RipG6 (RSc1356) from the R. pseudosolanacearum strain Pe_1 (Prokchorchik et al., 2020) and SlRLCK-VIII-6 (Solyc12g098820) coding sequences (Sakamoto et al., 2012) were synthesized (Cosmogenetech, Seoul, Korea). Golden gate-compatible modules flanked by BsaI restriction sites were amplified from synthetic gene fragments (Engler et al., 2008). The resulting PCR products were ligated into the entry vector pICH41021. The modules were assembled with epitope or fluorescent tags under the control of the cauliflower mosaic virus 35S promoter in the binary vector pICH86988. For Pseudomonas syringae-mediated effector delivery, RipG6 modules were assembled into the broad host range vector pBBR1 in fusion with AvrRps4 promoter (128 bp), AvrRps4 N-terminus (1-136 aa), and C-terminal 3xFLAG tag (Kovach et al., 1995; Sohn et al., 2007). The list of constructs used in this study is reported in Supplementary Table 1. Specific primers are listed in Supplementary Table 2.

Binary constructs were mobilized into Agrobacterium tumefaciens AGL1 strain by electroporation. Strains were grown at 28°C on LB medium supplemented with 100 μg/mL carbenicillin and 50 μg/mL kanamycin. The broad host range vector RipG6 construct was mobilized into P. syringae pv. tomato DC3000 effectorless mutant D36E (Wei et al., 2015) by triparental mating. Strains were grown at 28°C on King’s B medium supplemented with 50 μg/mL rifampicin, 100 μg/mL spectinomycin and 20 μg/mL gentamycin. Cells were collected from overnight liquid culture and resuspended in infiltration medium (10 mM MgCl₂ and 10 mM MES-KOH, pH 5.6 for A. tumefaciens; 10 mM MgCl₂ for P. syringae).

N. benthamiana plants were grown in a growth chamber at 25°C under long-day conditions (16 h light/8 h dark). A. thaliana ecotype Col-0, fls2 efr cerk1 (Gimenez-Ibanez et al., 2009) and cark6-1 mutant (Wang et al., 2019) were grown in a growth chamber at 22°C under short-day conditions (10 h light/14 h dark).

A. tumefaciens AGL1 cells grown overnight were centrifuged and resuspended in infiltration medium to reach OD₆₀₀ 0.1-0.5 depending on subsequent experiments as previously reported (Kim et al., 2023). The suspensions were infiltrated into fully expanded leaves of 5-week-old N. benthamiana plants using a needless syringe.

Yeast transformations were performed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA, USA). Yeast strain EGY48 carrying the lacZ-bearing reporter plasmid pSH18-34 was transformed with pLexA construct containing RipG6 and transformants were selected on synthetic defined (SD) minimal agar media lacking uracil and histidine (Clontech, Mountain View, CA, USA). Yeast strain RFY206 was transformed with pB42-AD construct containing SlRLCK-VIII-6 and transformants were selected on SD media lacking tryptophan. Bait and prey transformants were resuspended together in yeast extract peptone dextrose broth (Difco; Becton, Dickinson and Company, Sparks, MD, USA) and grown overnight for mating. Mated cells were spotted on SD media lacking histidine, uracil, leucine, and tryptophan containing raffinose, galactose (Sigma-Aldrich, St. Louis, MO, USA), and X-gal (LPS solution, Daejeon, Korea).

Confocal microscopy was conducted using a SP8X confocal laser scanning microscope (Leica Microsystems, Buffalo Grove, IL, USA) with a 40x water-immersion objective. YFP and mCherry fluorophores were excited with a 488 nm argon laser and a 561 nm argon laser, respectively. Emission was collected in the following channels: 520-540 nm (YFP) and 590-610 nm (mCherry). Image processing was performed with LasX software.

Binary constructs of RipG6 fused with the N-terminal part of the YFP (YFPn) at the C-terminus and SlRLCK-VIII-6 fused with the C-terminal part of the YFP (YFPc) at the C-terminus were mobilized into A. tumefaciens AGL1. Equal volumes of A. tumefaciens carrying RipG6-YFPn (OD₆₀₀ 0.4) and free YFPc or SlRLCK-VIII-6-YFPc (OD₆₀₀ 0.4) were mixed and infiltrated to N. benthamiana leaves. Yellow fluorescence was detected at 48 hpi.

