Arabidopsis AtERF014 acts as a dual regulator that differentially modulates immunity against Pseudomonas syringae pv. tomato and Botrytis cinerea

ERF transcription factors play critical roles in plant immune responses. Here, we report the function of AtERF014, a nucleus-localized transcriptional activator, in Arabidopsis immunity. Expression of AtERF014 was induced by Pseudomonas syringae pv. tomato (Pst) and Botrytis cinerea (Bc). AtERF014-overexpressing (OE) plants displayed increased Pst resistance but decreased Bc resistance, whereas AtERF014-RNAi plants exhibited decreased Pst resistance but increased Bc resistance. After Pst infection, expression of salicylic acid (SA)-responsive genes AtPR1 and AtPR5 in AtERF014-OE plants and of a jasmonic acid/ethylene-responsive gene AtPDF1.2 in AtERF014-RNAi plants was intensified but expression of AtPDF1.2 in AtERF014-OE plants and of AtPR1 and AtPR5 in AtERF014-RNAi plants was weakened. After Bc infection, expression of AtPR1 and AtPR5 in AtERF014-OE plants was attenuated but expression of AtPR1, AtPR5 and AtPDF1.2 in AtERF014-RNAi plants was strengthened. Pathogen- and flg22-induced ROS burst, expression of PTI genes and SA-induced defense were partially suppressed in AtERF014-RNAi plants, whereas pathogen-induced ROS and flg22-induced immune response were strengthened in AtER014-OE plants. Altered expression of AtERR014 affected expression of pectin biosynthetic genes and pectin content in AtERF014-RNAi plants was decreased. These data demonstrate that AtERF014 acts as a dual regulator that differentially modulates immunity against Pst and Bc in Arabidopsis.

Scientific RepoRts | 6:30251 | DOI: 10.1038/srep30251 The AP2/ERF superfamily is a large plant-specific TF family 18 and is characterized by the presence of one or two AP2/ERF domains that consist of 58 or 59 conserved amino acid residues 19 . The AP2/ERF superfamily can be divided into three subfamilies, namely AP2, RAV and ERF, and 122 out of 147 members in Arabidopsis are ERF TFs 18,20,21 . The ERFs can act as transcription activators or repressors; for example, Arabidopsis AtERF1, AtERF2 and AtERF5 are activators while AtERF3, AtERF4 and AtERF7 are repressors 22 . Generally, ERF TFs regulate the expression of genes, whose promoters harbor at least one core GCC box, and most of these GCC box-containing genes are defense-related 19,[23][24][25] .
AtERF014, a member of group II in the ERF family 21 , was recently reported to be involved in pectin biosynthesis 43 . However, the biological function of AtERF014 remains elusive. The present study focused on the function of AtEFR014 in disease resistance to P. syringae pv. tomato DC3000 and B. cinerea. Functional analyses using AtERF014-OE and AtERF014-RNAi lines suggest that AtERF014 acts as a positive regulator of resistance against Pst DC3000 but functions as a negative regulator of resistance to B. cinerea. Furthermore, AtERF014 is involved in flg22-induced PTI response and SA-induced defense response. Our data demonstrate that AtERF014 is a dual regulator that differentially modulates immunity against P. syringae pv. tomato DC3000 and B. cinerea in Arabidopsis.

Results
AtERF014 is responsive to pathogen infection and defense signaling hormones. To explore the involvement in disease resistance, we examined whether expression of AtERF014 could be induced by pathogen infection and defense signaling hormones such as SA and JA. As shown in Fig. 1A, the transcript level of AtERF014 increased as early as 12 hr post-inoculation (hpi), peaked with ~6 folds at 24 hpi, maintained at relatively higher level at 48 hpi and then decreased to basal level at 72 hpi after infection by Pst DC3000. Similar kinetics of change in the transcript level of AtERF014 was observed in B. cinerea-infected plants, showing a peak of 7 folds at 24 hpi (Fig. 1B). In addition, the expression of AtERF014 was also induced by SA and MeJA but showed different patterns. In SA-treated plants, the transcript level of AtERF014 increased rapidly at 3 hr post-treatment (hpt), peaked with 6 folds at 12 hpt and maintained at relatively higher level until 24 hpt (Fig. 1C). By contrast, the transcript level of AtERF014 in methyl jasmonate (MeJA)-treated plants increased gradually and peaked with 6 folds at 24 hpt (Fig. 1C). These data suggest that AtERF014 is responsive to pathogen infection and defense signaling hormones.
