Expression of Vitis amurensis VaERF20 in Arabidopsis thaliana Improves Resistance to Botrytis cinerea and Pseudomonas syringae pv. Tomato DC3000

Ethylene response factor (ERF) transcription factors play important roles in regulating immune responses in plants. In our study, we characterized a member of the ERF transcription factor family, VaERF20, from the Chinese wild Vitis genotype, V. amurensis Rupr “Shuangyou”. Phylogenetic analysis indicated that VaERF20 belongs to group IXc of the ERF family, in which many members are known to contribute to fighting pathogen infection. Consistent with this, expression of VaERF20 was induced by treatment with the necrotrophic fungal pathogen Botrytis cinerea (B. cinerea) in “Shuangyou” and V. vinifera “Red Globe”. Arabidopsis thaliana plants over-expressing VaERF20 displayed enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. Patterns of pathogen-induced reactive oxygen species (ROS) accumulation were entirely distinct in B. cinerea and PstDC3000 inoculated plants. Examples of both salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) responsive defense genes were up-regulated after B. cinerea and PstDC3000 inoculation of the VaERF20-overexpressing transgenic A. thaliana plants. Evidence of pattern-triggered immunity (PTI), callose accumulation and stomatal defense, together with increased expression of PTI genes, was also greater in the transgenic lines. These data indicate that VaERF20 participates in various signal transduction pathways and acts as an inducer of immune responses.


Introduction
In order to protect themselves from a wide variety of pathogens, plants have evolved an innate immunity system, comprising two inter-connected components: pathogen/microbe-associated molecular pattern (PAMP or MAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [1,2]. PTI and ETI play important roles in defense networks, which are mediated by kinases and the production of pathogenesis-related (PR) proteins [2][3][4]. ETI is often accompanied by programmed cell death (PCD), also called the hypersensitive response (HR), which reduces the spread of an invading pathogen [2]. Moreover, ETI can also take part in an active defense response, such as the production of reactive oxygen species [5,6]. Botrytis cinerea, a kind of necrotrophic fungal pathogen, and the causal agent of grey mold disease, can produce the ROS in its plant hosts [7]. Also, ROS play an important role in the interaction between Botrytis cinerea and its plant hosts [8,9].
After infection by a pathogen, plants can activate hormone-mediated signaling pathways to defend against disease and various defense networks are known to be regulated by the phytohormones function of VaERF20. Finally, we measured stomatal aperture and callose accumulation in epidermal peels following flagellin fragment (flg22) and lipopolysaccharides (LPS) treatments in order to obtain deeper insight into the role of VaERF20 in bacterial resistance.

B. cinerea Inoculation of Grape Induces VaERF20 Expression
To investigate the potential roles of VaERF20 in responses to pathogen infection, we measured the expression levels of VaERF20 in both the Chinese wild Vitis genotype, V. amurensis Rupr "Shuangyou" and V. vinifera cv "Red Globe" following inoculation with B. cinerea (Figure 1). VaERF20 transcript levels peaked at 4 h post-inoculation (hpi) and increased in both genotypes after B. cinerea infection compared to the control. over-expressed it in A. thaliana to examine its potential function in resistance to B. cinerea and PstDC3000. We also evaluated the expression of SA, JA and ET related genes after inoculation by B. cinerea and PstDC3000 to elucidate the function of VaERF20. Finally, we measured stomatal aperture and callose accumulation in epidermal peels following flagellin fragment (flg22) and lipopolysaccharides (LPS) treatments in order to obtain deeper insight into the role of VaERF20 in bacterial resistance.

B. cinerea Inoculation of Grape Induces VaERF20 Expression
To investigate the potential roles of VaERF20 in responses to pathogen infection, we measured the expression levels of VaERF20 in both the Chinese wild Vitis genotype, V. amurensis Rupr "Shuangyou" and V. vinifera cv "Red Globe" following inoculation with B. cinerea (Figure 1). VaERF20 transcript levels peaked at 4 h post-inoculation (hpi) and increased in both genotypes after B. cinerea infection compared to the control. and V. vinifera cv "Red Globe" (B) following inoculation with Botrytis cinerea. Data represent the means ± standard deviation from three independent experiments. Asterisks indicate statistical significance (** p < 0.01, one-way ANOVA) between treatment and mock.