Leaf tissues were frozen and ground in liquid nitrogen. Total proteins were extracted in GTEN buffer (10% glycerol, 50 mM Tris-HCl pH 7.5, 2 mM EDTA pH 8, 150 mM NaCl) supplemented with 5 mM DTT, 0.5% IGEPAL (Sigma-Aldrich), 1% PVPP and cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). Extracts were clarified by centrifugation at 15,000 ×g for 10 min at 4°C and filtered through MiraCloth (Millipore, Billerica, MA, USA). Filtered extracts were mixed with 3x SDS sample buffer and denatured at 96°C for 10 min. For immunoprecipitation, protein extracts were incubated with anti-FLAG-M2 agarose beads (Sigma-Aldrich), GFP-Trap or RFP-trap agarose beads (ChromoTek, Planegg-Martinsried, Germany) for 2 h at 4°C. Beads were washed three times with GTEN buffer supplemented with 5 mM DTT, 0.5% IGEPAL, and cOmplete protease inhibitor cocktail. Bound proteins were released in 3x SDS sample buffer at 96°C for 10 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membrane were probed with anti-FLAG (Sigma-Aldrich) anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA), anti-mCherry or anti-actin antibodies (Agrisera, Vännäs, Sweden) followed by secondary anti-mouse-horseradish peroxidase antibodies (Sigma-Aldrich). Immunoblots were developed using SuperSignal West substrate (Thermo Scientific, Waltham, MA, USA) and detected with an Azure 400 CCD imager (Azure Biosystem, Dublin, CA, USA).

A. tumefaciens carrying pTRV1 and pTRV2 (Choi et al., 2021; Liu et al., 2002) at OD₆₀₀ 0.5 were co-infiltrated into 2-week-old N. benthamiana leaves. N. benthamiana plants were further grown for 4-5 weeks before the experiments. To design NbRLCK-VIII-6.1/2/3 silencing fragment, coding sequences of SlRLCK-VIII-6 homologs in N. benthamiana were analyzed with the SolGenomics Network VIGS tool (http://vigs.solgenomics.net). Specific primers are listed in Supplementary Table 2.

N. benthamiana leaf disks were collected using a 5 mm biopsy punch. The leaf disks were recovered on 150 μL of distilled water overnight. The next day, the water was replaced with 100 μL of assay solution containing 100 μM luminol (Sigma-Aldrich), 2 μg of horseradish peroxidase (Sigma-Aldrich) and 50 nM of flg22 (Peptron, Daejeon, Korea) as an elicitor. Luminescence was measured in relative light unit (RLU) for 75 min using SYNERGY HTX multi-mode microplate reader (BioTek, Winooski, VT, USA).

RNA was isolated from N. benthamiana or A. thaliana leaf disks collected 10 h after Pst D36E infiltration or 1 h after treatment with water or flg22 peptide (Peptron). Total RNA was extracted using TRI reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Residual genomic DNA was removed by treatment with DNAseI (Sigma-Aldrich). One μg of RNA was used as template for cDNA synthesis using Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR analysis was conducted with GoTaq qPCR Master Mix (Promega, Madison, WI, USA) using a CFX Connect real-time system (Bio-Rad Laboratories, Hercules, CA, USA). Specific primers are listed in Supplementary Table 2.

Fully expanded leaves of 5-week-old A. thaliana plants were infiltrated with Pst DC3000 suspension adjusted at OD₆₀₀ 0.0001. Leaf discs were collected from the infiltrated area at 2 days post-inoculation. The samples were ground in 10 mM MgCl₂ and serial dilutions plated on King’s B medium supplemented with the appropriate antibiotics. Colony-forming units were enumerated after 48 h incubation at 28°C.

All the statistical analyses were conducted with the GraphPad Prism 10 software (GraphPad Software, San Diego, CA, USA) using merged data from at least two or three independent experiments, variance analysis and appropriate post hoc tests as indicated in figure legends.

