Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1)

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles

Multiple downy mildew effectors target the stress‐related NAC transcription factor LsNAC 069 in lettuce

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector‐target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast‐two‐hybrid (Y2H) screening. Interaction of the lettuce tail‐anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N‐terminal NAC domain but depends on the C‐terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail‐anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC‐domain (LsNAC069ΔNAC). Relocalization was significantly reduced in the presence of the Ser/Cys‐protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co‐expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses

Multiple downy mildew effectors target the stress‐related NAC transcription factor LsNAC 069 in lettuce

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector‐target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast‐two‐hybrid (Y2H) screening. Interaction of the lettuce tail‐anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N‐terminal NAC domain but depends on the C‐terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail‐anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC‐domain (LsNAC069ΔNAC). Relocalization was significantly reduced in the presence of the Ser/Cys‐protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co‐expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses

Comparison of expression profiles of genes <i>AT3G01345</i>, <i>AT3G27473</i>, <i>AT3G30720</i>, <i>AT3G30820</i>, <i>AT4G25530 and AT5G34850</i> in MET1 lines.

<p>T3 seeds are labelled in blue, T4 seeds are labelled in orange.</p

RT-PCR analysis of four genes with dense methylation in MET1 transformants with (+) and without the transgene (-).

<p>Lines A1 and A2 express a catalytically active MET1 transgene, lines I1 and I2 express a catalytically inactive MET transgene. The mean and the standard error are shown for three biological replicates each tested in three technical replicates. Values on the y-axis represent the log2-fold-difference compared to the control line.</p

Shoot and root phenotypes in wildtype control plants, in MET1 transformants (+) and in lines derived from MET1 transformants, from which the transgene has been removed (-).

<p>Lines A1 and A2 express a catalytically active MET1 transgene, lines I1 and I2 express a catalytically inactive MET transgene. Images were taken eight weeks after stratification. The scale bar for shoot images indicates 5cm, the scale bar for root images indicates 10mm.</p

Comparison of expression profiles of genes <i>AT3G01345</i>, <i>AT3G27473</i>, <i>AT3G30720</i>, <i>AT3G30820</i>, <i>AT4G25530 and AT5G34850</i> in the <i>met1-1</i> mutant and <i>met1-1 RE</i>.

<p>The mean and the standard error are shown for three biological replicates each tested in three technical replicates. Values on the y-axis represent the fold-difference compared to the control line.</p

ChIP analysis of genes <i>At3G27473</i>, <i>At3G01345</i>, <i>At3G30720</i> and At5G34850 for H3K9me2, H3K4me3 and H4 acetylation marks.

<p>The means and the standard errors are shown for three biological replicates each tested in three technical replicates. Values on the y-axis represent the fold-difference of histone mark levels compared to the control line.</p

DNA methylation analysis of regions (S3 Fig) of genes <i>AT3G01345</i>, <i>AT3G27473</i>, <i>AT3G30720</i>, <i>AT5G34850</i> in <i>MET1</i> transformants (+) and in lines derived from <i>MET1</i> transformants, from which the transgene has been removed (-).

<p>Lines A expresses a catalytically active <i>MET1</i> transgene, line I1 expresses a catalytically inactive <i>MET</i> transgene. Red bars denote CG methylation, blue bars CHG methylation and green bars CHH methylation.</p

Induction of epigenetic variation in Arabidopsis by over-expression of <i>DNA METHYLTRANSFERASE1 (MET1)</i> - Table

Induction of epigenetic variation in Arabidopsis by over-expression of <i>DNA METHYLTRANSFERASE1 (MET1)</i> - Tabl