AtERF014 is a transcriptional activator that is localized in nucleus. The biochemical characters of AtERF014 protein were examined by analyzing the transactivation activity in yeast and subcellular localization in planta. In transactivation activity assay, yeasts carrying pBD-AtERF014, pBD-GAL4 (a positive control) or pBD empty vector (a negative control) grew on SD/Trpmedium but only yeasts carrying pBD-AtERF014 or pBD-GAL4 exhibited β -galactosidase activity, as revealed by the production of blue pigment after addition of X-α -gal (Fig. 1D). In subcellular localization assay, the GFP alone was seen ubiquitously in cells without specific compartmental localization, whereas the GFP::AtERF014 fusion was solely localized in nucleus, co-localized with the known nucleus marker RFP-H2B protein 44 (Fig. 1E). These results indicate that AtERF014 may be a transcriptional activator that is localized in nucleus.

Altered expression of AtERF014 does not affect the growth and development in AtERF014-OE and
AtERF014-RNAi plants. To better understand the biological function of AtERF014, we generated transgenic lines with overexpression or RNA interfering (RNAi)-mediated suppression of AtERF014. Two homozygous single-copy AtERF014-OE and AtERF014-RNAi lines (T3 generation) were chosen for further studies. The transcript levels of AtERF014 in AtERF014-OE lines were 6.56 and 5.84 times higher than that in WT while the transcript levels in AtERF014-RNAi lines were 18% and 29% of that in WT ( Fig. 2A). The AtERF014-OE and AtERF014-RNAi plants grew and developed normally, indistinguishable from WT, at vegetable and reproductive stages (Fig. 2B). Thus, it is likely that altered expression of AtERF014 does not affect the growth and development in AtERF014-OE and AtERF014-RNAi plants, implying a limited role for AtERF014 in growth and development. Altered expression of AtERF014 affects the basal immunity in AtERF014-OE and AtERF014-RNAi plants against Pst DC3000. Having shown that the expression of AtERF014 was induced by Pst DC3000, we first examined whether altered expression of AtERF014 affected the resistance of AtERF014-OE and AtERF014-RNAi plants to this bacterial pathogen. At 4 days post-inoculation (dpi), typical Pst DC3000-provoked disease with chlorotic lesion was seen in WT plants (Fig. 3A). At the same time, AtERF014-RNAi plants displayed much severe disease with extensive chlorotic lesion while AtERF014-OE plants showed less severe disease (Fig. 3A). Accordingly, bacterial populations in AtERF014-RNAi plants were 7 and 21 folds higher while the populations in AtERF014-OE plants were 13 and 32 times lower, as compared to those in WT at 2 and 4 dpi, respectively (Fig. 3B). These data indicate that overexpression of AtERF014 leads to an increased resistance while suppression of AtERF014 results in a decreased resistance against Pst DC3000.
Altered expression of AtERF014 affects the basal immunity in AtERF014-OE and AtERF014-RNAi plants against B. cinerea. We next assessed whether altered expression of AtERF014 affected the resistance of AtERF014-OE and AtERF014-RNAi plants to B. cinerea, a necrotrophic fungus causing grey mold disease. After infection, typical B. cinerea-provoked disease symptom was seen in WT at 4 dpi (Fig. 4A). More severe disease symptoms were observed on leaves of AtERF014-OE plants but less disease was seen on leaves of AtERF014-RNAi plants, compared with that in WT (Fig. 4A). Similarly, the AtERF014-OE plants supported greater growth of B. cinerea, showing 1.4 and 1.1 times higher, while the AtERF014-RNAi plants supported lower growth, resulting in 60% of reduction, as compared with that in WT, at 48 hpi, respectively (Fig. 4B). Chlorophyll Yeasts harboring pBD-AtERF014, pBD empty vector (a negative control) and pBD-GAL4 (a positive control) were grown on SD/Trp − medium and β -galactosidase activity was examined by addition of X-α -gal. (E) AtERF014 is localized in nucleus. Leaves of Nicotinana benthamiana plants were collected at 24 hr after infiltration of agrobacteria and photos were taken in dark field for GFP and RFP, bright field for cell morphology and in combination (merged), respectively.