VaERF20 Bioinformatic and Sequence Analysis
Phylogenetic analysis indicated that, of the protein sequences examined, VaERF20 is most closely related to AtERF98 and OsERF89 (Figure 2), both of which belong to group IXc (B3) [27]. The importance of some genes from this group in the defense against pathogens has been shown [38]. The VaERF20 nucleotide and deduced amino acid sequences are shown in Supplementary Data S1 and S2, with conserved domains highlighted by red arrows. Expression analysis of VaERF20 in Chinese wild Vitis, V. amurensis Rupr "Shuangyou" (A) and V. vinifera cv "Red Globe" (B) following inoculation with Botrytis cinerea. Data represent the means ± standard deviation from three independent experiments. Asterisks indicate statistical significance (** p < 0.01, one-way ANOVA) between treatment and mock.

VaERF20 Bioinformatic and Sequence Analysis
Phylogenetic analysis indicated that, of the protein sequences examined, VaERF20 is most closely related to AtERF98 and OsERF89 (Figure 2), both of which belong to group IXc (B3) [27]. The importance of some genes from this group in the defense against pathogens has been shown [38]. The VaERF20 nucleotide and deduced amino acid sequences are shown in Supplementary Data S1 and S2, with conserved domains highlighted by red arrows.

VaERF20 Transcript Profile When Over-Expressed in A. thaliana Lines
The potential biological function of VaERF20 in resistance to biotic stress was investigated by over-expressing it under the CaMV 35S promoter-driven in A. thaliana over-expressing plants. A total of 60 independent transgenic lines were generated and confirmed by PCR analysis. Three homozygous T3 lines with the strongest resistance to B. cinerea and PstDC3000 infection (L1, L2 and L3) were selected for further study. The results of qRT-PCR analysis showing the expression levels VaERF20 in the transgenic lines are shown in Figure 3, indicating that the transgene is expressed correctly in over-expressing lines. After B. cinerea and PstDC3000 inoculation, the transcript levels of VaERF20 in these three transgenic lines were 2.7-4.2 times higher than Col-0 at 0 hpi.

VaERF20 Transcript Profile When Over-Expressed in A. thaliana Lines
The potential biological function of VaERF20 in resistance to biotic stress was investigated by over-expressing it under the CaMV 35S promoter-driven in A. thaliana over-expressing plants. A total of 60 independent transgenic lines were generated and confirmed by PCR analysis. Three homozygous T3 lines with the strongest resistance to B. cinerea and PstDC3000 infection (L1, L2 and L3) were selected for further study. The results of qRT-PCR analysis showing the expression levels VaERF20 in the transgenic lines are shown in Figure 3, indicating that the transgene is expressed correctly in over-expressing lines. After B. cinerea and PstDC3000 inoculation, the transcript levels of VaERF20 in these three transgenic lines were 2.7-4.2 times higher than Col-0 at 0 hpi.

VaERF20 Transcript Profile When Over-Expressed in A. thaliana Lines
The potential biological function of VaERF20 in resistance to biotic stress was investigated by over-expressing it under the CaMV 35S promoter-driven in A. thaliana over-expressing plants. A total of 60 independent transgenic lines were generated and confirmed by PCR analysis. Three homozygous T3 lines with the strongest resistance to B. cinerea and PstDC3000 infection (L1, L2 and L3) were selected for further study. The results of qRT-PCR analysis showing the expression levels VaERF20 in the transgenic lines are shown in Figure 3, indicating that the transgene is expressed correctly in over-expressing lines. After B. cinerea and PstDC3000 inoculation, the transcript levels of VaERF20 in these three transgenic lines were 2.7-4.2 times higher than Col-0 at 0 hpi.

