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Researchers Find Role for Dynamic Methylation in Arabidopsis Immunity

NEW YORK (GenomeWeb News) – DNA methylation contributes to Arabidopsis immunity, helping to dynamically regulate some of the genes that the plants use to ward off bacterial infection, according to a study appearing online last night in the Proceedings of the National Academy of Sciences.

Following preliminary experiments illustrating that methylation-deficient Arabidopsis thaliana mutants have enhanced bacterial resistance, a California research team led by investigators at the Salk Institute for Biological Studies used MethylC-sequencing to characterize typical A. thaliana plants exposed to the either pathogenic Pseudomonas syringae bacteria or a harmless strain of P. syringae. They also did similar genome-wide methylation single-base profiling on plants treated with salicylic acid, the hormone the plant uses to rally defenses against such interlopers.

Results of the study uncovered differentially methylated regions in the genomes of plants facing each stressor. In plants infected with pathogenic bacteria or treated with salicylic acid, for example, the team saw differentially methylated sites — often lower-than-usual methylation — near plant defense genes that were more highly expressed under these conditions.

The salicylic acid signaling hormone also led to methylation changes at some transposon sequences, which coincided with a jump in the production of small, interfering RNAs that can, in turn, influence transposon and coding gene expression.

"Our unbiased, genome-wide approach uncovered unique aspects of stress-induced dynamic DNA methylation changes, including a striking relationship among hypomethylation, biogenesis of specific siRNAs, and transcriptional derepression at some transposons," Joseph Ecker, who holds a Salk International Council Chair in Genetics, and his colleagues wrote.

As in animals, DNA methylation in Arabidopsis is known to contribute to key developmental and/or cell type specification processes, the researchers explained, including stable gene silencing, transposon silencing, and gene imprinting.

Although the methyltransferase and glycosylase enzymes that add and remove methyl groups to and from cytosine nucleotides have been found in the plant, they added, there are questions about how dynamic cytosine methylation remains once development and cellular differentiation are complete.

"Although it remains unclear if widespread alterations in DNA methylation analogous to gene imprinting can be elicited by stress," they wrote, "plants subjected to heat stress display transient changes in nucleosome density, as well as transcriptional derepression, at some repetitive elements, indicating that the epigenetic landscape can be dynamically modified."

For the current study, researchers first focused on A. thaliana plants infected with the P. syringae pv. tomato DC3000 pathogen, looking at whether methylation might have a role in response to this infection.

In their preliminary experiments, for instance, the team found that Arabidopsis mutants with methylation defects that were infected with P. syringae pv. tomato not only showed differential expression of several pathogen response genes compared to typical, wild type plants, but were also more resistant to the infection.

Using the MethylC-sequencing method that they have applied in previous methylation studies, Ecker and his colleagues went on to do genome-wide, single base level methylation profiling on DNA from the leaves of untreated, wild type A. thaliana plants and plants that had been infected with P. syringae pv. tomato for five days.

In the process, they generated four methylomes representing two infected and two uninfected plants at a depth of between 8.2 and 12.1X per cytosine across 93 to 95 percent of the cytosines in the plant's genome.

While the researchers did not find wholesale cytosine methylation changes following infection, they did identify specific regions of the genome that were differentially methylated, with many of these falling in gene-rich parts of the A. thaliana genome.

There were also differences in the prevalence of differential methylation depending on cytosine's sequence context and the type of sequence considered, the team found.

For example, differentially methylated regions involving cytosine bases that neighbored guanine tended to be enriched in intergenic regions, particularly upstream of transcriptional start sites. On the other hand, differentially methylated sites involving cytosines in the so-called CHH sequence context were more often differentially regulated at transposable element sequences.

Similar methylome profiling experiments on plants exposed to non-virulent bacteria or to the signaling hormone salicylic acid indicated that plants respond to these stresses with differential methylation as well. The extent of this differential methylation and the sequences involved varies depending on the specific stressor involved, researchers reported, though differentially expressed regions in both pathogenic bacteria and salicylic acid treatment were enriched near plant defense genes.

When the team folded in information from Illumina messenger RNA and small RNA sequencing, it found evidence that lower-than-usual methylation, in particular, seemed to prompt elevated expression of nearby genes in A. thaliana. Meanwhile, differential methylation of transposon sequences in plants exposed to salicylic acid showed ties to enhanced production of 21-nucleotide siRNAs.

"Our data support a model whereby DNA methylation imparts persistent control over some defense genes during non-stressful conditions," study authors wrote, "but, in response to environmental stimuli, can change dynamically to alter gene expression."

Those involved in the study cautioned that more research is needed to understand why specific genes are regulated in this manner, to determine which cells are involved in these processes, and to track methylation dynamics during infection.

"[A] detailed temporal analysis of the methylation dynamics from the onset of infection, for which our study provides a framework, will be necessary to fully understand dynamic methylation in the context of disease progression," they wrote.

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