Arabidopsis Elongator subunit 2 positively contributes to resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola

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1 The Plant Journal (2015) 83, doi: /tpj Arabidopsis Elongator subunit 2 positively contributes to resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola Chenggang Wang 1, Yezhang Ding 1, Jin Yao 2, Yanping Zhang 3, Yijun Sun 2, James Colee 4 and Zhonglin Mou 1, * 1 Department of Microbiology and Cell Science, University of Florida, P.O. Box , Gainesville, FL USA, 2 Department of Microbiology and Immunology, University of Buffalo, Buffalo, NY 14203, USA, 3 Interdisciplinary Center for Biotechnology Research, University of Florida, P.O. Box , Gainesville, FL 32610, USA, and 4 Department of Statistics, University of Florida, P.O. Box , Gainesville, FL 32611, USA Received 20 May 2015; revised 9 July 2015; accepted 15 July 2015; published online 28 July *For correspondence ( zhlmou@ufl.edu). SUMMARY The evolutionarily conserved Elongator complex functions in diverse biological processes including salicylic acid-mediated immune response. However, how Elongator functions in jasmonic acid (JA)/ethylene (ET)- mediated defense is unknown. Here, we show that Elongator is required for full induction of the JA/ET defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) and for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. A loss-of-function mutation in the Arabidopsis Elongator subunit 2 (ELP2) alters B. cinerea-induced transcriptome reprogramming. Interestingly, in elp2, expression of WRKY33, OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59), and PDF1.2 is inhibited, whereas transcription of MYC2 and its target genes is enhanced. However, overexpression of WRKY33 or ORA59 and mutation of MYC2 fail to restore PDF1.2 expression and B. cinerea resistance in elp2, suggesting that ELP2 is required for induction of not only WRKY33 and ORA59 but also PDF1.2. Moreover, elp2 is as susceptible as coronatine-insensitive1 (coi1) and ethylene-insensitive2 (ein2) to B. cinerea, indicating that ELP2 is an important player in B. cinerea resistance. Further analysis of the lesion sizes on the double mutants elp2 coi1 and elp2 ein2 and the corresponding single mutants revealed that the function of ELP2 overlaps with COI1 and is additive to EIN2 for B. cinerea resistance. Finally, basal histone acetylation levels in the coding regions of WRKY33, ORA59, and PDF1.2 are reduced in elp2 and a functional ELP2-GFP fusion protein binds to the chromatin of these genes, suggesting that constitutive ELP2-mediated histone acetylation may be required for full activation of the WRKY33/ORA59/PDF1.2 transcriptional cascade. Keywords: Elongator, COI1, EIN2, plant immunity, jasmonic acid, ethylene, gene expression, Arabidopsis thaliana. INTRODUCTION As sessile organisms, plants have evolved sophisticated defense signaling networks to survive the challenge of pathogens with diverse lifestyles. These defense signal networks are predominantly regulated by a group of signal molecules such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Depending upon the lifestyle of the invader, plants synthesize one or more of these primary defense signal molecules to turn on the signaling pathways that are the most effective for fending off the invading pathogens (Pieterse et al., 2009). After being synthesized, SA, JA, and ET are perceived by their receptors and the signals are transduced by components in their The Plant Journal 2015 John Wiley & Sons Ltd respective signaling pathways, ultimately leading to defense-associated transcriptional responses. JA and ET cooperate in regulating resistance to necrotrophic fungal pathogens (Thomma et al., 2001; Kunkel and Brooks, 2002; Glazebrook, 2005). They synergistically activate the JA/ET pathway defense marker gene PLANT DEFENSIN1.2 (PDF1.2). JA also activates a group of wound-responsive genes, such as VEGATATIVE STORAGE PROTEIN1 (VSP1), VSP2, and JASMONATE RESPONSIVE1 (JR1), which are regulated by the transcription factor MYC2, whereas ET suppresses these genes (Rojo et al., 1999; Lorenzo et al., 2004). The transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-Like1 (EIL1), 1019

2 1020 Chenggang Wang et al. which regulate most of the ET responsiveness, are signaling hubs of JA/ET cooperation (Chao et al., 1997; Solano et al., 1998; Alonso et al., 2003; An et al., 2010). JA and ET signaling converge at EIN3/EIL1, turning on genes encoding APETALA2/ETHYLENE RESPONSIVE FACTORs (AP2/ ERFs), including ORA59 and ERF1, which in turn activate PDF1.2 expression (Solano et al., 1998; Lorenzo et al., 2003; Pre et al., 2008; Zarei et al., 2011; Zhu et al., 2011). Overexpression of ORA59 and ERF1 has been shown to elevate basal expression of PDF1.2 and resistance to necrotrophic fungal pathogens (Berrocal-Lobo et al., 2002; Lorenzo et al., 2003; Pre et al., 2008), suggesting their predominant role in JA/ET-mediated defense signaling. The transcription factor WRKY33 is also an important regulator of resistance against necrotrophic fungal pathogens. Expression of the WRKY33 gene is highly inducible by Botrytis cinerea infection (AbuQamar et al., 2006), but the induction does not require the JA and ET signaling components CORONATINE-INSENSITIVE1 (COI1) and EIN2, respectively (Xie et al., 1998; Alonso et al., 1999; Zheng et al., 2006), suggesting that WRKY33 may be activated by a JA/ET-independent defense pathway. Mutations in WRKY33 compromise basal resistance to B. cinerea and Alternaria brassicicola, whereas overexpression of WKRY33 results in enhanced resistance to these pathogens and elevated basal expression of PDF1.2 (Zheng et al., 2006). WRKY33 appears to directly control the expression of ORA59 during the later stages of pathogen infection (Birkenbihl et al., 2012), but how WRKY33 activates defense gene expression still remains elusive. In eukaryotes, RNA polymerase II (RNAP II) catalyzes the transcription of protein-encoding genes to synthesize precursors of mrna. An RNAP II interacting complex called Elongator was first purified from yeast (Otero et al., 1999), and was later identified in human and Arabidopsis cells (Hawkes et al., 2002; Kim et al., 2002; Nelissen et al., 2010). In yeast, Elongator