Overexpression of VaERF20 in Transgenic A. thaliana Increases Resistance to B. cinerea
To examine the role of VaERF20 in disease resistance, the three VaERF20 over-expressing lines were inoculated with B. cinerea. In Col-0 wild type (WT) plants, typical B. cinerea-provoked lesions were seen at 3 dpi, while the VaERF20 over-expressing A. thaliana lines exhibited no significant lesions ( Figure 4A,E) and the proportion of large lesions were less than in Col-0 plants ( Figure 4F). Histochemical staining revealed that the transgenic lines had higher levels of cell death ( Figure 4B), O 2− ( Figure 4C) and H 2 O 2 ( Figure 4D) accumulation than the WT plants.
were inoculated with B. cinerea. In Col-0 wild type (WT) plants, typical B. cinerea-provoked lesions were seen at 3 dpi, while the VaERF20 over-expressing A. thaliana lines exhibited no significant lesions ( Figure 4A,E) and the proportion of large lesions were less than in Col-0 plants ( Figure 4F). Histochemical staining revealed that the transgenic lines had higher levels of cell death ( Figure 4B), O 2− ( Figure 4C) and H2O2 ( Figure 4D) accumulation than the WT plants.
Expression levels of defense-related genes were assessed by qPCR in response to B. cinerea inoculation. The expression of PDF1.2, which is involved in the JA/ET-mediated signaling pathway, showed a significant increase after infection ( Figure 4G). In the transgenic plants, the expression was up-regulated by 20-fold compared to WT at 72 h after B. cinerea inoculation ( Figure 4G). The expression of the pathogenesis-related gene 1 (AtPR1) and lipoxygenase-3 (AtLOX3), which play major roles in the SA-dependent disease resistance response and JA biosynthesis, respectively, also increased post infection ( Figure 4G). AtORA59, which belongs to the ERF family and has previously been shown to respond transcriptionally to pathogen challenge, showed peak transcript levels at 48 hpi in the three OE lines ( Figure 4G). The expression levels of VaERF20 increased in the three transgenic lines following B. cinerea inoculation ( Figure 3A).  Expression levels of defense-related genes were assessed by qPCR in response to B. cinerea inoculation. The expression of PDF1.2, which is involved in the JA/ET-mediated signaling pathway, showed a significant increase after infection ( Figure 4G). In the transgenic plants, the expression was up-regulated by 20-fold compared to WT at 72 h after B. cinerea inoculation ( Figure 4G). The expression of the pathogenesis-related gene 1 (AtPR1) and lipoxygenase-3 (AtLOX3), which play major roles in the SA-dependent disease resistance response and JA biosynthesis, respectively, also increased post infection ( Figure 4G). AtORA59, which belongs to the ERF family and has previously been shown to respond transcriptionally to pathogen challenge, showed peak transcript levels at 48 hpi in the three OE lines ( Figure 4G). The expression levels of VaERF20 increased in the three transgenic lines following B. cinerea inoculation ( Figure 3A).

Overexpression of VaERF20 in Transgenic A. thaliana Improves Resistance to Pst DC3000
Three transgenic A. thaliana lines and Col-0 plants were inoculated with PstDC3000 to elucidate whether VaERF20 plays a role in bacterial resistance. At 3 dpi, disease symptoms were more severe in Col-0 plants, showing symptoms of chlorosis, while the three transgenic A. thaliana lines exhibited no such symptoms ( Figure 5A). Accordingly, when bacterial populations were tested at 3 dpi, the quantities of bacteria in the three transgenic lines were lower than in Col-0 ( Figure 5E). In contrast to B. cinerea infection, the frequency of cell death ( Figure 5B) and the degree of O 2− ( Figure 5C) and H 2 O 2 ( Figure 5D) accumulation were higher in the three transgenic A. thaliana lines compared to Col-0.

