# Three ARID proteins involved in chromatin remodeling PEAT complexes are targeted by the Ralstonia solanacearum effector PopP2 and contribute to bacterial wilt disease

Léa Monge-waleryszak, Maxime Girard, Mélanie Carcagno, Raphaël Culerrier, Céline Vicédo, Yves Martinez, Claire Vérin, Yohann Couté, Valérie Pacquit, Laurent Deslandes

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Léa Monge-waleryszak, Maxime Girard, Mélanie Carcagno, Raphaël Culerrier, Céline Vicédo, et al. Three ARID proteins involved in chromatin remodeling PEAT complexes are targeted by the Ralstonia solanacearum effector PopP2 and contribute to bacterial wilt disease. The Plant Journal, 2025, 122 (3), pp.e70205. 10.1111/tpj.70205. hal-05090319

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The Plant Journal (2025) 122, e70205

doi: 10.1111/tpj.70205

# Three ARID proteins involved in chromatin remodeling PEAT complexes are targeted by the *Ralstonia solanacearum* effector PopP2 and contribute to bacterial wilt disease

Léa Monge-Waleryszak<fnref n="1" />, Maxime Girard<fnref n="1" />, Mélanie Carcagno<fnref n="1" />, Raphaël Culerrier<fnref n="1" />, Céline Vicédo<fnref n="1" />, Yves Martinez<fnref n="2" />,
Claire Vérin<fnref n="3" />, Yohann Couté<fnref n="3" />, Valérie Pacquit<fnref n="1" />,* and Laurent Deslandes<fnref n="1" />,†

Received 11 September 2024; revised 17 March 2025; accepted 22 April 2025.

*For correspondence (e-mail valerie.pacquit@inrae.fr and laurent.deslandes@inrae.fr).

## SUMMARY

Like many gram-negative phytopathogenic bacteria, *Ralstonia solanacearum* uses a type III secretion system to deliver into host cells a cocktail of effector proteins that can interfere with plant defenses and promote infection. One of these effectors, the nuclear-targeted PopP2 acetyltransferase, was reported to inhibit many defensive WRKY transcription factors through acetylation. To gain a better understanding of the mechanisms by which PopP2 might exert its virulence functions, we searched for other PopP2-interacting partners. Here we report the identification of the *Arabidopsis thaliana* AT-Rich Interaction Domain protein 3 (ARID3) and its close homologs, ARID2 and ARID4, as additional targets of PopP2. These ARID proteins are core components of the chromatin remodeling PEAT complexes that regulate gene expression through histone (de) acetylation and deubiquitination. In yeast, PopP2 binds the conserved C-terminal part of ARID2/3/4, which contains an α-crystallin domain putatively involved in their homo-oligomerization. ARID2/3/4 behave as substrates of PopP2 acetyltransferase activity, which causes the acetylation of several lysine residues conserved between these three proteins and located near their α-crystallin domain. Interestingly, while PopP2 negatively affects ARID3 and ARID4 self-interactions in planta, it promotes the interaction of ARID3 and ARID4 with PWWP1, another component of PEAT complexes, with which PopP2 can also interact. This study also reveals that disruption of ARID2/3/4 results in reduced growth of *R. solanacearum*. Overall, our data are consistent with a model in which PopP2 targets several components of PEAT complexes to interfere with their epigenetic regulatory functions and promote *Ralstonia* infection in *Arabidopsis*.

**Keywords:** YopJ acetyltransferase, PopP2, XopJ6, *Ralstonia solanacearum*, *Xanthomonas campestris*, *Arabidopsis thaliana*, ARIDs, PEAT complexes, susceptibility factor, vascular pathogen.

## INTRODUCTION

In nature, microbial pathogens and their hosts are engaged in a co-evolutionary arms race that is orchestrated by intricate molecular network strategies (Delplace et al., 2022). The plant immune system mostly relies on two layers of pathogen recognition and involves different types of immune receptors. The first layer is activated by cell surface-localized receptors, known as pattern recognition receptors (PRRs), that can recognize conserved pathogen-associated molecular patterns (PAMPs) and initiate PAMP-triggered immunity (PTI). In most cases, this initial layer of defense is sufficient to prevent pathogen colonization (Yuan et al., 2021). However, adapted pathogens

deliver virulence effector proteins into host cells, where they operate as immune suppressors by manipulating specific host targets to dampen PTI and promote infection (Wang et al., 2022). To counteract this effector-triggered susceptibility (ETS), plants have evolved a second layer of the immune system involving intracellular immune receptors, mainly belonging to the nucleotide-binding domain and leucine-rich repeat-containing protein (NLR) family. NLR proteins, through diverse modes of recognition, can sense specific effectors and initiate a robust immune response called effector-triggered immunity (ETI) that potentiates PTI responses (Ngou et al., 2021; Yuan et al., 2021). In order to get insight into the molecular

<footnote n="1">¹Laboratoire des Interactions Plantes-Microbes-Environnement (LIPME), Université de Toulouse, INRAE, CNRS, Castanet-Tolosan F-31326, France,</footnote>

<footnote n="2">²TRI-FRAIB Imaging Platform Facilities, FRAIB, Université de Toulouse, CNRS, UPS, Castanet-Tolosan 31320, France, and</footnote>

<footnote n="3">³Univ. Grenoble Alpes, INSERM, CEA, UA13 BGE, CNRS, CEA, FR2048, Grenoble 38000, France</footnote>
mechanisms underlying host-pathogen interactions and to develop innovative strategies for combating plant pathogens, it is essential to elucidate the virulence activities deployed by pathogen effectors to promote infection.

Bacterial wilt strains in the *Ralstonia solanacearum* species complex (RSSC) are considered very devastating bacterial pathogens affecting economically important crops worldwide. Indeed, the ability of the RSSC to infect a wide range of host species (over 200), its extensive geographical distribution, and its heightened aggressiveness in warm and humid climates contribute to its status as a significant threat to global agriculture (Vailleau & Genin, 2023). *R. solanacearum* infects its host via the roots and proliferates in xylem vessels where it produces exopolysaccharides that block water and mineral transport, resulting in the development of wilting disease symptoms and ultimately the death of the infected plant (Kai, 2023; Xue et al., 2020). The type III secretion system (T3SS) is one of the main determinants of *R. solanacearum* virulence (Landry et al., 2020). The T3SS is a membrane-anchored, needle-like protein complex that enables the bacteria to inject type III effectors (T3Es) directly into host cells (Galán & Waksman, 2018). The strains of the RSSC possess a large repertoire of 50-75 T3Es that interfere with various host functions and immunity to promote infection (Vailleau & Genin, 2023). PopP2, also referred to as RipP2, is one of the best-characterized *R. solanacearum* T3E as it plays a major role in bacterial virulence and multiplication in several host plants, including tomato, eggplant, beans, and in the model plant *Arabidopsis thaliana* (Le Roux et al., 2015; Macho et al., 2010).

PopP2 belongs to the Yersinia outer protein J (YopJ) T3Es family, hereafter referred to as YopJ T3Es, which are produced by diverse animal and plant bacterial pathogens (Ma & Ma, 2016). The majority of the YopJ T3Es possess a conserved catalytic triad required for their acetyltransferase activity, which is associated with their virulence functions (Cheong et al., 2014; Ma & Ma, 2016). YopJ T3Es use this enzymatic activity to acetylate host proteins on critical residues, thereby affecting their function and/or stability. For example, several YopJ T3Es from animal pathogens, such as YopJ and VopA, inhibit protein kinases associated with the MAPK-dependent signaling pathway by acetylating key serine and threonine residues in their activation loop, thus leading to the suppression of proinflammatory responses (Ma & Ma, 2016). In plants, the YopJ T3E HopZ3 from *Pseudomonas syringae* acetylates and disrupts the functions of several key immune components (Chakraborty, 2021; Lee et al., 2015). HopZ3 also acetylates other *P. syringae* T3Es to suppress their immune-inducing activities (Jeleńska et al., 2021; Lee et al., 2015). HopZ1a, another YopJ T3E from *P. syringae*, promotes bacterial infection through acetylation of the jasmonate (JA) ZIM-domain (JAZ) proteins, lifting the repression of the JA-dependent

signaling pathway and consequently leading to the suppression of salicylic acid (SA)-mediated defenses (Jiang et al., 2013). In addition, HopZ1a-triggered acetylation of tubulin causes destruction of plant microtubule networks, disruption of the plant secretory pathway, and suppression of cell wall-mediated defense (Lee et al., 2012). In *A. thaliana*, PopP2 dampens basal immune responses by acetylating critical lysine residues in the DNA-binding domain of defensive WRKY transcription factors (TFs), thus inhibiting their *trans*-regulating functions needed for defense gene expression (Le Roux et al., 2015; Sarris et al., 2015). The same acetylation strategy is used by the *Xanthomonas campestris* pv. *campestris* (Xcc) XopJ6 effector, recently identified as the closest homolog of PopP2 within the YopJ family (Lauber et al., 2024). These non-exhaustive examples highlight the diversity of host components targeted by YopJ T3Es to interfere with eukaryotic processes and promote pathogen infection.

To counteract the virulence functions of YopJ T3Es, some plants have evolved decoy proteins that mimic true effector targets and whose acetylation triggers activation of NLR-mediated immune responses. For instance, HopZ1a-triggered immunity involves the Arabidopsis HopZ-activated Resistance 1 (ZAR1) NLR protein, which can detect HopZ1a-triggered acetylation of the pseudokinase HopZ-ETI-deficient 1 (ZED1), serving as a decoy (Lewis et al., 2010, 2013). In the case of PopP2 and XopJ6, both effectors trigger activation of the Arabidopsis RPS4/RRS1-R immune receptor complex by acetylating a WRKY decoy domain directly integrated into RRS1-R (Lauber et al., 2024; Le Roux et al., 2015; Sarris et al., 2015; Williams et al., 2014).

Overall, these findings indicate that YopJ effectors can have multiple targets, but as there is no obvious similarity in the protein sequences of the different substrates identified, it is difficult to predict the number, nature, and function of host proteins that can be manipulated by YopJ proteins. Therefore, to decipher the extent of the virulence functions of YopJ T3Es, a detailed inventory of their substrates is necessary. Hence, to better understand how *R. solanacearum* YopJ T3E PopP2 could exert its virulence functions, we screened by yeast two-hybrid (Y2H) a cDNA library from *A. thaliana* using PopP2 as bait to identify additional putative PopP2-interacting partners. This Y2H screening led to the identification of a prey protein corresponding to a truncated form of the AT-Rich Interaction Domain protein 3 (ARID3). This protein was selected for further characterization as it had not previously been identified as a target of YopJ T3Es.

