing plants appeared to be growing normally (Figure 3D). The rate of diseased plants and the DI of the transgenic plants were significantly lower than those of the control (Figure 3E), showing that ectopic overexpression of GhMLP28 conferred increased tolerance to black shank disease in tobacco plants. GhMLP28 Interacts with GhERF6 To elucidate the molecular mechanism by which GhMLP28 may act in the defense responses, pull-down screening in combination with the mass spectra analysis was conducted to identify proteins that interact with GhMLP28. Total proteins extracted from cotton root were used as prey and 63 His-tagged GhMLP28 proteins were used as bait. The interacting proteins were collected and separated on 1D or 2D SDS-PAGE gels. The isolated protein bands or spots were subjected to mass spectra analysis. Ten proteins/peptides (Supplemental Table 1) were identified by this approach and four proteins that appeared with higher frequency, including the nucleotide binding site-leucine rich repeat protein (NBS-LRR), calcium-dependent protein kinase (CDPK), phosphospecific binding protein (14-3-3), and ERF were chosen as candidate proteins for subsequent interaction analysis. Yeast two-hybrid assay was then conducted to verify the interactions between candidate proteins and GhMLP28. Of the four proteins, only GhERF6 could interact with GhMLP28 (Figure 4A). The interaction between GhMLP28 and GhERF6 was further confirmed by immunoblot assay using purified recombinant proteins (Figure 4B). The luciferase (Luc) complementation imaging (LCI) assay was carried out to assess the interaction of GhMLP28 and GhERF6 in plant cells. As shown in Figure 4C and 4D, Luc signal was Molecular Plant 8, , March 2015 ª The Author

4 Interaction of MLP28 and ERF6 in Defense Response Figure 2. Increased Susceptibility of GhMLP28-Silenced Cotton Plants to V. dahliae. (A) Analysis of GhMLP28 expression levels. Total RNAs were extracted from leaves at 14 days post agroinfiltration and the expression level of GhMLP28 in VIGS plant was compared with that of control plant (transfected with TRV:00). (B) Disease symptom of GhMLP28-silenced plants infected by V. dahliae. (C) Rate of diseased plant (%) and disease index of the control and GhMLP28-silenced plants. Error bars in (C) indicate the SD (n = 80) of three biological replicates. Asterisks indicate statistically significant differences as determined by Student s t-test (*P < 0.05). detectable only when NLuc-GhMLP28 and CLuc-GhERF6 were co-transformed into the tobacco (N. benthamiana) leaf cells and only trace activities were detected with NLuc-GhMLP28/CLuc, CLuc-GhERF6/Nluc, and NLuc/CLuc controls. These results indicate that GhMLP28 and GhERF6 interact in plant cells as well. GhERF6 Binds to the GCC Box and Acts as a Transcription Factor It is known that ERF proteins bind to the GCC-box element in the promoter of some plant genes and act as transcription factors to regulate the expression of these genes. The DNA-binding ability of GhERF6 was tested by electrophoretic mobility shift assay (EMSA) using double repeats of the GCC box as the probe. The result showed that GhERF6 formed a complex with the labeled probe and the signal was gradually decreased by the addition of increasing amounts of unlabeled probe (Figure 5A), demonstrating that GhERF6 could bind specifically to the GCC box in vitro. The transcription factor activity of GhERF6 was examined using a dual-luciferase reporter (DLR) assay system in Arabidopsis protoplasts (Ohta et al., 2001). The coding sequence of GhERF6 was fused to the DNA sequence encoding the GAL4 DNA-binding domain under the control of the 35S promoter. Isolation and transformation of Arabidopsis protoplasts were performed as described by He et al. (2007). As shown in Figure 5B, GhERF6 was able to activate the reporter expression in vivo. GhMLP28 Enhances the GCC Box-Binding Activity of GhERF6 In Vitro Given that GhERF6 functions as a transcription factor, interaction of GhMLP28 with GhERF6 may have an effect on its activity. 