Overexpression of VaERF20 in Transgenic A. thaliana Improves Resistance to Pst DC3000
Three transgenic A. thaliana lines and Col-0 plants were inoculated with PstDC3000 to elucidate whether VaERF20 plays a role in bacterial resistance. At 3 dpi, disease symptoms were more severe in Col-0 plants, showing symptoms of chlorosis, while the three transgenic A. thaliana lines exhibited no such symptoms ( Figure 5A). Accordingly, when bacterial populations were tested at 3 dpi, the quantities of bacteria in the three transgenic lines were lower than in Col-0 ( Figure 5E). In contrast to B. cinerea infection, the frequency of cell death ( Figure 5B) and the degree of O 2− ( Figure 5C) and H2O2 ( Figure 5D) accumulation were higher in the three transgenic A. thaliana lines compared to Col-0.
The expression levels of certain disease resistance related genes have also been shown to be affected within 72h of inoculation with this pathogen, and we therefore examined their expression profiles after PstDC3000 inoculation. The expression of the A. thaliana JA signaling-related gene, AtLOX3, increased significantly by 72 hpi in the transgenic lines compared to Col-0 plants ( Figure  5F). In a similar pattern to that resulting from B. cinerea infection, AtPDF1.2, AtPR1 and AtORA59 ( Figure 5F) were all significantly up-regulated post-inoculation, while AtPR1 expression peaked at 48 hpi and was ~6-fold higher in the transgenic lines than in Col-0. The expression of AtPDF1.2 did not change at 24 hpi, but subsequently decreased and was still higher in the transgenic lines than in WT, while AtORA59 expression rose gradually and peaked at 72 hpi. Finally, VaERF20 expression levels increased more in the three transgenic lines than in Col-0 ( Figure 3B).  Data represent the means ± standard deviation from three independent experiments. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01, one-way ANOVA) between n transgenic A. thaliana and Col-0 samples.
The expression levels of certain disease resistance related genes have also been shown to be affected within 72h of inoculation with this pathogen, and we therefore examined their expression profiles after PstDC3000 inoculation. The expression of the A. thaliana JA signaling-related gene, AtLOX3, increased significantly by 72 hpi in the transgenic lines compared to Col-0 plants ( Figure 5F). In a similar pattern to that resulting from B. cinerea infection, AtPDF1.2, AtPR1 and AtORA59 ( Figure 5F) were all significantly up-regulated post-inoculation, while AtPR1 expression peaked at 48 hpi and was~6-fold higher in the transgenic lines than in Col-0. The expression of AtPDF1.2 did not change at 24 hpi, but subsequently decreased and was still higher in the transgenic lines than in WT, while AtORA59 expression rose gradually and peaked at 72 hpi. Finally, VaERF20 expression levels increased more in the three transgenic lines than in Col-0 ( Figure 3B).

B. cinerea Growth on Transgenic A. thaliana and Col-0 Plants
We next characterized differences in growth of B. cinerea on the resistant transgenic lines and susceptible Col-0 plants ( Figure 6). The degree of infection was more substantial in Col-0 and while no fungal growth was apparent at 4 hpi, germ tubes were detected on Col-0 leaves at 6 hpi. In contrast, almost no growth was observed in the transgenic lines, and B. cinerea conidia in the three transgenic lines germinated more slowly than in WT plants at 8

B. cinerea Growth on Transgenic A. thaliana and Col-0 Plants
We next characterized differences in growth of B. cinerea on the resistant transgenic lines and susceptible Col-0 plants ( Figure 6). The degree of infection was more substantial in Col-0 and while no fungal growth was apparent at 4 hpi, germ tubes were detected on Col-0 leaves at 6 hpi. In contrast, almost no growth was observed in the transgenic lines, and B. cinerea conidia in the three transgenic lines germinated more slowly than in WT plants at 8 hpi. From 12 hpi, infection hyphae in Col-0 plants were also observed and their numbers increased until 18 hpi. Overall, hyphal growth was substantially perturbed in the transgenic lines compared to Col-0 at 18 hpi.

