SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex

SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex Xuexi Dou, Daiki Matsumoto, Ryutaro Tao, Soichiro Nishiyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9513049/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In Prunus , self-incompatibility (SI) is controlled by S-RNases and pollen-expressed F-box proteins, whereas the molecular processes governing S-RNase regulation in pollen remain incompletely understood. Here, we characterized PavSDJ, a novel pollen protein from sweet cherry ( Prunus avium ), as a candidate factor involved in pollen-side S-RNase-associated processes. Sequence and structural analyses identified PavSDJ as a type III J-protein. Phylogenetic analyses placed PavSDJ within a distinct SDJ-like sublineage of the type III J-protein group, separate from a closely related sister lineage. Consistent with this divergence, PavSDJ was strongly expressed in anthers and pollen, whereas its sister gene was broadly expressed across organs. Transient expression assays indicated that PavSDJ localized predominantly to the cytosol. Biochemical analyses showed that PavSDJ associated with recombinant PavS-RNases in pollen extracts and in reconstituted pull-down assays, without obvious allele preference. Proteomic analysis of PavSDJ co-immunoprecipitates from pollen extracts identified a complex including PavSLFL6 and PavSSK1. Reconstitution assays further showed that PavSDJ promoted the co-precipitation of PavSLFL6 with S-RNase. These findings identify PavSDJ as a candidate pollen-side factor in the Prunus SI pathway and provide evidence that a specialized J-protein may contribute to SI-related protein complex assembly. J-protein Prunus avium self-incompatibility SLFL6 S-RNase pollen Figures Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Key Message PavSDJ is a pollen-expressed type III J-protein that binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex in Prunus . It may function as a general modifier involved in the GSI system of Prunus. Introduction Self-incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilization and inbreeding, thereby promoting outcrossing and helping maintain genetic diversity in flowering plants (McCubbin and Kao 2000; Gibbs 2014). Among homomorphic SI systems, S-RNase-based gametophytic self-incompatibility (GSI) has been widely studied in several eudicot lineages, including the Solanaceae and the Rosaceae. This system is characterized by the uptake of stylar S-RNases into pollen tubes, where these cytotoxic ribonucleases inhibit incompatible pollen tube growth(Kao and Tsukamoto 2004; Tao and Iezzoni 2010; Zhang et al. 2024). Although the S-locus determinants define allele specificity, accumulating evidence across species indicates that S-RNase-based GSI relies on additional accessory factors that modulate the intracellular fate and activity of S-RNase after pollen–pistil contact (McClure et al. 2011). In Prunus (Rosaceae), the S-RNase–based GSI system is controlled by a single multiallelic S locus encoding the pistil determinant S-RNase and the pollen determinant S haplotype-specific F-box protein (SFB) (Kao and Tsukamoto 2004; Tao and Iezzoni 2010). A characteristic of Prunus SI is its “self-recognition” system, a single pollen-expressed SFB allele is genetically linked to and recognizes its cognate stylar S-RNase allele. This leads to self-pollen rejection in a one-to-one manner (Entani et al. 2003; Tao and Iezzoni 2010). This model contrasts with the “collaborative non-self recognition” system described in the Solanaceae and in the tribe Maleae (e.g., Malus and Pyrus ) of the subfamily Amygdaloideae of the Rosaceae, where multiple pollen F-box proteins, including SLFs in the Solanaceae and SFBBs in Maleae, collectively recognize and detoxify non-self S-RNases (Sassa et al., 2010). Although the concepts of self- and non-self recognition provide a framework for understanding haplotype-specific pollen–pistil interactions in Prunus , they do not fully explain how S-RNase is handled after entering the pollen tube. In particular, it remains unclear which pollen-side factors and protein complexes regulate its stability, localization, accessibility, and interaction with ubiquitin-related machinery. Indeed, studies in several S-RNase–based GSI systems have highlighted that non- S -locus factors can shape critical steps of the SI pathway, including S-RNase binding, compartmentalization, redox regulation, and protein degradation (McClure et al. 2011). In the Solanaceae, for example, biochemical, genetic and cell biological studies have supported that the pollen-side response depends not only on the S locus F-box proteins, SLFs, but also on non-S factors including SSK1 and CUL1, in the SCF SLF uniquitin ligase machinery (Williams et al. 2015; Sun et al. 2018). In addition, its stylar factors such as 120K, NaTrxh, NaStEP, and HT-B have been reported to contribute to S-RNase activity and intracellular dynamics, including compartmentalization and pollen tube rejection outcomes (Cruz-Garcia et al. 2005; Hancock et al. 2005; Goldraij et al. 2006; Jiménez-Durán et al. 2013; Torres-Rodríguez et al. 2020; Cruz-Zamora et al. 2020). In Maleae, several non- S components have been associated with SCF complex assembly or with S-RNase regulation within pollen tubes (Xu et al. 2013; Minamikawa et al. 2014; Meng et al. 2014; Li et al. 2018). By comparison, the network of accessory factors in Prunus remains less comprehensively defined, despite the availability of core SCF-related components such as SSK1, SBP1, and CUL1 that interact with pollen F-box proteins (Zeng et al. 2019; Matsumoto et al. 2012, 2016). Moreover, the Prunus S locus is structurally simpler than the corresponding loci in the Solanaceae and Maleae, comprising a compact region with S-RNase and single SFB genes rather than multiple F-boxes (Matsumoto and Tao 2016; Claessen et al. 2019; Wu et al. 2020). Its flanking SLFL genes, which are orthologous to the pollen determinant F-box genes in Solanaceae and Maleae, are located near but outside the S locus and have been proposed to function as general detoxification factors rather than haplotype-specific determinants (Akagi et al., 2016; Matsumoto and Tao 2016). Thus, the compact S locus architecture of Prunus may mainly dedicate to the allele-specific recognition, whereas the downstream processes mediated by S-RNase likely may require additional non- S locus