402 Molecular Plant 8, , March 2015 ª The Author To assess this possibility, the binding of GhERF6 to GCC box was conducted in the presence of GhMLP28. As shown in Figure 6A, GhERF6 bound to GCC box, while GhMLP28 on its own did not; when 2.5 mm GhMLP28 was present in the reaction, an up-shifted band appeared; when the concentration of GhMLP28 was gradually increased, the lower band became weaker while the intensities of the upper protein-dna bands were substantially enhanced; only the up-shifted band could be viewed when the two proteins were present in the reaction at a 1:4 ratio, and further increase of GhMLP28 concentration to 2- to 4-fold higher than that of GhERF6 resulted in a stronger up-shifted band signal. To determine whether GhMLP28 was present in the upshifted band, an anti-his antibody was added into the binding mixture, after which the band was found to further shift up (Figure 6B). These results indicated that GhMLP28 could bind to GhERF6 and enhanced its GCC box-binding activity in vitro. GhMLP28 Stimulates the Transcription Factor Activity of GhERF6 Because the defense-related gene PDF1.2 can be transcriptionally regulated by ERFs in multiple organisms (Chakravarthy et al., 2003; Zarei et al., 2011), we chose this gene as a putative target to assess the roles of GhERF6 and GhMLP28 using a transient expression assay in vivo. A sequence containing two GCC-box elements in the promoter of a PDF1.2 gene was isolated from cotton genomic DNA and subsequently inserted into vector pgwb435 with a Luc reporter gene. The Agrobacterium cells harboring the indicated plasmids (Figure 7B) were simultaneously injected into tobacco leaves. Two days later, the expression of GhMLP28 and GhERF6 was confirmed by qrt-pcr (Figure 7A) and Luc expression was examined. As

5 Interaction of MLP28 and ERF6 in Defense Response Molecular Plant Figure 3. Enhanced Disease Tolerance of the Tobacco Plants Overexpressing GhMLP28. (A) Expression levels of GhMLP28 driven by the 35S promoter in transgenic tobacco lines (L1, L2, and L3). Histone 3 was used as an internal control. (B) Necrotic lesion area in leaf inoculated with V. dahliae in wild-type and GhMLP28-overexpressing (L2) tobacco plants. (C) Quantitative measurement of the necrotic lesion area shown in (B). (D) Symptoms of wild-type and GhMLP28 transgenic plants (L2) inoculated with P. parasitica for 10 days. (E) Rate of diseased plant (%) and disease index of wild-type and transgenic plants. Error bars indicate the SD of three biological replicates, n =30in(C) and n =48in(E). Asterisks indicate statistically significant differences as determined by Student s t-test (*P < 0.05).WT, wild-type. seen in Figure 7B and 7C, on its own, the GhPDF1.2 promoter drove Luc expression weakly; when co-transformed with 35S:GhERF6, the fluorescence signal was about 7-fold higher; fluorescence intensity also increased when 35S:GhMLP28 was co-transformed with GhPDF1.2 Pro :Luc, likely caused by the presence of tobacco ERF6 homolog(s), which might act in concert with GhMLP28 and stimulate the PDF1.2 promoter activity; when cells containing 35S:GhERF6, 35S:GhMLP28 and GhPDF1.2 Pro :Luc, respectively, were simultaneously injected, the fluorescence intensity of Luc was much higher and reached a level about 6-fold more than that in the cells co-transformed with GhPDF1.2 Pro :Luc and 35S:GhERF6. These results demonstrate that GhERF6 can activate PDF1.2 promoter activity and GhMLP28, in turn, can enhance this activity of GhERF6 in vivo. In line with these data, in vitro EMSA showed that GhERF6 was able to bind to the promoter region of the GhPDF1.2 gene (Supplemental Figure 2). It has been reported that transient expression of two ERFs of sweet potato preferentially activated expression of the PR5 gene but had no obvious effect on other PR genes, such as PR1, PR2, PR3, and PR4 (Kim et al., 2012). Therefore, we also tested the binding of GhERF6 to a PR5 promoter containing two GCC boxes (Supplemental Figure 3) and the roles of GhERF6 and GhMLP28 on regulation of the promoter activity (Supplemental Figure 4), similar results were obtained as described for the PDF1.2 promoter. Transcriptional Dependence of Candidate GCC-Box Genes on GhERF6 and GhMLP28 The above results raised the possibility that expression of some GCC-box genes such as PDF1.2 and PR5 may rely on the functions of GhERF6 and GhMLP28. To see if this is true, Luc expression driven by the PDF1.2 promoter was tested in cotton leaves following knockdown of either GhMLP28 or GhERF6 expression by VIGS. As shown in Figure 8A and 8B, fluorescence intensity was high when the Luc reporter gene was driven by PDF1.2 promoter, although this was lower than that driven by the strong 35S promoter; following knockdown of GhERF6, PDF1.2 Pro -driven Luc expression was substantially inhibited, indicating a requirement of GhERF6 for the promoter activity; following silencing of GhMLP28, the fluorescent signal was decreased to about a quarter of the control value, suggesting that GhMLP28 function was also required for the full PDF1.2 promoter activity. In a parallel experiment, the dependence of PR5 promoter activity on GhMLP28 and GhERF6 was also confirmed in GhMLP28- or GhERF6-silenced cotton plants (Supplemental Figure 5). To exclude the possibility that knockdown of GhMLP28 might affect the expression of GhERF6 and thus caused the decreased Luc expression, we examined