Overexpression of VaERF20 Causes Callose Accumulation in Transgenic A. thaliana in Response to Different Treatments
The cell wall polymer, callose, plays an important role in plant innate immunity at the early time-points of infection, as it can act as a physical barrier to repress pathogen elicitors [39]. When the DC3000 bacteria or the defense response elicitors flg22 and LPS were separately applied to the transgenic lines and Col-0, and the plants treated with the callose-binding stain aniline blue, we observed more callose in the transgenic lines than in WT (Figure 7).

Overexpression of VaERF20 Causes Callose Accumulation in Transgenic A. thaliana in Response to Different Treatments
The cell wall polymer, callose, plays an important role in plant innate immunity at the early time-points of infection, as it can act as a physical barrier to repress pathogen elicitors [39]. When the DC3000 bacteria or the defense response elicitors flg22 and LPS were separately applied to the transgenic lines and Col-0, and the plants treated with the callose-binding stain aniline blue, we observed more callose in the transgenic lines than in WT (Figure 7).

Stomatal Closure Immunity Response
Stomatal closure is an important part of the induced plant innate immunity response [40]. We measured leaf stomatal aperture size in all three transgenic lines and Col-0 plants at 1 hpi and 3 hpi ( Figure 8A,B). Stomata were predominantly closed in the transgenic lines and Col-0 at 1 hpi after PstDC3000, flg22 or LPS treatments. However, stomata reopened in Col-0 plants during the third hour after incubation with DC3000, flg22 or LPS, while stomatal apertures in the transgenic lines still decreased.
To obtain a better understanding of the role of VaERF20 in bacterial resistance, we measured the expression level of genes involved in this process. FRK1, a flg22-induced receptor kinase and the transcriptional regulator WRKY53 both participate in PTI [41]. The expression levels of AtFRK1 ( Figure 8C,D) and AtWRKY53 ( Figure 8E,F) were both up-regulated after injection with PstDC3000, flg22 and LPS compared to the mock controls, as was the expression of both genes compared to WT.

Stomatal Closure Immunity Response
Stomatal closure is an important part of the induced plant innate immunity response [40]. We measured leaf stomatal aperture size in all three transgenic lines and Col-0 plants at 1 hpi and 3 hpi (Figure 8A,B). Stomata were predominantly closed in the transgenic lines and Col-0 at 1 hpi after PstDC3000, flg22 or LPS treatments. However, stomata reopened in Col-0 plants during the third hour after incubation with DC3000, flg22 or LPS, while stomatal apertures in the transgenic lines still decreased.
To obtain a better understanding of the role of VaERF20 in bacterial resistance, we measured the expression level of genes involved in this process. FRK1, a flg22-induced receptor kinase and the transcriptional regulator WRKY53 both participate in PTI [41]. The expression levels of AtFRK1 ( Figure 8C,D) and AtWRKY53 ( Figure 8E,F) were both up-regulated after injection with PstDC3000, flg22 and LPS compared to the mock controls, as was the expression of both genes compared to WT.

Discussion
The AP2/ERF gene family has previously been reported to be involved in the regulation of plant disease resistance pathways [18,29,42]. In this study, we cloned the ERF20 gene from Chinese wild V. amurensis Rupr "Shuangyou", and showed by sequence analysis that the protein has the conserved ERF domains (Supplementary Data S1 and S2) and share similarities with its counterpart in V. vinifera "Pinot Noir". Phylogenetic analysis indicated that VaERF20 belongs to ERF group IXc (B3). The B3 group plays a significant role in defense responses. One member, ERF1, enhanced disease resistance to Fusarium oxysporum in A. thaliana [29]. The A. thaliana AtERF5, AtERF6, AtERF15 and ORA59 genes play redundant roles in the defense against B. cinerea [18,22,28,43], while AtERF15, induces immunity against PstDC3000 and B. cinerea [38]. We observed that overexpression of VaERF20 in A. thaliana increased resistance to B. cinerea and PstDC3000 compared to WT plants, and may also contribute to enhanced PTI responses after PstDC3000, flg22 and LPS  A, B) Statistical analysis of stomatal apertures (width/length; µm) following 1h and 3h of incubation. Data represent the means± standard deviation from three independent experiments with at least 60 stomata per sample. Asterisks indicate statistical significance (** p < 0.01, one-way ANOVA) between transgenic A. thaliana and Col-0. (C-F) Expression analysis of stomatal immunity response related genes in transgenic A. thaliana lines and Col-0 plants at 1h and 3h following inoculation with PstDC3000, flg22 or LPS. Data represent the means ± standard deviation from three independent experiments. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01, one-way ANOVA) between transgenic A. thaliana lines and Col-0.