proteins for their assembly, stabilization or regulation. Among the candidate pollen-side factors, SLFL-related proteins have also been proposed to participate in S-RNase-associated processes, although the roles of individual members remain unresolved in Prunus . Consistent with this view, a Prunus -specific M locus factor, encoded by MGST at chromosome 3, distinct from the S locus on chromosome 6, had been reported as an essential factor to SI behavior in multiple Prunus species (Akagi et al. 2016; Ono et al. 2018; Ono et al. 2022). In addition, PavAct1 has been suggested to be related to target protein in S-RNase cytotoxicity (Matsumoto and Tao 2012). Together, these findings suggest that the outcome of SI in Prunus is influenced by additional factors at loci beyond the S locus with canonical S determinants. In our previous work to comprehensively identify pollen proteins that bound to silkworm-expressed recombinant S-RNase in sweet cherry ( Prunus avium ), pollen-expressed DnaJ-like protein was isolated (S-RNase–binding DnaJ-like protein, SDJ; Matsumoto and Tao 2019). This finding suggested a potentially new connection between S-RNase regulation and molecular chaperone-like modules. SDJ belongs to the large and diversified family of J-domain proteins (DnaJ/Hsp40), in which members typically function as regulators of protein interaction, folding, or complex assembly (Craig et al. 2006; Kampinga and Craig 2010). Canonical DnaJ-like proteins are commonly classified into three types ( Kampinga and Craig 2010), the type III J-proteins are the most structurally diverse, sharing only the J-domain with Types I and II J-proteins, and in many cases, this domain is also relatively divergent (Hennessy et al. 2000; Ajit Tamadaddi and Sahi 2016). Accordingly, they have frequently evolved lineage- and pathway-specific roles than the canonical housekeeping roles of modulating Hsp70 chaperone function (Hennessy et al. 2000; Ajit Tamadaddi and Sahi 2016). These facts raise the possibility that SDJ may act as a pathway-specialized, adaptor- or chaperone-like factor that contributes to the assembly or stabilization of an S-RNase–associated protein complex in Prunus pollen. In this study, we characterized PavSDJ from sweet cherry using sequence/phylogenetic analyses, expression profiling, and subcellular localization. We also examined its biochemical interactions with S-RNase and pollen proteins, and discussed its role in an S-RNase–associated protein complex in Prunus pollen. Materials and Methods Plant materials Sweet cherry ( Prunus avium ) ‘Satonishiki’ ( S haplotypes: S 3 S 6 ) was used in this study. Anthers were collected from flower buds just before anthesis and then dehisced by overnight incubation in the presence of silica gel desiccant. Pollen was collected by filtering through a 100-μm nylon mesh and stored with silica gel desiccant at −80°C. Other floral organs from flower buds and young leaves were also collected during spring, frozen in liquid nitrogen and stored at -80°C. Quantitative RT-PCR Total RNA was extracted by the cold phenol method as described previously (Tao et al. 1999). First-strand cDNA was synthesized from total RNA using a PrimeScript ® RT reagent kit with gDNA Eraser (Takara Bio, Otsu, Japan). Quantitative RT-PCR was conducted using gene-specific primers for PavSDJ (XM_021972310.1; Fw: 5’-CGAAGGCCAGACAAGAGCTT-3’, Rv: 5’-AAAGTCCTCAGGCTCCATCATG-3’) and PavSDJ-sister (XM_021972277.1; Fw: 5’-TCACGTGTCTGCCTAGCTG-3’, Rv: 5’-ACGCAGAATCTATCTTGGTGTCA-3’). Reactions were carried out using SYBR® Premix Ex Taq™ (Takara Bio) on a LightCycler® Nano system (Roche Diagnostics, Basel, Switzerland) under the following cycling conditions: a denaturation step at 95°C for 30 s, followed by 45 cycles of 95°C for 5 s and 60°C for 20 s. Gene-specific amplifications were verified by melting-curve analysis. A sweet cherry ubiquitin homolog, PavUBQ (Fw: 5’- TGATCCTTGTGGTTCCATCC-3’, Rv: 5’- CATCCATCAGCCAAGTACGA-3’) was used as an internal control. Two technical replicates were analyzed for each of three biological replicates. Sequence analysis of SDJ Protein sequence of SDJ was obtained from National Center for Biotechnology Information (NCBI) database (RefSeq accession XP_021828002.1; corresponding UniProtKB accession A0A6P5TLJ7). Conserved domains were annotated using InterProScan and NCBI-CD-search. The presence and integrity of the J-domain were assessed by identifying the canonical HPD motif and surrounding conserved residues within the predicted J-domain region. Additional sequence features were analyzed based on the primary amino acid sequence. The hydropathy profile of PavSDJ was calculated using the Kyte–Doolittle algorithm with a sliding window of 9 residues, in order to assess the distribution of hydrophobic regions along the protein sequence. The predicted three-dimensional structural model of SDJ was retrieved from the AlphaFold Protein Structure Database (AlphaFold DB) based on its sequence. Model confidence was evaluated using the predicted local distance difference test (pLDDT) score, and regions with pLDDT values >70 were considered to be predicted with relatively high confidence (Jumper et al. 2021; Varadi et al. 2022). Structural visualization and figure preparation were performed using PyMOL (Version 2.1, Schrödinger, LLC). Phylogenetic analysis To determine the family assignment of PavSDJ, representative type I, type II, and type III J-proteins from Arabidopsis thaliana (listed in Supplementary Table 1) were analyzed together with PavSDJ (Rajan and Silva 2009; Pulido and Leister 2018). To further examine the evolutionary relationships of PavSDJ-like proteins, homologous sequences were retrieved by BLASTP search using PavSDJ as the query against the Phytozome v13 database (https://phytozome-next.jgi.doe.gov/), with an E-value cutoff of 1e −25 . Retrieved sequences included homologs from peach ( Prunus persica ), strawberry ( Fragaria vesca ), apple ( Malus domestica ), soybean ( Glycine max ), poplar ( Populus trichocarpa ), grapevine ( Vitis vinifera ), cucumber ( Cucumis sativus ), Arabidopsis thaliana , Aquilegia coerulea , maize ( Zea mays ), and rice ( Oryza sativa ). Protein sequences were aligned using MUSCLE implemented in MEGA version 6.0 (Tamura et al. 2013), and phylogenetic trees were constructed using the Maximum Likelihood (ML) method or Neighbor-Joining (NJ) method in IQ-TREE and MEGA with 1,000 bootstrap replicates. Pollen protein extraction