is composed of six subunits (ELP1 P6) with ELP1 ELP3 forming the core subcomplex, ELP4 ELP6 the accessory subcomplex, and ELP3 being the catalytic subunit with histone acetyltransferase activity (HAT) (Winkler et al., 2002). The six subunits act together as a functional unit and lack of any one subunit compromises the integrity and function of the complex. The Arabidopsis Elongator has a similar structure to that in yeast and consists of six subunits: ELP1/ELONGATA2 (ELO2), ELP2, ELP3/ELO3, ELP4/ELO1, ELP5, and ELP6 (Nelissen et al., 2010). Note that the acronym ELP has been used previously to describe EDM2-like proteins in Arabidopsis (Eulgem et al., 2007). Although it has been shown that histone acetylation levels at several auxin- and defense-related genes are reduced in elp/elo mutants (Nelissen et al., 2010; Wang et al., 2013), the catalytic HAT activity of ELP3/ELO3 has not been demonstrated. Elongator has been implicated in multiple different cellular processes such as histone modification, exocytosis, a-tubulin acetylation, trna modification, and zygotic paternal genome demethylation (Hawkes et al., 2002; Winkler et al., 2002; Huang et al., 2005; Rahl et al., 2005; Creppe et al., 2009; Okada et al., 2010). Loss-of-function mutations in Elongator lead to diverse morphological, physiological, and pathological phenotypes in yeast, humans, or Arabidopsis (Otero et al., 1999; Anderson et al., 2001; Jablonowski et al., 2001; Krogan and Greenblatt, 2001; Slaugenhaupt et al., 2001; Nelissen et al., 2005, 2010; Chen et al., 2006; Zhou et al., 2009; Versees et al., 2010; Xu et al., 2012). The Arabidopsis elp/elo mutants accumulate higher levels of JA and ET and express higher levels of several JA biosynthesis and JA- or ET-responsive genes than wild type (Nelissen et al., 2010). These results suggest that Elongator might negatively contribute to the JA/ET-mediated defense signaling. Surprisingly, here we found that ELP2 plays opposite roles in the ETHYLENE-RESPONSE FAC- TOR1 (ERF1)/OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59) branch and the MYC2 branch of the JA pathway and is required for JA/ET-mediated and WRKY33- activated defense responses against the necrotrophic fungal pathogen B. cinerea. We showed that the function of ELP2 overlaps with COI1 and is additive to EIN2 in mediating resistance to B. cinerea. Finally, we found that histone acetylation levels in WRKY33, ORA59, and PDF1.2 are reduced in elp2 and that ELP2 is associated with the chromatin of these genes. Taken together, these results suggest that constitutive ELP2-mediated histone acetylation may be needed for proper activation of the WRKY33/ORA59/PDF1.2 transcriptional cascade. RESULTS Mutations in ELP/ELO genes inhibit JA- and ET-induced defense gene expression Previous works have shown that the Elongator subunits ELP2 and ELP3/ELO3 function in SA-mediated signaling (Defraia et al., 2010, 2013). To test whether Elongator is also required for JA/ET-induced defense gene expression, we treated elp/elo mutants with the ET precursor 1- aminocyclopropane-1-carboxylic acid (ACC), the JA derivative methyl jasmonate (MeJA), and their combination by transferring 10-day-old seedlings grown on half-strength Murashige and Skoog (½MS) (Murashige and Skoog, 1962) plates onto ½MS plates supplemented with or without 0.1 mm ACC and/or 0.1 mm MeJA. Two days later, the induction of three JA/ET-inducible genes PDF1.2, BASIC CHITINASE (CHIB), and HEVEIN-LIKE (HEL) was examined by RNA gel blot analysis. In the absence of ACC and MeJA, expression of PDF1.2 and CHIB was not detected in the wild type and elp/elo plants, whereas HEL was weakly expressed in the elp/elo seedlings (Figure 1), probably due to the elevated JA/ET content (Nelissen et al., 2010).

3 Function of Elongator in necrotroph resistance 1021 Figure 1. ACC- and MeJA-induced defense gene expression in elp/elo mutants. ACC- and MeJA-induced expression of PDF1.2, CHIB, and HEL in WT and elp/elo plants. Ten-dayold seedlings grown on ½MS medium were transplanted onto ½MS medium ( ) or ½MS medium supplemented with 0.1 mm ACC, 0.1 mm MeJA, or both (ACC+MeJA). Total RNA was extracted from plant tissues except roots collected 2 days later and subjected to RNA gel blot analysis. The UBQ5 gene was used as a loading control. ACC, MeJA, and their combination all induced PDF1.2, CHIB, and HEL expression. While the elp/elo mutations inhibited ACC-, MeJA- and ACC/MeJA-induced PDF1.2 expression, induction of CHIB and HEL by ACC alone appeared to be enhanced in the elp/elo mutants (Figure 1). Conversely, induction of CHIB and HEL by MeJA was decreased in some elp/elo mutants (elo2, elp2, and elp5). When both ACC and MeJA were added, the expression levels of CHIB and HEL were comparable to those in the wild type. These results indicate that Elongator is required for full induction of some JA/ET-inducible defense genes. Mutations in ELP/ELO genes suppress B. cinerea-induced defense gene expression and compromise resistance to both B. cinerea and A. brassicicola To test whether Elongator is essential for fungal pathogeninduced defense gene expression, we inoculated 4-weekold soil-grown elp/elo and wild type plants with the necrotrophic fungal pathogen B. cinerea, and monitored the expression of PDF1.2, CHIB, and HEL at different time points post-inoculation. At 0 h post-inoculation (hpi), expression of all three genes was not detected (Figure 2a), suggesting that they are not constitutively expressed in soil-grown elp/elo mutants. At 12 hpi, both PDF1.2 and HEL were induced in the wild type, whereas in the elp/elo plants expression of PDF1.2 was still not detectable and the induction of HEL was obviously decreased (Figure 2(a)). At 24 and 48 hpi, while the three genes were significantly activated in the wild type, their induction in the elp/elo plants was inhibited to different extents (Figure 2a). These results clearly demonstrate that Elongator is required for full induction of PDF1.2, CHIB, and HEL during B. cinerea infection. We then tested the susceptibility of elp/elo plants to B. cinerea and another necrotrophic fungal pathogen A. brassicicola. The disease severity was measured by the size of the lesions, and the growth of pathogen was estimated by normalizing the B. cinerea