Discussion
The AP2/ERF gene family has previously been reported to be involved in the regulation of plant disease resistance pathways [18,29,42]. In this study, we cloned the ERF20 gene from Chinese wild V. amurensis Rupr "Shuangyou", and showed by sequence analysis that the protein has the conserved ERF domains (Supplementary Data S1 and S2) and share similarities with its counterpart in V. vinifera "Pinot Noir". Phylogenetic analysis indicated that VaERF20 belongs to ERF group IXc (B3). The B3 group plays a significant role in defense responses. One member, ERF1, enhanced disease resistance to Fusarium oxysporum in A. thaliana [29]. The A. thaliana AtERF5, AtERF6, AtERF15 and ORA59 genes play redundant roles in the defense against B. cinerea [18,22,28,43], while AtERF15, induces immunity against PstDC3000 and B. cinerea [38]. We observed that overexpression of VaERF20 in A. thaliana increased resistance to B. cinerea and PstDC3000 compared to WT plants, and may also contribute to enhanced PTI responses after PstDC3000, flg22 and LPS application. This suggests that it plays a role in the defense against both necrotrophic fungal and bacterial pathogens.
In pathogen-challenged plants, array recognition mechanisms and signal transduction defense-signal pathways are associated with a complex signaling cross-talk involving SA, JA and ET [44][45][46][47]. It has been proposed that the SA pathway is involved in resistance to biotrophic pathogens, such as PstDC3000. Conversely, the JA and ET signaling pathways have been shown to be more effective against necrotrophic pathogens, such as B. cinerea and F. oxysporum [29,43,48,49]. Most studies have reported that the SA and JA/ET signaling pathways have mutually antagonistic interactions in regulating plant defense responses [46,[50][51][52]. Interestingly, our data showed that the expression of both SA-and JA/ET-responsive defense genes was influenced after B. cinerea and PstDC3000 inoculation. That conclusion is consistent with studies of AtERF1 and AtERF15, which showed that these genes are induced by JA and ET as well as SA [38,53].
ROS have been shown to play an important role in plant responses to pathogen attack [5], and ROS accumulation is known to benefit B. cinerea infection [54] by facilitating programmed cell death and promoting infection, resulting in larger disease lesions [55,56]. Our results corresponded well with these findings. VaERF20 transgenic lines showed enhanced resistance to B. cinerea compared to Col-0 plants, reflected in disease phenotype or conidial growth changes, and accumulated less ROS than did the Col-0 plants post-infection. This was different from PstDC3000 inoculation, where the VaERF20 over-expressing lines had increased resistance, but where the pathogen-induced ROS were higher in transgenic A. thaliana lines. The PstDC3000 results correlate well with previous studies showing that pathogen-induced ROS increase immunity to this pathogen [57].
To obtain a better understanding of the effect of VaERF20 over-expression in A. thaliana, we also examined the change in transgenic A. thaliana and Col-0 plants to defense response elicitors. Callose accumulation as a downstream signal of PTI showed diverse patterns in the two genotypes. Compared to WT, overexpression of VaERF20 resulted in more callose accumulation post-inoculation with PstDC3000. We observed that injection with flg22 and LPS also caused accumulation of more callose. A previous study has shown that callose can rapidly form a physical defense to protect cells from external injury [58]. Our result further suggests that VaERF20 helps prevent pathogen spread. Another defense mechanism is stomatal defense, which has been shown to be important for PTI during bacterial challenge. We observed that the stomata of the transgenic A. thaliana plants remained closed for longer than in Col-0 plants, indicating that VaERF20 over-expression caused an innate immunity response. We measured the expression of two PTI marker genes, AtFRK1 and AtWRKY53, which were both fostered in transgenic A. thaliana more than in Col-0. These results not only further associate VaERF20 with the innate immune response, but also support the hypothesis that VaERF20 enhances defense against B. cinerea and PstDC3000.
In conclusion, our data demonstrate that ERF20 from Chinese wild V. amurensis Rupr "Shuangyou" can improve plant disease resistance. VaERF20 over-expressing lines showed enhanced resistance to B. cinerea and PstDC3000 infection and activated SA and JA/ET signaling. Our findings suggest that VaERF20 is activated in plant responses to biotic stresses. Future studies will examine the transcriptional networks and regulatory mechanisms in which VaERF20 operates in immune responses to pathogens.