Pollen was cultured in a pollen germination medium [PGM; 50 mM MES-NaOH (pH 6.5), 3 mM Ca(NO 3 ) 2 , 0.8 mM MgSO 4 , 1 mM KNO 3 , 1.6 mM H 3 BO 3 , 15% polyethylene glycol 4000, and 300 mM sucrose] for 3 h at 20°C (Hiratsuka et al. 2001). Germinated pollen was collected by centrifugation at 500 × g for 5 min, and homogenized in the immunoprecipitation (IP) buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.3% (v/v) Triton X-100] containing 1 × cOmplete™ Protease Inhibitor Cocktail (Roche Diagnosis, Mannheim, Germany). After centrifugation at 18,000 × g for 15 min, the supernatant was filtered through a 0.45-μm cellulose acetate membrane (Advantec, Tokyo, Japan). Protein concentration was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. Co-immunoprecipitation of PavSDJ from pollen extract Four microliters of Pierce™ Protein A/G Magnetic Beads (Thermo Fisher, Waltham, MA, USA) were incubated with 6 μg of anti-PavSDJ antibody for 30 min at room temperature in IP buffer. The anti-PavSDJ antibody was raised against the synthetic peptide SQDYWIHTDTT (amino acids 93–103) and affinity-purified using the antigen peptide. After one wash with IP buffer, 5 mg of crude pollen extract was added to the beads, and the mixture was incubated at 4°C for 2 h. The beads were then washed five times with IP buffer, and used for the recovery of innate PavSDJ complex or mass spectrometric analysis. As a parallel antibody control, purified anti-PavS6-RNase antibody was used in the same co-immunoprecipitation procedure. Because S-RNase is a pistil-expressed determinant and no specific endogenous PavS6-RNase antigen was expected to be present in the mature pollen extracts under our assay conditions, this control was used to estimate nonspecific proteins recovered through the antibody/IP procedure. For the recovery of innate PavSDJ complex, the beads were incubated with 1 mg mL⁻¹ antigen epitope in 50 mM tris-HCl (pH 7.5) and 150 mM NaCl for 20min for three times, and eluted proteins were diafiltered with Milli-Q water using Amicon Ultra 0.5 centrifugal filters (Millipore, Billerica, MA, USA). For proteomic analysis, bead-bound proteins were subjected to nanoLC-MS/MS analysis by Kazusa Genome Technologies (Kisarazu, Japan). Each sample was digested with trypsin and analyzed using an UltiMate 3000 RSLCnano LC system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Data were analyzed using Scaffold DIA (Proteome Software Inc., Portland, OR, USA) against the database of Prunus avium UniProtKB/Swiss-Prot database (UP000515124). Transient recombinant protein expression in tobacco leaves cMyc-tagged PavSDJ, cMyc-tagged PavSDJ-sister and HA-tagged S-locus F-box like 6 (PavSLFL6) was expressed in Nicotiana benthamiana leaves using the agroinfiltration method as described previously, with slight modification (Matsumoto and Tao 2016). The coding sequences (CDSs) of PavSDJ and PavSDJ-sister were amplified from ‘Satonishiki’ pollen cDNA using PrimeSTAR ® HS DNA Polymerase (Takara Bio) and further fused with cMyc tag sequence at their N-terminus by PCR. The resulting PCR products were cloned into the pGWB2 vector (Nakagawa et al. 2007) using an In-Fusion HD cloning kit (Takara Bio). The previously constructed pGWB2 constructs cloning HA-PavSLFL6 and SFB-interacting Skp1 like 1 (PavSSK1), whose co-expression enhances accumulation of PavSLFL protein, were also used in this study (Matsumoto and Tao 2016, 2019). Agrobacterium tumefaciens strain LBA4404 (Takara Bio) was transformed with either of the pGWB2 constructs or pBIN61 turnip crinkle virus coat protein ( TCVCP ). Transformed Agrobacterium strains were cultured separately in LB medium to OD 600 0.3–0.5 and resuspended in an MS medium containing 100 μM acetosyrigone to a final OD 600 of 2.0. After an Agrobacterium strain transformed with pGWB2 construct and that transformed with pBIN61 were mixed equally, two to three leaves of N. benthamiana plants at the six-leaf stage were agroinfiltrated. Plants were then grown for 4–5 days at 18°C. Agroinfiltrated leaves were homogenized with a pestle in the immunoprecipitation (IP) buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM 2-mercaptoethanol, 0.3% (v/v) Triton X-100] containing 1 × cOmplete™ Protease Inhibitor Cocktail (Roche Diagnosis, Mannheim, Germany). The protein concentration was determined as described above. Co-immunoprecipitation using recombinant PavS-RNases The recombinant PavS 3 -, S 4 -, S 6 -RNase tagged with DDDDK at the N-terminus were expressed with the silkworm-baculovirus system (Sysmex, Kobe, Japan) and purified using a DDDDK-tagged protein purification kit (MBL & Biological Laboratories, Nagoya, Japan), as described previously (Matsumoto and Tao 2019). For pull-down assays, 1 mg crude protein extract from pollen or agroinfiltrated tobacco leaves was incubated with 1 μg recombinant S-RNase and 10 μl DDDDK-tagged protein purification gel for 2 h at 4°C. After four washes with IP buffer, bead-bound proteins were eluted with a Laemmli SDS sample buffer [0.2 M tris-HCl (pH 6.8), 2% (w/v) SDS, 0.85 M 2-mercaptoethanol, and 10% (v/v) glycerol]. SDS-PAGE and protein detection Protein samples were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were visualized by silver staining using Sil-Best Stain One (Nacalai Tesque, Kyoto, Japan) or blotted onto PVDF membranes (Millipore) for immunoblot analysis. Anti-DDDDK mouse monoclonal antibodies (MBL & Biological Laboratories), anti-HA mouse monoclonal antibodies (TANA2, MBL & Biological Laboratories), anti-Myc rabbit ployclonal antibodies (MBL & Biological Laboratories) and antiserum against PavSDJ were used as the primary antibodies. Immune complexes were detected using an ECL Advance Western Blotting Detection Kit (GE Healthcare) and the LAS-500 system (Cytiva, Tokyo, Japan). Exposure time in detection was adjusted according to the signal intensity of the target protein. Subcellular localization analysis A PavSDJ PCR fragment was fused GFP at its C-terminus and cloned into the pGWB2 vector using an In-Fusion HD cloning kit (Takara Bio). The resulting pGWB2-PavSDJ-GFP was introduced into Agorbacterium strain EHA105. Agroinfiltration against N. benthamiana leaves was conducted as described above. Three days after inoculation, leaf discs (approximately 5 mm square) were immersed in 1 mg L⁻¹ Propidium Iodide (PI) solution for 10 min. Subcellular localization (GFP fluorescence) of PavSDJ-GFP was observed using a FLUOVIEW