the mrna level of GhERF6 in GhMLP28-silenced cotton plants, no significant change was detected. These data support that both GhMLP28 and GhERF6 act in the transcriptional regulation of PDF1.2 and PR5 genes in cotton cells and imply that the transcriptional activation activity of GhERF6 is coupled with GhMLP28 function. Unfortunately, we failed to generate the erf6 mlp28 double mutant by VIGS and were unable to show the effect of GhMLP28 on the function of GhERF6 in cotton leaf cell. To further test the transcriptional dependence of PDF1.2 and PR5 genes on GhERF6 and GhMLP28, we measured the expression levels of these two genes in GhMLP28- or GhERF6-silenced Molecular Plant 8, , March 2015 ª The Author

6 Interaction of MLP28 and ERF6 in Defense Response Figure 4. Interaction between GhMLP28 and GhERF6. (A) Yeast two-hybrid analysis of GhMLP28 and GhERF6 interaction. AD/T-BD/Lam and AD/T-BD/p53 represent the negative and positive control, respectively. b-galactosidase activity was tested by X-gal filter assay. (B) Immunoblot assay of GhMLP28 and GhERF6 interaction. GST-tagged GhERF6 proteins were incubated with excess GhMLP28 proteins and the samples were subjected to native polyacrylamide gel separation. The bands were detected with anti-gst antibody. (C) LCI assay of the interaction between GhMLP28 and GhERF6. Luminescence imaging of N. benthamiana leaves was performed 48 h after co-infiltration with the same amount of Agrobacterium cells harboring constructs indicated on the left panel. (D) Quantification of relevant Luc activities in (C). Error bars represent the SD (n > 30) of three biological replicates. Asterisks indicate statistically significant differences as determined by Student s t-test (**P < 0.01). plants. As shown in Figure 9A, the transcripts of both genes were decreased in GhERF6-silenced, and to a lesser extent in GhMLP28-silenced cotton plants. Accordingly, we found that overexpression of GhMLP28 in tobacco led to increased expression of NtPDF1.2 and NtPR5 genes (Figure 9B). These results suggest that PDF1.2 and PR5 expressions are dependent on both GhERF6 and GhMLP28. Dependence of GhMLP28 Nuclear Distribution on GhERF6 The intracellular distribution of GhMLP28 was determined to further dissect its cellular function. First, we looked at the subcellular localization of GhMLP28 proteins in transgenic tobacco BY2 cells. Figure 10A shows that GhMLP28-GFP fusion proteins were accumulated predominantly in the nucleus and weaker fluorescence could be detected in the cytoplasm as well, supporting that GhMLP28 functions as a transcriptional regulator. To verify the functional link of GhMLP28 with GhERF6, we investigated the association of its nuclear distribution with the presence of GhERF6 by immunoblot analysis. As shown in Figure 10B, GhMLP28 proteins were present in both the nucleus and cytoplasm of cotton root cells; in GhERF6-silenced cotton plant, however, the amount of GhMLP28 proteins in the nucleus was significantly lower than in the control plants, whereas the amount of proteins in the cytoplasm was not obviously altered. 404 Molecular Plant 8, , March 2015 ª The Author In parallel, we also examined if such a dependence of GhMLP28 on GhERF6 was also true when the plants were infected by the pathogen. After challenging with V. dahliae, the accumulation of GhMLP28 proteins in both the nucleus and cytoplasm of root cells increased to higher levels compared with the unchallenged plant, but in GhERF6-silenced plants, the abundance of GhMLP28 proteins in the nucleus was much lower than in the control root cells (Figure 10B). These results indicate that the distribution or accumulation of GhMLP28 proteins in nucleus is dependent on the presence of GhERF6 proteins. DISCUSSION GhMLP28 Is a Defense-Related Protein in Cotton The plant-specific MLPs belong to the Bet v 1 protein family. Although a number of studies have reported that the expression of MLP genes is responsive to pathogen invasion, the biological function of this protein family in defense responses is poorly understood. Previously, we identified GhMLP28 as a pathogenresponsive gene (Qu et al., 2005). We also detected the induced expression of MLPs in cotton root in response to V. dahliae invasion by proteomic analysis (Wang et al., 2011). Accordingly, MLPs were found to be expressed at higher levels in cotton plant tolerant to Verticillium wilt in several other studies (Chen and Dai, 2010; Zhang et al., 2012b). Consistent