Plant Materials and Growth Conditions
The Chinese wild Vitis genotype, V. amurensis Rupr "Shuangyou" and V. vinifera cv. "Red Globe" used for B. cinerea inoculation were obtained from the grape germplasm resources repository at the Northwest A&F University, Yangling, Shaanxi, China. A. thaliana ecotype Columbia-0 (Col-0) and transgenic lines were grown at 22 • C under long-day (16 h light/8 h dark) and 70% relative humidity conditions. All experiments used 4-week old A. thaliana and were repeated three times. Samples were immediately frozen in liquid nitrogen and stored at −80 • C until further use.

B. cinerea Inoculation of Grape
B. cinerea was isolated as previously described [37]. Briefly, conidia cultured on Potato Glucose Agar medium for 3 weeks were washed with distilled water. Conidia suspensions were pre-germinated for 2 h at 22 • C and a concentration of 105 spores/mL was used. Detached grape leaves were placed on 0.8% agar in trays and sprayed with B. cinerea conidia suspension. After inoculation, all leaves were incubated at 22 • C and a high humidity of 90-100% in the dark for the first 24 h and then in a light/dark (12/12 h) regime. Control leaves were sprayed with distilled water and incubated under the same conditions as the inoculated leaves. Samples were collected at 4, 8, 18 and 36 hpi (hours post-inoculation) for further analysis.

Generation and Characterization of Transgenic Lines
Total grape RNA from leaves infected with B. cinerea was extracted using the E.Z.N.A. ® Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). First-strand cDNA was synthesized using the PrimeScript TM RTase 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China) and diluted 6-fold with ultrapure water for subsequent analysis. The gene coding sequence (CDS) was obtained from the Grape Genome Browser (12×) (http://www.genoscope.cns.fr) and is also available through the National Center for Biotechnology Information (NCBI) (http://blast.ncbi. nlm.nih.gov/Blast.cgi) database. The VaERF20 CDS was amplified by PCR using the gene-specific primers F (5 -GCTCTAGAATGGAAGGAGGAGGGAGGAGG-3 , XbaI site underlined) and R (5 -GGGGTACCCTAATTATTCCCTCTTCTATCACCCTGTG-3 , KpnI site underlined), the PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and the sequence verified by sequencing. The CDS was then inserted in the plant expression vector, pCambia 2300, downstream of the CaMV 35S promoter (Cambia, Brisbane, QLD, Australia).
The above construct was introduced into Agrobacterium tumefaciens (strain GV3101) using the floral dip method [59]. T0 seeds were harvested and grown on MS medium (pH 5.8, 10 g/L sucrose, 8 g/L agar) supplemented with 75 mg/L kanamycin. The T3 homozygous lines, L1, L2 and L3, which had the strongest resistance to B. cinerea and PstDC3000, as well as highVaERF20 expression, were selected from 60 independent lines.

Bioinformatic Analysis
The ERF protein sequences from rice and A. thaliana were as described in a previous report [27]. Phylogenetic analysis was performed using the MEGA 7.0 software and the neighbor joining (NJ) method [60,61] with 1000 bootstrap replicates.