FV3000 confocal laser microscope system (Olympus, Japan). The transient expression assay was repeated more than three times to confirm the protein localization. Results Sequence features and structural characteristics of PavSDJ The PavSDJ protein from Prunus avium (XP_021828002.1) is predicted to be a 355-amino-acid protein with a molecular mass of approximately 42 kDa. Its encoding gene is located on chromosome 8, distinct from the S locus on chromosome 6 and from MGST on chromosome 3. Domain annotation identified a conserved J-domain at the N-terminus (residues 24–84), including the canonical HPD (His-Pro-Asp) motif at positions 51–53, a hallmark of J-domain proteins that can stimulate Hsp70 ATPase activity (Fig 1a; Hennessy et al. 2000; Kampinga and Craig 2010). PavSDJ lacks the glycine/phenylalanine (G/F)-rich region and the cysteine-rich zinc-finger motifs typical of type I/II J-proteins, supporting its classification as a type III J-protein with a canonical J-domain. Structural analysis of the C-terminal region revealed features consistent with a potential client-interaction surface. Two clusters of hydrophobic residues were identified between positions 286 and 328 (Fig 1b). Secondary structure predictions combined with AlphaFold suggested the N-terminal region contains a canonical J-domain with the conserved HPD motif, consistent with a four-helix bundle, and the C-terminal region shows a structurally distinct segment with an extended terminal segment, which may contribute to an interaction-related surface (Fig 1c) (Dyson et al. 2006; Marinko et al. 2019). Evolutionary p osition of PavSDJ To place PavSDJ within the plant J-protein repertoire, we first compared PavSDJ with representative Arabidopsis thaliana type I, type II, and type III J-proteins. In this broader classification tree (Supplementary Fig 1), PavSDJ clustered within the type III J-protein assemblage, in proximity to the Arabidopsis type III reference DNAJC75 (NP_192673.1), supporting its assignment as a type III J-protein. We next examined the relationships among PavSDJ-like proteins in a focused phylogenetic analysis. Notably, the retrieved SDJ-like sequences were resolved into three major clades (Clades I–III), all of which belong to the type III J-protein/SDJ-like group rather than corresponding to the three canonical J-protein families (Fig 2; Supplementary Fig 2). Within this framework, PavSDJ was placed in Clade III, where it grouped with the peach homolog Prupe.8G091400.1 and the strawberry homolog Fvesca mrna21834.1. A closely related but distinct neighboring branch within the same clade contained Prupe.8G091300.1, Fvesca mrna21835.1, and apple homologs, and was therefore designated as the SDJ sister gene group. Thus, SDJ occupies a distinct sublineage within Clade III, whereas Malus/Pyrus -related homologs were associated more closely with the sister branch than with the PavSDJ branch itself. Together, these phylogenetic patterns are consistent with divergence between PavSDJ and its sister lineage. Expression patterns and divergence from the SDJ sister gene To examine whether PavSDJ and its sister gene differ in expression and biochemical behavior, we compared their transcript abundance across tissues and tested their interaction with recombinant S-RNase. qRT-PCR showed that PavSDJ transcripts accumulated predominantly in anther and pollen, with little to no detectable expression in vegetative organs (leaf) and most floral tissues (sepal, petal, ovary, pistil) (Fig 3a). In contrast, the PavSDJ sister gene displayed broad expression across organs, with relatively higher transcript levels in non-reproductive tissues (Fig 3a). Consistent with these divergent expression profiles, PavSDJ and its sister gene differed in S-RNase association in a recombinant co-immunoprecipitation assay. DDDDK-tagged PavS6-RNase precipitated cMyc-tagged PavSDJ, whereas the cMyc-tagged sister protein was not detectably co-precipitated under the same conditions (Fig 3b). Recovery of the bait protein was confirmed by anti-DDDDK immunoblotting. These results support functional divergence between PavSDJ and its sister gene, with PavSDJ displaying a pollen-enriched expression pattern and S-RNase association. Subcellular localization of PavSDJ Transient expression of PavSDJ–GFP in Nicotiana benthamiana epidermal cells revealed GFP fluorescence distributed throughout the cytoplasm, with no clear enrichment at the cell wall (Fig 4). The cytosolic distribution is consistent with the expectation that PavSDJ could access cytoplasmic protein interaction partners. While this heterologous system does not directly report localization in pollen tubes, the observed cytosolic distribution complements the pollen-enriched expression pattern and is compatible with a role in intracellular S-RNase-associated processes. Proteomic profiling of PavSDJ co-immunoprecipitates identifies an SLFL6/SSK1-associated module To identify proteins associated with endogenous PavSDJ in pollen, we performed co-immunoprecipitation (co-IP) from ‘Satonishiki’ pollen extracts using anti-PavSDJ polyclonal antibody, in parallel, purified anti-PavS6-RNase antibody was used as a antibody control, as no specific endogenous S-RNase target was expected in the pollen extracts under our assay conditions. Silver staining of the co-IP eluates revealed a prominent band at ~42 kDa in the anti-PavSDJ pull-down, consistent with the predicted molecular weight of PavSDJ, together with additional co-precipitated or non-specific bands (Fig 5). The control pull-down showed a distinct banding pattern, indicating that the two antibodies recovered different protein populations under the tested conditions. Table 1 Top 15 proteins identified in MS/MS analysis of the co-IP using anti-SDJ pAb against the pollen extract of 'Satonishiki' Proteins preferentially identidfied in co-IP 1 Accessions Molecular mass (kDa) Peak intensities in co-IP Peak intensities in control 2 Enrichment ratio PavSDJ1 A0A6P5TLJ7 41 2.3.E+10 6.6.E+07 342.8 PavSLFL6 A0A6P5SY21 49 6.9.E+09 6.9.E+06 1004.5 PavSSK1 A0A6P5RK47 21 5.5.E+09 7.6.E+07 71.9 60S acidic ribosomal protein P2-2-like isoform X1 A0A6P5S8S2 12 2.4.E+09 1.6.E+08 15.4 60S ribosomal protein L13a-4 A0A6P5RWM2 24 1.5.E+09 1.4.E+08 10.9 60S ribosomal protein L12 A0A6P5TU86 18 1.3.E+09 1.1.E+08 12.3 60S ribosomal protein L10-like A0A6P5RWC6 25 1.2.E+09 1.2.E+08 10.4 60S ribosomal protein L7-2 A0A6P5RV38 28 1.2.E+09 8.4.E+07 14.5 60S acidic ribosomal protein P0 A0A6P5RIH1 34 1.2.E+09 8.3.E+07 13.8 60S ribosomal protein L3 A0A6P5RYC9 44 1.1.E+09 9.3.E+07 11.4 60S ribosomal protein L22-3 A0A6P5RJF4 14 1.0.E+09 6.2.E+07 16.7 60S ribosomal protein L34-like A0A6P5SV33 14 1.0.E+09 1.0.E+08 9.8 50S ribosomal protein L23, chloroplastic A0A6P5SJW1 18 9.0.E+08 1.3.E+08 7.1 60S ribosomal protein L4-like A0A6P5REB2 45 8.8.E+08 6.9.E+07 12.8 60S ribosomal protein L14-2-like A0A6P5RNY9 15 8.5.E+08 8.1.E+07 10.5 Total intensities 9.7.E+10 1.2.E+11 1 Proteins with a 4-fold or greater increase in signal intensity by co-IP using anti-PavSDJ antibody are shown. 