The ARID protein family constitutes a group of transcription factors and chromatin regulators, characterized by a highly conserved ARID domain, found in a wide range of proteins throughout eukaryotic kingdoms, and that mainly enables the recognition of specific AT-rich DNA
sequences (Korn & Schlundt, 2022). In *A. thaliana*, the ARID family has 10 members divided into four subgroups depending on the presence of additional domains, besides the ARID domain, in their C-terminal region (Zhu et al., 2008). ARID3, with its close homologs ARID2 and ARID4, forms the ACD subgroup, so named because they contain an α-crystallin domain (ACD), also referred to as the Hsp20 domain, which is thought to play a role in protein-protein interactions (Waters & Vierling, 2020). Interestingly, ARID2/3/4 were all shown to be core components of the chromatin remodeling PEAT complexes, which regulate gene expression epigenetically through histone modifications at targeted loci (Tan et al., 2018; Zheng et al., 2023).

These ARID proteins therefore represent promising effector targets that would enable a pathogen like *Ralstonia* to interfere with host epigenetic mechanisms. The targeting of chromatin-related components by nuclear effectors has been already documented, predominantly in plant-oomycetes and plant-nematodes interactions, as a potent virulence strategy to modulate host transcriptional reprogramming during infection and facilitate host invasion (Chen et al., 2024; Kong et al., 2017; Li et al., 2018; Vijayapalani et al., 2018). Whether and how type III effectors produced by plant bacterial pathogens also target chromatin-related components remains largely unknown (Harris et al., 2023). Here we investigated the targeting of ARID3 by PopP2 at the molecular level and determined whether its two close homologs, ARID2 and ARID4, are also manipulated by this bacterial effector. Our study reveals that these three ARID proteins behave as direct substrates of PopP2 acetyltransferase activity and that they contribute to *Ralstonia* infection. By influencing protein-protein interactions between components of PEAT, PopP2 is hypothesized to function as a modulator of the assembly of this chromatin remodeling complex.

## RESULTS

### PopP2 interacts with the C-terminal part of ARID2, ARID3, and ARID4 in yeast

PopP2 was previously reported to dampen basal immunity by acetylating a wide range of defensive WRKY TFs (Le Roux et al., 2015). We hypothesized that PopP2 could exert its virulence functions by targeting other nuclear components. To identify such targets, a large-scale yeast two-hybrid (Y2H) screening of an *A. thaliana* cDNA library from 10-day-old Arabidopsis Col-0 seedlings was previously performed using PopP2 fused to a GAL4 DNA-binding domain (BD) as a bait (BD-PopP2) (Bernoux et al., 2008). This screening identified, among others, a truncated cDNA encoding the last 136 residues of the C-terminus of the protein ARID3 (residues 237–398 of ARID3, hereafter designated ARID3<sub>Cterm</sub>) (Figure 1).

Physical interaction between PopP2 and ARID3 was further confirmed by using full-length ARID3 fused to the GAL4 activation domain (AD) as a prey protein in Y2H assays (Figure 1). No interaction was detected when ARID3 or ARID3C<sub>term</sub> were co-expressed with an unrelated bait protein, the murine p53 protein (BD-p53). The proper accumulation of the different bait and prey proteins was verified by immunoblot analysis performed on total yeast protein extracts (Figure S1a,b).

Next, we delimited the ARID3 subdomains that could interact with PopP2. For this, three additional AD-fusion constructs were generated, encoding either the N-terminus of ARID3 (ARID3<sub>1-92</sub>), the ARID DNA-binding domain of ARID3 (ARID3<sub>93-236</sub>), or both of them (ARID3<sub>1-236</sub>). Yeast cells co-expressing BD-PopP2 with these three additional AD-constructs did not grow on selective media, suggesting that PopP2 interacts with ARID3 only through binding on ARID3<sub>Cterm</sub> (Figure 1; Figure S1a,b).

In the ARID family, ARID3 and its close homologs ARID2 and ARID4 form the ACD subgroup (Zhu et al., 2008) and are all components of the chromatin remodeling PEAT complexes (Tan et al., 2018). ARID2/3/4 have variable N-terminal regions, but their ARID domain and C-terminal regions, containing an ACD and a loop connecting the ARID and the ACD domains, are conserved. Whether the C-terminal region of the ARID proteins is a ubiquitous target of PopP2 was therefore evaluated by testing the ability of PopP2 to interact with ARID2 and ARID4 C-terminus in yeast (Figure 1). A control prey protein corresponding to ARID1, a member of another ARID protein subfamily, devoid of ACD at its C-terminus, was included to verify the specificity of the interactions detected. Interestingly, whereas no interaction could be detected with full-length ARID2 and ARID4 in yeast, ARID2<sub>Cterm</sub> (residues 620–786 of ARID2) and ARID4<sub>Cterm</sub> (residues 296 to 457 of ARID4) were found to interact with PopP2, as observed with ARID3<sub>Cterm</sub> (Figure 1; Figure S1a,b). The failure to detect the interaction of full-length ARID2 and ARID4 with PopP2 in Y2H assays could be due to an incorrect folding of these proteins in yeast cells. These data suggest that ARID2, ARID3, and ARID4 represent host proteins whose C-terminus part is targeted by PopP2.

Recently, the XopJ6 from *Xcc* that corresponds to the closest homolog of PopP2 in the YopJ family was shown to target and acetylate WRKY TFs, similarly to PopP2 (Lauber et al., 2024). We therefore tested whether XopJ6 could also interact with ARID3. Y2H assays using the catalytic unit of XopJ6 were fused to the BD domain (BD-XopJ6<sub>224-565</sub>). The results of our Y2H assay indicated that the XopJ6 catalytic unit also binds to ARID3<sub>Cterm</sub> (Figure S1c,d), thereby suggesting that PopP2 and XopJ6 not only share WRKY TFs as common targets but also ARID3. However, in pursuing this study, we have chosen to focus our investigation on the targeting of ARID proteins by PopP2.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** PopP2 interacts with ARID2, ARID3, and ARID4 in yeast.</figcaption></figure>

(a) Schematics of full-length and truncated versions of ARID proteins used in yeast two-hybrid (Y2H) assays in (b). Proteins were N-terminally fused with the GAL4 activation domain (AD) and expressed as preys in yeast. The first and last amino acid positions of the different ARID proteins used in the Y2H assay are indicated. The ARID domain is in yellow, the ACD (α-crystallin domain) is in green, and the ELM domain of ARID1 is in blue.

(b) Serial dilutions of yeast cells co-transformed with different combinations of prey (AD) constructs with PopP2 N-terminally fused with the GAL4 DNA-binding domain (BD) as a bait construct. The various AD-fusion proteins are presented in (a). The murine p53 protein was used as a negative control (BD-p53) for interaction with AD-fusion proteins and as a positive control for interaction with the SV40 large T-antigen (AD-T). Transformed yeast cells were grown on selective media (SD/-Trp/-Leu [-TL] or SD/-Trp/-Leu/-His supplemented with 5 mM 3-amino-1,2,4-triazole [3-AT] [-TLH + 5 mM 3AT]). Plates were incubated at 28°C after plating and photographed 5 days later. This experiment was repeated three times with similar results. Production of the different bait and prey proteins in yeast cells was verified by immunoblot analysis (see Figure S1).

## PopP2 physically interacts with ARID2, ARID3 and ARID4 in planta

As core components of the nuclear PEAT complexes, the transient expression in *Nicotiana benthamiana* leaves of ARID2, ARID3, and ARID4 C-terminally fused to enhanced green fluorescent protein (eGFP) confirmed their nuclear localization. Interestingly, unlike ARID2 and ARID3, which accumulate exclusively in the nucleus, ARID4 showed a nucleocytoplasmic localization (Figure 2; Figure S2). The nuclear co-localization of these ARID5-eGFP and PopP2 C-terminally fused to the mCherry fluorescent protein (PopP2-mCherry) is consistent with their targeting by the effector in this subcellular compartment.

Interaction between ARID proteins and PopP2 was further investigated using a split-luciferase complementation (SLC) assay enabling the monitoring of protein-protein interactions in living *N. benthamiana* cells. In this assay, two proteins of interest are fused to either the amino-terminal or carboxyl-terminal half of luciferase, Nluc and Cluc, respectively. In case of interaction, the two halves of luciferase can be reconstituted to form a functional luciferase enzyme producing luminescence upon

addition of its substrate, the luciferin. A triple HA and a triple Flag epitope were added at the N-terminus of the Nluc and Cluc moieties, respectively, enabling the immunodetection of the transiently expressed proteins. Given that PopP2 binds the C-terminus of the immune receptor RRS1-R (RRS1-R<sub>Cterm</sub>) (Le Roux et al., 2015), co-expression of PopP2-3HA-Nluc with RRS1-R<sub>Cterm</sub>-3Flag-Cluc was used as a positive control (Figure 3). This SLC assay revealed that PopP2 interacts *in planta* with full-length ARID2, ARID3, and ARID4, but not with ARID1. As negative controls, co-expression of Cluc-fused ARID2/3/4 proteins with GFP-3HA-Nluc led to significantly lower signals (*P* < 0.0001) than those obtained with PopP2-3HA-Nluc. Immunoblot analysis using anti-Flag antibodies showed that the ARID2 fusion protein accumulates at lower levels than ARID3 and ARID4 fusion proteins (Figure S3), which could explain the lower luminescent signal obtained upon co-expression of PopP2 with ARID2, compared with those observed with ARID3 and ARID4. Together, these data indicate that PopP2 physically interacts with ARID2, ARID3, and ARID4, likely by targeting their C-terminus part, as indicated by our Y2H data (Figure 1).

<caption>**Figure 2.** ARID2, ARID3, and ARID4 co-localize with PopP2 in the plant nucleus.</caption>

ARID2-eGFP, ARID3-eGFP, and ARID4-eGFP were transiently expressed in *Nicotiana benthamiana* leaves either alone or with PopP2-mCherry. Confocal fluorescence imaging was performed 48 h post-agro-infiltration. Scale bars = 50 µm. The expression of the different fusion proteins in *N. benthamiana* was checked by immunoblot analysis (see Figure S2).
<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** PopP2 physically interacts with ARID2, ARID3, and ARID4 *in planta*.</figcaption></figure>

Quantitative measurement of luciferase-derived signals shows that PopP2 interacts with all three ARIDs in living *Nicotiana benthamiana* cells. For this split luciferase complementation (SLC) assay, PopP2 fused to the N-terminal part of the luciferase (PopP2-3HA-Nluc) was expressed either with GFP or with ARID2/3/4 proteins all fused to the C-terminal part of the luciferase (X-3Flag-Cluc). The co-expression of PopP2-3HA-Nluc with RRS1-R<sub>Cterm</sub>-3Flag-Cluc was used as a positive control for protein-protein interaction. The co-expression of GFP-3HA-Nluc with ARID2/3/4-3Flag-Cluc served as negative controls. Leaf samples were collected 48 h post-agro-infiltration. Bars indicate mean absolute luminescence unit ± SEM. Three independent replicates were performed (for each replicate, *n* = 24). Data points displayed on the graph are colored depending on the replicate they were collected from. Statistical analysis was performed using one-way ANOVA with Tukey's test. ****P < 0.0001; ns, non-significant. The expression of the different fusion proteins in *N. benthamiana* was checked by immunoblot analysis (see Figure S3).