RipG family effectors collectively contribute to R. solanacearum virulence in host plants (Angot et al., 2006; Remigi et al., 2011; Wang et al., 2016). Although mutant strains lacking individual member of the RipG family are not impaired for growth in planta, further functional characterization of individual effector can be achieved using engineered type III delivery from P. syringae pv tomato DC3000 effectorless strain (Pst D36E) (Sohn et al., 2007; Wei et al., 2015) and heterologous expression in the alternative host N. benthamiana (Jeon et al., 2025; Remigi et al., 2011). We first tested whether RipG6 delivery in leaf cells could affect immune-marker gene expression. Bacterial perception in plants induces a transcriptional reprograming oriented towards defense responses (Navarro et al., 2004; Zipfel et al., 2004). In N. benthamiana leaf, NbACRE132 and NbCYP71D20 expression is rapidly induced by flg22 treatment (Jeon et al., 2020; Segonzac et al., 2011) and was markedly increased 10 h post-infiltration with Pst D36E strain carrying the empty vector, reflecting PAMP detection and activation of PTI (Fig. 1A). Conversely, NbACRE132 and NbCYP71D20 transcript accumulation in tissues infiltrated with Pst D36E (RipG6) strain was not distinguishable from that in tissues infiltrated with the buffer only. As RipG6 protein delivery in plant tissue was confirmed by immunoblotting (Supplementary Fig. 1A), these results indicate that RipG6 delivery in minute amount can suppress PTI. A similar approach using Pst type III secretion system for RipG family effector delivery in A. thaliana also demonstrated that RipG4 alone could suppress PAMP-triggered callose deposition (Remigi et al., 2011) emphasizing that these surrogate delivery assays are relevant to characterize the contribution of individual members of the RipG family to R. solanacearum virulence in plants.

RipG family effectors are proposed to act through the recruitment of host SCF ubiquitin-ligase complexes via their F-box domain interaction with the host adaptor subunit Skp1 (Angot et al., 2006; Dahal et al., 2018). To assess the role of RipG6 F-box domain, we predicted RipG6 structure using AlphaFold3 (Abramson et al., 2024) and generated RipG6^ΔF-box expression construct by deleting 62 residues from Pro84 to Lys145 in RipG6 coding sequence (Supplementary Fig. 2A and 2B). Then, we compared flg22-induced NbACRE132 and NbCYP71D20 expression in N. benthamiana tissues expressing the FLAG-GFP control, RipG6 or RipG6^ΔF-box (Fig. 1B). As observed upon RipG6 delivery by Pst D36E, RipG6 but not RipG6^ΔF-box transient expression significantly reduced the induction of NbCYP71D20 expression 1 h after flg22 treatment. A similar trend was observed for NbACRE132, although this gene induction in RipG6^ΔF-box -expressing tissues did not reach the level observed in the GFP-expressing control tissues. As both proteins accumulated to a similar amount in N. benthamiana tissues (Supplementary Fig. 1B), these results indicate that RipG6 suppression of PTI predominantly depends on the presence of the F-box domain. Among the RipG family, RipG1, G2, G6 and G7 have been confirmed to interact with the N. benthamiana homolog of A. thaliana Skp1 (NbSkp1) through the F-box domain (Dahal et al., 2018). We therefore hypothesized that RipG6 could target a regulator of plant immune signaling pathway for ubiquitin-proteasome mediated degradation, hence impairing immune-related gene expression. Only a couple of chloroplastic proteins have been reported as interactors of RipG2 and RipG7 in yeast two-hybrid assays (Dahal et al., 2018). Considering the sequence and functional divergence of RipG family effectors (Remigi et al., 2011), it is expected that each member could target different host proteins. However, bona fide targets of RipG family effectors remain to be identified (Landry et al., 2020).

