Formaldehyde-assisted isolation of regulatory DNA elements from Arabidopsis leaves.

Eukaryotic gene transcription is associated with the eviction of nucleosomes and the formation of open chromatin, which enables the recruitment of transcriptional coactivators and other regulatory factors. Open chromatin is thus a hallmark of functional regulatory DNA elements in genomes. In recent years, formaldehyde-assisted isolation of regulatory elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes, particularly yeast, human, and mouse. However, it has proven challenging to adapt the FAIRE protocol for use on plant material, and the few available protocols all have their drawbacks (e.g., applicability only to specific developmental stages). In this Protocol Extension, we describe a reliable FAIRE protocol for mature Arabidopsis (Arabidopsis thaliana) leaves that adapts the original protocol for use on plants. The main differences between this protocol extension and the earlier FAIRE protocol are an increased formaldehyde concentration in the chromatin crosslinking buffer, application of a repeated vacuum to increase crosslinking efficiency, and altered composition of the DNA extraction buffer. The protocol is applicable to leaf chromatin of unstressed and stressed plants and can be completed within 1 week. Here, we also describe downstream analysis using qPCR and next-generation sequencing. However, this Protocol Extension should also be compatible with downstream hybridization to a DNA microarray. In addition, it is likely that only minor adaptations will be necessary to apply this protocol to other Arabidopsis organs or plant species.

Isolation of Open Chromatin Identifies Regulators of Systemic Acquired Resistance.

Upon local infection, plants activate a systemic immune response called systemic acquired resistance (SAR). During SAR, systemic leaves become primed for the superinduction of defense genes upon reinfection. We used formaldehyde-assisted isolation of regulatory DNA elements coupled to next-generation sequencing to identify SAR regulators. Our bioinformatic analysis produced 10,129 priming-associated open chromatin sites in the 5' region of 3,025 genes in the systemic leaves of Arabidopsis (Arabidopsis thaliana) plants locally infected with Pseudomonas syringae pv. maculicola Whole transcriptome shotgun sequencing analysis of the systemic leaves after challenge enabled the identification of genes with priming-linked open chromatin before (contained in the formaldehyde-assisted isolation of regulatory DNA elements sequencing dataset) and enhanced expression after (included in the whole transcriptome shotgun sequencing dataset) the systemic challenge. Among them, Arabidopsis MILDEW RESISTANCE LOCUS O3 (MLO3) was identified as a previously unidentified positive regulator of SAR. Further in silico analysis disclosed two yet unknown cis-regulatory DNA elements in the 5' region of genes. The P-box was mainly associated with priming-responsive genes, whereas the C-box was mostly linked to challenge. We found that the P- or W-box, the latter recruiting WRKY transcription factors, or combinations of these boxes, characterize the 5' region of most primed genes. Therefore, this study provides a genome-wide record of genes with open and accessible chromatin during SAR and identifies MLO3 and two previously unidentified DNA boxes as likely regulators of this immune response.

Innate immune memory in plants.

The plant innate immune system comprises local and systemic immune responses. Systemic plant immunity develops after foliar infection by microbial pathogens, upon root colonization by certain microbes, or in response to physical injury. The systemic plant immune response to localized foliar infection is associated with elevated levels of pattern-recognition receptors, accumulation of dormant signaling enzymes, and alterations in chromatin state. Together, these systemic responses provide a memory to the initial infection by priming the remote leaves for enhanced defense and immunity to reinfection. The plant innate immune system thus builds immunological memory by utilizing mechanisms and components that are similar to those employed in the trained innate immune response of jawed vertebrates. Therefore, there seems to be conservation, or convergence, in the evolution of innate immune memory in plants and vertebrates.