ActinA (BcActA) or A. brassicicola CutinaseA (AbCutA) DNA against the Arabidopsis Actin2 (AtActin) DNA (Dhawan et al., 2009). Four days after inoculation, the size of B. cinerea-caused lesions on the elp/elo plants was more than 3.8 times as large as that on the wild type (Figure 2b,c), and the amount of B. cinerea DNA on the elp/elo plants was more than 25 times as much as that on the wild type (Figure 2d). Similarly, the size of A. brassicicola -caused lesions on the elp/ elo plants was more than 3.3 times as large as that on the wild type (Figure 2e,f), and the amount of A. brassicicola DNA on the elp/elo plants was more than 5.8 times as much as that on the wild type (Figure 2g). Therefore, Elongator is required for resistance to these two different necrotrophic fungal pathogens. The elp2 mutation alters B. cinerea-induced transcriptome reprogramming To determine how Elongator regulates fungal pathogen-induced transcriptional changes at the genome level, we conducted a microarray experiment on B. cinerea-infected elp2 and wild type plants (NCBI GEO Series number GSE48207). Three independent experiments were conducted and the results were analyzed to identify genes that exhibited a two-fold or higher induction or suppression with a low q-value ( 0.05) in elp2 and the wild type. Consistent with previous microarray analyses (AbuQamar et al., 2006; Rowe et al., 2010; Windram et al., 2012), B. cinerea infection-induced profound transcriptional changes in the wild type plants. The numbers of genes that were up- and down-regulated two-fold or more at 48 hpi (1490 and 1837, respectively) were close to those (1458 and 1602, respectively) reported by Rowe et al. (2010), confirming the transcriptome changes induced by B. cinerea infection. Interestingly, although elp2 also exhibited drastic transcriptional changes upon B. cinerea infection, the kinetics of the transcriptional reprogramming in elp2 appeared to be slower than that in the wild type (Figure 3a). While the numbers of genes up- and down-regulated in the wild type were highest at 24 hpi, in elp2 they showed different patterns for up- and down-regulated genes. For up-regulated genes, they reached the highest at 24 hpi, and for down-regulated genes they were higher at 48 hpi. The numbers of genes up- and down-regulated in elp2 were smaller than those in the wild type at all the tested time points except 48 hpi, suggesting that the elp2 genome might respond more slowly to B. cinerea infection than that of the wild type. To compare the B. cinerea infection-induced transcriptome changes in elp2 and the wild type, we used linear model methods to identify genes whose expression

4 1022 Chenggang Wang et al. Figure 2. Induction of JA/ET-responsive genes and resistance to necrotrophic pathogens in elp/elo mutants. (a) Botrytis cinerea-induced expression of PDF1.2, CHIB, and HEL in WT and elp/elo plants. Four-week-old soil-grown plants were inoculated with B. cinerea spores, and the inoculated leaves were collected and subjected to total RNA extraction and RNA gel blot analysis. The UBQ5 gene was used as a loading control. (b, c) Symptoms (b) and size (c) of the necrotic lesions formed on B. cinerea -infected WT and elp/elo plants. In (b), photographs were taken 4 days post-inoculation. In (c), lesion sizes on 72 leaves measured in three independent experiments were combined and analyzed as a one-way ANOVA, blocked by experiment. The resulting mean and standard error (SE) are presented. a,b Different letters above the bars indicate significant differences (P < ). (d) Biomass of B. cinerea on WT and elp/elo plants. Data represent the mean qpcr amplification of the B. cinerea ActinA (BcActA) DNA relative to the Arabidopsis Actin2 DNA from three independent samples. a,b,c,d Different letters above the bars indicate significant differences (P < 0.05, one-way ANOVA). (e, f) Symptoms (e) and size (f) of the necrotic lesions formed on Alternaria brassicicola-infected WT and elp/elo plants. In (e), photos were taken 4 days postinoculation. In (f), data were analyzed and presented as in (c). a,b,c Different letters above the bars indicate significant differences (P < 0.02). (g) Biomass of A. brassicicola on WT and elp/elo plants. Data represent the mean qpcr amplification of the A. brassicicola Cutinase (AbCutA) DNA relative to the Arabidopsis Actin2 DNA from three independent samples. a,b,c Different letters above the bars indicate significant differences (P < 0.05, one-way ANOVA). changes in elp2 and the wild type were significantly different (q < 0.05) at least at one time point after B. cinerea infection. A total of 9403 genes were identified and used for principal component analysis (PCA) to outline the difference between elp2 and the wild type at the transcriptome level. The results are shown by principal component score plotting (Figure 3b). The abscissa is the first principal component (PC1), and the ordinate is the second (PC2) or the third principal component (PC3). Each plot in Figure 3(b) represents the corresponding transcriptome. PCA revealed that the three highest ranked principal components accounted for 69% of the total variance within the dataset. The first principal component, accounting for 36% of total variance, not only resolved the time points, but also separated elp2 from the wild type, particularly at 6, 12, and 24 hpi. At these time points, the wild type transcriptome clearly deviated further away from the initial state than the elp2 transcriptome, indicating that the wild type transcriptome changed more dramatically than the elp2 transcriptome in response to B. cinerea infection. We further analyzed the induction dynamics of several B. cinerea inducible genes including ORA59, ERF1, WRKY33, and PDF1.2. As shown in Figure 3(c), induction of ORA59 and PDF1.2 at 12 hpi in elp2 was significantly lower than in the wild type (Figure 3c), indicating that activation of these two defense genes was delayed and decreased in the elp2 mutant. Conversely, although linear model analysis revealed that induction of ERF1 and WRKY33 at 6 hpi in elp2 was significantly lower than in the wild type, this result may not have biological relevance, because expression of these two genes fluctuated in the wild type and/or elp2 (Figure 3c).