## PopP2 affects the homo-oligomerization of ARID3 and ARID4 *in planta*

Previously, the pairwise interactions between several PEAT components revealed that ARID3 and ARID4 self-interact in yeast (Tan et al., 2018). We therefore investigated whether the targeting of ARID3 and ARID4 by PopP2 could affect their ability to form homo-oligomers. As a pre-requisite, we first checked that ARID3 and ARID4 self-interaction could be monitored *in planta* using the SLC assay. Thus, both full-length ARID3 and ARID4 proteins were C-terminally fused with 3Flag-Cluc or 3HA-Nluc. As expected, a significant increase in luminescence was detected when ARID3-3Flag-Cluc was transiently co-expressed with ARID3-3HA-Nluc (Figure 4a; Figure S4a), indicating that ARID3 forms homo-oligomers. By contrast, no increase in luminescence was observed upon co-expression of ARID3 with the negative control GFP in this SLC assay. Similar results were obtained with ARID4 (Figure 4b; Figure S4a).

To investigate whether PopP2 could modulate ARID3 and ARID4 homo-oligomerization, the Cluc and Nluc fusion proteins of ARID3 or ARID4 were co-expressed with 3HA-tagged PopP2 or the unrelated β-glucuronidase (GUS) protein, serving as a negative control. A significant decrease in luminescence was monitored only when PopP2 was

present, suggesting that PopP2 interferes with ARID3/ARID3 and ARID4/ARID4 self-interactions (Figure 4c; Figure S4b). This could be the result of either direct binding of the effector to their C-terminal part, thus preventing self-interactions, and/or a consequence of PopP2 acetyl-transferase activity. It is worth noting that PopP2-C321A can also physically interact with both ARID3<sub>Cterm</sub> and full-length ARID3 in yeast, like PopP2 (Figure S4c,d; Figure 1). Interestingly, the reduction of homo-oligomerization of ARID3 and ARID4 was significantly less severely affected in the presence of a catalytic mutant of PopP2 (PopP2-C321A). These data suggest that PopP2 acetyltransferase activity plays a role in the PopP2-triggered reduction of ARID3 and ARID4 self-interactions.

## PopP2 promotes the association of PWWP1 with ARID3 and ARID4

ARID2, ARID3, and ARID4 were previously shown to directly interact within the PEAT complexes with three PWWP proteins (PWWP1/-2/-3) (Tan et al., 2018). These PWWP proteins contain a Pro-Trp-Trp-Pro sequence motif and interact with several histone-modifying enzymes (Godwin et al., 2024; Tan et al., 2018; Zheng et al., 2023). Since PopP2 affects ARID3 and ARID4 self-interactions (Figure 4c), we therefore assessed PopP2's impact on ARIDs/PWWP1 interactions. Given that PWWP1 interacts
<figure><img src="image_6.png" /><figcaption>**Figure 4.** PopP2 negatively affects ARID3 and ARID4 homo-oligomerization in *planta*.</figcaption></figure>

(a, b) Quantitative measurement of luciferase-derived signals shows that ARID3 and ARID4 homo-oligomerize in *planta*. Luciferase activity detected in *Nicotiana benthamiana* leaves transiently expressing ARID3 (a) or ARID4 (b) fused to either the N-terminal part (Nluc) or the C-terminal part (Cluc) of the luciferase (ARID3/4-3HA-Nluc and ARID3/4-3Flag-Cluc, respectively). The co-expression of ARID3/4-3HA-Nluc with GFP-3Flag-Cluc or GFP-3HA-Nluc with ARID3/4-3Flag-Cluc served as negative controls.

(c) PopP2 affects ARID3 and ARID4 homo-oligomerization in *planta*. Luciferase activity in *N. benthamiana* leaves transiently expressing ARID3 or ARID4 fused to the N-terminal part (Nluc) and the C-terminal part (Cluc) of the luciferase upon transient co-expression with 3HA-tagged GUS, PopP2, or C321A. Leaves transiently expressing GFP fused to both Nluc and Cluc in the presence of 3HA-tagged GUS, PopP2, or C321A served as negative controls. Transient co-expression with GUS served as a negative control. In (a-c), leaf samples were collected 48 h post-agro-infiltration. Bars indicate mean absolute luminescence unit ± SEM. Three independent replicates were performed (for each replicate *n* = 24). Data points displayed on the graph are colored depending on the replicate they were collected from. Statistical analysis was performed using one-way ANOVA with Tukey's test. ***P* < 0.001, ****P* < 0.0001. The proper expression of each fusion protein in *N. benthamiana* was checked by immunoblot analysis (see Figure S4).

with several PEAT components and particularly with ARIDs
with a very high score (Tan et al., 2018; Zheng et al., 2023),
we focused our analysis on ARID3/PWWP1 and ARID4/
PWWP1 interactions.

Firstly, the interaction of ARID3 or ARID4 with
PWWP1 was verified by using the SLC assay, as shown
by an increase of luminescence observed upon co-
expression of PWWP1-3Flag-Cluc with either ARID3-3HA-
<figure><img src="image_7.png" /><figcaption>**Figure 5.** PopP2 promotes the association of ARID3 and ARID4 with PWWP1 in planta.

Luciferase activity was detected in *Nicotiana benthamiana* leaves transiently co-expressing ARID3 or ARID4 fused to the N-terminal part of the luciferase enzyme (ARID3/4-3HA-Nluc) together with PWWP1 fused to the C-terminal part of the luciferase enzyme (PWWP1-3Flag-Cluc) upon transient co-expression with 3HA-tagged GUS, PopP2, or PopP2-C321A. Leaves transiently co-expressing GFP-3HA-Nluc and PWWP1-3Flag-Cluc served as negative controls. Transient co-expression with GUS served as a negative control. Leaf samples were collected 48 h post-agro-infiltration. Bars indicate mean absolute luminescence unit ± SEM. Three independent replicates were performed (for each replicate *n* = 24). Data points displayed on the graph are colored depending on the replicate they were collected from. Statistical analysis was performed using one-way ANOVA with Tukey's test. ****P < 0.0001. The proper expression of each fusion protein in *N. benthamiana* was checked by immunoblot analysis (see Figure S5a).</figcaption></figure>

Nluc or ARID4-3HA-Nluc, but not with the control protein
GFP-3HA-Nluc. Co-expression of ARID3-3HA-Nluc with
PWWP1-3Flag-Cluc led to an increase of luciferase activ-
ity only in the presence of wild-type PopP2, but not with
PopP2-C321A or GUS (Figure 5), suggesting that PopP2
enzymatic activity promotes ARID4/PWWP1 association.
Interestingly, the SLC assay revealed that while PopP2
also facilitates ARID4/PWWP1 association, this occurs
independently of its acetyltransferase activity, as indi-
cated by the increase in luciferase activity resulting from
the co-expression of ARID4-3HA-Nluc with PWWP1-3Flag-
Cluc in the presence of either PopP2 or PopP2-C321A. In
contrast, the two PopP2 variants did not induce any
increase in luminescence upon co-expression of PWWP1-
3Flag-Cluc proteins with the non-interacting GFP-3HA-
Nluc protein, highlighting the specificity of the action of
PopP2 on the interaction between PWWP1 and ARID3 or
ARID4.

Immunodetection of the different proteins of interest
showed that both PWWP1 and ARID3 accumulated at
higher levels when co-expressed with PopP2, but not with
GUS or PopP2-C321A (Figure S5a). Therefore, it is also

possible that the increase in luminescence interpreted as a
stimulation of PWWP1/ARID3 association by PopP2 is
rather the consequence of the enhanced accumulation of
PWWP1 and ARID3 induced by wild-type PopP2. However,
the increase in luminescence observed for ARID4/PWWP1
with both PopP2 and PopP2-C321A was not strictly corre-
lated with an increase of their protein accumulation levels,
especially with the catalytic mutant (Figure S5a). Overall,
our data strongly suggest that PopP2 does indeed stimu-
late the interaction of ARID3 and ARID4 with PWWP1, likely
in a PopP2 acetyltransferase activity-dependent manner for
ARID3/PWWP1.

Since PopP2 promotes certain ARID/PWWP1 interac-
tions, we speculated that PopP2 might bind and physically
stabilize these hetero-oligomers by a molecular mecha-
nism that remains unknown. Consistent with this hypothe-
sis, a physical interaction between PopP2 and PWWP1 was
indeed detected in a Y2H assay (Figure S5b). Taken
together, our data demonstrate that PopP2 can bind at
least two different types of proteins belonging to the PEAT
complexes, likely to interfere with PEAT oligomerization
status and functions.
<figure><img src="image_8.png" /><figcaption>**Figure 6.** ARID2, ARID3, and ARID4 are acetylated by PopP2 *in planta*.</figcaption></figure>

(a) ARID2-eGFP, ARID3-eGFP, and ARID4-eGFP were transiently expressed with 3HA-tagged active PopP2 or C321A catalytic mutant in *Nicotiana benthamiana* leaves. Samples were collected at 48 h post-agro-infiltration. Proteins isolated from crude extracts (CE) were immunoblotted with anti-HA (α-HA) and anti-GFP (α-GFP) antibodies. GFP-tagged ARID proteins were immunoprecipitated on GFP-trap agarose beads (IP-GFP) and analyzed by immunoblotting with anti-GFP (α-GFP) and anti-acetylated-lysine (α-Ac-K) antibodies. Ponceau S staining indicates similar protein amounts loaded in the different lanes.

(b) Recombinant GST-ARID3-His6 fusion protein was expressed with 3HA-tagged active PopP2 or C321A catalytic mutant in *Escherichia coli*. Protein extracts (CE) were immunoblotted with anti-His6 (α-His6) antibodies. GST-ARID3-His6 was purified on Ni-NTA agarose beads (Ni-NTA pull-down) and analyzed by immunoblotting with anti-His6 (α-His6) and anti-acetylated-lysine (α-Ac-K) antibodies. These experiments were conducted three times with similar results.

