with the anti-myc antibody greatly enriched ACS2 and ACS6 promoter regions containing the W-boxes. In contrast, the IgG control antibody failed to enrich either gene promoter. This result demonstrates that WRKY33 directly binds to the promoters of ACS2 and ACS6 in vivo, suggesting that WRKY33 is the transcription factor downstream of the MPK3/MPK6 cascade involved in the activation of ACS2 and ACS6 expression. High levels of ACS6 gene expression elevate B. cinerea– induced ethylene production To provide further direct evidence in support of the role of ACS6 gene activation in B. cinerea-induced ethylene production, we transformed a DEX-inducible promoter-driven ACS6 (GVG-ACS6) construct into the acs2-1/acs6-2/acs7-1 mutant background and then compared the ethylene induction in acs2-1/acs6-2/acs7-1/ GVG-ACS6 plants with and without DEX treatment. Two independent lines (#5 and #12) with different levels of ACS6 Figure 9. WRKY33 transcription factor binds to the promoter of ACS2 and ACS6 genes in vivo. (A) The promoters of ACS2 and ACS6 genes are rich in W-boxes, the cis-element binding sites of the WRKY transcription factor. A diagram indicates the number and relative position of the W-boxes in the promoters of the ACS2 and ACS6 genes. Line arrows indicate the position of primers used for qPCR after chromatin immunoprecipitation (ChIP). Positions of the predicted transcriptional starting sites are indicated by arrows with turning lines and negative numbers. The T-DNA insertion site in the SALK_090423 acs6-2 allele, which locates in the promoter region of ACS6 gene, is also indicated. (B) ChIP-qPCR analysis was performed using DD/4myc- WRKY33WT plants generated from the cross of wrky33/4myc-WRKY33WT with DD lines. Input chromatin was isolated from two-week-old seedlings 12 hr after DEX treatment. Epitope-tagged WRKY33-chroma- tin complex was immunoprecipitated with an anti-myc antibody. A control reaction was processed side-by-side using mouse IgG. ChIP- and input-DNA samples were quantified by real-time qPCR using primers specific to the promoters of ACS2 (left panel) and ACS6 (right panel) genes. ChIP results are presented as percentage of input DNA. Error bars indicate standard deviations (n = 3). doi:10.1371/journal.pgen.1002767.g009 Dual-Level Regulation of Ethylene Biosynthesis transgene induction after DEX treatment were used for this experiment to establish a correlation between the levels of ACS6 gene expression and the levels of ethylene induction. As shown in Figure 10A, without DEX treatment, both GVG-ACS6 transgenic lines produced about the same levels of ethylene after B. cinerea treatment in comparison to the acs2-1/acs6-2/acs7-1 triple mutant. In the presence of DEX, which induced ACS6 expression (Figure 10B), the ethylene production was greatly enhanced. The higher level of ACS6 induction in line #5 in the presence of DEX correlated with a higher level of ethylene induction than that in line #12. Furthermore, ethylene induction in Line #5 was higher than that in the wild type, indicating that transgene induction after DEX treatment not only complements the loss of ACS6, but also compensates the loss of ACS2 and ACS7 genes. In the absence of B. cinerea infection, DEX treatment only slightly elevated the ethylene production (Figure S5) to a level similar to the basal level ethylene production of Col-0 (,10 nL/g FW in 24 hrs). This low-level ethylene production is likely a result of high-level ACS6 gene induction after DEX treatment (and associated higher-level of de novo ACS6 protein synthesis) in combination with the basal level activity of MPK6, which can phosphorylate and stabilize ACS6 protein. MPK6 has very low basal activity even in the absence of stress/pathogen infection [16]. This is consistent with our previous conclusion that the overexpression of ACS6 gene in the absence of MPK3/MPK6 activation is not sufficient to induce ethylene production due to the lack of phosphorylation stabilization [16]. As a result, we can conclude that the high level of ethylene production seen in acs2-1/acs6-2/acs7-1/GVG-ACS6 lines after DEX and B. cinerea treatment is a combination of high level of gene expression (as a result of DEX treatment), and phosphorylation stabilization due to MPK3/ MPK6 activation by B. cinerea infection. Our attempts to identify the T-DNA insertion line in the coding region of the ACS6 gene (SALK_025672, acs6-1) failed to reveal a true mutant plant from the seeds received. As a result, we have been using the SALK_090423 line (acs6-2), which has a T-DNA insertion 170 bp upstream of the ATG start codon (Figure 9A)[16,28]. In the past, we routinely used the double DCt method to quantify gene expression in real-time PCR analysis, which indicated the acs6-2 mutant allele as a knockout mutant (Figure 11B, upper panel). However, a more careful analysis performed in this study revealed that it is actually a knockdown mutant with an elevated basal level expression. As shown in Figure 11B (lower panel), acs6-2 seedlings showed a higher basal level of ACS6 expression, but no increase in its transcripts was detected after B. cinerea infection. In contrast, no transcript was detected in the acs6-1 mutant before and after B. cinerea infection. Side-by-side comparison demon- strated that ethylene production levels in acs6-2 and acs6-1 single mutants after B. cinerea inoculation were similar (Figure 11A). In contrast, acs2-1/acs6-2 seedlings produce higher levels of ethylene than acs2-1/acs6-1 (both have the same acs2 mutant allele)(Figure S2). The observable difference between acs6-2 and acs6-1 alleles in the acs2-1 mutant background could be due to the reduction of total ethylene production in the absence of ACS2 gene, which makes it possible to observe a small difference. These results suggest that acs6-2 mutant allele is not a mutant as acs6-1 allele, and that the high level induction of ACS6 is important to pathogen-induced ethylene production. Together with the gain-of-function evidence shown in Figure 10, we can conclude that ACS6 gene activation plays an essential role in promoting ethylene production in plants challenged by pathogens. Discussion Plants challenged by pathogens, especially necrotrophs such as B. cinerea, produce very high levels of ethylene, a critical event in PLoS Genetics | www.plosgenetics.org 8 June 2012 | Volume 8 | Issue 6 | e1002767