Context of this chapter

A significant transcriptional reprogramming is well-known to occur in plants within minutes following pathogen or PAMP perception; specifi- cally ∽5% (∽1,100 genes) of the Arabidopsis genome is differentially regu-

lated after elicitation with flg22 (Moore et al., 2011; Navarro et al., 2004; Thilmony et al., 2006; Zipfel et al., 2004). The majority of these genes (966) were found to be upregulated within 30 minutes of elicitation, whereas only 202 genes were down-regulated, leading to a significant net increase in gene expression after PAMP perception. These transcriptional changes were specific to PAMP-mediated perception asmutants lacking a functional FLS2 receptor, failed to initiate a significant transcriptional re- sponse (Zipfel et al., 2004).

Considering the degree of transcriptional reprogramming, it is rea- sonable to hypothesise that significant changes occur in the chromatin structure to allow an overall increase in the transcription levels. Chro- matin organisation is established, maintained and altered according to developmental cues and environmental stimuli by the combined action of a variety of chromatin remodelling mechanisms. Nucleosome sliding (Al- varez et al., 2010), covalent modification of histones (Lusser, 2002) and histone variants exchange (Kumar and Wigge, 2010) are the major mech- anisms responsible for the remodelling of chromatin.

In recent years, advances in confocal microscopy techniques have facilitated the in vivo study of chromatin dynamics and chromatin-associ- ated proteins in great detail to determine important aspects of transcrip- tion and chromatin regulation such as histone mobility and transcription factor binding properties (Mueller et al., 2010). A well-established method known as Fluorescence Recovery After Photobleaching (FRAP) has been key in exploring the dynamics of fluorescent-tagged proteins in-

volved in cellular processes, including chromatin binding and remodel- ling. In FRAP experiments, cells are stably or transiently transfected with a protein of interest fused to a fluorescent protein such as GFP. Once ex- pression is established as a visible fluorescence signal through a confocal microscope, a targeted high-power laser beam at the same wavelength of excitation, photo-bleaches a region of interest (ROI) leading to irre- versible removal of the fluorescent signal in the selected area.

Figure 3.1. Principles of Fluorescence Recovery After Photobleaching (FRAP) ex- periments. (a) A. thaliana leaf expressing a 35S::GFP-H2B construct. A fluorescent nu- cleus is bleached and the recovery of fluorescence at the region of interest (ROI, white arrow) is recorded over time. Multiple slices of the nucleus at the z-axis are obtained to generate a vertical stack of images for each time point, which are later compressed into one image per time point during analysis (image was obtained by E. Mastorakis); (b) DNA (black line) is wrapped around histones (yellow and red circles). Fluorescence recovery of GFP-tagged histones depends on the chromatin structure, which can be highly dynam- ic. A more relaxed conformation of chromatin allows faster exchange of nearby histones (red) within the chromatin fraction; (c) fluorescence intensity of the bleached spot (ROI, shown by white arrow) is calculated and plotted after accounting for total loss of fluo- rescence due to bleaching from image acquisition. Complete recovery cannot be achieved due to some permanently photo-bleached molecules, known as the ‘immobile’ fraction . ‘b’ and ‘c’ were obtained from Mistel et al (2001). 


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As fluorescent molecules move dynamically inside the living cell, fluorescence in the photo-bleached ROI can recover at a rate that directly depends on the mobility of the protein of interest. Time-lapse imaging immediately after bleaching allows the rate of fluorescence recovery to be measured providing quantitative information on the mobility of the pro- tein and by extension, its protein binding properties (Fig. 3.1) (Misteli, 2001).

In functional terms, chromatin status is an important determinant of the accessibility of chromatin-associated proteins such as transcription factors and the RNA polymerase machinery (Alajem et al., 2015; Gaspar- Maia et al., 2011; Gaspar-Maia et al., 2009). In various eukaryotic model organisms the process of cellular differentiation is accompanied by global changes in chromatin organisation ranging from a euchromatic state in undifferentiated cells, such as embryonic stem cells, to a heterochromatic state in differentiated cells, such as somatic cells (Kouzarides, 2007). In plants, cell differentiation is also accompanied by changes in chromatin structure, while histone modifications have also been implicated in this process (Rosa et al., 2014). For example, the Arabidopsis thaliana root con- sists of the meristematic zone found at the tip, where cells actively divide and are highly undifferentiated. Above the meristematic zone, cell divi- sion stops and cells increase in length throughout the elongation zone and start to differentiate until they reach complete differentiation status in the maturation zone (Fig. 3.2a) (Dolan et al., 1993). Using the FRAP method, Rosa et al (2014) demonstrated a decrease in histone mobility from the meristematic to the differentiation zone. Interestingly, cells treated with Trichostatin (TSA), a chemical inhibitor of several histone deacetylases, resulted in faster histone mobility and higher histone acety- lation levels, whereas in the histone acetyltransferase mutant hag1-6 low- er histone acetylation levels led to slower histone mobility. Most notably, hyperacetylation caused by TSA not only increased histone mobility but

also altered the expression of a meristem marker (RHD6) in cells from the differentiation zone (Rosa et al., 2014). These results implicated histone acetylation in the process of differentiation, but most importantly provid- ed a functional link between chromatin mobility and histone modifica- tions in plants, whereby higher acetylation levels are correlated with higher histone mobility and a more relaxed chromatin conformation.