Scientific RepoRts | 6:30251 | DOI: 10.1038/srep30251 content in AtERF014-OE plants decreased gradually with disease development and exhibited 30-35% lower while chlorophyll content in AtERF014-RNAi plants decreased slowly and showed 6-9% higher, than that in WT, at 10 dpi (Fig. 4C). Furthermore, more AtERF014-OE plants died (38-41% of death) while more AtERF014-RNAi plants survived (7-11% of death), as compared to that in WT (16% of death) (Fig. 4D). These results indicate overexpression of AtERF014 weakens while suppression of AtERF014 strengthens the resistance against B. cinerea. Altered expression of AtERF014 affects Pst DC3000-and B. cinerea-induced defense response in AtERF014-OE and AtERF014-RNAi plants. To examine whether altered expression of AtERF014 affected the defense response, we analyzed and compared the patterns for accumulation of reactive oxygen species (ROS) and expression of defense genes among WT, AtERF014-OE and AtERF014-RNAi plants after infection by Pst DC3000 or B. cinerea. In in situ detection of ROS assays, accumulation of superoxide anion and H 2 O 2 in leaves was detected by nitroblue tetrazolium (NBT) 45 and 3, 3-diaminobenzidine (DAB) staining 46,47 , respectively. Without pathogen challenge, no difference in accumulation of superoxide anion and H 2 O 2 was seen among WT, AtERF014-OE and AtERF014-RNAi plants (Fig. 5A,B). At 24 hr after infection by Pst DC3000 or B. cinerea, accumulation of superoxide anion and H 2 O 2 in inoculated leaves increased obviously except the case of H 2 O 2 in B. cinerea-infected leaves (Fig. 5A,B). However, more staining for superoxide anion and H 2 O 2 in AtERF014-OE leaves while less in AtERF014-RNAi leaves was observed, as compared to that in WT (Fig. 5A,B). On the other hand, the expression levels of some selected defense genes including AtPR1, AtPR3, AtPR5 and AtPDF1.2 in AtERF014-OE and AtERF014-RNAi plants were comparable to those in WT without infection with Pst DC3000 or B. cinerea (Fig. 5C,D). As compared with those in WT, higher expression levels of AtPR1 and AtPR5 and a lowered expression level of AtPDF1.2 in AtERF014-OE plants were observed, whereas lowered expression levels of AtPR1 and AtPR5 and a higher expression level of AtPDF1.2 in AtERF014-RNAi plants were detected, at 24 hr after infection by Pst DC3000 (Fig. 5C,D). By contrast, lowered expression levels of AtPR1 and AtPR5 and a higher expression level of AtPDF1.2 in AtERF014-OE plants were seen, while higher expression levels of AtPR1 and AtPR5 and a lowered expression level of AtPDF1.2 in AtERF014-RNAi plants were observed, as compared to those in WT at 24 hr after infection with B. cinerea (Fig. 5C,D). There was no difference in pathogen-induced expression of AtPR3 among WT, AtERF014-OE and AtERF014-RNAi plants (Fig. 5C,D). Taken together, these Altered expression of AtERF014 affects flg22-triggered immune response in AtERF014-OE and AtERF014-RNAi plants. To examine whether AtERF014 has a function in Arabidopsis PTI response, we compared the occurrence of flg22-induced ROS burst and flg22-induced expression of PTI marker genes among WT, AtERF014-OE and AtERF014-RNAi plants. In ROS burst assay, no significant ROS burst was observed in WT, AtERF014-OE and AtERF014-RNAi leaves without treatment of flg22 (Fig. 6A). Compared with a significant flg22-induced ROS burst in WT leaves at 6 min, much enhanced flg22-induced ROS bursts were seen in AtERF014-OE leaves while only a much reduced ROS burst was detected in AtERF014-RNAi leaves after addition of flg22 (Fig. 6A). Similarly, the expression levels of FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (AtFRK1) and AtWRKY53, two well-known PTI-responsive genes 48,49 , in AtERF014-OE and AtERF014-RNAi leaves were comparable to those in WT without flg22 treatment or at 0 hr after addition of flg22 (Fig. 6B,C), suggesting that altered expression of AtERF014 did not affect the expression of these two PTI genes. At 60 min after treatment, flg22 significantly upregulated the expression of AtFRK1 and AtWRKY53 in WT, AtERF014-OE and AtERF014-RNAi leaves with distinct patterns (Fig. 6B,C). As compared with the levels in flg22-treated WT leaves, the flg22-upregulated expression of AtFRK1 and AtWRKY53 in AtERF014-OE leaves was further increased, resulting in 1.4-1.6 times and 4-5 times of increases, whereas the expression of these two genes in AtERF014-RNAi leaves was markedly suppressed, leading to 25-30% and 50-60% of reduction (Fig. 6A,B). These data indicate that overexpression of AtERF014 strengthens while suppression of AtERF014 attenuates the flg22-induced PTI response, implying a function for AtERF014 in PTI response in Arabidopsis.