Pathogen Inoculation Assays in A. thaliana
A B. cinerea spore concentration of 2.0 × 10 6 mL −1 in sterile water with 4% maltose and 1% peptone [62] was used for inoculation assays. Ten ml conidia suspension was applied to 60 detached leaves from 25 four-week old transgenic and Col-0 A. thaliana plants and incubated at 22 • C under a 16 h light/8 h dark cycle with a humidity of 90-100% [63]. Lesion diameter and percentage of lesion areas were determined daily until 3 days. Samples were collected at 0, 24, 48, and 72 hpi for further use and experiments were repeated three times.
P. syringae pv. tomato DC3000 was cultivated at 28 • C in King's B liquid medium overnight with shaking at 200 rpm [38]. The bacterial inoculum was re-suspended in 10 mM MgCl 2 containing 0.02% Silwet L-77 to an OD600 of 0.02. Four-week old transgenic and Col-0 A. thaliana plants were soaked in DC3000 cell suspension for 10 min, and covered with a transparent plastic film to maintain a high humidity for 24 h [64]. To measure bacterial populations, leaf discs of inoculated leaves were rinsed with sterile water then homogenized in 100 µL 10 mM MgCl 2 , then the solution were gradually diluted and one hundred microliters of the resulting diluted solution were plated on KB agar plates as previously described [64]. Samples were collected at 0, 24, 48, and 72 hpi for further use and experiments were repeated three times.
To measure the stomatal response, 4-week old transgenic and Col-0 A. thaliana plants were exposed to 100 µmol·m −2 ·s −1 light for 3 h to ensure that 80% of the stomata were open. The epidermis of the leaves was immersed in 10 mM MgCl 2 , DC3000 suspension with OD = 0.02, 5 µM flg22 (Flagellin Fragment, Anaspec, Fremont, CA, USA) and 0.1 mg/mL LPS (lipopolysaccharides, Sigma, St. Louise, MO, USA) as previously described [63]. At least 30 fully expanded young leaves from 12 four-week old transgenic and Col-0 A. thaliana plants were peeled epidermis for each treatment. Transverse length and longitudinal width of stomata were measured using Image-J and samples were collected and observed under a microscope (Olympus BX51, Tokyo, Japan) at 1 and 3 h after treatment [63].
For the callose accumulation assay, 4-week old transgenic and Col-0 A. thaliana leaves were infiltrated with MgCl2, PstDC3000, flg22 and LPS using a 1 mL needleless syringe, as described above. The injected leaves were examined for callose accumulation 18 hpi by aniline blue staining as previously described [65]. All experiments were repeated three times.

Quantitative Real-Time PCR (qRT-PCR)
Total A. thaliana RNA from tissue inoculated with B. cinerea and DC3000 was extracted and first-strand cDNA was synthesized as described above. qRT-PCR was conducted using SYBR Green (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions, using the following thermal profile: 95 • C for 30 s, 40 cycles of 95 • C for 5 s, and 60 • C for 30 s with StepOnePlusTM Real-Time PCR system (Applied Biosystems AB, Life Technologies Holdings Pte Ltd., Foster, CA, USA). Melt-curve analyses was performed using a program of 40 PCR cycles with 95 • C for 15 s, and then a constant increase from 60 • C to 95 • C. VvActin (GenBank accession number AY680701) or AtActin1 (TAIR: AT2G37620) were used as reference genes. Specific primers used for qRT-PCR are listed in Supplementary Table S1. Relative gene expression levels were calculated using the 2 −∆∆Ct method [38] and each reaction performed with three technical and three biological replicates.

Statistical Analysis
Data represent the means ± standard deviation from three independent experiments and were calculated using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Statistical significance (one-way ANOVA) was determined using the SPSS Statistics 17.0 software package (IBM China Company Ltd., Beijing, China). All experiments were repeated in triplicate as independent analyses.