<i>Ralstonia solanacearum</i> Acetyltransferase <scp>RipU</scp> Hijacks <scp>SlJAR1</scp> to Inhibit Jasmonic Acid Signalling and Facilitate Pathogen Infection

Ralstonia solanacearum delivers numerous Type III effectors (T3Es) to cause bacterial wilt. These T3Es serve as molecular probes for identifying plant immunity components (Islam et al. 2024). We previously identified the T3E RipU from R. solanacearum strain P380. When delivered by a T3E-deficient Pseudomonas mutant D36E, RipU suppressed pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and induced cell death in Nicotiana benthamiana (Cong et al. 2023). However, whether RipU regulates immunity in tomato, the natural host of strain P380, remains unknown. To investigate the function of RipU in tomato, we infiltrated leaves with D36E harbouring empty vector (EV) or RipU. RipU suppressed reactive oxygen species (ROS) burst and induced cell death with increased ion leakage compared to EV (Figure 1a–c). Moreover, ripU-overexpressing (OE-RipU) tomato plants exhibited suppressed flg22-induced ROS production and decreased resistance to both R. solanacearum P380 and Pst DC3000 (Figure 1d, Figure S1a–c). Soil-drench inoculation showed that the ripU mutant attenuated virulence in tomato and Arabidopsis thaliana, which was restored by complementation (Figure S2a–d). Consistently, the mutant displayed impaired bacterial growth in A. thaliana, a defect also restored upon complementation (Figure S2e). These results demonstrate that RipU suppresses tomato immunity and is essential for R. solanacearum virulence. We performed RNA sequencing on tomato leaves infiltrated with D36E harbouring EV or RipU to characterise the transcriptional defence response. The six RNA-seq libraries generated approximately 263 million reads with a 97% genome mapping rate. RipU treatment upregulated 2704 genes (7.94% of genome) and downregulated 4404 genes (12.92% of genome) compared to EV (≥ 2-fold change, p < 0.05; Figure S3a). Differentially expressed genes (DEGs) analysis revealed that a set of immune-associated genes was downregulated by RipU (Figure S3b,c). Additionally, both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed significant enrichments in plant hormone response among downregulated genes (Figure 1e, Figure S3d,e). We next investigated whether RipU altered the expression of genes associated with jasmonic acid (JA), a plant hormone that mediates resistance against R. solanacearum (Jiang et al. 2025). Reverse transcription quantitative PCR (RT-qPCR) revealed that RipU inhibited the expression of SlJAR1, SlMYC2 and SlLOX2 (Figure 1f). Consistent with this, RipU significantly reduced endogenous JA levels in tomato leaves compared to EV (Figure 1g). Furthermore, overexpression of ripU significantly decreased levels of JA-Isoleucine (JA-Ile), a key bioactive jasmonate synthesised by SlJAR1, in tomato roots (Figure 1h) (Suza et al. 2010). We next examined whether RipU altered the levels of JA in tomato roots during pathogen infection. The ΔripU mutant inoculation resulted in greater JA accumulation than the wild-type (WT) P380 strain. This effect was reversed by complementation with ripU (comp-ripU) (Figure 1i). These results suggest that RipU inhibits JA accumulation. Given that many T3Es inducing cell death possess acetyltransferase activity (Ma and Ma 2016), we tested RipU in an in vitro assay and confirmed its autoacetylation activity (Figure 1j). However, RipU shows low sequence conservation with known acetyltransferase T3Es and lacks the conserved catalytic cysteine (Figure S4). Based on the central role of SlJAR1 in JA signalling (Suza et al. 2010), we hypothesized RipU targets it via acetylation. We confirmed the physical interaction between RipU and SlJAR1 in planta using split-luciferase complementation (Split-LUC) and co-immunoprecipitation (Co-IP) assays (Figure 1k,l). We next employed immunoblotting with anti-acetyl-lysine (Ac-K) antibody to investigate whether RipU could acetylate SlJAR1. Acetylated SlJAR1-Flag was detected with RipU-GFP but not GFP alone (Figure S5). These results suggest that SlJAR1 may be an acetylation substrate of RipU. Our structural modelling of RipU indicated that the amino acids at positions 203–205 (NVS residues) are likely critical sites. We mutated these three amino acids to alanine to generate RipUΔNVS and assessed its activity. Immunoblot analysis with anti-Ac-K antibody showed that the acetylation level of Flag-SlJAR1-His was attenuated when co-expressed with RipUΔNVS-GST compared to RipU-GST in Escherichia coli cells (Figure 1m). These results indicate that RipU acetylates SlJAR1 partially depend on the NVS residues. Importantly, mutation of the NVS residues also impaired the interaction between RipU and SlJAR1 (Figure 1n,o). Furthermore, we investigated whether RipU contributed to R. solanacearum virulence via its acetyltransferase activity. Mutation of NVS residues in RipU compromised its ROS-suppressive and cell death-inducing activities in both N. benthamiana and tomato leaves (Figure 1a–c, Figure S6). We next quantified the bacterial populations of R. solanacearum in tomato xylem. In line with the established role of RipU in R. solanacearum pathogenesis (Hiles et al. 2024), we observed a significant reduction of the ΔripU mutant in bacterial replication (Figure 1p). This reduction was fully rescued by comp-ripU and only partially rescued by complementation with ripUΔNVS (comp-ripUΔNVS) (Figure 1p). These results indicate that the acetyltransferase activity of RipU is required for full bacterial virulence. In summary, RipU is a unique T3E acetyltransferase that suppresses JA-mediated immunity by targeting SlJAR1. It lacks homology and the catalytic cysteine of known T3E acetyltransferases, revealing a novel hormone-sabotage strategy for bacterial wilt control. This research was supported by grants from the National Natural Science Foundation of China (32560627; 32472514), the Guangxi Natural Science Foundation (2024GXNSFAA010356) and the Beijing Innovation Consortium of the Agriculture Research System (BAIC04-2025). The authors have nothing to report. The authors declare no conflicts of interest. All data supporting this study are included in the paper and its Supporting Information S1. Data S1: pbi70522-sup-0001-Supinfo.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.