2 Purified anti-PavS6-RNase rabbit antibody was used in control. Precipitated proteins detected by LC–MS/MS were quantified by protein peak intensities, and enrichment ratios were calculated as the ratio of peak intensity in the anti-PavSDJ co-IP to that in the control IP (Table 1). Proteins showing a ≥4-fold higher peak intensity in the anti-PavSDJ co-IP relative to the control are listed in Table 1. Among these proteins, PavSDJ was strongly enriched (enrichment ratio 342.8), supporting efficient recovery of the bait protein. Notably, PavSLFL6 and PavSSK1 were among the most enriched non-bait proteins (enrichment ratios of 1004.5 and 71.9, respectively), and no other SLFL family members were detected other than PavSLFL6 under our experimental conditions. In addition, multiple ribosomal proteins displayed moderate enrichment (enrichment ratios ~7.1–16.7) (Table 1). These results support the view that PavSDJ is associated with an SLFL6/SSK1-containing module in pollen, suggesting a possible role in the organization of S-RNase-associated protein complexes. PavSDJ associates with recombinant S-RNases in pollen extracts and promotes co-precipitation of PavSLFL6 with S-RNase To test whether PavSDJ associates with S-RNase in the non-allelic specific manner, pollen protein extracts were incubated with recombinant DDDDK-tagged PavS3-, PavS4-, or PavS6-RNase, followed by pull-down with anti-DDDDK beads and immunoblot detection. PavSDJ was recovered in the DDDDK pull-downs for all three S-RNase alleles (Fig 6a, anti-PavSDJ), signal bands are of the same size with the pollen extract input control. These data support that PavSDJ can associate with multiple S-RNase alleles. To further confirm the PavSDJ containing protein complex binds to S-RNase directly, we prepared PavSDJ-containing eluates by anti-PavSDJ immunoprecipitation from pollen extracts and incubated them with recombinant DDDDK-PavS6-RNase prior to anti-DDDDK pull-down. PavSDJ was detected in the DDDDK pull-down when DDDDK-PavS6-RNase was included (Fig 6b), supporting that PavSDJ present in the PavSDJ-enriched fraction remains competent for S-RNase association. Together, the pollen-extract and PavSDJ-enriched assays support that PavSDJ associates with S-RNase in a pollen-derived biochemical context. We next examined whether PavSDJ influences the association of PavSLFL6 with S-RNase in a reconstituted pull-down assay. DDDDK-PavS6-RNase was used as a bait, cMyc-tagged PavSDJ was stably recovered in the pull-down both in the presence and absence of HA-tagged PavSLFL6 (Fig 6c, anti-cMyc). In contrast, HA-PavSLFL6 alone showed little to no recovery with PavS6-RNase, whereas clear co-precipitation of HA-PavSLFL6 was observed when PavSDJ was included (Fig 6c, anti-HA). Bait recovery was confirmed by anti-DDDDK immunoblotting. These results indicate that PavSDJ is sufficient for detectable association with PavS6-RNase in this assay and that PavSDJ promotes the co-precipitation of PavSLFL6 with S-RNase. Discussion In this study, we characterize PavSDJ as a pollen-expressed cytosolic type III J-protein in sweet cherry and provide biochemical evidence linking PavSDJ to S-RNase association and to an SLFL6/SSK1-containing pollen interaction module. By integrating sequence/phylogenetic analyses, expression profiling, co-immunoprecipitation coupled with mass spectrometry, and recombinant pull-down assays, we establish a framework in which PavSDJ serves as an S-RNase–binding factor that can facilitate recruitment of PavSLFL6 to an S-RNase–containing complex. While the present data do not directly demonstrate a causal role in SI outcomes, they nominate PavSDJ as a candidate pollen-side component relevant to S-RNase regulation and provide a mechanistic hypothesis that can guide future functional tests. 4.1 PavSDJ combines a conserved J-domain with a divergent C-terminal region that may support specialized interactions PavSDJ was appeared to contain a conserved N-terminal J-domain with the canonical HPD motif, consistent with type III J-protein classification. Unlike type I/II J-proteins, PavSDJ lacks the G/F-rich and zinc-finger regions, suggesting a streamlined architecture that may favor pathway-specific protein interactions. The C-terminal region of PavSDJ contains hydrophobic clusters and a short C-terminal motif that may contribute to client binding or partner recruitment, although these hypotheses require further experimental validation. Such features are consistent with the broader theme that type III J-proteins frequently evolve specialized interaction modules beyond canonical Hsp70 co-chaperone functions (Ajit Tamadaddi and Sahi 2016). 4.2 PavSDJ associates with multiple PavS-RNase alleles without strong allele preference, consistent with a general S-RNase–binding factor in pollen One of the key findings of this study is that PavSDJ was repeatedly consistently recovered in pull-down assays from pollen extracts of sweet cherry ‘Satonishiki’, using multiple recombinant S-RNase alleles ( S 3 , S 4 , and S 6 ) as baits (Fig 6a). Notably, S 3 and S 6 correspond to the S haplotypes of ‘Satonishiki’, whereas S 4 represents a non-self (foreign) S haplotype. Under our assay conditions, PavSDJ co-precipitated with all three S-RNases in an S haplotype-independet manner. This interpretation is further supported by the reconstitution assays using recombinant proteins Specifically, cMyc–PavSDJ was robustly pulled down by recombinant PavS6-RNase, whereas its recombinant sister protein (cMyc–PavSDJ-sister) was not detectably recovered under the same conditions (Fig 3b, 6c). Together, the lack of a strong allele-specific preference, combined with the specificity difference between PavSDJ and its sister protein suggest that PavSDJ is likely to be a general factor that engages S-RNase after its entry into the pollen tube. This is consistent with its pollen-enriched expression and cytosolic localization. 