5 Function of Elongator in necrotroph resistance 1023 Figure 3. Botrytis cinerea-induced transcriptome changes in elp2. (a) Dynamic changes in the numbers of genes that are up- or downregulated in WT and elp2 plants after Botrytis cinerea infection. (b) Scores of the principal component analysis of transcriptome data of WT and elp2 at different time points after B. cinerea infection. The distances between the samples were calculated as described in Experimental Procedures. Principal components 1, 2, and 3 were chosen for best visualization of differences between samples. (c) Induction kinetics of ORA59, ERF1, WRKY33, and PDF1.2 in WT and elp2 plants after B. cinerea infection. An asterisk shows that the induction of the gene at the indicated time point in elp2 was significantly different from that in the wild type (q < 0.05, linear model analysis). The elp2 mutation differentially influences the WRKY33, ORA59, and MYC2 transcriptional cascades To identify genes whose expression requires ELP2 during B. cinerea infection, we analyzed the genes that were differentially expressed between elp2 and the wild type. Genes that displayed a two-fold or bigger difference in the expression levels with a low q-value ( 0.05) were selected for further analysis. Interestingly, expression of many

6 1024 Chenggang Wang et al. fungal defense-related genes, including PAD3, CYP71A13, CYP79B2, GRXS13, GRX480, ATG18a, GLIP1, WRKY70, BIK1, BAK1, and TGA3 (Glawischnig et al., 2004; AbuQamar et al., 2006; Veronese et al., 2006; Ferrari et al., 2007; Kemmerling et al., 2007; Nafisi et al., 2007; Ndamukong et al., 2007; Kwon et al., 2009; La Camera et al., 2011; Lenz et al., 2011; Windram et al., 2012), was not significantly affected by the elp2 mutation. Conversely, the expression of the three transcription factor genes WRKY33, ORA59, and ERF1, which have also been implicated in fungal resistance (Lorenzo et al., 2003; Zheng et al., 2006; Pre et al., 2008), was decreased in elp2 compared with the wild type after B. cinerea infection (Table 1). Consistent with this, expression of many of the WRKY33- and/or ORA59-regulated genes including PDF1.2 and HEL were inhibited in elp2 (Table 1). Interestingly, the induction of a group of genes that are controlled by the transcription factor MYC2, including VSP1, VSP2, and JR1, was enhanced in elp2 compared to the wild type after B. cinerea infection (Table 1). The microarray data also indicated that the MYC2 gene itself was up-regulated in elp2 at 3 hpi [log 2 (fold change) = 1.462, q < 0.001]. Taken together, these results indicate that ELP2 oppositely contributes to the transcriptional cascades mediated by WRKY33/ORA59/ERF1 and MYC2. Overexpression of WRKY33 or ORA59 does not restore PDF1.2 expression and resistance in elp2 Since PDF1.2 is directly regulated by ORA59, which in turn is partially controlled by WRKY33 (Pre et al., 2008; Birkenbihl et al., 2012), the decreased induction of PDF1.2 in elp2 might be due to reduced expression of WRKY33 or ORA59. To test this, we transformed a 35S:WRKY33 or a 35S:ORA59 transgene into elp2 and examined if overexpression of WRKY33 or ORA59 could rescue the expression pattern of PDF1.2 and restore resistance in elp2. A representative transgenic line for each transgene was crossed with wild type Columbia (Col-0) plants to move the transgene into Col-0 background. The resulting 35S:WRKY33 and 35S: ORA59 transgenic lines exhibited previously described morphological phenotypes, namely, early flowering and serrated leaves for 35S:WRKY33 and a dwarf phenotype for 35S:ORA59 (Figure S1) (Zheng et al., 2006; Pre et al., 2008). In the transgenic plants (in either elp2 or Col-0 background), the expression levels of WRKY33 or ORA59 were significantly higher than those in the wild type before B. cinerea infection (Figure 4a,e). Note that, in Figure 4(a), due to the large variances of the expression data of WRKY33 in 35S: WRKY33 and 35S:WRKY33 elp2, one-way ANOVA analysis did not reveal the significant difference between elp2 and the wild type. When the expression data of WRKY33 in elp2 and the wild type were compared using Student s t-test, significant differences were detected between elp2 and the wild type (Figure S2), which confirmed the microarray results (Table 1). As reported previously (Zheng et al., 2006; Pre et al., 2008), overexpression of WRKY33 or ORA59 elevated basal transcript levels of PDF1.2 in the Col-0 background, but it did not do so in the elp2 background (Figure 4b,f). In fact, the expression levels of PDF1.2 in the 35S:WRKY33 elp2 and 35S:ORA59 elp2 plants were comparable with those in elp2 both before and after B. cinerea infection (Figure 4b,c,f,g). Moreover, although overexpression of ORA59 slightly and significantly increased resistance to B. cinerea, which again confirmed the previous result (Pre et al., 2008), resistance of the 35S:ORA59 elp2 plants to B. cinerea was comparable to that of elp2 (Figure 4d,h). Resistance of the 35S:WRKY33 elp2 plants was also comparable to that of elp2 (Figure 4d,h). These results together indicate that overexpression of either WRKY33 or ORA59 is not able to restore PDF1.2 expression and resistance in elp2. Suppression of the MYC2 transcriptional cascade does not restore PDF1.2 expression and resistance in elp2 It has been well documented that the transcription factor MYC2 positively regulates the expression of wound- and insect-responsive genes such as VSP1, VSP2, and JR1, and negatively regulates the expression of pathogen defense genes including PDF1.2 and HEL (Lorenzo et al., 2004; Kazan and Manners, 2013). Interestingly, in