**PopP2 induces acetylation of ARID2, ARID3, and ARID4 on multiple lysine residues in planta**

PopP2 is known to modify the functions of various defensive WRKY TFs by acetylation of critical lysine residues (Le Roux et al., 2015; Sarris et al., 2015). Thus, we tested whether ARID2, ARID3, and ARID4 were acetylated in the presence of PopP2 *in planta*. ARID2, ARID3, and ARID4 fused to the enhanced GFP tag (ARIDs-eGFP) were individually co-expressed transiently in *N. benthamiana* with PopP2-3HA or PopP2-C321A-3HA (Figure 6a). As a positive control for this assay, NbWRKY8-GFP, a known substrate of PopP2 acetyltransferase activity (Le Roux et al., 2015),

was used (Figure S6). ARIDs-eGFP proteins were purified from total plant protein extracts using a GFP affinity matrix. Immunoprecipitated GFP-tagged proteins were analyzed by immunoblots probed with anti-GFP and anti-acetyl-lysine (anti-Ac-K) antibodies. While similar amounts of each GFP fusion protein co-expressed with PopP2 or PopP2-C321A were purified, Lys-acetylated forms of ARID2, ARID3, ARID4, and the control protein NbWRKY8-GFP were detected only in the presence of catalytically active PopP2, but not with the inactive PopP2-C321A (Figure 6a; Figure S6). This indicates that PopP2 promotes the acetylation of these three ARIDs on lysine residues.
To identify the residues acetylated in the presence of PopP2, ARID-eGFP proteins (ARID2-, ARID3-, and ARID4-eGFP) were produced in *N. benthamiana* leaves with either PopP2-3HA or PopP2-C321A and then were immunopurified and subjected to mass spectrometry (MS)-based proteomic analyses. These experiments revealed several acetylated lysine (Lys) residues in the different ARID proteins, some of them with a higher identification rate in samples co-expressing PopP2-3HA compared with PopP2-C321A-3HA, with 10, 11, and 7 Lys-acetylated residues in ARID2, ARID3, and ARID4, respectively (Table S1; Figure 7). Some of them are located just upstream of the ARID domain of ARID2 and ARID3 (ARID2<sup>K434/K447</sup> and ARID3<sup>K79/K86</sup>), in the ARID domain of ARID2 and ARID4 (ARID2<sup>K542</sup>, ARID4<sup>K208</sup>), and in the ARID2/3/4<sub>Cterm</sub> (ARID2<sup>K753</sup>, ARID3<sup>K367</sup> and ARID4<sup>K426</sup>, which corresponds to a lysine conserved in the three ARIDs) (Figure 7). Interestingly, of all the PopP2-induced acetylation sites identified, nine correspond to Lys residues that are conserved between the three ARID proteins, with eight of them located in the C-terminal part of ARIDs, consistent with the ability of PopP2 to bind this sub-region in our Y2H assay (Figure 1). Of these eight residues, six are located in a region between the ARID and the ACD and two are located within the ACD (Figure 7). A limited number of acetylated serine and threonine residues were detected as well, none being specifically identified from samples containing active PopP2 (Table S1), suggesting that PopP2 acetyltransferase activity is not implicated in the modification of these residues, by contrast to the acetylated lysines described above.

## ARID3 is a direct substrate of PopP2 acetyltransferase activity

We next investigated whether PopP2 was able to directly acetylate its interacting ARID partners. For this purpose, we used a bacterial acetylation assay that has previously been successfully used to characterize direct PopP2 substrates. Indeed, expressed in *Escherichia coli*, PopP2 undergoes auto-acetylation and is capable of acetylating critical Lys residues in the WRKY DNA-binding domain of WRKY transcription factors in a manner analogous to that observed *in planta* (Le Roux et al., 2015; Tasset et al., 2010). Here, each ARID protein was expressed as a fusion with an N-terminal glutathione S-transferase (GST)

tag and a C-terminal six-histidine residue (His<sub>6</sub>) tag (GST-ARIDs-His<sub>6</sub>), together with either PopP2-3HA or PopP2-C321A-3HA. Despite several attempts, only the recombinant GST-ARID3-His<sub>6</sub> could be produced in a soluble form and with a suitable yield for purification on a Ni-NTA affinity matrix. Immunoblot analysis of purified GST-ARID3-His<sub>6</sub> revealed the presence of acetylated ARID3 only when co-expressed with wild-type PopP2, but not with the catalytically inactive PopP2-C321A (Figure 6b). The presented data substantiate the fact that ARID3 behaves as a direct substrate of PopP2 acetyltransferase activity.

MS-based proteomic analyses performed on purified GST-ARID3-His<sub>6</sub> co-expressed with either active PopP2 or inactive PopP2-C321A in *E. coli* confirmed PopP2-dependent acetylation of 7 of the 11 modified lysine residues identified in ARID3-eGFP co-expressed with active PopP2 in *N. benthamiana*: K79, K259, K261, K268, K271, K273, and K281 (Figure 7; Table S1). Notably, three of these seven residues (K261, K268 and K271), all located in a predicted loop between the ARID and the ACD domains, were found to be acetylated by PopP2 in the three ARID proteins in *N. benthamiana*. Extrapolation of these data strongly suggests that ARID2 and ARID4, which interact physically with PopP2 (Figures 1 and 3) and which are both acetylated *in planta* in its presence (Figure 6a), could also be considered as direct substrates of PopP2.

The ubiquitous targeting of the conserved lysine residues by PopP2 acetyltransferase activity suggests that the acetylation of the C-terminal region of the ARID proteins may be critical for PopP2 to manipulate their functions. To explore the effect of Lys-acetylation on ARIDs functions, we designed an ARID3 acetyl-mimetic mutant, named ARID3-11KQ, in which each of the 11 lysines acetylated in the presence of PopP2 was replaced with a glutamine (Q), a polar but uncharged residue classically used to mimic the charge neutralization of acetyl-lysine. ARID3-11KQ, like ARID3, physically interacts with PopP2 through its C-terminal domain in yeast (Figure S7a,b), indicating that the substitutions introduced did not compromise its ability to interact with PopP2. In addition, as expected, acetyl-mimetic ARID3-11KQ was no longer detected as being acetylated on Lys residues *in planta* (Figure S7c). However, SLC-based measurements in living plant cells showed that the interaction between ARID3-11KQ and PopP2 was

**Figure 7.** PopP2 causes acetylation of ARID2, ARID3, and ARID4 on multiple lysine residues. Schematic diagram of domain structures and sequence alignment of ARID2, ARID3, and ARID4. Conserved residues in ARID2, ARID3, and ARID4 are indicated in bold. The ARID domain and the ACD are boxed in yellow and green, respectively. ARID<sub>Cterm</sub> indicates the C-terminal region of ARIDs shown to interact with PopP2 in the Y2H assay. Acetylated lysine residues reproducibly identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics are boxed in red. Conserved lysine residues bearing a PopP2-induced acetylation are underlined. Mapping of these acetylated sites derived from the analysis of ARID2-eGFP, ARID3-eGFP, and ARID4-eGFP (transiently expressed with 3HA-tagged active PopP2 or PopP2-C321A catalytic mutant in *Nicotiana benthamiana* [–Nb]), and GST-ARID3-His<sub>6</sub> (co-expressed with 3HA-tagged PopP2 or C321A in *Escherichia coli* [–Ec]). Purified proteins were digested, and the derived peptides were analyzed by LC–MS/MS. Data were obtained from 2 to 3 replicates for each protein. Acetylated Lys residues identified in peptides with a SC<sub>WT</sub> PopP2/SC<sub>PopP2-C321A</sub> ratio ≥0.5 in at least one replicate were considered to be residues preferentially acetylated by active PopP2 (see also Table S1).
ARID2-Nb 1 MENLTEIESTMESLTEMESERVEQGTDKIEIGSGEKRQDDVKETENENSGERVGEEAPVREHEDSPCLIVIEEGTSLASLE
ARID3-Nb 1 ---MVD--------TEMQEQDVTFGA--------------------------------------------
ARID3-Ec 1 ---MVD--------TEMQEQDVTFGA--------------------------------------------
ARID4-Nb 1 --------MMADTEMQEQDVPSGTKG--------------------------------VVEEP---SELE
TEM V G

ARID2-Nb 81 EVTNADDLPKIDDEKNSQFETSPHPSPSPSVALDTEEGLINPTAEDTVEENIVSSEVSSDILKDDGDAVEVDRDTAEVQE
ARID3-Nb
ARID3-Ec
ARID4-Nb 28 KDLNSIERPK--------------------------------SPVPEDT------THTLDSDV-----------------

ARID2-Nb 161 ETANIPESKLSEDTGSPHHHADILMVQEKAAEEHDMIASGDHEEFPVNPDNKHSEENQSPHHHANNVMEQDQAAEEREI
ARID3-Nb 16 --------LVETKYLEEE-----------------PLEPENDHNP------------------------
ARID3-Ec 16 --------LVETKYLEEE-----------------PLEPENDHNP------------------------
ARID4-Nb 53 --------HLSDA-----------------------PI--------------------------ANQI-----------------P

ARID2-Nb 241 SPGEHKEIPANPDTKVVEENNDRIDEGEANNLNLAGDGSGAVDHDYLTKTELDKVLEVPGSETISKLEDRPSEHLSSETM
ARID3-Nb 36 ------SEIP----------QPLLLGDGQANN-------GHG-------------------------------------------M
ARID3-Ec 36 ------SEIP----------QPLLLGDGQANN-------GHG-------------------------------------------M
ARID4-Nb 64 --------EANE-----------------EVGGQNSV---------------------------
AN

ARID2-Nb 321 NVEKELEMPAVEILPDNDKNSDVAVGVSGSDDNVVSLPASQTSSDRDEGMITVDAEPTEDMKLDVPDSKLVTDTTVDS
ARID3-Nb 56 NGG-------------------AVGV-------------------------------------------VDH
ARID3-Ec 56 NGG-------------------AVGV-------------------------------------------VDH
ARID4-Nb 76 -----------------DGRNGDV-----------------DQSEKKITSDGGQEE----------------TTL--

ARID2-Nb 401 TNNKDAHVEANTERQDNSSALVLNDANNESAPVIRVPGFYVASSNISEARGSGDLNNGVHKIVRTPPVFDGTMRAKRSF
ARID3-Nb 66 SERKTRRVQMLSPITEGENA-------------------------------------------KKRTW
ARID3-Ec 66 SERKTRRVQMLSPITEGENA-------------------------------------------KKRTW
ARID4-Nb 101 ---------------------GESNPLKGDPS---------------------SPHVPEESVKKKWTW

ARID2-Nb 481 LLLDAS--DG-NEIGTEEDQSAFMKELDSFFRERNMDFKPPKFYGEGLNLCKLWRAVTRLGGYDVTGSKLWRQVGESFR
ARID3-Nb 92 LLDSEA--QGTDEAGTPVEQVAFLREVEAFYKESFLEFKPPKFYGQPLNILKLWRAVVNLGGYEVVTTNKLWRQVGESFN
ARID3-Ec 92 LLDSEA--QGTDEAGTPVEQVAFLREVEAFYKESFLEFKPPKFYGQPLNILKLWRAVVNLGGYEVVTTNKLWRQVGESFN
ARID4-Nb 127 LLSDAEAREV-DEAGAPQDQEAFIKEVEAFNKENFLEFKAPKFYGQPLNCKLKWRAVIKLGGYDVVTTSKLWRQVGESFH
LL E G Q AF E F E FK PKFYG LN LKLWRAV LGGY VT KLWRQVGESF

ARID2-Nb 558 PPKTCTTVSWTFRGFYEK---------------------ALLEYERHKVSEGELQIP------LPLELEPMNID------
ARID3-Nb 170 PPKTCTTVSYTFRNFYEK---------------------ALLEYEKCLRNGELNLPGSTLILSSSVEKEPSS------
ARID3-Ec 170 PPFTCTTVSYTFRNFYEK---------------------ALLEYEKCLRNGELNLPGSTLILSSSVEKEPSS------
ARID4-Nb 206 PPFTCTTVSWTFRIFYEKNFIFGLSTEYALILMRYLFSLALLEYEKHLRONGELNLP------GSASLPSSGIEEASS
PPKTCTTVS TFR FYEK