4.3 PavSDJ promotes recruitment of PavSLFL6 to S-RNase and links to an SLFL6/SSK1 module A central finding of this study is that PavSDJ promoted co-precipitation of PavSLFL6 with S-RNase in a reconstituted assay: SLFL6 was not recovered with S-RNase alone but was robustly recovered when PavSDJ was included. Together with the co-IP/MS enrichment of SLFL6 and SSK1 in anti-PavSDJ co-IP/MS analyses, these results support the association of PavSDJ with an SLFL6/SSK1-containing module. Because SSK1 is a Skp1-like component that interacts with pollen F-box proteins in Prunus (Zeng et al. 2019; Matsumoto et al. 2016), this network is compatible with a model in which PavSDJ facilitates the assembly or stabilization of S-RNase–associated complexes that include F-box–related partners. In the broader context of S-RNase-based GSI, pollen tubes are likely to require accessory factors that influence S-RNase stability, accessibility, localization, or interaction competence after S-RNase entry. From this perspective, PavSDJ may act as an adaptor- or chaperone-like general binder that stabilizes S-RNase and/or facilitates its productive association with downstream pollen proteins, rather than conferring allele specificity per se. In addition, multiple ribosomal proteins showed moderate enrichment in the anti-PavSDJ co-IP. Given their high cellular abundance and frequent recovery in proteomic datasets, these proteins may in part represent common co-purifying components under the present experimental conditions. However, their detection is also noteworthy from a broader perspective of the J protein concept. For example, specialized cytosolic J-proteins such as Zuo1 and Jjj1 associate with 60S ribosomal particles and function in nascent polypeptide regulation or ribosome biogenesis (Kaschner et al. 2015; Hong et al. 2014). The enrichment of ribosomal proteins raises the possibility that PavSDJ operates in a ribosome-proximal chaperone environment. Previous studies proposed Prunus SLFL proteins as candidate general inhibitors because of their interactions with tested S-RNases (Matsumoto et al. 2012, 2019), however, their S-RNase-binding behavior may not be uniformly aligned. Matsumoto et al (2016) proposed that, PavSLFL1 bound only PavS3-RNase, PavSLFL2 bound all four tested PavS-RNases (PavS1-, PavS3-, PavS4-, and PavS6-RNase), and PavSLFL3 showed no detectable binding to any of the tested S-RNases, and in the subsequent study of Matsumoto et al (2019), PavSLFL6 was further identified but did not show detectable binding to the four tested PavS-RNases. This study demonstrated that PavSDJ promoted the binding of PavSLFL6 to S-RNase. Against this background, rather than indicating that PavSLFL6 is itself a strong direct binder of S-RNase, our results suggest that PavSLFL6 may not be a strong direct binder of S-RNase on its own. Instead, they are more consistent with a model in which PavSDJ facilitates or stabilizes the assembly of PavSLFL6 into an S-RNase-associated complex. Consistent with this view, we observed that PavSDJ exhibits broad binding affinity toward various S-RNases, rather than showing a preference for specific alleles. However, our current data are not yet sufficient to elucidate the specific biochemical events following S-RNase binding mediated by PavSDJ. Mechanistically, PavSDJ may provide an additional interaction surface for SLFL6, alter the conformation or accessibility of S-RNase, or stabilize protein assemblies by preventing protein aggregation. These possibilities are consistent with known modes of action for specialized J-proteins (Kampinga and Craig 2010), but remain to be tested in vivo. 4.4 Divergence from the SDJ sister gene supports pollen-enriched specialization of PavSDJ Phylogenetically, PavSDJ and its sister protein occupy adjacent but distinct branches within the type III J-protein group. Homologs from Prunus and Fragaria are present in both branches, and their separation is consistent with a duplication event predating the divergence of these rosaceous lineages, followed by subsequent sequence divergence (Lallemand et al. 2020). The closer association of Malus/Pyrus -like homologs with the sister branch, rather than with the PavSDJ branch itself, suggests differential retention, gene loss, and/or divergence among rosaceous lineages (Panchy, Lehti-shiu, and Shiu 2016), even though further support from synteny and broader taxon sampling is still required. PavSDJ and its sister gene display strikingly distinct expression patterns: PavSDJ was enriched in pollen/anther, whereas the sister gene was broadly expressed across organs. Combined with their contrasting S-RNase association in recombinant assays, these patterns support functional divergence following gene duplication. This is consistent with pollen-lineage specialization, similar to that previously observed for the non- S modifier MGST (Ono et al. 2018). Such neofunctionalization is plausible within the context of pollen–pistil interactions, where selective pressures can drive rapid evolution of reproductive proteins and their interaction interfaces (Swanson and Vacquier 2002; Assis 2018). Collectively, our results support a working model in which PavSDJ is a cytosolic, pollen-expressed type III J-protein that associates with S-RNase in an allele-independent manner. It facilitates the recruitment of PavSLFL6, likely within an SSK1-containing SCF-related module, into an S-RNase-associated complex. These findings suggest that PavSDJ may regulate S-RNase in pollen by facilitating its interaction with partner proteins. Although this study primarily relies on biochemical and interaction assays, it provides a foundational framework for future studies to elucidate the underlying mechanism in vivo. Furthermore, the evolutionary divergence between PavSDJ and its sister lineage, together with their contrasting expression and biochemical profiles, suggests that specialized type III J-proteins have been co-opted into reproductive protein-processing pathways in the Rosaceae. Declarations Author contribution statement DM and XD conceived the study, performed experiments, analyzed data, data interpretation and wrote the manuscript. DM contributed to experimental design, supervision of the study and served as corresponding author. RT, DM and SN contributed to study design, discussion, and manuscript revision. All authors read and approved the final manuscript. Acknowledgments none. Funding Declaration This work was supported by the Grant-in-Aid for Scientific Research (A) with JSPS KAKENHI Grant Number 24H00510 of RT. Competing Interests The authors declare no competing interests. References Ajit Tamadaddi C, Sahi C (2016) J domain independent functions of J proteins. 