7 Interaction of MLP28 and ERF6 in Defense Response Molecular Plant Figure 5. DNA-Binding and Transcription Activation Activity of GhERF6. (A) EMSA analysis of the binding of GhERF6 to the GCC box. GhERF6 proteins were incubated with biotin-labeled probe (23 AGCCGCC) in the absence or presence of 2- to 8-fold of unlabeled probes for 30 min. (B) DLR assay of the transcription factor activity of GhERF6 in Arabidopsis protoplast. The empty vector prt-bd and prt-bd-vp16 were used as negative or positive control, respectively. Error bars represent the SD of three biological replicates with three technical repeats each. Asterisks indicate statistically significant differences as determined by Student s t-test (*P < 0.05, **P < 0.01). observation of induced expression of MLP genes/proteins in response to pathogen invasion encouraged us to further investigate the biological role of this protein in the defense response in cotton. By virtue of loss- and gain-of-function approaches, our results demonstrate that knockdown of GhMLP28 expression by VIGS abolished the disease tolerance in cotton and that ectopic overexpression of the GhMLP28 gene enhanced disease tolerance of the transgenic tobacco plants. These results showed that the function of GhMLP28 was tightly associated with plant defense response. Previously, Chen and Dai (2010) reported their work on the identification and characterization of Gh-MLP, the same gene as GhMLP28. The authors did not observe an obvious change in disease tolerance when the gene was overexpressed in Arabidopsis. The reason for this discrepancy could be due to different experimental procedures for pathogen inoculation as well as the different host plants used in the two studies. Interestingly, the authors found that the function of Gh-MLP was related to salt stress tolerance in Arabidopsis. Further studies are warranted to see if the involvement of GhMLP28 in response to abiotic stress shares a similar molecular basis to response to biotic stress. Figure 6. Enhancement of GCC Box-Binding Activity of GhERF6 by GhMLP28. (A) EMSA analysis of the dose effect of GhMLP28 proteins on the binding activity of GhERF6 to the GCC box. GhERF6 proteins were incubated with a biotin-labeled probe (23 AGCCGCC) in the presence of His-tagged GhMLP28 proteins with indicated concentrations. (B) EMSA test for the presence of GhMLP28 in the up-shifted bands shown in (A). Anti-His antibody against His-tagged GhMLP28 was added in the reaction. The bands were detected by the method as in (A). GhMLP28 Is an Interaction Partner of GhERF6 Yeast two-hybrid, immunoblot, and LCI assays revealed that GhMLP28 interacts with GhERF6. It is well established that ERF proteins function as transcription factors and are involved in various biological processes (Licausi et al., 2013). After assessment of the transcription factor activity of GhERF6, we addressed whether GhMLP28 is involved in GhERF6- mediated transcriptional regulation. Indeed, EMSA showed that GhMLP28 could enhance the GCC box-binding activity of GhERF6 in a dose-dependent manner. EMSA, DLR, and transient expression assays demonstrated that GhERF6 acts as a transcription factor and that GhMLP28 can enhance its transcriptional activation activity. Several ERF proteins have been shown to form stable complexes with other transcriptional regulators such as Sin3 and SAP18, which influence the activity of ERF transcription factors (Song et al., 2005; Song and Galbraith, 2006). Here, we show that GhMLP28 can interact with GhERF6 and enhance the transcriptional activation activity of GhERF6 in plant cells. Our study added Molecular Plant 8, , March 2015 ª The Author

8 Interaction of MLP28 and ERF6 in Defense Response Figure 7. Transient Expression Assay on GhMLP28-Enhanced Transcriptional Activation Activity of GhERF6. (A) Expression levels of GhERF6 and GhMLP28 in tobacco leaves transformed with indicated constructs in (B). (B) Luminescence signal on N. benthamiana leaves. Luminescence imaging was performed 48 h after co-infiltration with the same amount of Agrobacterium cells harboring constructs indicated on the left panel. (C) Luminescence intensity in N. benthamiana leaves measured by IndiGo software. Error bars in (C) represent the SD (n = 60) of three biological repeats. Asterisks indicate statistically significant differences as determined by Student s t-test (*P < 0.05, **P < 0.01). a novel defense-related member in the group of proteins that interact with ERFs and affect their activities. Interestingly, we observed that the accumulation of GhMLP28 proteins in nucleus was dependent on the presence of GhERF6. Although the underlying mechanism for such a link is currently unknown, it is apparent that these two proteins share a synergetic relationship in the process of defense against V. dahliae infection. Interaction of GhMLP28 with GhERF6 Contributes to Disease Protection In cotton, a number of studies have shown that the expression of MLPs is responsive