ARID2-Nb 605 NQASGSGRARRDAASRAMQGWHSQRLNGNGEVDSDPAIKDNL--VLHQREQIGTTPGLLRKR--AAEHGATNAI-HV
ARID3-Nb 222 HQGSGSGRARRDSAARAMQGWAQRLVGSGEVTAPAVDGL--ISTPHKL--SIGLQHHPQQTSMDHVVTNE----
ARID3-Ec 222 HQGSGSGRARRDSAARAMQGWAQRLVGSGEVTAPAVDGL--ISTPHKL--SIGLQKHPQQTSMDHVVTNE----
ARID4-Nb 281 HQASGSGRTRRDAARAMQGWHSQRLLLGSGEVTEPIVFGLNSTPKQNLNIGV---QKQKTTTGMDLVFSHE----
Q SGSGR RRD A RAMQGWH QRL G GEV P K K L K K K K

ARID2-Nb 680 SKSMLDVTVVDVGPPADWVKINVQRTQDCFEVYALVPGLVREEVRVQSDPAGRLVISGEPENPMNPWGATPFKVVSLPT
ARID3-Nb 294 ADKQLAAEVVVDPGVADWVKINVKESKDSFEIFALVPGLLRRKEVRIQSDPAGKVVITGQPEQLDNPWGITPFKKIVDLSA
ARID3-Ec 294 ADKQLAAEVVVDPGVADWVKINVKESKDSFEIFALVPGLLRRKEVRIQSDPAGKVVITGQPEQLDNPWGITPFKKIVDLSA
ARID4-Nb 353 SEKQSTAEEVIDVGPPADWVKINVRETKDCFEIFALVPGLLREEVRVQSDPAGRLVIAGQPEQLDNPWGITPFKKIVNFPA
V DVGP ADWVKINV D FE ALVPGL R EVR QSDPAG VI G PE NPWG TPFKK V

ARID2-Nb 760 RIDPHHTSAVVTLNGQLFVRVPLEQLE
ARID3-Nb 374 RIDPLHTSAVMSMHGRLFIRVPFEQ--
ARID3-Ec 374 RIDPLHTSAVMSMHGRLFIRVPFEQ--
ARID4-Nb 433 RIDPLHTSAVVSLHGRLFVRVPFEQ--
RIDP HTSAV G LF RVP EQ
disturbed (Figure S7d,e). This discrepancy with the Y2H
data could be explained by mislocalization of ARID3-11KQ.
This acetyl mimetic variant indeed exhibited a nucleocyto-
plasmic distribution, contrasting with the nuclear localiza-
tion of wild-type ARID3 and PopP2 as well (Figure S8a,b).
This finding suggests that the substitutions introduced
impede the capacity of ARID3 to specifically accumulate in
the nucleus, thus limiting interactions with nuclear compo-
nents. In this context, the monitoring of both ARID3-11KQ
self-association and the reduction of its interaction with
PWW1 using SLC assays must be interpreted with caution
(Figure S7f-i), as the possible consequences of acetyl-
mimetic mutations on ARID3 interacting properties cannot
be dissociated from the mislocalization of the protein that
they cause.

The *arid2/3/4* triple mutant exhibits reduced susceptibility
to *R. solanacearum* GMI1000

To determine whether ARID2, ARID3, and ARID4 play a role
in plant response to R. solanacearum, we used Arabidopsis
plants carrying mutations in the genes encoding these pro-
teins. Since ARID2/3/4 have been shown to function redun-
dantly in transcriptional regulation (Tan et al., 2018), we
decided to focus our analysis on the phenotypical charac-
terization of the previously published triple mutant
arid2/3/4. As described previously (Tan et al., 2018), while
the development of arid2/3/4 is stopped at the cotyledon
stage in seedlings grown on MS medium, this mutant is
able to continue its development up to flowering and seed
production when grown in soil. Upon root inoculation of 4-
week-old plants grown in soil, the R. solanacearum
GMI1000 strain producing wild-type PopP2 caused weaker
and delayed disease wilting symptoms in the arid2/3/4 tri-
ple mutant compared with wild-type Col-0 plants
(Figure 8a). Survival curve analysis confirmed this result,
showing a significantly higher survival rate for the arid2/3/4
mutant compared with Col-0 (Figure 8b,c). Consistent with
these observations, plant colonization, measured as bacte-
rial titers in aerial plant tissues, was also severely compro-
mised in the arid2/3/4 mutant (Figure 8d), indicating that
ARID2/3/4 contribute to R. solanacearum infection. How-
ever, does this contribution depend on the presence of
PopP2? We thus investigated the phenotypical response of
arid2/3/4 plants to the ΔpopP2 mutant strain, whose viru-
lence on Col-0 was reported to be attenuated (Le Roux
et al., 2015). Interestingly, the ΔpopP2 mutant did not show
additional virulence attenuation in the arid2/3/4 triple
mutant (Figure 8a,b), indicating that abrogation of
ARID2/3/4-dependent functions is deleterious to the devel-
opment of bacterial wilt disease, regardless of whether
PopP2 is produced or not. This leads us to speculate that
PopP2 might be manipulating ARID2/3/4 functions to create
a physiological context that promotes the pathogenicity of
R. solanacearum.

DISCUSSION

The diversity of host plant components targeted by patho-
gen effectors highlights the sophistication of the virulence
strategies employed by phytopathogens to defeat host
immune systems. In the repertoire of R. solanacearum
T3Es, PopP2 is considered a major virulence factor
involved both in the virulence and the fitness of this bacte-
rial pathogen (Landry et al., 2020; Macho et al., 2010).
There is increasing evidence that PopP2, as several YopJ
T3Es, might target multiple host proteins to promote its
virulence, in addition to its well-known targets, the WRKY
TFs. Identifying host components targeted by PopP2 is
therefore crucial to elucidate its biological activities. In this
study, we have identified novel targets of PopP2, namely
ARID3 and its two closest homologs in A. thaliana, ARID2
and ARID4, which have previously been shown to be part
of the PEAT protein complexes that regulate gene tran-
scription through modulation of chromatin condensation
(Tan et al., 2018).

PopP2 acetyltransferase uses ARID2/3/4 proteins as
substrates

To demonstrate the physical interaction between PopP2
and ARID2, ARID3, and ARID4, we used two complemen-
tary approaches able to detect protein-protein interactions
in vivo, that is, yeast two-hybrid (Y2H) and split-luciferase
complementation (SLC) assays. The SLC assays are partic-
ularly useful for studying protein-protein interactions in
living plant cells because they preserve the native subcellu-
lar microenvironment, are highly sensitive, and allow
quantitative results to be obtained. Using these two
approaches, the physical interaction between PopP2 and
ARID2, ARID3, and ARID4 was clearly demonstrated. Fur-
thermore, the C-terminus of these three ARID proteins has
been identified as sufficient for physical interaction with
PopP2. This C-terminal part contains an α-crystallin domain
(ACD) which comprises a compact seven-stranded β-
sandwich of approximately 90–100 amino acids that forms
the structural core of small Heat Shock Proteins (sHSPs)
(Waters & Vierling, 2020). Plant sHSPs, originally described
as heat shock-induced, can behave as protein chaperones
that oligomerize and bind other proteins, protecting them
from various stress-induced damages (Groenen et al.,
1994; Park & Seo, 2015). Due to the massive evolutionary
expansion of sHSPs families in plants, ACD-containing pro-
teins are now numerous and can harbor supplementary
domains, hence providing putative additional functions
(Scharf et al., 2001; Waters & Vierling, 2020). Whether
PopP2 has the ability to target other ACD-containing pro-
teins or if it does specifically target the ACD of ARID2/3/4
proteins remains to be investigated. Given that PopP2
interacts with and acetylates ARID2/3/4, a MS-based prote-
omic analysis was conducted on immunoprecipitated
<figure><img src="image_9.png" /><figcaption>**Figure 8.** The *arid2/3/4* triple mutant displays reduced susceptibility to *Ralstonia solanacearum* GMI1000. Root inoculation assays were performed on *Arabidopsis thaliana* wild-type Col-0 plants and the *arid2/3/4* triple mutant plants using *R. solanacearum* strains GMI1000 or *ΔpopP2*.</figcaption></figure>

(a) Disease index (DI) indicating the average wilting symptoms scored daily with a scale from 0 (no symptom) to 4 (100% of wilted leaves). Error bars indicate SEM. Statistical analysis was performed on areas under the curves (AUC) using a pairwise Wilcoxon test. ****P < 0.0001; ns, non-significant.

(b) Survival rate: The disease index (DI) was transformed into binary data to obtain survival rates: DI < 3/4 was defined as "0" (survival) and DI ≥ 3/4 was defined as "1" (death event). Statistical analysis was performed using a Log-rank test. ****P < 0.0001; ns, non-significant. DI (a) and survival curves (b) are a composite of four independent biological replicates (n ≥ 30 per condition per replicate).

(c) Representative phenotypes of *A. thaliana* Col-0 and *arid2/3/4* triple mutant plants at 8 days post-inoculation with *R. solanacearum* GMI1000.

(d) Growth of *R. solanacearum* GMI1000 in Col-0 and *arid2/3/4* triple mutant. GMI1000 quantification was determined in the aerial part of root-inoculated *A. thaliana* Col-0 and *arid2/3/4*, as described in (a). Bacterial samples were collected for Dlmean(Col-0) = 1. The presented plot is a composite from three independent biological replicates (n = 6 plants per condition in each replicate). Data points displayed on the graph are colored depending on the replicate they were collected from. Error bars indicate SD. Statistical analysis was performed using a Wilcoxon rank test. ****P < 0.0001.
ARIDs co-expressed *in planta* with catalytically active or inactive PopP2. Although members of the YopJ family can target and modify a range of host components by acetylating specific serine, threonine, and/or lysine residues (Ma & Ma, 2016), ARID2/3/4 were found to be preferentially acetylated in the presence of the active PopP2 only on lysine residues. Interestingly, many of these acetylated lysine residues are conserved in ARID2/3/4 and map to a region located between the ARID and ACD domains. It is noteworthy that three of these conserved lysines were also identified as targeted by active PopP2 in recombinant ARID3 produced in *E. coli*, thereby substantiating their direct acetylation by PopP2. Furthermore, one additional conserved lysine residue, located within the ACD of ARID2/3/4, was found to be acetylated by PopP2 *in planta*. Collectively, these data indicate that ARID2/3/4 represent previously undescribed direct substrates of PopP2 enzymatic activity.