Suppression of AtERF014 attenuates the SA-induced defense response in AtERF014-RNAi plants.
To explore whether AtERF014 is required for SA-induced defense response, we evaluated the SA-induced resistance between AtERF014-RNAi and WT plants by comparing disease phenotype and bacterial population in water-and SA-treated plants after inoculation with Pst DC3000. SA-treated WT plants showed less disease than water-treated plants at 4 dpi (Fig. 7A). Disease in SA-treated AtERF014-RNAi plants was less than that in water-treated plants but was much severe than that in SA-treated WT plants (Fig. 7A). Accordingly, in planta growth of Pst DC3000 in inoculated leaves was consistent with the disease severity observed. At 0 dpi, no significant difference in bacterial growth was seen between AtERF014-RNAi and WT plants treated with or without 1 mM SA. At 3 dpi, the bacterial titers in inoculated leaves of water-treated WT, AtERF014-RNAi-1 and AtERF014-RNAi-2 plants increased to 1.10 × 10 7 CFU/cm 2 , 1.12 × 10 8 CFU/cm 2 and 2.04 × 10 8 CFU/cm 2 , respectively, whereas the titers in inoculated leaves of SA-treated WT, AtERF014-RNAi-1 and AtERF014-RNAi-2 plants were 8.51 × 10 5 CFU/cm 2 , 2.29 × 10 7 CFU/cm 2 and 2.88 × 10 7 CFU/cm 2 , respectively, showing 12.1, 5.6 and 6.7 times lower than those in corresponding water-treated plants (Fig. 7B). The expression levels of AtPR1 and AtPR5 in AtERF014-RNAi plants were comparable to those in WT without SA treatment; their expression was significantly induced by SA in both AtERF014-RNAi and WT plants (Fig. 7C). However, the SA-induced expression of AtPR1 and AtPR5 in AtERF014-RNAi plants was weakened as compared to those in WT (Fig. 7C). These results indicate that suppression of AtERF014 weakens the SA-induced defense response in AtERF014-RNAi plants.

Altered expression of AtERF014 affects the pectin content and expression of pectin biosynthetic genes in AtERF014-OE and AtERF014-RNAi plants.
It was previously reported that overexpression of AtERF014 affected the expression of pectin biosynthetic genes and pectin deposition in cultivated Arabidopsis cells 42 . We thus analyzed and compared the pectin contents and expression levels of the pectin biosynthetic gene in WT, AtERF014-OE, and AtERF014-RNAi plants. As shown in Fig. 8A, the pectin contents in AtERF014-RNAi plants were measured to be 13.06 and 13.15 mg/g FW, respectively, significantly lower than that in WT (15.87 mg/g FW); however, the pectin contents in AtERF014-OE plants, measured as 16.57 and 16.29 mg/g FW, respectively, were comparable to that in WT. Accordingly, the expression levels of AtASX2, AtGAE1, AtMUM4, AtQUA1 and AtUGP1, which are involved in pectin biosynthesis [50][51][52][53][54][55] , in AtERF014-OE plants were significantly higher but the levels in AtERF014-RNAi plants were markedly lower than those in WT (Fig. 8B).
These results indicate that altered expression of AtERF014 affects the pectin biosynthetic pathway, leading to altered pectin contents in AtERF014-OE and AtERF014-RNAi plants.