to V. dahliae infection. Likewise, more than 200 pathogen-responsive ESTs representing ERFs have been identified in cotton roots challenged with V. dahliae (Zhang et al., 2013), and overexpression of an ERF gene (GbERF) conferred increased disease resistance in transgenic tobacco plants (Qin et al., 2006). Using the VIGS system, we found that knockdown of GhMLP28 expression resulted in increased susceptibility of the plant to V. dahliae infection, and we also observed that GhERF6 silencing led to decreased Verticillium wilt tolerance (Supplemental Figure 6). Taken together, these results indicate that both proteins participate in the defense response against V. dahliae in cotton. Several studies have reported the transcriptional activation of PDF1.2 genes by ERFs, a regulation that was related to improved disease tolerance of the plants. Although less is known about the regulation of PR5 genes by ERFs, evidence on this aspect is emerging. For example, Kim et al. (2012) showed that transient expression of two ERF genes in tobacco leaves resulted in increased transcription of the PR5 gene. Thus, we chose these two genes as candidate targets to test the transcriptional regulatory functions of GhERF6 and GhMLP28. Our results showed that GhERF6 could bind to the promoters of PDF1.2 and PR5 and activate their transcriptional activity, 406 Molecular Plant 8, , March 2015 ª The Author and that GhMLP28 could enhance such functions of GhERF6 both in vivo and in vitro. Consistently, we observed that expression levels of PDF1.2 and PR5 genes were remarkably reduced in GhERF6- and GhMLP28-VIGS plants. Based on our results, we speculate that the PDF1.2 and PR5 genes may represent two direct targets subjected to GhMLP28-GhERF6- mediated transcriptional regulation and that the altered susceptibility or tolerance to V. dahliae attack in the GhMLP28-silenced or -overexpressing plants may be attributed, at least partially, to the changed expression levels of these two genes. Apart from PDF1.2 and PR5, other GCC-box genes may also be regulated by GhMLP28 and GhERF6, and functionally important for Verticillium wilt tolerance in cotton plants. GhMLP28-Mediated Defense May Represent a Broad- Spectrum Response in Plants In addition to Verticillium wilt, we also observed increased disease tolerance against black shank when GhMLP28 was ectopically expressed in tobacco plants. In addition, the expression of PDF1.2 and PR5 genes was upregulated in GhMLP28-overexpressing tobacco plants. These results suggest that GhMLP28- mediated defense may represent a more general process in higher plants. Like Verticillium wilt, black shank is caused by the fungus P. parasitica var. and therefore it is possible that the GhMLP28-mediated defense response is important in protection against fungi-caused diseases. Further studies are required to assess this possibility. METHODS Plant Materials and Growth Conditions The seeds of BD18, a Verticillium wilt-tolerant breeding line of upland cotton (G. hirsutum), were kindly provided by Prof. Guiliang Jian (Institute of Plant Protection, CAAS). The seeds were sown in soil and plants were grown in a greenhouse under 16 h light/8 h dark conditions at 28 C. The plants were irrigated with Murashige and Skoog nutrient solution weekly.

9 Interaction of MLP28 and ERF6 in Defense Response Molecular Plant Figure 8. Effect of GhMLP28/GhERF6 Silencing on PDF1.2 Promoter-Driven Luc Expression. (A) Transient expression assay of Luc in GhMLP28-or GhERF6-silenced cotton leaves. Top leaves (similar size) of GhERF6-or GhMLP28-silenced plants were injected with an equal amount of Agrobacterium cells harboring GhPDF1.2 Pro :Luc or 35S:Luc. Luminescence imaging of cotton leaves was performed 48 h after infiltration with the constructs indicated on the left panel. (B) Luminescence intensity in cotton leaves measured by IndiGo software. Error bars indicate the SD (n = 60) of three biological repeats. Asterisks indicate statistically significant differences as determined by Student s t-test (*P < 0.05, **P < 0.01). For collection of cotton roots, the plants were grown under hydroponic growing conditions as described by Qu et al. (2005). The seeds of tobacco (N. benthamiana and N. tabacum) were sown in soil and plants were grown in a greenhouse under the conditions according to Wu et al. (2012). Pathogen Cultivation and Inoculation The V. dahliae strain V991, a highly aggressive defoliating isolate, was used. Fungal colonies were grown on potato dextrose agar plate for 1 week at 26 C. Spores were inoculated into Czapek medium and harvested at the 5th day. The roots of cotton seedlings grown under hydroponic conditions for 14 days were rinsed in sterile water and infected by