Acetylations are post-translational modifications that
can significantly alter the function of a protein by modify-
ing its properties (i.e. hydrophobicity, solubility, and sur-
face characteristics). These modifications may influence
protein conformation, stability, subcellular localization,
and interactions with other molecules (Christensen
et al., 2019; Finkemeier et al., 2011; Xing & Poirier, 2012).
PopP2 was reported to dampen basal immune responses
through acetylation of key lysine residues in the WKRY
DNA-binding domain of multiple defensive WKRY tran-
scription factors, causing inhibition of their trans-
regulating function involved in defense gene expression
(Le Roux et al., 2015). Hence, PopP2 acetylation of con-
served lysine residues in the C-terminal region of
ARID2/3/4 suggests that PopP2 also employs this mecha-
nism to interfere with ARID2/3/4 properties. It is therefore
tempting to speculate that acetylation of critical lysine resi-
dues near and within the ACD of ARIDs could trigger a con-
formational change that alters their DNA-binding
properties, which would be reminiscent of what has been
described for the WRKY TFs acetylated by PopP2. PopP2
could also use acetylation to affect their ability to interact
with other nuclear proteins, and in particular those that are
part of the PEAT complexes.

PopP2 interacts with several components of PEAT complexes

Given that ARID2/3/4 proteins have previously been shown
to self-interact, probably through their ACD (Bondino
et al., 2012; Li et al., 2015; Waters & Vierling, 2020), and
also to associate with PWWP proteins of PEAT complexes
(Tan et al., 2018), we investigated the effect of PopP2 on
these protein-protein interactions. Due to the difficulty of
expressing ARID2 at a sufficient level in N. benthamiana,
we focused our analysis on ARID3 and ARID4.
Interestingly, the SLC assay revealed that ARID3 and ARID4
homo-oligomerization was more affected in the presence

of active PopP2 than with the catalytically inactive PopP2
mutant. As both PopP2 variants are able to physically inter-
act with ARID3, and considering that ARID3 was identified
here as a direct substrate of PopP2, our results support a
model in which PopP2 acetyltransferase activity interferes
with ARID3 and ARID4 homo-oligomerization. In addition,
our SLC assays confirmed the previously reported interac-
tions between PWWP1 and ARID3/4 (Tan et al., 2018;
Zheng et al., 2023), and showed that PopP2 promotes the
interaction of PWWP1 with both ARID3 and ARID4. Further-
more, PWWP1, which interacts with PopP2 in the Y2H
assay, might represent an additional effector target within
PEAT complexes. These PopP2-facilitated interactions
between PWWP1 and ARIDs differ slightly depending on
the ARID protein and appear to depend not only on PopP2
acetyltransferase activity but also on the presence of the
effector, whether catalytically active or not. We hypothe-
size that PopP2, apart from its enzymatic activity that
would modulate these interactions through acetylation of
particular residues, could also promote ARIDs/PWWP1
associations simply by physically interacting with these
proteins. To evaluate the role of acetylated lysine residues
on the interaction properties of ARID3, we generated an
acetyl-mimetic ARID3-11KQ mutant in which the 11 lysine
residues acetylated by PopP2 *in planta* were replaced by
glutamines (K → Q). This mutant was included in SLC
assays to investigate its ability to interact with both PopP2
and PWWP1, and to self-associate. Unfortunately, the
nucleocytoplasmic localization of ARID3-11KQ makes it dif-
ficult to determine the precise effect of the introduced
mutations on ARID3 interaction properties. For example,
the lower level of interaction measured between ARID3-
11KQ and its nuclear partners could simply be due to a
reduction in the nuclear pool of ARID3-11KQ compared
with that of ARID3. Since the nuclear localization of wild-
type ARID3 seems not to be affected in the presence of
active PopP2, it is very likely that the mislocalization of
ARID3-11KQ is caused by the substitution of lysine resi-
dues that are critical for its nuclear accumulation, rather
than by its acetyl mimetic state. However, we cannot
exclude the possibility that multiple forms of ARID3 acety-
lated by PopP2 on a range of lysine residues coexist in the
nucleus, where only a small fraction of ARID3 acetylated
on 11 lysine residues at a time would be impaired in its
ability to accumulate in the nucleus. Whether the mislocali-
zation of ARID3-11KQ has any biological significance
would require further investigation.

Based on limited structural data currently available, it
was not possible to confidently predict the structure of the
ARID3 variants or that of a multi-protein complex involving
ARID3, PWWP1, and PopP2. Overall, further structure-
function studies are needed to precisely identify the Lysine
residues critical for ARID2/3/4 functions and the biological
consequences of these acetylations.
It is noteworthy that the well-characterized BRIGHT protein, that belongs to a sub-group of ARID proteins in animals, can also self-associate through its C-terminal part located downstream of its ARID domain (Wilsker et al., 2005). Interestingly, BRIGHT was shown to form homodimers, while it does exist as a tetramer upon DNA binding (Herrscher et al., 1995; Nixon et al., 2004). Similarly, the C-terminal part of the ARID protein LjSIP1 from *Lotus japonicus*, a close homolog of ARID2/3/4, is required for LjSIP1 self-association and association with other proteins (Zhu et al., 2008). LjSIP1 was hypothesized to form a dimer in the absence of its target loci (Zhu et al., 2008). It is therefore tempting to hypothesize that PopP2 could manipulate PEAT functions through regulating ARID2/3/4 oligomerization levels and additionally modulate the subsequent associations with other PEAT components like PWWP1.

**Do PEAT complexes serve as hubs of pathogen effectors to promote host infection?**

To investigate the contribution of ARIDs in *A. thaliana* infected by *R. solanacearum*, the *arid2/3/4* mutant previously described by Tan et al. (2018), was root-inoculated with the GMI1000 strain. The reduced susceptibility of the *arid2/3/4* mutant to the bacteria indicates that ARID2/3/4 contribute to *Ralstonia* infection and should therefore be considered as susceptibility factors to this bacterial pathogen. Given that PopP2 affects two components of the PEAT complexes, it is worth considering that PEAT complexes may be involved in the virulence of *R. solanacearum*. In *A. thaliana*, the PEAT complexes transcriptionally regulate over 8000 genes by recruiting the histone acetyltransferases HAM1/2 and histone deacetylases HDA6/9 to regulate histone H4 acetylation levels and, subsequently, chromatin accessibility at these specific loci (Tan et al., 2018; Zheng et al., 2023). Transcriptomic data generated from 12-day-old seedlings of null mutants in PEAT components showed that among the 8000 genes differentially regulated by the PEAT complexes at early stages of plant growth, around a few hundred genes are involved in defense responses (Tan et al., 2018; Zheng et al., 2023). Although our root-inoculation assays were performed at a later stage of development (e.g. 4-week-old plants), we hypothesize that the hijacking of these chromatin-remodeling complexes by an effector like PopP2 would represent a potent virulence strategy, allowing *R. solanacearum* to modulate the expression of host genes to favor infection. The exact number and nature of the genes modulated by PEAT complexes in infected plants remains unknown and requires further investigation.

A substantial body of evidence derived from a diverse array of pathogens indicates that manipulation of host epi-genetic regulation represents a conserved strategy to dampen host immune responses (Harris et al., 2023; Schator et al., 2021). In plants, a few effectors able to modify

chromatin condensation were recently discovered in nematodes, oomycetes, and fungi (Harris et al., 2023). For example, the nematode *Heterodera schachtii* effector 32E03 epigenetically regulates plant gene expression by direct inhibition of the *A. thaliana* histone deacetylase AtHDT1 involved in rRNA transcriptional regulation through chromatin modifications (Vijayapalani et al., 2018). The oomycete *Phytophthora sojae* PsAvh52 effector interacts with the soybean histone acetyltransferase GmTAP1 and relocates it to the nucleus, where it can promote transcriptional activation of putative susceptibility genes through the acetylation of histones H2A and H3 (Li et al., 2018). By contrast, the *P. sojae* effector PsAvh23 targets a component of a histone acetyltransferase (HAT) complex to suppress histone H3 acetylation and the activation of defense genes, promoting pathogen infection (Kong et al., 2017).

Pathogen interference with the PEAT complexes might represent a virulence strategy used by at least two different vascular phytopathogenic bacteria. Indeed, the Xop6 effector from Xcc was identified here as interacting with ARID3, like PopP2. This suggests that these two closely related YopJ T3Es share common host targets in addition to the defensive WRKY TFs (Lauber et al., 2024). However, it remains unclear whether XopJ6 manipulates ARID proteins in a similar manner and whether it is also able to target other components of the PEAT complexes. More generally, whether PEAT remodeling complexes serve as hubs for phytopathogen effectors to promote infection is a key question to be addressed in the near future.

**EXPERIMENTAL PROCEDURES**

**Bacterial strains, yeast strains, and growth conditions**

*Agrobacterium tumefaciens* strains were grown at 28°C in liquid YEB medium supplemented with adequate antibiotics: gentamicin (15 µg ml⁻¹) and carbenicillin (25 g ml⁻¹) for pAM-PAT-35S transformed GV3103 strains; gentamicin (15 µg ml⁻¹) and spectinomycin (50 µg ml⁻¹) for pB7-35S GV3101 transformed strains; tetracyclin (5 µg ml⁻¹) and Kanamycin (25 µg ml⁻¹) for pBIN transformed C58C1 strains. *E. coli* Rosetta (DE3) strains (Sigma Aldrich, Darmstadt, Germany) were grown at 37°C in Luria-Bertani rich medium supplemented with adequate antibiotics. *R. solanacearum* strains were grown for 3 days at 28°C on solid φ medium supplemented with 5 g L⁻¹ glucose and 0.004% triphenyl tetrazolium chloride for GMI1000, and also supplemented with gentamicin 7.5 µg ml⁻¹ for Δ*popP2*. Strains were then grown in liquid φ medium at 28°C under shaking to OD₆₀₀nm = 1 before dilution for inoculation assays at OD₆₀₀nm = 0.01 (10⁷ CFU ml⁻¹). For yeast-two-hybrid assays, the *S. cerevisiae* AH109 strain was used (Takara Bio, San Jose, CA, USA).

**Plant material**

*Nicotiana benthamiana* plants were grown on soil at 21°C and 60% relative humidity, under long day conditions (16 h light/8 h dark). *Agrobacterium tumefaciens*-mediated transient expression experiments were performed on leaves of 4-week-old plants. *Arabidopsis thaliana* plants Col-0 and *arid2/3/4* triple mutant were
grown on Jiffy-7 substrate pellets (Jiffy Products International AS, Norway) at 22°C and 60% relative humidity, under short day conditions (8 h light/16 h dark). Root inoculations with *R. solana-cearum* GM1000 and *ΔpopP2* strains were performed on 3-week-old plants. The *arid2/3/4* triple mutant was generated by crossing the single mutants *arid2* (SALK_026835), *arid3* (SALK_022359), and *arid4* (SALK_007400) (Tan et al., 2018).

