**3. Histone methylation changes associated with biotic stress conditions**

Biotic stress is the result of the damage done to plants by insects or pathogens, such as bacteria or fungi. Plant pathogens are generally divided into two distinct categories: biotrophs, which colonize living plant tissue and obtain nutrients from living host cells, and necrotrophs, which depend on dead host tissue for nutrients and reproduction. To fend off pathogens with different infection strategies, plants have evolved complex defence mechanisms. Classically, the pathogen-sensing machinery induces signalling cascades that promote the accumulation of hormones such as salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) [34]. These hormones then orchestrate the overall plant defence reaction locally and systemically by inducing the transcriptional activation of defence genes through an intricate signalling network. In this part, we highlight recent examples illustrating how histone methylations condition major steps leading to immunity, ranging from initial pathogen perception to hormonal homeostasis changes for antimicrobial effector expression.

### **3.1. Histone methylation/demethylation in the defence against biotrophic pathogens**

(H3K4me3 and H3K36me3) are generally enriched at actively transcribed genes, whereas H3K27me3 is associated with repressed genes and H3K9me2 and H4K20me1 are enriched at constitutive heterochromatin and silenced transposons [18]. For histone arginine methylation, a definitive role has not yet been clearly established. However, because the level of symmetric H3R2me2 and H4R3me2 was negatively correlated with the level of H3K4me3, a well-known mark reflecting active transcription, high levels of H3R2me2 and H4R3me2 are thought to cause transcriptional repression [19–21]. In contrast, asymmetric H4R3me2 was associated

Histone methylation is relatively stable and can be established on lysine and arginine by two distinct families of enzymes, the histone lysine methyltransferases (HKMTs), all containing the evolutionary conserved catalytic SET domain in plants [24], and the protein arginine methyltransferases (PRMTs) [25], respectively. As a counterpart, methyl groups on histone can also be removed by at least two evolutionarily conserved classes of histone demethylases, the lysine-specific demethylase1 (LSD1) type and the Jumonji C (JmjC) domain-containing demethylases [26]. Histone methyltransferases and demethylases are well conserved in angiosperms and have been identified and classified on the basis of phylogenetic analyses and domain organization in several plants, including *Arabidopsis*, maize, tomato, rice, grapevine and *Brassica rapa*, [27–32]. However, cellular and molecular functions of many of these

Although histone acetylation can directly modulate the chromatin structure, arginine and lysine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, named readers, needed to 'translate' the code in order to determine the functional and structural outcome of the corresponding PTMs. Just as there are a large number of PTMs on histone tails, there are also numerous protein domains that recognize and bind to particular PTMs on these tails. For example, PTM-recognition domains such as plant homeo‐ domain (PHD) fingers, chromodomains and Tudor domains all recognize methylated lysine

**3. Histone methylation changes associated with biotic stress conditions**

hormonal homeostasis changes for antimicrobial effector expression.

Biotic stress is the result of the damage done to plants by insects or pathogens, such as bacteria or fungi. Plant pathogens are generally divided into two distinct categories: biotrophs, which colonize living plant tissue and obtain nutrients from living host cells, and necrotrophs, which depend on dead host tissue for nutrients and reproduction. To fend off pathogens with different infection strategies, plants have evolved complex defence mechanisms. Classically, the pathogen-sensing machinery induces signalling cascades that promote the accumulation of hormones such as salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) [34]. These hormones then orchestrate the overall plant defence reaction locally and systemically by inducing the transcriptional activation of defence genes through an intricate signalling network. In this part, we highlight recent examples illustrating how histone methylations condition major steps leading to immunity, ranging from initial pathogen perception to

with gene activation [22, 23].

34 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

modifiers have not yet been addressed.

residues [33].

The phytohormone SA plays an important role in plant defence, from the induction of pathogen resistance (*PR*) genes against biotrophic bacteria (e.g. *Pseudomonas syringae*) to the establishment of systemic acquired resistance (SAR) [35]. Several studies suggested that the SA signalling pathway is notably controlled by histone methylation. Under normal growth conditions, *Arabidopsis* mutants for *SNI1* (*Suppressor of NPR1, Inducible*), a negative regulator of SAR required to dampen the basal expression of *PR* genes, presented an increased H3K4me2 on *PR1* [36]. Rather than being a constitutive mark of transcription, H3K4me2 was proposed to be involved in the fine-tuning of tissue-specific expression [37]. Using the functional SAanalogue S-methyl benzo [1,2,3] thiadiazole-7-carbothioate (BTH), an increased level of H3K4me2 on *PR1* was observed in wild-type plants 48 h after treatment and was not detected in mutants. Interestingly, when expressed in yeast, SNI1 also repressed transcription, sug‐ gesting a highly conserved mechanism of transcriptional repression. These results together with the structural similarity of SNI1 with armadillo repeat (ARM) proteins (i.e. a motif known to mediate protein–protein interactions) imply that SNI1 may form a scaffold for interaction with proteins that modulates the chromatin structure of *PR* genes, thus repressing their transcription. In addition, the presence of H3K4me2 detected on *PR1* before induction suggested that this mark is readily in place, providing the appropriate chromatin configuration for the efficient induction of *PR1* upon need. Using a similar approach, Alvarez-Venegas et al. [38] reported no significant changes in levels of H3K4me2 and H3K4me3 on *PR1* 24 h after the SA treatment [38]. This discrepancy may reflect differences in experimental conditions. Indeed, the action of the so-called 'SA-analog' BTH on gene transcription is significantly broader than the action of SA itself [39]. Moreover, samplings were performed 48 h versus 24 h after treatment. Together, because the H3K4 methylation increase does not occur immediately after the induction of *PR1*, this mark may not be directly related to the transcriptional induction itself, but later, for the maintenance/reinforcement of *PR1* expression.

The ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) is a H3K4 trimethyltransferase providing basal resistance against *Pseudomonas syringae pv. tomato* (*Pst*); [40]. Despite being not induced by either *Pst* infection or SA, ATX1 positively and directly regulates the expression of the transcription factor WRKY70 through H3K4 trimethylation at the WRKY70 promoter. In addition, *atx1* mutant shows induced expression of the JA-inducible *THI1.2* gene and the reduced *PR1* expression without detectable changes in their chromatin, resulting in impaired resistance to *Pst* infection. Since the transcriptional factor WRKY70 was positioned at the convergence nod of the SA and JA signalling pathways, activating the SA-responsive *PR1* gene and repressing the JA inducible genes [41], ATX1 was proposed to indirectly regulate *PR1* and *THI1.2* through *WRKY70*. SET DOMAIN GROUP 8 (SDG8), another HKMT encoding the major *Arabidopsis* H3K36 di- and trimethyltransferase [42], was also involved in the plant-defence against *Pst*, but it was more upstream than ATX1 [43]. Indeed, SDG8 sustains the basal transcription of particular *R* genes (*RPM1* or *LAZ5*) by maintaining a basal level of H3K36me3, another histone mark tightly associated with active transcription. SDG8 is also required for the transcriptional induction of these *R* genes upon BTH treatment or *Pst* inoculation. How‐ ever, this induction occurs without any detectable increase of H3K36me3. Therefore, in resting plants, SDG8 may establish a 'permissive' chromatin structure at some *R* genes by methylating H3K36, thus ensuring their basal expression and their transcriptional inducibility upon need. Similarly as *atx1* and *sdg8* mutants, loss-of-function mutants for the putative HKMT *SDG7* were also found to be more sensitive to *Pst* infection than wild-type plants [44]. The expression of other *R* genes seems to be under the control of histone methylation. Indeed, enhanced downy mildew 2 (EDM2) impacts disease resistance by controlling levels of H3K9me2 at an alternative polyadenylation site in the immune receptor gene *RPP7*, thus regulating the balance between full-length *RPP7* transcripts and prematurely polyadenylated transcripts, which do not encode the *RPP7* immune receptor [45, 46]. EDM2, as an epigenetic 'reader', contains two stretches of atypical PHD-finger motifs known to dock specifically several forms of methylated or unmethylated lysine residues on histones [47]. Besides this, EDM2 was also proposed to cooperate within a large protein complex with EMSY-like (AtEML) members, harbouring an Agenet domain related to the Tudor domain family of epigenetic 'readers' [48].

Apart from *Arabidopsis*, little is known about the regulatory role of histone methylation in the defence against pathogen attack in other plant species. In rice, the JmjC protein gene *JMJ705* encoding a histone lysine demethylase that specifically reverses H3K27me2/3 was found induced during infection with the bacterial pathogen *Xanthomonas oryzae*. JMJ705 was further involved in the dynamic removal of the basal H3K27me3 over defence-related genes, thereby increasing their basal expression and/or potentiating their higher expression upon biotic stress. Interestingly, the *JMJ705* overexpression resulted in an enhanced resistance to the bacterial pathogen, while its mutation reduces the plant resistance [49].

#### **3.2. Histone methylation/demethylation in the defence against necrotrophic pathogens**

While to combat biotrophic pathogens the plant activates mainly the SA signalling pathway, the activation of the JA/ET signalling pathway is prominent to mediate defences against necrotrophic pathogens and herbivorous insect attacks [50]. The involvement of histone methylation in the defence against necrotrophic pathogens is far less documented as compared with the defence against biotrophic pathogens. Besides being more susceptible to *Pst* [43], *sdg8* mutants were also reported to be more sensitive to necrotrophic fungal pathogens such as *Alternaria brassicicola* (*Alt*) and *Botrytis cinerea* [51]. This increased susceptibility was the consequence of the inefficient transcriptional induction of different genes along the JA/ET signalling pathway that was correlated with a stably weak level of H3K36me3 at these genes. Inversely, in wild-type plants, H3K36me3 together with gene expression were increased upon *Alt* infection or stimulation with exogenous MeJA. Under resting conditions, a similarly weak level of H3K36me3 was correlated with a reduced basal expression in *sdg8*. On that account, H3K36 methylation was proposed to act as a 'permissive' mark correlated with gene activity and readily in place at a subset of JA/ET signalling-related genes to raise their rapid and efficient transcriptional induction when required [52]. Interestingly, a stable and very low level of H3K27me3 was detected in defence effector genes. Because H3K27me3 is often associated with epigenetic silencing [53], this low H3K27me3 level may provide these genes with a reduced probability for undesired silencing, thus participating in the reactivity of plants to pathogen infections.