root-dip inoculation into spore suspension (10 5 spores ml 1 ) for 30 min, then harvested at 0, 1, 2, 3, 4, or 5 days after inoculation for RNA or protein extractions. To infect VIGS cotton plants, the spores of V991 were adjusted to a concentration of 10 5 spores ml 1 with sterile distilled water and injected into the hypocotyl, 1 cm under the cotyledons, at a dose of 3 ml per plant. DI was calculated according to the method described by Wang et al. (2004). Inoculation of tobacco leaf was performed based on the method described by Munis et al. (2010). The pathogen of tobacco black shank, P. parasitica, was grown at 24 Con a millet medium plate (5% millet and 0.8% agar, w/v). The mycelia were ground in a mortar and mixed with soil at a ratio of 1:1000 (w/w). The mixture was used to inoculate the 5-leaf-stage tobacco plants in soil. DI was calculated according to the method described by Nichols and Rufty (1992). VIGS A fragment of GhMLP28 or GhERF6 cdna was amplified and cloned into the plasmid ptrv2 according to the method described by Liu et al. (2002). The recombinant plasmids were transformed into A. tumefaciens strain GV3101. Cotton gene silencing was performed following the procedure described by Gao et al. (2011a). Cotyledons of 14-day-old seedlings were injected with a mixture (1:1 ratio, v/v) of Agrobacterium cultures (OD 600 = 1) harboring the ptrv1 and ptrv2-ghmlp28/ptrv2-gherf6 plasmid using a needleless syringe. Alternatively, seedlings were transfected with the mixture by vacuum infiltration according to Qu et al. (2012). A trial experiment was performed to test the efficiency of VIGS under our experimental conditions. The cdna of the GhCLA1 gene (Pang et al., 2013) encoding 1-deoxy-D-xylulose-5-phosphate synthase was cloned into ptrv2 vector and the construct was transformed into A. tumefaciens strain GV3101. The culture was co-inoculated with that of GV3101 harboring ptrv1 into the cotyledon of cotton plants and the albino phenotype was examined (Supplemental Figure 7). Tobacco Transformation The coding sequence of GhMLP28 was cloned under the control of 35S promoter in the plant expression vector ppzp111 (Hajdukiewicz et al., 1994). The resulting plasmid ppzp111-ghmlp28 was introduced into the A. tumefaciens strain EHA105. Transgenic tobacco plants were generated by Agrobacterium-mediated leaf-disc transformation (Horsch et al., 1985). Phylogenetic Analysis The neighbor-joining method was used to produce the phylogenetic tree of GhMLP28 and MLPs in other plants using the MEGA program version 4.0 (Tamura et al., 2007). qrt-pcr Analysis Total RNAs were extracted from cotton or tobacco plants using TRIzol reagent (Invitrogen, CA), and samples were treated with DNase I (TaKaRa Bio, DaLian, China). Two micrograms of total RNA were reverse transcribed using a cdna synthesis kit (Toyobo, Japan) according to the manufacturer s instructions. qrt-pcr was carried out using a SYBR Green Real-Time PCR Master Mix (Toyobo) on the DNA Engine Opticon 2 Real-Time PCR Detection System (MJ Research). The tobacco actin or cotton histone 3 gene was used as an internal control. The primers used in the assay are listed in Supplemental Table 2. Protein Expression and Purification The coding sequences of GhMLP28 and GhERF6 were amplified using the specific primers shown in Supplemental Table 2 and cloned into plasmid Molecular Plant 8, , March 2015 ª The Author

10 Interaction of MLP28 and ERF6 in Defense Response Figure 9. Expression of PDF1.2 and PR5 Genes in GhMLP28-Overexpressing and GhMLP28-Silenced Plants. (A) Expression levels of GhPDF1.2 and GhPR5 in GhERF6- or GhMLP28-silenced cotton plants. (B) Expression levels of NtPDF1.2 and NtPR5 in GhMLP28 transgenic tobacco lines (L1, L2, and L3). Error bars indicate the SD of three technical replicates within one biological experiment. Three biological repeats were performed. pet28a and pgex6p-1, respectively. The plasmid constructs were introduced into the BL21 (DE3) strain of Escherichia coli by electroporation. The bacterial cells harboring pet28a-ghmlp28 plasmid were cultured in Luria-Bertani (LB) medium supplemented with 100 mg l 1 kanamycin. His-tagged GhMLP28 proteins were purified using Ni-NTA beads (Qiagen, CA). BL21 cells harboring the pgex6p-1-gherf6 construct were cultured in LB medium supplemented with 100 mg l 1 ampicillin. The GST-tagged recombinant GhERF6 proteins were purified by glutathione Sepharose 4B (GE Healthcare) according to the manufacturer s instructions. Pull-Down Screening Assay and Mass Spectrum-Based Protein Identification The in vitro pull-down screening assay was performed according to the protocol provided with the Pull-Down HIS Protein Kit (Invitrogen). Histagged GhMLP28 proteins