## Construction of plasmids

Standard DNA cloning methods (Sambrook et al., 1989), PCR, and Gateway technology (Invitrogen) were used for plasmid construction. All primer sequences are listed in Table S2. PCR products flanked by the attB sites were recombined into the pDONR207 vector (Invitrogen) via a BP reaction to create the corresponding entry clones with attL sites (pENTR). The pENTR-RRS1-R<sub>Cterm</sub>, pENTR-PopP2, pENTR-PopP2-C321A, pENTR-Xop<sub>6224-565</sub> (pDONR207 vector backbone) clones used in this study have been previously described (Lauber et al., 2024; Le Roux et al., 2015). Full-length PWWP1 and ARID cDNA clones (ARID1, ARID2, ARID3, and ARID4) and the truncated versions of ARIDs (ARID<sub>2Cterm</sub>, ARID3<sub>Cterm</sub>, ARID4<sub>Cterm</sub>, ARID3<sub>1-92</sub>, ARID3<sub>93-236</sub>, ARID3<sub>1-236</sub>) flanked with the AttB1 and AttB2 recombination sites were amplified from cDNA (Col-0) using PrimeStar HS DNA polymerase from Takara Bio Inc. (Otsu, Japan). The introduction of the acetyl mimetic mutations in ARID3-11KQ variant was performed by gene synthesis (the nuclease sequence of ARID3-11KQ flanked with AttB1 and AttB2 sites is indicated in Table S2). The pENTR-PopP2-3HA and pENTR-PopP2-C321A-3HA were generated by two-step PCR-based methods with PopP2-3HA and PopP2-C321A-3HA flanked with Ncol and AvrII restriction sites. Ncol/AvrII-digested fragments were then introduced by ligation in Ncol/AvrII-digested pETDuet to produce the pETDuet-PopP2-3HA and pETDuet-PopP2-C321A-3HA plasmids used in the bacterial acetylation assay. The pDEST pBIN-35S-GWY-3HA-Nluc and pBIN-35S-GWY-3Flag-Cluc vectors used in the SLC assay were obtained by insertion of a blunt-end chloramphenicol/ccdB resistance gateway cassette (Invitrogen) in SnaBI-digested pBIN-35S-3HA-Nluc and pBIN-35S-3Flag-Cluc (with the Nluc and Cluc moieties corresponding to the residues 1-416 and 398-550 of the firefly luciferase, respectively). The pDEST pB7-35S-GWY-mCherry vector is derived from the pB7FWG2-35S-GWY-eGFP with replacement of the eGFP coding sequence with the sequence of the mCherry using PCR-based methods. The inserts cloned in pENTR vectors were then recombined in pDEST vectors via LR reaction (Invitrogen). All plasmids used for cloning in this study were transformed into *E. coli* strain DH5α using electroporation (0.1 cm cuvette, 1.8 kV pulse, 25 µF and 200 Ω) and selected on LB agar medium with appropriate antibiotics. Plasmid DNAs were extracted with the Wizard DNA plasmid purification kit (Promega) and sequenced using either Sanger-based sequencers or a MinION NanoSeq sequencer (Oxford Nanopore Technology) with the resulting sequencing data processed thanks to the Long read Plasmid and PCR assembly pipeline NanoSeq release v0.1.3 (Carlier & Moreau, 2024).

## Yeast two-hybrid assays

Constructs for yeast two-hybrid analysis were prepared in the MatchMaker GAL4 two-hybrid system (Clontech) using the pGBG-GWY (BD fusion) and pGAD-GWY (AD fusion) vectors to express bait and prey proteins, respectively. All expressed proteins N-terminally fused with either the BD or AD domain of GAL were tagged with a c-myc or an HA epitope, respectively. Bait and prey plasmids were introduced in the AH109 yeast strain by using the LiAc/Salmon Sperm carrier DNA/PEG method (Gietz &

Schiestl, 2007). Transformed cells were plated on minimal SD-Trp-Leu (SD-TL) medium and incubated for 3-4 days at 30°C. Transformed yeast cells were incubated in liquid SD-TL medium under shaking at 30°C overnight. Cell density was checked at OD<sub>600nm</sub> and adjusted to final OD<sub>600nm</sub> = 0.5 in sterile ultrapure water. A dilution gradient was performed (from 1 to 10<sup>-3</sup>) to plate the cells on different media: non-selective (SD-Trp-Leu) and selective (SD-Trp-Leu-His + 5 mM 3-AT, where 1,2,4-triazole [3-AT] serves as competitive inhibitor of *HIS3* gene product), and incubated for 4 to 5 days at 30°C. Protein expression in yeast cells was evaluated following incubation of the different yeast strains under shaking at 30°C overnight in SD-TL media. For each combination of bait/prey proteins, the cell density of an overnight culture was adjusted to OD<sub>600nm</sub> = 1. Total yeast proteins were extracted according to a LiAc/NaOH pre-treatment protocol as previously described (Zhang et al., 2011). Samples were denatured for 3 min at 95°C in Laemmli buffer (2x) supplemented with β-mercaptoethanol 5% before separation by SDS-PAGE and immunoblotting with anti-c-myc-HPR and anti-HA-HPR conjugated antibodies.

## Agrobacterium-mediated transient expression assay in *N. benthamiana*

For transient expression in *N. benthamiana* leaf epidermal cells, *A. tumefaciens* strains were grown in liquid YEB medium containing appropriate antibiotics for 16 h at 28 °C under shaking. Cells were harvested by centrifugation and resuspended in infiltration medium (10 mM MES pH 5.6, 10 mM MgCl₂, 150 µM acetosyringone) at OD<sub>600nm</sub> = 0.25. For co-expression, bacterial suspensions carrying individual constructs were mixed in a 1:1 ratio (total OD<sub>600nm</sub> = 0.5). When three different *A. tumefaciens* strains were mixed, a 1:1:1 ratio was used (total OD<sub>600nm</sub> = 0.75). After incubation at room temperature for 1 h, bacteria were infiltrated into the leaves of 4-week-old *N. benthamiana* plants using a needleless syringe. Plants were incubated for 48 h in growth chambers under controlled conditions before harvesting leaf disc samples.

## Confocal microscopy

Agrobacterium-mediated transient expression in *N. benthamiana* was performed using strains carrying the PopP2-mCherry, ARID2-eGFP, ARID3-eGFP, ARID3-11KQ-eGFP, and ARID4-eGFP constructs. Confocal microscopy observations and sampling for immunoblot analysis were conducted at 48 h post-Agrobacterium infiltration in *N. benthamiana* leaves. For protein subcellular localization, GFP and mCherry fluorescence was analyzed with a confocal laser scanning microscope (TCS SP8; Leica) using a ×25 water immersion objective lens (numerical aperture 0.95; HCX PL APO CS2). GFP and mCherry fluorescence were excited with the 488/561 nm ray lines of the lasers and recorded in one of the confocal channels in the 500-550/580-620 nm emission range, respectively. The images were acquired in the sequential mode using Leica LAS X software (version 3.0). The signal (detector gain) was adjusted on the sample transiently expressing the ARIDx-eGFP fusion protein alone and then kept unchanged or lower if saturated for the sample co-expressing also PopP2-mCherry.

## Split-luciferase complementation assay

The different Nluc and Cluc fusion proteins were transiently expressed in *N. benthamiana* leaves using Agrobacterium-mediated transformation. For luminescence quantification, single 4 mm punch disks were harvested 48 h later and placed into wells of a 96-well plate. Samples were washed with 100 µl of ultrapure sterile water for 5 min and then incubated in 100 µl of luciferin
substrate (1 mM, XenoLight D-Luciferin; Perkin Elmer, Waltham, MA, USA) as previously described (Chen et al., 2008; Yu et al., 2020). After 10 min of incubation, light emission was quantified in each well for 5 sec using a luminometer (PerkinElmer; VICTOR Nivo). For each interaction tested, 24 technical replicates were performed (24 leaf disks/plate i.e., eight leaf disks per plant from three plants per biological replicate). Each experiment was replicated at least three times independently. As a positive control, PopP2-3HA-Nluc and RRS1-R<sub>Cterm</sub>-3Flag-Cluc proteins were used, based on the previously reported physical interaction between these two proteins (Le Roux et al., 2015).

## Purification and immunoblot analysis of proteins transiently expressed in *N. benthamiana*

For Agrobacterium-mediated transient expression experiments in *N. benthamiana*, protein samples were prepared from four leaf discs (8 mm diameter) harvested 48 h after Agrobacterium-mediated transformation and homogenized in 800 µl of ice cold buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 2 mM DTT, 1× Plant protease cocktail inhibitor (Sigma), 0.2% Triton). Protein extracts were rotated for 10 min at 4°C and centrifuged at 13 000 g for 2 min at 4°C to collect the supernatant. Fifty microliters of the protein extract was denatured at 100°C for 3 min in 50 µl of 2 × laemmli buffer (CE, crude extract). For immunoprecipitation of GFP-tagged proteins, the remaining supernatant was transferred to a new tube and incubated with 8 µl of pre-equilibrated GFP-trap agarose beads (Chromotek) for 50 min at 4°C under stirring. Beads were washed three times with 800 µl of ice cold buffer before denaturation in 2 × laemmli buffer at 95°C for 3 min (IP-GFP).

Protein samples were analyzed by immunoblotting (SDS-PAGE using 4-15% pre-casted gels [Bio-Rad]). After migration, proteins were transferred on a nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). Transferred proteins were visualized by Ponceau S red staining (0.5% Ponceau S [w/v], 5% anhydrous acetic acid). PageRuler Prestained Protein Ladder (Thermofischer) was used to estimate protein molecular weights. Membranes were blocked in a 2% milk TBST (Tris Buffer Saline-Tween-20; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) solution before immunodetection. The following primary antibodies were used in this study: anti-c-myc-HRP (mouse monoclonal, clone 9E10, dilution 1:10 000; Roche), anti-HA-HRP (mouse monoclonal, clone 3F10, dilution 1:5000; Roche), anti-Flag-M2-HRP (mouse monoclonal, dilution 1:5000; Sigma), anti-GFP (mouse monoclonal, dilution 1:3000; Roche), anti-Acetylated Lysine (Ac-K-103, dilution 1:2000; Cell Signaling Technology), anti-His6-HRP (mouse monoclonal, dilution 1:25 000; Roche). The appropriate horseradish peroxidase-conjugated secondary antibody was applied to the membranes: goat anti-mouse IgG-HRP (Bio-Rad; dilution 1:10 000, for detection of anti-GFP antibodies); goat anti-mouse IgG2a-HRP (Bio-Rad; dilution 1:5000, for detection of anti-Ac-K antibodies). Immunodetections were performed using Clarity Western ECL substrate reagent (Bio-Rad, Hercules, CA, USA). Chemiluminescence was captured using a G:BOX system (Syngene, Iselin, NJ and Baltimore, MD, USA).