Discussion
Fifteen ERF TFs are classified into group II 21 and some of them have recently been functionally characterized. Overexpression of AtERF012 (DREB26) resulted in deformed plants, indicating an involvement in developmental processes 56 . AtERF06 (AtRAP2.1) was found to function as a transcriptional repressor that plays a role in abiotic stress response 57 . AtERF011 (AtDEAR1) acts as an upstream regulator that mediates plant defense and freezing stress responses in Arabidopsis 40 while AtERF018 seems to be involved in JA signaling pathway in response to wounding 58 . In the present study, we explored in detail the biological function of AtERF014 in immunity against different pathogens using AtERF014-OE and AtERF014-RNAi lines and found that AtERF014 acts as a dual regulator that differentially modulates immunity against Pst DC3000 and B. cinerea. These findings, together with a Group II of the ERF subfamily consists of three subgroups, IIa, IIb, and IIc 21 . ERF TFs in this group all contain a common AP2/ERF domain at N-terminal and a conserved motif CMII-1 at C-terminals but also have several additional conserved motifs 21 . The six IIa members contain a CMII-2 motif that is similar to the EAR (ethylene response factor-associated amphiphilic repression) motif, which functions as a repression domain 22,59 . For instance, AtERF06 (AtRAP2.1) and AtERF011 (AtDEAR1) in subgroup IIa were experimentally demonstrated to be active transcriptional repressors that repress the transcription of some stress-responsive genes whose promoters harbor DRE (dehydration-responsive elements)/CRT (dehydration-responsive element) cis-elements 40,57 . By contrast, the members in subgroup IIb, except AtERF#015, have a CMII-3 motif at the C-terminal instead of the EAR-like CMII-2 motif 21 . ERF TFs lacking an EAR motif are expected to act as transcriptional activators. We found that AtERF014 displayed transactivation activity in yeasts (Fig. 1D), implying that AtERF014 may be a transcriptional activator. Another member of subgroup IIb, AtERF012 (DREB26), was also found to have transactivation activity in yeast 56 . Thus, it seems likely that the group II ERFs have distinct biochemical activity and can act as transcriptional activators or repressors. Further transactivation assays in in vivo systems like Arabidopsis protoplasts or tobacco leaves are required to confirm the transactivation activity of AtERF014 and its closely related members in group II.
We showed in the present study that AtERF014 plays differential functions in immunity against Pst DC3000 and B. cinerea, demonstrating that AtERF014 is a dual regulator of immunity against these two pathogens. It is generally accepted that immune response against (hemi)biotrophic pathogens such as Pst DC3000 is modulated (B,C) flg22-induced expression of the PTI marker genes AtFRK1 (B) and AtWRKY53 (C). Data presented are the means ± standard deviation from three independent experiments and * above the columns indicate significant differences at p < 0.05 level between AtERF014-OE/AtERF014-RNAi and WT plants.
Scientific RepoRts | 6:30251 | DOI: 10.1038/srep30251 through the SA signaling while immune response against necrotrophic pathogens like B. cinerea is regulated by the JA/ET signaling [60][61][62] . Antagonistic interaction between the SA and JA/ET signaling pathways often occurs and allows plants to mount appropriate defense responses against different invading pathogens 60,62-64 . The expression of AtERF014 was induced by Pst DC3000 and B. cinerea as well as by SA and MeJA (Fig. 1A-C). However, overexpression of AtERF014 led to increased resistance to Pst DC3000 but decreased resistance to B. cinerea, whereas suppression of AtERF014 resulted in decreased resistance to Pst DC3000 but increased resistance to B. cinerea (Figs 3 and 4), indicating that AtERF014 is a positive regulator of immunity to Pst DC3000 but a negative regulator of immunity to B. cinerea. This is similar to the functions of AtERF1, AtERF5 and AtERF6, which have opposite functions in immunity against Pst DC3000 and necrotrophic fungi including B. cinerea and Alternaria brassicicola 26,32,33 . The expression of SA-responsive and JA-responsive defense genes exhibited opposite patterns in AtERF014-OE and AtERF014-RNAi plants after infection of Pst DC3000 (Fig. 5C), indicating that AtERF014 is involved in modulating an antagonistic interaction between the SA and JA/ET signaling pathways, resulting in the expression of corresponding defense genes. It was previously shown that some of the B3 group members including ERF1, AtERF5, AtERF6, AtERF14 and ORA59 act as regulators of the JA/ET signaling pathway 26,27,[30][31][32]42 . In contrast, only a few