Receptor-like cytoplasmic kinases (RLCKs) are essential components of the phospho-relay signal downstream of pattern-recognition receptor activation (Bender and Zipfel, 2023). Members of the RLCK-VII subfamily are targeted and modified by diverse pathogen effectors (Breit-McNally et al., 2022). In a previous work, we screened one-on-one interaction between 127 tomato (Solanum lycopersicum) RLCKs and 40 effectors conserved in R. pseudosolanacearum Korean isolates, including RipG6 (Choi et al., 2026; Prokchorchik et al., 2020; Sakamoto et al., 2012). Among positive interactions, we found that RipG6 could interact with SlRLCK-VIII-6 (Fig. 2A). SlRLCK-VIII-6 structural prediction revealed that the conserved kinase domain is framed by short flexible regions (Supplementary Fig. 2C and 2D). As some RLCK-VIII subfamily members are involved in development or immune signaling pathways (Liang and Zhou, 2018; Schwizer et al., 2017), we further assessed RipG6/SlRLCK-VIII-6 interaction using transient expression system in N. benthamiana. SlRLCK-VIII-6-YFP fusion protein was localized to the cell periphery when co-expressed with free mCherry (Supplementary Fig. 3). RipG6-mCherry fusion protein showed a nucleo-cytoplasmic distribution, which overlapped with SlRLCK-VIII-6 at the periphery in cells expressing both proteins. The proximity between the two proteins was further confirmed using a split-YFP fluorescence complementation assay (Fig. 2B). Both live imaging experiments suggest that RipG6 and SlRLCK-VIII-6 could localize to the same compartment and interact at the periphery of the cell. We also performed co-immunoprecipitation assays with RipG6-mCherry and SlRLCK-VIII-6-YFP, demonstrating that RLCK-VIII-6-YFP was found in complex with RipG6-mCherry but not with FLAG-mCherry control (Fig. 2C). Altogether, these results support the physical association or close proximity of RipG6 with SlRLCK-VIII-6 in plant cells. RipG6 nucleo-cytoplasmic localization correlates with the subcellular localization of the N. benthamiana homolog of Skp1 (Jia et al., 2016) and also places the effector in proximity with cell periphery-localized SlRLCK-VIII-6. SlRLCK-VIII-6/Solyc12g098820 is the most highly expressed member of this subfamily in tomato roots (Supplementary Fig. 4), supporting the relevance of this interaction in natural infection settings. From this, we hypothesized that through interaction with SlRLCK-VIII-6 via the LRR domain and with Skp1 via the F-box domain, RipG6 might promote SlRLCK-VIII-6 ubiquitination and affect SlRLCK-VIII-6 stability.

We tested SlRLCK-VIII-6 protein stability when co-expressed with RipG6, RipG6^ΔF-box or FLAG-mCherry as a control (Fig. 3). SlRLCK-VIII-6-YFP protein accumulation was reduced correlatively with RipG6-mCherry accumulation but stable in presence of FLAG-mCherry or RipG6^F-box-mCherry (Fig. 3A and 3B, middle panels). The same co-expression experiments were repeated independently and SlRLCK-VIII-6 accumulation was quantified after normalization with the anti-actin signal (Fig. 3C). This analysis confirmed that SlRLCK-VIII-6 accumulation was significantly reduced in presence of RipG6, supporting the hypothesis that targets of RipG family effectors can be destabilized, likely through SCF complex-mediated ubiquitination (Angot et al., 2006; Landry et al., 2020). Of note, we observed high molecular weight signals for SlRLCK-VIII-6 in several immunoblots (as in Fig. 2C, top panel), suggesting that SlRLCK-VIII-6 might be polyubiquitinated in presence of RipG6. However, as RipG6 would require a host SCF complex to promote SlRLCK-VIII-6 ubiquitination, further biochemical investigations, including in vitro ubiquitination assays are challenging. Nonetheless, as we could demonstrate the destabilization of SlRLCK-VIII-6 in presence of RipG6 and in an F-box-dependent manner, we propose that SlRLCK-VIII-6 is a relevant biological target of this effector in tomato roots.