bound to an Ni-NTA affinity column were used as bait and total proteins extracted from cotton roots were used as prey. Interacting proteins were separated on 1D or 2D SDS-PAGE gels. Protein bands or spots were excised manually from the gels followed by in-gel digestion with trypsin (Promega, Madison, MI) for 24 h at 37 C. The mass spectra analysis was carried out according to the method of Zhao et al. (2010). Yeast Two-Hybrid Assay The Matchmaker Gold Yeast Two-Hybrid System was used according to the manufacturer s instructions (Clontech, Palo Alto, CA). The coding region of GhMLP28 was cloned into the BD vector pgbkt7 and the resulting construct BD-GhMLP28 was used as bait. The coding region of ERF6, LRR, , or CDPK genes of cotton was cloned into the AD vector pgadt7 to produce the prey constructs. The bait and each of the prey constructs were co-transformed into the yeast strain AH109 (Clontech). Interactions were visually detected using an X-gal filter assay. Immunoblot Analysis Roots of wild-type and GhERF6-silenced cotton plants challenged with or without V. dahliae were ground into fine powders in liquid nitrogen. The nuclear and cytoplasmic proteins were extracted according to the method described by Han et al. (2013). After quantification by Bradford assay (Bio- Rad protein assay kit), 20 mg of nuclear or cytoplasmic proteins was subjected to SDS-PAGE. An immunoblot experiment was performed using the antibodies (1:2000 dilution) raised against GhMLP28, b-actin (EarthOx, San Francisco, CA) (1:5000 dilution) and Histone 3 (EarthOx) (1:5000 dilution) as the primary antibodies, and horseradish peroxidaseconjugated goat anti-rabbit/mouse IgG (1:3000 dilution; Sungene Biotechnology, Tian Jin) as the secondary antibody. EMSA EMSA was carried out using biotin-labeled probes and the Pierce Light Shift Chemiluminescent EMSA kit (Thermo Scientific, Rockford, IL). Histagged GhMLP28 and GST-tagged GhERF6 proteins were expressed and purified as described above. Promoter sequences and synthesized DNA fragments containing two tandem repeats of the GCC box were used as probes. The binding reaction was carried out in a 20-ml reaction mixture at room temperature for 30 min and then applied onto a 6% native polyacrylamide gel in 0.53 Tris-borate/EDTA buffer. The bands were detected according to the instructions provided with the EMSA kit. Firefly LCI Assay LCI assay was conducted using an Agrobacterium-mediated transient expression system (Chen et al., 2008). Briefly, the ORFs of GhMLP28 and GhERF6 were ligated to the DNA sequence encoding the C-terminal end of dissected Luc in the vector pcambia-cluc, and the resulting constructs were named CLuc-GhERF6 and CLuc-GhMLP28, respectively. The ORFs of GhMLP28 and GhERF6 were also ligated to the coding region of the N-terminal end of dissected Luc in plasmid pcambia-nluc; the resulting constructs were named NLuc-GhMLP28 and NLuc-GhERF6, respectively. The empty vectors were transformed into the A. tumefaciens strain GV3101 as mock constructs. Equal amounts of Agrobacterium cultures harboring each of the CLuc and NLuc constructs at an OD 600nm = 1.0 in the infiltration buffer (10 mm MES ph 5.6, 10 mm MgCl 2, and 200 mm acetosyringone) were mixed and injected into fully expanded tobacco (N. benthamiana) leaves by using a needleless syringe. The tobacco plants were grown in the dark for 24 h and then exposed to a 16 h light/8 h dark cycle for 48 h at 23 C. The detached leaves were sprayed with 1 mm luciferin (Promega), and the Luc signal was captured with a low-light cooled charge-coupled device camera (Night owl LB985, Berthold Technologies, Germany) and relative Luc activity was measured. Quantitative analysis was performed using the IndiGo software (Berthold Technologies). DLR Assay The coding region of GhERF6 was cloned into the expression vector prt- BD to generate the BD-GhERF6 effecter plasmid. Isolation and transformation of Arabidopsis protoplasts were performed as described by He et al. (2007). 53 Gal4-Luc was used as reporter. The DLR assay was conducted according to the method described by Ohta et al. (2001). Renilla Luc gene was used as an internal control. The co-transformed cultures were placed in the dark for 16 h at 24 C. The Luc assay was carried out using the Promega DLR assay system and values were measured by the GloMax luminometer (Bio-Rad). Promoter Isolation and Transient Expression Assay The promoter sequence of the GhPDF1.2 or GhPR5 gene was isolated by PCR-based genome walking. Primers were designed based on the EST sequences of GrPDF1.2 or GhPR5. The gateway cloning system was used following the instruction manual (Invitrogen). The 1134 bp 408 Molecular Plant 8, , March 2015 ª The Author 2015.