elp2, the MYC2 transcriptional cascade was enhanced compared with the wild type during B. cinerea infection, whereas ERF1, ORA59, and their target genes including PDF1.2 and HEL were inhibited (Table 1). It is therefore possible that inhibition of the ORA59/ERF1 branch of the JA signaling pathway in elp2 during pathogen infection is simply caused by enhanced expression of MYC2. To test this hypothesis, we constructed an elp2 jin1 (jasmonate-insensitive1) double mutant (jin1 has a mutation in the MYC2 gene) and tested its responses to B. cinerea infection. Consistent with the microarray data, the expression of MYC2 and two MYC2 target genes VSP2 and JR1 was significantly increased at 3 hpi in elp2 (Figure 5a c), and the expression levels of VSP2 and JR1 at 48 hpi were also higher in elp2 than in the wild type (Figure 5b,c). In jin1 and elp2 jin1 plants, the jin1 mutation largely eliminated the expression of MYC2 and completely blocked the induction of VSP2 and JR1 (Figure 5a c), indicating that elp2-mediated enhancement of the MYC2 transcriptional cascade depends on MYC2. Conversely, removal of MYC2 by the jin1 mutation did not have significant effects on elp2-mediated suppression of PDF1.2 expression and susceptibility to B. cinerea (Figure 5d f), indicating that inhibition of the ORA59/ERF1 branch in elp2 is not due to enhanced induction of the MYC2 branch. ELP2 is a major contributor to B. cinerea resistance and its function overlaps with COI1 and is additive to EIN2 for this resistance To test the relationship between ELP2 and the JA/ET signaling pathways, we generated elp2 coi1 and elp2 ein2

7 Function of Elongator in necrotroph resistance 1025 Table 1 Defense genes that are differentially expressed between elp2 and wild type during Botrytis cinerea infection elp2/wild type (WT) 6 h 12 h 24 h 48 h AGI locus Gene name Log 2 (FC) q Value Log 2 (FC) q Value Log 2 (FC) q Value Log 2 (FC) q Value AGI description Major transcription factor genes At1g06160 ORA < <0.001 OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF 59 At3g23240 ERF ETHYLENE RESPONSE FACTOR 1 At2g38470 WRKY WRKY DNA-binding protein 33 ORA59-, ERF1-, and/or WRKY33-regulated genes At5g44420 PDF < < <0.001 PLANT DEFENSIN 1.2 At2g26020 PDF1.2b < PLANT DEFENSIN 1.2b At5g44430 PDF1.2c < < <0.001 PLANT DEFENSIN 1.2c At1g75830 PDF < PLANT DEFENSIN 1.1 At2g26010 PDF < < <0.001 PLANT DEFENSIN 1.3 At3g04720 HEL < <0.001 HEVEIN-LIKE At2g Chitinase, putative At3g < < < <0.001 Legume lectin family protein At3g < < <0.001 Legume lectin family protein At2g26560 PLP <0.001 PATATIN-LIKE PROTEIN 2 At4g11650 OSM OSMOTIN 34 At5g <0.001 Curculin-like lectin family protein At1g <0.001 Disease resistance protein, putative MYC2-regulated genes At5g24780 VSP < VEGETATIVE STORAGE PROTEIN 1 At5g24770 VSP < <0.001 VEGETATIVE STORAGE PROTEIN 2 At3g16470 JR <0.001 JASMONATE RESPONSIVE 1 At3g28740 CYP81D < <0.001 CYTOCHROME P450, FAMILY 81, SUBFAMILY D, POLYPEPTIDE 11 At1g19570 DHAR <0.001 DEHYDROASCORBATE REDUCTASE At3g09940 MDHAR < <0.001 MONODEHYDROASCORBATE REDUCTASE double mutants, and evaluated the responses of wild type, elp2, coi1, ein2, and the double mutants to B. cinerea infection. Consistent with COI1 and EIN2 being key regulators of B. cinerea resistance, both coi1 and ein2 showed significantly larger B. cinerea lesions and supported significantly more pathogen growth than the wild type (Figure 6a f). Interestingly, the elp2 mutant appeared to be as susceptible as coi1 and ein2 to this fungal pathogen. Although the size of the lesions on elp2 was slightly but significantly smaller than that on coi1 (Figure 6b), the amount of B. cinerea DNA on elp2 was comparable with that on coi1 (Figure 6c). On the other hand, elp2 and ein2 exhibited similar sizes of disease lesions and produced similar amounts of B. cinerea DNA (Figure 6e,f). These results indicate that, like COI1 and EIN2, ELP2 is also a major contributor to resistance against B. cinerea. The B. cinerea disease severity was further enhanced in the double mutants elp2 coi1 and elp2 ein2. Both double mutants displayed significantly larger lesions than their corresponding single mutants (Figure 6b,e). The double

8 1026 Chenggang Wang et al. Figure 4. Characterization of 35S:WRKY33 elp2 and 35S:ORA59 elp2 plants. (a c) Expression of WRKY33 (a) and PDF1.2 (b and c) in WT, elp2, 35S:WRKY33, and 35S:WRKY33 elp2 plants after B. cinerea infection. Expression levels were monitored using qpcr and normalized against UBQ5. a.b.c Different letters above the bars indicate significant differences (P < 0.05, one-way ANOVA). The statistical comparisons were performed among genotypes separately for each time point. (d) Size of the necrotic lesions formed on B. cinerea -infected WT, elp2, 35S:WRKY33, and 35S:WRKY33 elp2 plants. Lesion sizes on 72 leaves measured in three independent experiments were combined and analyzed as a two-way ANOVA, blocked by experiment. The resulting mean and SE are presented. a,b Different letters above the bars indicate significant differences (P < ). No significant interaction was detected between elp2 and 35S:WRKY33 (P = ). (e g) Expression of ORA59 (e) and PDF1.2 (f, g) in WT, elp2, 35S:ORA59, and 35S:ORA59 elp2 plants after Botrytis cinerea infection. Data were analyzed as in (a). (h) Size of the necrotic lesions formed on B. cinerea -infected WT, elp2, 35S:ORA59, and 35S:ORA59 elp2 plants. Lesion sizes were analyzed and presented as in (d). a,b,c Different letters above the bars indicate significant differences (P < ). No significant interaction was detected between elp2 and 35S:ORA59 (P = ).