In addition to developmental cues, biotic or abiotic stress can also lead to rapid changes in chromatin dynamics, mediated by different chromatin remodelling processes. Studies with cultured animal cells have shown that the perception of PAMPs such as lipopolysaccharide (LPS), synthetic bacterial lipoproteins (sBLP) and fungus-derived molecules lead to nucleosome eviction and a rapid increase of histone H3 acetylation and phosphorylation serving as positive marks for defence gene expression. For example, the promoter of IL-2 gene (encoding cytokines involved in innate defence), is occupied by a single nucleosome, which is evicted upon LPS perception (Weinmann et al., 1999) and this is also correlated with a general increase in H3 acetylation and phosphorylation at promot- ers of defence genes (Weinmann et al., 2001). There has been no report linking the rapid deposition of activating histone marks with PAMP per- ception in plants. However, several reports showed the role of acetylation in abiotic stress responses. For example, H3K9, H3K14 and H4 acetyla- tion, which are known to activate transcription (Lusser et al., 2001), were enriched on the promoter and coding regions of salt-stress inducible genes before and after high salt treatment (Kaldis et al., 2011). Other re- ports have shown that treatment with the synthetic analogue of SA re- sulted in histone acetylation of defence genes in leaves distal to the infec- tion (Jaskiewicz et al., 2011). In addition, the mutant lacking ADA2b, a major interactor of the histone acetyltransferase HAG1 (Hark et al., 2009), displayed lower levels of H3K9/14 and H4 acetylation compared to wild type, although it should be noted that these findings are in the con-

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text of salt-stress, a type of abiotic stress (Kaldis et al., 2011). Along the same lines, HAG1 has been found to play a role in light-regulated gene expression (Benhamed et al., 2006; Benhamed et al., 2008), further sup- porting the dynamic nature of histone acetylation in a variety of biological processes.

The acetylation levels are balanced by the antagonistic actions of HATs and HDACs. Interestingly, genetic evidence suggests that the his- tone deacetylase HDA19 works opposite HAG1 in the regulation of light- responsive genes. Furthermore, hda19 mutants have a shorter hypocotyl and higher H3K9, H3K27, H4K5 and H4K8 levels on those genes, while double mutants lacking both HAG1 and HDA19 show a reversal of this photomorphogenic phenotype (Benhamed et al., 2006). However, this is not the only report suggesting an antagonistic relationship between HAG1

and HDA19, which were found to interact genetically, acting on the same

pathway for shoot differentiation (Long et al., 2006). Strangely, although there is no evidence for a direct role of HAG1 in plant immunity, the role of HDA19 in plant immunity has become more clear in the past few years. An important process regulated by HDA19 is the repression of SA responses. Specifically, PR1 and PR2 (PATHOGENESIS RELATED) genes, which are normally induced after infection by Pseudomonas syringae or treatment with PAMPs and salicylic acid are more highly expressed in

hda19 mutants (Makandar et al., 2006). The enrichment of histone acety-

lation at the promoters of these genes was also important for their up- regulation. Mutants lacking HDA19 showed increased resistance to Pst DC3000 along with increased SA content and PR gene expression (Mosh- er et al., 2006). Mutating the SA-biosynthetic gene SA-INDUCTION DEFI-

CIENT 2 (SID2) or introducing the bacterial encoded gene for an SA-de-

grading salicylate hydroxylase (NahG) into wild type plants leads to lower SA levels and increased bacterial growth (Delaney et al., 1994; Wilder- muth et al., 2001). The same sid2/NahG mutations in hda19 mutant

background reversed the resistant phenotype of hda19 mutants, suggest- ing that SA is essential for the resistance conferred by the lack of HDA19 (Choi et al., 2012). In this line of thinking it is unknown whether a his- tone acetyltransferase is working opposite HDA19 to locally restore the acetylation levels at the promoters of SA genes such as PR1 and PR2 to ensure their activation upon infection. In all, these responses result in global transcriptomic changes, which have been described in detail (Zipfel et al., 2004), however, it still remains unclear how chromatin remodelling affects the expression of defence responsive genes. This chapter explores the role of histone acetylation as a chromatin remodelling mechanism in the context of plant immunity.