## Bacterial acetylation assay

Single colonies of *E. coli* Rosetta (DE3) cells (Novagen) co-transformed with pCDF-GST-ARID3-His6 and either pETDuet-PopP2-3HA or pETDuet-PopP2-C321A-3HA were grown in 50 ml of LB medium containing carbenicillin (50 µg ml⁻¹), spectinomycin (50 µg ml⁻¹) and chloramphenicol (30 µg ml⁻¹) at 37°C to an OD₆₀₀nm = 0.6 and induced with 125 µM IPTG (isopropyl-β-thiogalactopyranoside) for 4 h 30 min at 28°C. Pelleted cells were

concentrated 25 times in ice-cold protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Aldrich), 1 mM sodium butyrate, 0.1% Triton, 5 mM imidazole) and lysed using a French press. After centrifugation, supernatants were incubated with 100 µl of pre-equilibrated Ni-NTA agarose beads (Qiagen) for 2 h at 4°C with rotation. Beads were recovered by centrifugation (5000 g for 10 min at 4°C) and then washed three times in 10 ml of protein extraction buffer for 10 min at 4°C under stirring. Purified proteins were denatured in laemmli buffer (2×) for 3 min at 95°C.

## Mass spectrometry-based proteomic analyses

For identification of acetylated residues in ARIDs, protein samples from *N. benthamiana* and from *E. coli* were used. Whole *N. benthamiana* leaves co-expressing eGFP-tagged ARID proteins with PopP2-3HA or PopP2-C321A-3HA were collected at 48 hpi. Total proteins were extracted from 3 to 4.5 g of fresh material (FM) ground in liquid nitrogen and resuspended in protein extraction buffer (1 ml for 1 g FM, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 2 mM DTT, 1 × plant protease cocktail inhibitor [Sigma], 0.2% Triton, 10 mM sodium butyrate). Total protein extracts were centrifuged and the supernatant (protein extract) was filtered on miracloth. About 50 µl of the protein extract was sampled, mixed in 50 µl of 2 × laemmli buffer and denatured at 95°C for 3 min (CE, crude extract). The remaining protein extract was incubated under shaking at 4°C with pre-equilibrated GFP-trap agarose beads (Chromotek GmbH, Planegg, Germany) for 1 h. GFP-trap agarose beads were collected after centrifugation and the supernatant was discarded. GFP-trap agarose beads were washed four times in protein extraction buffer. Immunoprecipitated proteins were resuspended in 2 × laemmli buffer and denatured at 95°C for 3 min. Affinity purified proteins (from *N. benthamiana* and Rosetta DE3 cells) were migrated on an SDS-PAGE gel. After Coomassie blue staining (InstantBlue Coomassie Protein Stain; Abcam Inc., Waltham, MA, USA), gel bands were cut out and proteins were digested in-gel using trypsin (Promega; sequencing grade) as previously described (Casabona et al., 2013), except that tris(2-carboxyethyl)phosphine hydrochloride was used instead of dithiothreitol. The resulting peptides were analyzed by online nanoliquid chromatography coupled to MS/MS (Ultimate 3000 RSLCnano and Q-Exactive HF; Thermo Fisher Scientific). For this purpose, the peptides were sampled on a precolumn (300 µm × 5 mm PepMap C18; Thermo Scientific) and separated in a 75 µm × 250 mm C18 column (Aurora Generation 3, 1.7 µm; IonOpticks) using an acetonitrile gradient for 35 min. The MS and MS/MS data were acquired using Xcalibur 2.9 (Thermo Fisher Scientific, Waltham, MA, USA).

Peptides and proteins were identified by Mascot (version 2.8; Matrix Science) through concomitant searches against the Uniprot database (*N. benthamiana* or *E. coli* BL21-DE3 taxonomies), a homemade database containing the sequences of recombinant proteins, and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin...). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set respectively at 10 and 20 ppm. Peptide modifications allowed during the search were: Carbamidomethyl (C, fixed), Acetyl (K, S, T, variable), Acetyl (Protein N-term, variable) and Oxidation (M, variable). The Proline software version 2.2.0 (Bouyssié et al., 2020) was used for the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥6 amino acids, false discovery rate of peptide-spectrum-match identifications <1% (Couté et al., 2020),
and minimum of one specific peptide per identified protein group). MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the dataset identifier PXD055620. Proline was then used to perform a spectral counting-based quantification of the identified protein groups. Only class I acetylated lysines (localization probability ≥0.75) identified in peptide-spectrum-matches with Mascot score >30 were further considered.

### R. solanacearum inoculation assay

Three-week-old *A. thaliana* Col-0 and *arid2/3/4* plants (n ≥ 30 for each line in each condition) were inoculated by soil-drenching with a *R. solanacearum* bacterial suspension (strains GMI1000 or Δ*popP2*) containing 10⁷ colony-forming units per ml (CFU ml⁻¹). After a 20-min incubation in the bacterial solution, plants were transferred to a new tray. Inoculated plants were incubated in a dedicated growth chamber (12 h day/12 h night, 27°C, 75% humidity). Symptoms development was rated daily for 12 days on a scale of 0-4 (disease index [DI]) relative to the percentage of wilted leaves on the aerial part of the plants (0 = 0% wilted, 1 = 25% wilted, 2 = 50% wilted, 3 = 75% wilted, 4 = 100% wilted). To assess survival rates, DI was converted into binary data considering DI ≥ 3 → 1 (death) and DI < 3 → 0 (survival). A Kaplan-Meier survival analysis was then performed on these binary data. For bacteria quantification in Arabidopsis leaves upon *R. solanacearum* inoculation, the aerial parts of Col-0 and *arid2/3/4* plants inoculated with *R. solanacearum* GMI1000 strain were collected (n = 6 for each genotype and for one biological replicate) at DI<sub>mean</sub> (Col-0) = 1. Leaf tissues were weighed, decontaminated in 70% ethanol for 3 min, and washed in two consecutive baths of sterile water. Aerial parts were then ground and suspended in sterile water (in a 1 ml:1 g Fresh Material ratio). CFUs were counted by spreading serial dilutions (between 10⁻¹ and 10⁻⁵, with two spreading replicates per sample) on solid SMSA medium (supplemented with 2.5 µg ml⁻¹ violet crystal, 12.5 µg ml⁻¹ bacitracin, 50 µg ml⁻¹ polymyxin B, 24 µg ml⁻¹ chloramphenicol, 0.25 µg ml⁻¹ penicillin G and 22 U ml⁻¹ nystatin) after 36 h of incubation at 28°C.

### Protein sequence alignment

The amino acid sequences of the ARID proteins (ARID2 [786 residues], ARID3 [398 residues] and ARID4 [457 residues]) were considered to perform a multiple sequence alignment using the Scoring Matrix Blosum 62 parameters in Clone Manager 9 Professional Edition software.

### Statistical analysis

For SLC assay-related measurements, data were analyzed and represented as the mean ± SEM using GraphPad Prism 10.0.3; the horizontal line in the graphs indicates the median. For split-luciferase-related data, one-way ANOVA tests were performed. Stars between two columns indicate statistically significant differences between different tests (****P < 0.0001; **P < 0.001; ns = non-significant).

For DI curves, the area under the curves was calculated using the Agricolae package in Rstudio, and statistical analysis was performed using a pairwise Wilcoxon rank test, adjusted with the Benjamini Hochberg method.

For the Kaplan-Meier survival analysis, curves were computed by PRISM, version 10 (GraphPad Software Inc., San Diego, CA, USA) and statistical analysis was performed using the Gehan-Breslow-Wilcoxon method.

For the bacterial growth analysis, plots were computed by PRISM, version 10 (GraphPad Software Inc.), and statistical analysis was performed using a Wilcoxon rank test. For all statistical analyses, the threshold for significance was set for P-value <0.05.

### ACCESSION NUMBERS

ARID1 (At2g46040), ARID2 (At2g17410), ARID3 (At1g20910), ARID4 (At1g76510), PopP2 (CAD14570), RRS1-R (At5g45260), NbWRK8 (BAI63296.1), PWWP1 (At3g03140), *arid2/3/4* (SALK_026835/SALK_022359/SALK_007400).

### AUTHOR CONTRIBUTIONS

LM-W, VP, and LD designed the research. LM-W, MG, MC, RC, CVi, YM, CVe, YC, VP, and LD performed research. LM-W, MG, CVe, YC, VP, and LD analyzed the data. LM-W, VP, and LD wrote the manuscript with inputs from all the authors. All authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

L.M.-W. was funded by a PhD grant from the French Ministry of National Education and Research. L.D. was supported by two research grants from the Agence Nationale pour la Recherche (ANR15-CE20-0016-01 and ANR-18-CE20-0015). This work was supported by the French Laboratory of Excellence project "TULIP" (ANR-10-LABX-41; ANR-11-IDEX-0002-02), the "École Universitaire de Recherche (EUR)" TULIP-GS (ANR-18-EURE-0019), the Région Occitanie, European funds (Fonds Européens de Développement Régional, FEDER), Toulouse Métropole and by the French Ministry of Research with the Investissement d'Avenir Infrastructures Nationales en Biologie et Santé program (ProFI, Proteomics French Infrastructure project, ANR-10-INBS-08), and FBI, France BioImaging infrastructure project (ANR-10-INBS-0004). The proteomic experiments were partially supported by Agence Nationale de la Recherche under projects ProFI (Proteomics French Infrastructure, ANR-10-INBS-08) and GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (ANR-17-EURE-0003). We also thank Dr Xin-Jian He for providing the *A. thaliana* *arid2/3/4* triple mutant. We also thank Dr Zhi-Min Zhang for useful discussions on this study. Some elements of the graphical abstract were created using content from BioRender.com.

### CONFLICT OF INTEREST

The authors declare no conflict of interest.

### DATA AVAILABILITY STATEMENT

All relevant data can be found within the manuscript and its supporting materials.

### SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.

**Figure S1.** PopP2 and XopJ6 physically interact with ARID3 C-terminal part (related to Figure 1).

**Figure S2.** Immunodetection of eGFP-tagged ARID2, ARID3, and ARID4 expressed alone or with PopP2-mCherry (related to Figure 2).
**Figure S3.** Immunodetection of different fusion proteins used in the SLC assay (related to Figure 3).

**Figure S4.** Immunodetection of split-luciferase fusion proteins (related to Figure 4) and Y2H data showing that PopP2-C321A associates with ARID3.

**Figure S5.** PopP2 associates with PWWP1 in yeast and promotes ARID3/PWWP1 and ARID4/PWWP1 associations (related to Figure 5).

**Figure S6.** *In planta* acetylation of ARID3 and NbWRKY8 in presence of active PopP2.

**Figure S7.** Interacting abilities of the acetyl-mimetic ARID3-11KQ variant towards itself, PopP2, and PWWP1.

**Figure S8.** ARID3-11KQ-eGFP is nucleo-cytoplasmic, by contrast to ARID3-eGFP that specifically accumulates in the plant nucleus.

**Table S1.** List of acetylated K, S, and T residues detected by LC-MS/MS analyses performed on ARID2, ARID3, and ARID4 proteins co-expressed with either PopP2 or PopP2-C321A.

**Table S2.** List of bacterial strains, plasmid constructs, and oligonucleotides used in this study.

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