9 Function of Elongator in necrotroph resistance 1027 Figure 5. Characterization of the elp2 jin1 double mutant. (a d) Expression of MYC2 (a), VSP2 (b), JR1 (c), and PDF1.2 (d) in WT, elp2, jin1, and elp2 jin1 (e2j1) plants. Expression levels were monitored using qpcr and normalized against UBQ5. Data represent the mean of three independent samples with standard deviation (SD). a,b,c Different letters above the bars indicate significant differences (P < 0.05, oneway ANOVA). The statistical comparisons were performed among genotypes for each time point. (e) Symptoms on rosette leaves of 4-week-old soilgrown plants inoculated with Botrytis cinerea. Photos were taken 4 days post-inoculation. (f) Size of the necrotic lesions formed on B. cinerea -infected WT, elp2, jin1, and elp2 jin1 plants. Lesion sizes on 72 leaves measured in three independent experiments were combined and analyzed as a twoway ANOVA, blocked by experiment. The resulting a,b, mean and standard error (SE) are presented. c Different letters above the bars indicate significant differences (P < ). The asterisk indicates a significant interaction between elp2 and jin1 (P < ), suggesting that elp2 suppressed jin1- mediated B. cinerea resistance. mutants also harbored significantly more biomass of the fungal pathogen than the single mutants, as estimated by the amounts of B. cinerea DNA (Figure 6c,f). A two-way ANOVA, blocked by experiment, of the lesion sizes revealed a significant interaction between elp2 and coi1 and no significant interaction between elp2 and ein2 (Figure 6b,e), indicating that the function of ELP2 overlaps with COI1 and is additive to EIN2 for B. cinerea resistance. Histone acetylation levels in WRKY33, ORA59, and PDF1.2 are reduced in elp2 To examine if ELP2 plays a role in maintaining basal histone acetylation levels in some JA/ET pathway defense genes, we analyzed the acetylation levels of histone H3 in WRKY33, ORA59, and PDF1.2 using chromatin immunoprecipitation (ChIP). Following formaldehyde crosslinking and cell lysis of wild type and elp2 leaf tissues, DNA-histone complexes were immunoprecipitated using a specific antibody against histone H3 acetylated at lysines 9 and 14 (H3K9/14ac). The amount of precipitated DNA was assessed using qpcr to determine the levels of histone H3K9/14ac. Consistent with the reduced basal expression and induction, levels of histone H3K9/14ac in the coding regions of WRKY33, ORA59, and PDF1.2 were significantly lower in elp2 than in the wild type (Figure 7). As histone acetylation is thought to promote transcription (Workman and Kingston, 1998), decreased basal histone acetylation may contribute to the delayed and/or reduced activation of these JA/ET pathway defense genes in elp2. ELP2-GFP is associated with the chromatin of WRKY33, ORA59, and PDF1.2 To test whether ELP2 is associated with the chromatin of WRKY33, ORA59, and PDF1.2, ChIP assays were performed with chromatin isolated from transgenic plants containing a 35S:ELP2-GFP transgene in the elp2 mutant background using an anti-gfp antibody. The ELP2-GFP fusion protein is functional, since it complements all the elp2 mutant phenotypes, including susceptibility to B. cinerea (Defraia et al., 2010) (Figure S3). Wild type Col-0 plants were used as negative controls, as the elp2 mutant is in the Col-0 genetic background. The same primer sets used in Figure 7 for histone H3K9/14ac assays were used for qpcr to estimate WRKY33, ORA59, and PDF1.2 chromatin enrichment. As shown in Figure 8, ELP2-GFP was significantly enriched in the coding regions of WRKY33, ORA59, and PDF1.2. Intriguingly, ELP2-GFP was also enriched in the promoter regions of ORA59 and PDF1.2, despite the fact that histone

10 1028 Chenggang Wang et al. Figure 6. Characterization of the elp2 coi1 and elp2 ein2 double mutants. (a, b) Symptoms and size of the necrotic lesions formed on Botrytis cinerea-infected WT, elp2, coi1, and elp2 coi1 (e2c1) plants. In (a), photographs were taken 3 days post-inoculation. In (b), lesion sizes on 72 leaves measured in three independent experiments were combined and analyzed as a twoway ANOVA, blocked by experiment. The resulting mean and SE are presented. a,b,c,d Different letters above the bars indicate significant differences (P < ). The asterisk indicates a significant interaction between elp2 and coi1 (P = ). (c) Biomass of B. cinerea on WT, elp2, coi1, and elp2 coi1 (e2c1) plants. Data represent the mean qpcr amplification of the B. cinerea ActinA (BcActA) DNA relative to the Arabidopsis Actin2 DNA from three independent samples. a,b,c Different letters above the bars indicate significant differences (P < 0.05, one-way ANOVA). (d, e) Symptoms and size of the necrotic lesions formed on B. cinerea -infected WT, elp2, ein2, and elp2 ein2 (e2e2) plants. In (d), photos were taken 4 days post-inoculation. In (e), lesion sizes were analyzed and presented as in (b). a,b,c Different letters above the bars indicate significant differences (P < ). No significant interaction was detected between elp2 and ein2 (P = ). (f) Biomass of B. cinerea on WT, elp2, ein2, and elp2 ein2 (e2e2) plants. Data were analyzed as in (c). H3K9/14ac levels in these regions are comparable between elp2 and the wild type. Although the underlying reasons for this discrepancy need further investigation, this result clearly indicates that ELP2-GFP binds to the chromatin of several ELP2-modulated JA/ET pathway defense genes. DISCUSSION Necrotrophic fungal pathogens cause devastating diseases on horticultural and agronomic crops. Unlike biotrophic pathogens, which rely on living tissues, necrotrophs kill host cells and proliferate on nutrients from dead or dying tissues. The interaction between necrotrophic pathogens and host plants is complex and involves both pathogen disease factors and plant immune regulators (Lai and Mengiste, 2013). In recent years, quantitative resistance and major defense regulators in host plants have been reported (Glawischnig