of ERFs, e.g. AtERF15 and AtERF5 33,35 , were shown to be involved in the SA signaling pathway. The involvement of AtERF014 in the SA signaling pathway can be further validated by the facts that the SA-induced Pst DC3000 resistance and SA-upregulated expression of defense genes were partially suppressed in AtERF014-RNAi plants (Fig. 7). The pathogen-induced ROS in AtERF014-OE plants (Fig. 5A,B) may function differentially in immunity against Pst DC3000 or B. cinerea. In the AtERF014-OE plants, the pathogen-induced ROS may largely contribute to signaling in immunity against Pst DC3000 65 , whereas this pathogen-induced ROS may benefit the infection by B. cinerea 66 , correlating with the altered resistance against these pathogens (Figs 3 and 4). Notably, B. cinerea did not induce significant accumulation of H 2 O 2 but caused remarkable accumulation of superoxide anion in infected Arabidopsis plants (Fig. 5A,B). It was suggested that the kinetics of ROS accumulation is the better indicator of defense response than the absolute levels of ROS in many interactions 66 . Quantitative analysis of the kinetics of ROS accumulation in AtERF014-OE and AtERF014-RNAi plants after pathogen infection should clarify the involvement of H 2 O 2 in AtERF014-mediated defense response against B. cinerea. In addition, the flg22-induced ROS burst and the expression of PTI marker genes AtFRK1 and AtWRKY53 were fostered in AtERF014-OE plants but was weakened in AtERF014-RNAi plants (Fig. 6), demonstrating that AtERF014 plays a role in innate immunity. AtERF5 and AtERF15 were also found to be involved in flg22-or chitin-induced PTI 33,35 .
Plant cell walls consist of three major components cellulose, hemicelluloses and pectins, and pectins make up approximately 50% of Arabidopsis leaf cell walls 67 . It was previously shown that overexpression of AtERF014 coordinately activated the pectin biosynthetic pathway genes and increases the pectin content in cultured Arabidopsis cells 43 . We found in the present study that the pectin content in AtERF014-RNAi plants was significantly decreased due to the downregulated expression of pectin biosynthetic gene such as AtASX2, AtGAE1, AtMUM4, AtQUA1 and AtUGP1 50-55 (Fig. 8). Although overexpression of AtERF014 upregulated the expression of these pectin biosynthetic genes, the pectin content in AtERF014-OE plants was comparable to that in WT (Fig. 8). This is contrary to the observation in cultured Arabidopsis cells, where overexpression of AtERF014 led to increased pectin content 43 .
Plant cell walls act as preformed structural barriers against pathogen entry and modifications of cell walls can induce plant defense responses, which are able to reduce pathogen penetration and growth 68 . In the present study, we found that suppression of AtERF014 resulted in reduced pectin content in AtERF014-RNAi plants as compared to WT (Fig. 8A); however, the contribution of the reduced pectin content to the resistance against Pst DC3000 and B. cinerea may differ (Figs 3 and 4). It seems reasonable that the reduced pectin content in AtERF014-RNAi plants may be responsible for the decreased resistance against Pst DC3000, as decrease in pectin content should weaken the cell wall strength and integrity and thereby make it more susceptible to Pst DC3000 infection. This is in agreement with a recent observation that mutation in GAE6, coding for a glucuronate 4-epimerase involved in the biosynthesis of pectin, impaired the immunity to P. syringae pv. maculicola ES4326 69 . However, the Arabidopsis powdery mildew-resistant (pmr) 5 and pmr6 mutant plants, whose cell walls contain enriched pectin, are fully susceptible to Pst DC3000, as the WT plants 70,71 , indicating that increased pectin content in pmr5 and pmr6 plants is not responsible for the resistance to this bacterial pathogen. On the other hand, most of the necrotrophic fungal pathogens like B. cinerea secrete a variety of cell wall-degrading enzymes including polygalacturonases that can degrade pectin in cell walls during pathogenesis. B. cinerea was also shown to metabolize pectin and probably utilize pectin as a carbon source during in planta growth 72,73 . In this regard, reduced pectin content in AtERF014-RNAi plants (Fig. 8A) may contribute to increased resistance to B. cinerea (Fig. 4A), as the AtERF014-RNAi plants supported less in planta growth of B. cinerea in comparison to that in WT (Fig. 4B). This finding is different from the observation that the gae1 gae6 plants, which are reduced in pectin Data presented are the means ± standard deviation from three independent experiments and * above the columns indicate significant differences at p < 0.05 level between AtERF014-RNAi and WT plants.
