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.

Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense.

Modern crop production calls for agrochemicals that prime plants for enhanced defense. Reliable test systems for spotting priming-inducing chemistry, however, are rare. We developed an assay for the high-throughput search for compounds that prime microbial pattern-induced secretion of antimicrobial furanocoumarins (phytoalexins) in cultured parsley cells. The screen produced 1-isothiocyanato-4-methylsulfinylbutane (sulforaphane; SFN), a secondary metabolite in many crucifers, as a novel defense priming compound. While elucidating SFN's mode of action in defense priming, we found that in Arabidopsis (Arabidopsisthaliana) the isothiocyanate provokes covalent modification (K4me3, K9ac) of histone H3 in the promoter and promoter-proximal region of defense genes WRKY6 and PDF12, but not PR1 SFN-triggered H3K4me3 and H3K9ac coincide with chromatin unpacking in the WRKY6 and PDF12 regulatory regions, primed WRKY6 expression, unprimed PDF12 activation, and reduced susceptibility to downy mildew disease (Hyaloperonospora arabidopsidis). Because SFN also directly inhibits Harabidopsidis and other plant pathogens, the isothiocyanate is promising for the development of a plant protectant with a dual mode of action.

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant.

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.