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J Mol Biol 368:230–243. https://doi.org/10.1016/j.jmb.2007.02.003 Zeng B, Wang J, Hao Q, Yu Z, Abudukayoumu A, Tang Y, Zhang X, Ma X (2019) Identification of a novel SBP1-containing SCFSFB complex in wild dwarf almond (Prunus tenella). Front Genet 10:1019. https://doi.org/10.3389/fgene.2019.01019 Zhang D, Li YY, Zhao X, Zhang C, Liu DK, Lan S, Yin W, Liu ZJ (2024) Molecular insights into self-incompatibility systems: from evolution to breeding. Plant Commun 5:100719. https://doi.org/10.1016/j.xplc.2023.100719 Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9513049","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":641638512,"identity":"764b9d57-aaeb-4b7c-a17f-f70e145774bd","order_by":0,"name":"Xuexi Dou","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Xuexi","middleName":"","lastName":"Dou","suffix":""},{"id":641638513,"identity":"dcd6f51e-0d4b-48b9-978c-c3f1b7dbef59","order_by":1,"name":"Daiki Matsumoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3RP2sCMRzG8Sc4uLRmvVvsW0g5cKr4VhIK51ZaCs4nP7guBde4+RIEwTnlQJfSWwO3nAidO3aSpiq45W4sNN8lIfAh/4BQ6C9mGEECfe7mX+c12YokcQamWxJ0fgclzIX4620LEjXWMikXjJ5y3PAMn7WPxO+KpET1MLA1o3mOW20wFj4ijCKjDtXzwJru/joHWwJp5CXljozbRa20ewhHRs3Eng6mljw7EtVIYrsj4UgSWTYl/RHd66LhLr1yvI+/UfX5rHijx8ndcPbymtY+cilSmfshd6TOVdpOgJ8/FehuWpJQKBT6J/0Ao1ZPY9/IM4oAAAAASUVORK5CYII=","orcid":"","institution":"Fukui Prefectural University","correspondingAuthor":true,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Matsumoto","suffix":""},{"id":641638514,"identity":"ba04c8ae-6a12-4727-b599-fb5445e3fa3b","order_by":2,"name":"Ryutaro Tao","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Ryutaro","middleName":"","lastName":"Tao","suffix":""},{"id":641638515,"identity":"19def69a-34de-40e8-81a9-040e314de5a1","order_by":3,"name":"Soichiro Nishiyama","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Soichiro","middleName":"","lastName":"Nishiyama","suffix":""}],"badges":[],"createdAt":"2026-04-24 06:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9513049/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9513049/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109432698,"identity":"34df7fcd-7e1b-4f6e-9e2f-86c3690900ff","added_by":"auto","created_at":"2026-05-18 05:29:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125903,"visible":true,"origin":"","legend":"\u003cp\u003eStructural features of PavSDJ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eSchematic representation of the PavSDJ protein showing the J-domain, the HPD motif, the C-terminal region, and the predicted hydrophobic segment near the C terminus. \u003cstrong\u003e(b)\u003c/strong\u003eKyte–Doolittle hydropathy plot of PavSDJ, with the dashed box indicating the C-terminal region with relatively increased hydropathy. Window size equals to 9. Positive values indicate hydrophobicity, whereas negative values indicate hydrophilicity. Two hydrophobic peaks greater than 1.5 were observed in the C-terminal region (residues 286–294 and 321–328), as highlighted by the red dotted boxes. (\u003cstrong\u003ec)\u003c/strong\u003e Predicted three-dimensional structure generated by AlphaFold2. In the left-side figure, blue color indicates higher confidence while red indicates lower confidence. In the middle figure, blue color contrast to green color in the middle is the J-domain, HPD motif are highlighted with red marks. In the right-side figure, orange molecules are the hydrophobic residues, C-terminal region is colored in light blue, it is a visualization of possible binding pockets from the C-terminus.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/8a65438a7a86da6bc92716b0.png"},{"id":109759612,"identity":"5111c764-aa3f-4946-ba64-209a4aafd50b","added_by":"auto","created_at":"2026-05-22 07:27:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163160,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships of PavSDJ and related proteins\u003c/p\u003e\n\u003cp\u003eA ML tree of clade III members of PavSDJ-like protein homologs in eudicots and monocots (according to supplementary Fig.1). The alignment of the deduced amino acids was conducted using MUSCLE. The deduced amino acid sequences were from peach (prefixed with Prupe), strawberry (Fvesca), apple (MDP), soybean (Glyma), poplar (Potri), grapevine (GSV), cucumber (Cucsa), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AT), tomato (Solyc), \u003cem\u003eAquilegia coerulea\u003c/em\u003e (Aqcoe), maize (Zm) and rice (LOC_Os). The Neighbor-joining tree was generated with 1,000 bootstrap replicates. Bootstrap values over 50 are shown. The ML tree was generated with 1000 bootstrap replicates. The clade I and clade II members of PavSDJ-like protein homologs in peach, Prupe.7G066900.1 and Prupe.8G013200.1, respectively, were defined as the outgroup. The PavSDJ-containing clade is indicated in red and the closely related sister lineage is indicated in blue. Numbers at nodes indicate bootstrap support values, and the scale bar represents amino acid substitutions per site.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/9d617b3ebfcf0a6f6418a209.png"},{"id":109799187,"identity":"7850bf7a-7a4e-480b-80a4-56d8c270aca2","added_by":"auto","created_at":"2026-05-22 15:26:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143480,"visible":true,"origin":"","legend":"\u003cp\u003ePavSDJ is pollen-enriched, whereas its sister gene is broadly expressed, and only PavSDJ associates with PavS6-RNase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRelative expression levels of PavSDJ and its sister gene in the indicated tissues. PavSDJ is highly expressed in anthers and pollen, whereas the sister gene is expressed broadly across floral and vegetative organs. \u003cstrong\u003e(b)\u003c/strong\u003eCo-immunoprecipitation assay testing association of cMyc-PavSDJ or cMyc-PavSDJ sister with DDDDK-PavS6-RNase. Anti-cMyc immunoblotting detected co-precipitation of PavSDJ but not the sister protein with PavS6-RNase, whereas anti-DDDDK immunoblotting confirmed recovery of the bait protein.