content, impaired the resistance to specific B. cinerea isolates and allowed enhanced in planta growth of these specific isolates 69 . The AtERF014OE plants had similar pectin content to WT (Fig. 8A) but showed enhanced susceptibility to B. cinerea and in planta fungal growth (Fig. 3A,B), indicating that utilization of pectin as a carbon source may not contribute to immunity against B. cinerea. It is likely that the pectin content itself may not be a contributing factor that determines the immunity to pathogens; instead, the degree and pattern of pectin modification such as methylesterification are critical to immunity against P. syringae [74][75][76] or B. cinerea 77,78 . It was previously shown that pectin fragments known as oligogalacturonides can trigger plant immune responses 69,79,80 . How AtERF014 regulates the biosynthesis of pectin and thereby the immunity against different pathogens need to be further investigated.
The involvement of ERFs in plant growth and development has been well-documented 81 . No morphological and developmental changes (Fig. 2) and altered expression of defense genes (Fig. 5C,D) were observed in the AtERF014-OE and AtERF014-RNAi plants, indicating that AtERF014 may not play a significant role in growth and development. This is in consistent with the observation that overexpression or suppression of AtERF5, AtERF6 or AtERF15 did not result in any distinguishable morphological and developmental changes to WT plants 33,35,82 .

Conclusion
Our data presented in this study demonstrate that AtERF014 is a nucleus-localized transcriptional factor that acts as a dual regulator, which differentially modulates immunity against Pst DC3000 and B. cinerea, probably through regulating pectin biosynthesis pathway. However, how AtERF014 regulates plant immunity against Pst DC3000 and B. cinerea is largely unknown. This question can be explored by the characterization of AtERF014-mediated regulon and its direct target genes. Comparative RNA-seq analysis of the AtERF014-RNAi and WT plants and ChIP-seq analysis of transgenic palnts with expression of epitope tagged AtERF014, with or without pathogen infection, will not only lead to the identification of AtERF014-dependent differentially expressed regulon, but also provide information on the AtERF014 binding sites at genome-wide level. Data from these analyses will definitely be helpful to characterize the direct target genes, putative pathways and transcriptional network that are regulated by AtERF014 during immune response against pathogens. Generation and characterization of transgenic lines. The AtERF014 cDNA was obtained by PCR amplification using primers AtERF014-orf-1F (5′ -ATG GTG AAA ACA CTT CAA-3′ ) and AtERF014-orf-1R (5′ -TCA GCA GAA GTT CCA TAA-3′ ) and cloned into pMD19-T vector. After confirmation by sequencing, the resulting plasmid pMD19-AtERF014 was used as templates for all experiments described below. For construction of overexpression vector, the AtERF014 coding sequence was amplified with a pair of primers AtERF014-orf-2F (5′ -GCG GAATTC ATG GTG AAA ACA CTT CAA AAG AC-3′ , a BamHI site underlined) and AtERF014-orf-2R (5′ -AGA GGGCCC GTA TAA TGA AAG TAG AAT CAG CAG-3′ , a SmaI site underlined) and inserted into pCAMBIA 99-1 under the control of CaMV 35S promoter. For construction of RNAi vector, a fragment was amplified with a pair of primers AtERF014-RNAi-F (5′ -AGA TTAATTAA CCATGG AAC ACT TCA AAA GAC ACC AAA GAG A-3′ , PacI and NcoI sites underlined) and AtERF014-RNAi-R (5′ -GCG GGATCC GGCGCGCC GAG TGC TGC GTC GTA GGC TCT AGC C-3′ , EcoRI and AscI sites underlined) and inserted into vector pGSA1165. Arabidopsis transformation was performed through the floral dip method 83 . Putative single-copy transgenic lines and homozygous lines were obtained by screening for a 3:1 segregation ratio of hygromycin-resistant (Hgr R ) character and 100% Hgr R phenotype in T2 and T3 generations on 1/2 MS medium supplemented with 50 μ g/mL Hgr, respectively.