As we could not directly investigate SlRLCK-VIII-6 function in tomato roots, due to the difficulties of generating tomato knock-out line and the likely negligeable effect of ripG6 deletion in R. solanacearum (Angot et al., 2006; Remigi et al., 2011), we decided to knock-down or knock-out SlRLCK-VIII-6 homologs in N. benthamiana and A. thaliana (Fig. 4, Supplementary Figs. 5 and 6). The RLCK-VIII subfamily contains 7 and 11 members in tomato and A. thaliana, respectively (Anthony et al., 2006; Sakamoto et al., 2012). Using BLASTp search, we identified 5 proteins with high homology to SlRLCK-VIII-6 in the N. benthamiana genome (Kurotani et al., 2025). The phylogenetic relationship between the corresponding proteins in RLCK-VIII subfamily revealed that SlRLCK-VIII-6 belongs to a monophyletic subgroup comprising A. thaliana CARK5/MAZZA and CARK6, as well as the 5 NbRLCK-VIII-6 homologs, with NbRLCK-VIII-6.1, NbRLCK-VIII-6.2 and NbRLCK-VIII-6.3 being more closely related to SlRLCK-VIII-6 (Supplementary Fig. 5). Considering the high degree of identity (over 98%) between NbRLCK-VIII-6.1, NbRLCK-VIII-6.2 and NbRLCK-VIII-6.3, we generated a silencing construct directed at all three genes and confirmed that their bulk expression was significantly reduced in N. benthamiana plants silenced with the TRV:NbRLCK-VIII-6.1/2/3 construct (Fig. 4A). To investigate the possible role of NbRLCK-VIII-6, we first measured flg22-induced ROS production in NbRLCK-VIII-6.1/2/3-silenced N. benthamiana plants (Fig. 4B and 4C). Although the onset of ROS production was similar to that of control EV-silenced plants, the amplitude and duration of this response were significantly reduced in NbRLCK-VIII-6.1/2/3-silenced plants, suggesting a positive regulatory role for these RLCKs in the signaling downstream of the PAMP perception. A significant reduction of NbACRE132 transcript accumulation was also observed in NbRLCK-VIII-6.1/2/3-silenced plants (Fig. 4D). NbCYP71D20 expression appeared not affected in NbRLCK-VIII-6.1/2/3-silenced plants. As PAMP perception leads to sequential waves of transcription (Bjornson et al., 2021), NbCYP71D20 maximal expression may occur earlier or later than the 1 h timepoint used in our experiment and a potential delay or reduction could not be observed in NbRLCK-VIII-6.1/2/3-silenced plants. Alternatively, NbCYP71D20 regulation may be controlled by distinct but overlapping regulatory pathways, as demonstrated for defense-marker genes in Arabidopsis thaliana (Boudsocq et al., 2020). Nonetheless, these experiments clearly indicate that SlRLCK-VIII-6 homologs in N. benthamiana contribute to early PTI signaling. Further, as two other SlRLCK-VIII, Pti1a and Pti1b (SlRLCK-VIII-2 and SlRLCK-VIII-7 in Supplementary Fig. 5), have previously been reported as positive regulators of flg22-triggered ROS production (Schwizer et al., 2017), our results highlight the conservation of a robust regulatory role among the members of this RLCK subfamily, although their connection with the RLCK-VII network downstream of PRRs remains to be explored.

In parallel, we investigated the possible involvement in immune signaling of the closest SlRLCK-VIII-6 homolog in A. thaliana, identified as At2g43230/CARK6 by the phylogenetic analysis (Supplementary Fig. 5). We selected homozygous individuals of the T-DNA insertion line SALK_203094/cark6-1 (Wang et al., 2019) and confirmed the lack of CARK6 transcript accumulation in these plants (Supplementary Fig. 6). We then tested the multiplication in planta of the virulent Pst DC3000 strain in wild-type (Col-0), cark6-1 and the PTI-deficient fls2 efr cerk1 plants (Gimenez-Ibanez et al., 2009) (Fig. 4E). Two days after inoculation, bacterial count were significantly higher in cark6-1 and fls2 efr cerk1 mutants than in wild-type plants. In accordance with our interpretation of the PTI assays in the NbRLCK-VIII-6.1/2/3 silenced plants, the enhanced susceptibility to bacterial infection of cark6-1 mutant is indicative of a positive contribution of CARK6 to immune signaling.

Because of the high sequence homology (>90%) between SlRLCK-VIII-6 and its homologs in N. benthamiana and A. thaliana, it is conceivable that SlRLCK-VIII-6 also contribute as a positive regulator of immunity signaling in tomato. Although we could not directly address the possible redundancy between NbRLCK-VIII-6 homologs in our assays, other researchers have reported the redundant involvement of CARK6 and CARK5/MAZZA in the negative regulation of flg22-induced ROS production (Gonçalves Dias et al., 2025). However, the redundancy with CARK6 is not observed for MAZZA regulation of the CLV1-signaling pathway (Blümke et al., 2021) and MAZZA is not involved in CARK6 regulation of ABA-signaling (Wang et al., 2019). Additionally, several RLCK-VIII/CARK members were reported to form homo and hetero-dimer in planta (Blümke et al., 2021; Gonçalves Dias et al., 2025; Li et al., 2022), adding another layer of complexity to the functional characterization of these proteins. Nonetheless, our experimental evidence suggests that RLCKVIII-6 may play a conserved positive regulatory role on immune signaling in plants and highlights their relevance as targets of a bacterial F-box containing effector.