Changes on AtSN1 methylation levels by Chop-PCR

Our results and others (Dowen et al., 2012; Yu et al., 2013) showed that several TGS-loci are transcriptionally activated during Pto-infection. Transcriptional activation of many of these loci is due to reduce DNA methylation levels, including that of Athila or AtSN1, the two loci displaying the strongest activation. To confirm that transcriptional activation of TGS loci was also due to DNA hypomethylation in our system, we used chop-PCR to analyse any changes on the DNA methylation status of AtSN1 on Pto-infected plants. Chop-PCR is an assay in which genomic DNA is subjected to digestion with a methylation-sensitive or methylation-dependent restriction endonuclease and then tested as a template for PCR amplification using primers flanking the restriction sites (Earley et al., 2010; Oakes et al., 2009).

McrBC is a restriction enzyme widely used for this type of analysis. It

recognizes the two half-sites of the form 5′-G/Am_{C-3′ that can be separated}

up to 2kb (5´…Pum_{C (N40-2000) Pu}m_{C…3´), with an optimal separation of 55-}

103 bp. McrBC is methylation-dependent and thus cuts methylated but not unmethylated DNA. The McrBC enzyme only requires two methylated half- sites within the PCR-amplified region to cleave DNA, despite the methylation status of other sites. If a genomic region becomes hypomethylated, the enzyme will cut to a lesser extend and this will increase the amount of DNA that can be amplified by PCR. Ten Arabidopsis plants were infiltrated with

Pto DC3000 (5x107 _{cfu/ml, night infection) and samples were taken at 0 and}

24hpi. Mock samples were included to determine wild-type levels of DNA methylation of AtSN1 and samples from ago4-2 plants were analysed as a positive control as this transposon becomes hypomethylated in this mutant (Agorio and Vera, 2007). Three leaves from each plant were independently macerated and frozen tissue was split in two to extract RNA and DNA. RNA was used to confirm transcriptional activation of AtSN1 by RT-qPCR, and genomic DNA used to perform the chop-PCR assay. Genomic DNA was digested with McrBC and a qPCR performed using primers flanking the region of AtSN1 that has been previously described to be most intensely methylated (Agorio and Vera, 2007; Xie et al., 2004; Yu et al., 2013). Although we could detect the transcriptional activation of AtSN1 on Pto-infected plants by 24 hpi (Figure 1.12A), we found no evidence of hypomethylation for the transposon

on the same infected plants (Figure 1.12B). To rule out the possibility that

DNA hypomethylation was taking place at an earlier time on the infection process, we repeated these assays analysing samples at 3 and 9 hpi. No evidence for hypomethylation of AtSN1 during Pto infection could be detected at these time points either (Figure 1.12C). This failure to detect DNA

hypomethylation of AtSN1 during Pto infection could be due to technical reasons, i.e. a low sensitivity of the chop-PCR assay, which could perhaps be solved by using other methylation-sensitive enzymes and other primers for

AtSN1 flanking their corresponding restriction sites, or due to the nature of

the assay. This later possibility is supported by the small differences in methylation levels found for AtSN1 by Yu and collaborators (Yu et al., 2013) following flg22 treatment. Flg22 treatment is expected to induce stronger changes than those taking place during infection since higher amounts of flagellin are thus presented to the plant. Furthermore, Yu and collaborators proposed that changes due to interaction with Pto occurs only in cells adjacent to bacteria, leading to leaf samples including cells with altered, as well as cells with normal DNA methylation levels, thus potentially diluting the changes and reducing the sensitivity of the assay. Any of these reasons could explain our negative results.

Figure 1.12. DNA methylation status of AtSN1 in Pto DC3000-infected Arabidopsis plants by Chop-PCR. (A and B) Arabidopsis plants were infected with Pto DC3000 and samples were harvested

at 0 and 24 hours post-inoculation (0 hpi and 24 hpi). Mock-inoculated plants (10 mM MgCl2) and

ago4-2 were included as negative and positive controls, respectively. Macerated tissue was split in two

and samples were processed for RT-qPCR on (A) and for chop-PCR on (B). (A) Accumulation of AtSN1

transcripts was analysed using RT-qPCR. Transcript levels were normalised to actin and the results presented as relative transcript accumulation compared to levels detected in plants inoculated with Pto DC3000 at 0 hpi. (B) Genomic DNA was digested with the methylation-dependent enzyme McrBC and

an AtSN1 fragment was amplified by PCR. A fragment from locus At3g18780 known to be non- methylated (Widman et al., 2009), was used to normalize the PCR amplified levels. The experiment was performed two times and one representative biological replicate is shown. Bars represent the mean values from 1 experiment with 3 (mock), 4 (0 hpi) and 10 (24 hpi) plants. Error bars represent the standard error. Mean values marked with the same letter were not significantly different from each other as established by One-way ANOVA, Benferroni PosHoc test (99% confidence intervals). (C)

Arabidopsis plants were infected with Pto DC3000 and samples were harvested at 3 and 9 hours post-

inoculation (3 hpi and 9 hpi). Mock-inoculated plants (10 mM MgCl2) and ago4-2 were included as

negative and positive controls, respectively. Genomic DNA was digested with the methylation- dependent enzyme McrBC and an AtSN1 fragment was amplified by PCR. A fragment from locus At3g18780 known to be non-methylated, was used to normalize the PCR amplified levels. Bars represent the mean values from 1 experiment with 3-4 plants. Error bars represent the standard error. Mean values marked with the same letter were not significantly different from each other as established by One-way ANOVA, Benferroni PosHoc test (99% confidence intervals).

1.5. Activation of a transcriptionally silent GUS transgene