Transactivation activity and subcellular localization assays. For transactivation activity assay, the
AtERF014 coding sequence was amplified by PCR with a pair of primers AtERF014-y-1F (5′ -AGT GAATTC ATG GTG AAA ACA CTT CAA-3′ , an EcoRI site underlined) and AtERF15-y-1R (5′ -AGT GTCGAC TCA GCA GAA GTT CCA TAA-3′ , a SalI site underlined) and inserted into pBD vector between EcoRI and SalI sites. The resulting plasmid pBD-AtERF014, pBD-GAL4 (as a positive control) and pBD empty vector (as a negative control) were introduced into yeast strain AH109 and the yeast transformants were plated and grown on SD/Trpmedium at 30 °C for 3 days. Transactivation activity was assessed according to the growth status on SD/Trpplate and the production of blue pigment after addition of X-α -gal. For subcellular localization assay, the AtERF014 coding sequence was obtained by PCR with a pair of primers AtERF014-s-1F (5′ -GCG TCTAGA ATG GTG AAA ACA CTT CAA-3′ , a XbaI site underlined) and AtERF15-s-1R (5′ -ATA CCCGGG TCA GCA GAA GTT CCA TAA-3′ , a SmaI site underlined), and inserted into vector pFGC-EGFP between XbaI and SmaI sites. The recombinant plasmid pFGC-GFP-AtERF014 and the empty vector pFGC-EGFP were transformed into Agrobacterium tumefaciens strain GV3101. Agrobacteria harboring pFGC-GFP-AtERF014 or pFGC-EGFP were infiltrated into leaves of N. benthamiana plants expressing a red nuclear marker protein RFP-H2B 44 and the agroinfiltrated leaves were Scientific RepoRts | 6:30251 | DOI: 10.1038/srep30251 collected 24 hr later. Fluorescence signals were excited at 488 nm and detected under a confocal laser scanning microscope (LSM 510 Meta, Zeiss, Oberkochen, Germany) using a 500-530 nm emission filter.

Disease assays and measurement of in planta pathogen growth. Pst DC3000 was grown in
King's B (KB) broth and collected by centrifugation, followed by re-suspending in 10 mM MgCl 2 solution to OD 600 = 0.002. The bacterial inoculation was performed by hand infiltration using 1-ml syringes without needle into rosette leaves and the inoculated plants were kept in sealed containers to facilitate disease development under high humidity. For quantification of in planta bacterial growth, leaf discs from inoculated leaves were collected at different time points and homogenized in 10 mM MgCl 2 . After a series of gradient dilutions, the homogenate was plated on KB plates supplemented with 25 μ g/mL rifampicin and bacterial colonies were counted at 2 days after incubation at 28 °C. B. cinerea strain BO5.10 (provided by Dr. Tesfaye Mengiste, Purdue University, USA) was grown on 2 × V8 agar 84 and spores were collected, resuspended in 1% maltose buffer to a final concentration of 2 × 10 5 spores/mL. Plant inoculation, measurement of chlorophyll content and recoding of dead plants were followed by previously described methods 84,85 . Measurement of in planta fungal growth was performed by analyzing the transcript level of B. cinerea BcActinA gene and comparing with the transcript level of an Arabidopsis actin gene as an internal control according to a reported protocol 35,85 .

Measurement of ROS burst and in situ detection of ROS accumulation. Measurement of ROS burst
was carried out using a luminol-based luminescence method 86 . Briefly, 4-mm leaf discs were floated in 200 μ L H 2 O overnight and then transferred into 100 μ L 100 nM flg22 solution containing 34 μ g/ml luminol (Sigma, St. Louis, MO, USA) and 20 μ g horseradish peroxidase (Sigma, St. Louis, MO, USA) or in solution without luminol as controls. Luminescence was recorded at a 2 min interval over 20 min using a Synergy HT plate reader (Biotek Instruments Inc. Winooski, VT, USA). In situ detection of superoxide anion and H 2 O 2 was carried out by NBT and DAB staining, respectively 45,46 . Accumulation of ROS in stained leaves was visualized by a digital camera.
Measurement of pectin content. Pectin content was determined using a Pectin Measurement kit (Keming Biotechnology Co., Suzhou, China) according to the manufacturer's instruction. Briefly, 100 mg of leaf tissues was immerged into 1 mL sterile water and then ground to homogenate. After centrifugation at 8000 × g at 4 °C for 10 min, the supernatant was used for measurement of pectin.

Statistical analysis.
All experiments were repeated independently for at least three times and data were subjected to statistical analysis using the Student's t-test at p = 0.05 level.