11 Interaction of MLP28 and ERF6 in Defense Response Molecular Plant Figure 10. Intracellular Distribution of GhMLP28 Proteins. (A) Subcellular localization of GhMLP28-GFP proteins in BY2 cells. The fluorescence of GFP- GhMLP28 was visualized under a fluorescent scope at 488 nm. n, nucleus. Bar, 25 mm. (B) Dependence of GhMLP28 nuclear distribution on the presence of GhERF6. Nuclear or cytoplasmic proteins were extracted from the roots of control (TRV:00) (lanes 1, 3, 5, and 7) or GhERF6- silenced cotton plants (lanes 2, 4, 6, and 8). Lanes 1, 2, 5, and 6, without inoculation; lanes 3, 4, 7, and 8, inoculated with V. dahliae; Histone 3, a marker of nuclear proteins; b-actin, a marker of cytoplasmic proteins; CBB, Coomassie brilliant blue staining of nuclear and cytoplasmic proteins. and 1093 bp promoter sequences of the two genes were respectively cloned into vector pgwb435 (Invitrogen) containing a Luc reporter gene to generate GhPDF1.2 Pro :Luc and GhPR5 Pro :Luc as reporters. The A. tumefaciens strain GV3101 harboring pgwb435-ghpdf1.2 Pro, pgwb435-ghpr5 Pro or pgwb435 was cultured in LB medium supplemented with 50 mg l 1 spectinomycin and 50 mg l 1 rifampicin. The coding sequence of GhMLP28 or GhERF6 was cloned into ppzp111 to generate 35S:GhMLP28 or 35S:GhERF6 as effectors. A. tumefaciens strain GV3101 containing ppzp111-ghmlp28, ppzp111-gherf6, or ppzp111 was cultured in the same medium supplemented with 50 mg l 1 chloramphenicol and 50 mg l 1 rifampicin. For expression assay in cotton, the Agrobacterium cells harboring pgwb435-pdf1.2 Pro or pgwb435-pr5 Pro were injected into leaves of the wild-type and GhMLP28- or GhERF6-silenced plants by using a needleless syringe. Agrobacterium cells harboring pbi121-luc driven by 35S promoter were injected into the other side of the same leaf for use as a positive control. The luminescence was measured using the same procedure described for the LCI assay. Relative luminescence was measured after 48 h. For the expression assay in tobacco, Agrobacterium cells harboring the indicated plasmids were mixed and treated with infiltration buffer for 3 h and then co-injected into the leaf cells. The luminescence was measured using the same procedure described for the LCI assay. Relative luminescence was measured after 48 h. Subcellular Localization The coding sequence of GhMLP28 was fused to that of GFP and cloned under the control of the 35S promoter in the plant expression vector ppzp111. The resulting plasmid ppzp111-ghmlp28-gfp was transformed into A. tumefaciens strain EHA105 and introduced into BY2 cells by Agrobacterium-mediated transformation (An, 1985). Cells were examined under a confocal laser microscope (Leica SP8, Leica Microsystems). ACCESSION NUMBERS Sequence data for the genes described in this study can be found in the GenBank/EMBL database under the following accession numbers: GhMLP28 (DQ ), GhERF6 (AY ), GhPR5 (AF ), NtPDF1.2 (X ), NtPR5 (X ) and the Phytozome database under the accession number: GrPDF1.2 (Gorai.002G ). SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online. FUNDING This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDB ACKNOWLEDGMENTS We thank Dr Yule Liu (Tsinghua University) for kindly providing us with VIGS vector. We are also grateful to Dr Jun Liu and Dr Jie Zhang (Institute of Microbiology, Chinese Academy of Sciences) for critical reading of the manuscript and helpful discussion. No conflict of interest declared. Received: September 12, 2014 Revised: November 6, 2014 Accepted: November 12, 2014 Published: December 27, 2014 REFERENCES An, G. (1985). High efficiency transformation of cultured tobacco cells. Plant Physiol. 79: Brown, R.L., Kazan, K., McGrath, K.C., Maclean, D.J., and Manners, J.M. (2003). A role for the GCC-box in jasmonate-mediated Molecular Plant 8, , March 2015 ª The Author