et al., 2004; AbuQamar et al., 2006; Veronese et al., 2006; Ferrari et al., 2007; Kemmerling et al., 2007; Nafisi et al., 2007; Ndamukong et al., 2007; Rowe and Kliebenstein, 2008; Kwon et al., 2009; La Camera et al., 2011; Lenz et al., 2011; Windram et al., 2012). However, mechanisms and processes underlying host responses to necrotrophs still remain to be fully understood. In this study, we analyzed defense responses against the necrotrophic fungal pathogens B. cinerea and A. brassicicola in Arabidopsis elp/elo mutants. Our results indicate that Elongator is required for proper transcription of certain defense genes and contributes significantly to resistance against the necrotrophic pathogens. Elongator is required for full induction of several defense genes by JA and ET. Previous work has shown that basal levels of JA and ET are significantly higher in elp/elo mutants than in wild type and that background expression levels of some JA biosynthesis and JA- or ETresponsive genes are also higher in the mutants (Nelissen et al., 2010). Interestingly, we found that mutations in Elongator dramatically inhibit JA- and ET-induced defense gene expression (Figure 1). The elp/elo mutations also significantly affect B. cinerea -induced transcriptional changes. In elp/elo mutants, B. cinerea -induced expression of ORA59, PDF1.2, CHIB, and HEL are delayed and/or decreased (Figures 2a and 3c). At the transcriptome level, the elp2

11 Function of Elongator in necrotroph resistance 1029 Figure 7. Histone H3 acetylation levels in WRKY33, ORA59 and PDF1.2. The position of the primers is relative to the initiation ATG codon. The relative amount of immunoprecipitated chromatin fragments (as determined by qpcr) from elp2 was compared with that from WT (arbitrarily set to 1). Data represent the mean of three independent samples with SD. An asterisk indicates a significant difference between elp2 and WT (P < 0.05, Student s t- test). mutation dramatically changes B. cinerea -induced transcriptional reprogramming. Compared with wild type, the numbers of genes that are up- or down-regulated in elp2 are significantly reduced at early time points after B. cinerea infection (Figure 3a). Therefore, ELP2 is likely required for the Arabidopsis genome to efficiently change its transcriptional profiles for defending against necrotrophic pathogens (Figure 3b). In agreement, elp/elo Figure 8. Association of ELP2-GFP with WRKY33, ORA59 and PDF1.2 chromatin. Chromatin was isolated from 10-day-old wild type Col-0 and 35S:ELP2-GFP elp2 seedlings and immunoprecipitated using an anti-gfp antibody. Immunoprecipitated DNA and input DNA were determined by qpcr. The y- axis indicates the relative enrichment of DNA fragments, which was calculated by normalizing immunoprecipitated DNA against input DNA. The x- axis indicates the position (relative to the initiation ATG codon) of the primers used in the qpcr. Data represent the mean of three independent samples with standard deviation (SD). mutants are significantly more susceptible than wild type to B. cinerea and A. brassicicola (Figure 2b g). ELP2 appears to differentially influence several wellcharacterized defense-related transcriptional cascades.

12 1030 Chenggang Wang et al. Induction of the ORA59 and ERF1 transcriptional cascades, which function downstream of JA and ET (Lorenzo et al., 2003; Pre et al., 2008), and the WRKY33 transcriptional cascade, which appears to be independent of JA and ET (Zheng et al., 2006), are inhibited in the elp2 mutant compared with wild type during B. cinerea infection (Table 1). Since overexpression of ORA59 or WRKY33 fails to restore the induction pattern of their target gene PDF1.2 in elp2 plants (Figure 4), ELP2 may be required for transcription activation of ORA59 and WRKY33 as well as their target gene PDF1.2. Conversely, induction of the MYC2 transcriptional cascade, which acts downstream of JA (Lorenzo et al., 2004), is enhanced in elp2 (Table 1). Removal of MYC2 by a jin1 mutation completely blocks the induction of its target genes JR1 and VSP2 (Figure 5a c), indicating that enhancement of the MYC2 transcriptional cascade in elp2 upon B. cinerea infection depends on the MYC2 gene. Although MYC2 negatively regulates ORA59 and ERF1 as well as their target genes during JA signaling (Dombrecht et al., 2007; Zander et al., 2010), removal of MYC2 does not restore PDF1.2 induction in elp2 plants (Figure 5d). Therefore, ELP2 seems to independently and oppositely contribute to the two different branches of the JA signaling pathway that regulate resistance to pathogens and insects, respectively. Unlike the coi1 and ein2 mutations, which almost completely block JA and ET signaling, respectively (Xie et al., 1998; Alonso et al., 1999), the elp2 mutation delays and/or decreases JA- and ET-mediated defense signaling against pathogens (Figure 1). However, the elp2 mutant is as susceptible as coi1 and ein2 to B. cinerea, indicating that ELP2 is also a major contributor to resistance against B. cinerea (Figure 6a f). Interestingly, the elp2 mutation significantly interacts with coi1 but not with ein2 in resistance to B. cinerea (Figure 6b,e), indicating that the function of ELP2 overlaps with COI1 and is additive to EIN2 with respect to B. cinerea. The Arabidopsis Elongator has been implicated in multiple cellular processes including histone acetylation, DNA methylation, and trna modification (Ding and Mou, 2015). In elp2, reduced histone acetylation levels in WRKY33, ORA59, and PDF1.2 are correlated with delayed and/or reduced induction of these defense genes during B. cinerea infection, suggesting that ELP2 may contribute to resistance against this pathogen through histone acetylation. Consistent with this hypothesis, histone acetylation has been emerging as a pivotal player in JA/ET signaling. Depending upon the regulatory enzyme, histone acetylation could be an activator or a repressor of JA/ET signaling and/or resistance to necrotrophic fungal pathogens (Devoto et al., 2002; Zhou et al., 2005; Wu et al., 2008; Zhu et al., 2011). Conversely, since DNA methylation affects JA/ET signaling and resistance to B. cinerea (see Figure S4) (Lopez et al., 2011; Luna et al., 2012), Elongator may also contribute to defense against fungal pathogens through DNA methylation. Additionally, recent work suggests that the defects of elp/elo mutants in auxin-controlled developmental processes are likely caused by deficiencies in trna maturation (Leitner et al., 2015). The elp/elo defense phenotypes may simply result from trna modification deficiencies. Clearly, further investigations are needed to pinpoint the exact mechanisms by which Elongator functions in plant immunity including resistance to necrotrophic fungal pathogens. EXPERIMENTAL PROCEDURES Plant materials and pathogen infection The wild type Arabidopsis thaliana (L.) Heynh. Columbia (Col-0) ecotype and the mutant alleles elo2 (SALK_004690), elp2-5 (Defraia et al., 2010), elo3 (GABI_555H06), elo1 (SALK_079193), elp5 (GABI_700A12), coi1-1 (Xie et al., 1998), ein2-1 (Alonso et al., 1999), and jin1-9 (SALK_017005) were used. Plant growth and pathogen infection were performed as previously described (Zhang et al., 2012). The B. cinerea strain B05 and A. brassicicola strain MUCL were used in this study. B. cinerea and A. brassicicola inoculation was performed as described by Pre et al. (2008) and Zhang et al. (2012). Lesion lengths and widths were measured before disease symptoms expanded beyond inoculated leaves using a caliper and the average of the length and the width was used to represent the size of a lesion. In each experiment, 24 plants per genotype were used for three sub-experiments. Each sub-experiment was performed in the same flat under the same clear plastic dome. In each flat, plants from different genotypes (eight plants per genotype) were randomly arranged. One leaf on each plant was inoculated and all leaves were inoculated with the same spore suspension. In total, 24 lesions (one on each plant) were measured and used for statistical analysis. RNA analysis RNA extraction, RNA gel blot analysis, reverse transcription, and real-time quantitative PCR (qpcr) analysis were carried out as described by Defraia et al. (2010). In each experiment, three independent biological samples (one sample per sub-experiment) were collected at each time point per genotype and analyzed. UBQ5 was used as the reference gene, since it is stably expressed (Gutierrez et al., 2008). New qpcr primers used in this study are listed in Table S1. Microarray analysis Microarray experiments were performed and data were processed and normalized as described previously (Wang et al., 2013). After normalization, instead of using Student s t-test as in Wang et al. (2013), a linear model was fitted on each gene for comparison. To control false discovery rate (FDR) and correct multiple hypothesis testing, a q-value was calculated and used to assess the significance of each test (Benjamini and Hochberg, 1995). To determine which genes were differentially expressed between elp2 and wild type across temporal states, the gene expression data were further analyzed using linear model methods with a consideration of factorial designs. Each gene was tested for difference in fold changes of elp2 from that of the wild type at 3, 6, 12, 24, and 48 hpi, respectively. The fold changes were calculated using the corresponding samples of 0 hpi as the reference. Multiple hypothesis testing was adjusted by calculating

13 Function of Elongator in necrotroph resistance 1031 q-values to control the FDR (Benjamini and Hochberg, 1995). The linear model analysis was performed using limma package in R (Smyth, 2004). In total, 9403 genes, whose changes in elp2 were significantly different (q-value < 0.05) from those in the wild type at least at one time point, were selected for PCA. PCA was performed using prcomp in R. Sample means were projected on to the first three principal components to visualize the distribution of each sample state. Chromatin immunoprecipitation Chromatin immunoprecipitation was performed as previously described (Wang et al., 2013). For ELP2-GFP, an anti-gfp antibody (ab290, Abcam) was used and the quantity of precipitated DNA corresponding to a specific gene region was assessed by qpcr and normalized to input DNA (Haring et al., 2007). Primers used for ChIP-qPCR are listed in Table S2. Statistical methods Statistical analyses were conducted using the one-way ANOVA in the IBM SPSS Statistics software ( ware/analytics/spss/products/statistics/) and the data analysis tools in Excel of Microsoft Office 2004 for Macintosh (Student s t-test: two samples assuming unequal variances). Lesion sizes measured in three independent experiments were combined and analyzed as a one-way or two-way ANOVA, blocked by experiment, using JMP 11 (JMP Software, jmp/). Other experiments were conducted three times with similar patterns and results from a representative experiment were presented. ACKNOWLEDGEMENTS We thank Dr Jeffery A. Rollins (University of Florida, USA) for the Botrytis cinerea strain B05, Dr Xinnian Dong (Duke University, NC, USA) for the Alternaria brassicicola strain MUCL 20297, Dr Robert Fischer (University of California, Berkeley, CA, USA) for rdd seeds, the Arabidopsis Biological Resource Center at The Ohio State University (Columbus, OH, USA) for met1-3, ddc, SALK_004690, SALK_079193, and SALK_ seeds, and the European Arabidopsis Stock Centre at The University of Nottingham (Nottingham, UK) for GABI_555H06 and GABI_700A12 seeds. This work was supported by a grant from the National Science Foundation (IOS ) awarded to Z.M. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Morphology of 35S:WRKY33, 35S:WRKY33 Atelp2, 35S: ORA59, and 35S:ORA59 Atelp2 plants Figure S2. Student s t-test of the expression data of WRKY33 in elp2 and wild-type plants. Figure S3. 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