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/e3ec7a1518db2be81ab6bbb8.png"},{"id":109760124,"identity":"2f27b934-5c41-42ad-9c54-5e5f5500ad5b","added_by":"auto","created_at":"2026-05-22 07:28:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180945,"visible":true,"origin":"","legend":"\u003cp\u003eSilver-stained proteins recovered by anti-PavSDJ co-immunoprecipitation from pollen extracts\u003c/p\u003e\n\u003cp\u003eProteins recovered by anti-PavSDJ co-IP and control IP are shown, and arrowheads indicate bands excised for LC–MS/MS analysis. Molecular mass markers are indicated at left.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/ad7a0edd77c59b4760b20cae.png"},{"id":109760763,"identity":"705887fd-5d98-4cab-862f-e9e82a97a9d0","added_by":"auto","created_at":"2026-05-22 07:29:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":265664,"visible":true,"origin":"","legend":"\u003cp\u003ePavSDJ associates with S-RNase and promotes PavSLFL6 to an S-RNase-containing complex\u003cbr\u003e\n \u003cstrong\u003e(a)\u003c/strong\u003e Pull-down or co-immunoprecipitation assay showing association of PavSDJ from pollen extract with recombinant DDDDK-tagged PavS3-, PavS4-, and PavS6-RNases. Anti-PavSDJ immunoblotting detected PavSDJ in the precipitated fractions, and anti-DDDDK immunoblotting confirmed recovery of the bait proteins. (\u003cstrong\u003eb)\u003c/strong\u003e Reconstituted assay showing recovery of PavSDJ-containing material with DDDDK-PavS6-RNase. PavSDJ in a PavSDJ-enriched pollen fraction was incubated with DDDDK-tagged PavS6-RNase, and co-immunoprecipitated using anti-DDDDK antibody gel beads. The bead-bound proteins were detected by the immunoblot. (\u003cstrong\u003ec)\u003c/strong\u003e Reconstituted assay showing that PavSDJ promoted co-precipitation of PavSLFL6 with PavS6-RNase. cMyc-PavSDJ and HA-PavSLFL6 were combined as indicated, immunoprecipitated with DDDDK-PavS6-RNase, and detected by immunoblotting with anti-cMyc and anti-HA antibodies.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/59c26c9c2c90f3764784cfb2.png"},{"id":109759730,"identity":"3d4fb1c4-8f3a-470e-bb75-91d8330e0932","added_by":"auto","created_at":"2026-05-22 07:27:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":539382,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/a81beda5-cbb1-4ad1-bde4-a7d66967144b.pdf"},{"id":109760764,"identity":"d3816fc7-8eb9-4c62-a9ea-0dbab0490838","added_by":"auto","created_at":"2026-05-22 07:29:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1210737,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-9513049/v1/7852b09524426afe8b47811d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex","fulltext":[{"header":"Key Message","content":"\u003cp\u003ePavSDJ is a pollen-expressed type III J-protein that binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex in \u003cem\u003ePrunus\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eIt may function as a general modifier involved in the GSI system of \u003cem\u003ePrunus.\u003c/em\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSelf-incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilization and inbreeding, thereby promoting outcrossing and helping maintain genetic diversity in flowering plants (McCubbin and Kao 2000; Gibbs 2014).\u0026nbsp;Among homomorphic SI systems, S-RNase-based gametophytic self-incompatibility (GSI)\u0026nbsp;has been widely studied in several eudicot lineages,\u0026nbsp;including the Solanaceae and the Rosaceae. This system is characterized by the uptake of stylar S-RNases into pollen tubes, where these cytotoxic ribonucleases inhibit incompatible pollen tube growth(Kao and Tsukamoto 2004; Tao and Iezzoni 2010; Zhang et al. 2024). Although the S-locus determinants define allele specificity, accumulating evidence across species indicates that S-RNase-based GSI relies on additional accessory factors that modulate the intracellular fate and activity of S-RNase after pollen\u0026ndash;pistil contact\u0026nbsp;(McClure et al. 2011).\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003ePrunus\u003c/em\u003e (Rosaceae), the S-RNase\u0026ndash;based GSI system is controlled by a single multiallelic \u003cem\u003eS\u003c/em\u003e locus encoding the pistil determinant S-RNase and the pollen determinant \u003cem\u003eS\u003c/em\u003e haplotype-specific F-box protein (SFB) (Kao and Tsukamoto 2004; Tao and Iezzoni 2010). A characteristic of \u003cem\u003ePrunus\u003c/em\u003e SI is its \u0026ldquo;self-recognition\u0026rdquo; system, a single pollen-expressed \u003cem\u003eSFB\u003c/em\u003e allele is genetically linked to and recognizes its cognate stylar \u003cem\u003eS-RNase\u003c/em\u003e allele. This leads to self-pollen rejection in a one-to-one manner (Entani et al. 2003; Tao and Iezzoni 2010). This\u0026nbsp;model\u0026nbsp;contrasts with the \u0026ldquo;collaborative non-self recognition\u0026rdquo;\u0026nbsp;system\u0026nbsp;described in the Solanaceae and in the tribe Maleae (e.g., \u003cem\u003eMalus\u003c/em\u003e and \u003cem\u003ePyrus\u003c/em\u003e) of the subfamily Amygdaloideae of the Rosaceae, where multiple pollen F-box proteins, including SLFs in the Solanaceae and SFBBs in Maleae,\u0026nbsp;collectively\u0026nbsp;recognize and\u0026nbsp;detoxify non-self S-RNases\u0026nbsp;(Sassa et al., 2010). Although the concepts of\u0026nbsp;self- and non-self recognition provide a framework for understanding haplotype-specific pollen\u0026ndash;pistil interactions in \u003cem\u003ePrunus\u003c/em\u003e,\u0026nbsp;they do not fully explain\u0026nbsp;how S-RNase is handled after entering the pollen tube. In particular, it remains unclear\u0026nbsp;which pollen-side factors and protein complexes regulate its stability, localization, accessibility, and interaction with ubiquitin-related machinery.\u003c/p\u003e\n\u003cp\u003eIndeed, studies in several S-RNase\u0026ndash;based\u0026nbsp;GSI\u0026nbsp;systems have highlighted that non-\u003cem\u003eS\u003c/em\u003e-locus factors can shape critical steps of the SI pathway, including S-RNase binding, compartmentalization, redox regulation, and protein\u0026nbsp;degradation\u0026nbsp;(McClure et al. 2011). In the Solanaceae, for example,\u0026nbsp;biochemical, genetic and cell biological studies\u0026nbsp;have supported that the pollen-side response depends\u0026nbsp;not only on the \u003cem\u003eS\u003c/em\u003e locus F-box proteins, SLFs, but also on non-S factors including SSK1 and CUL1, in the\u0026nbsp;SCF\u003csup\u003eSLF\u003c/sup\u003e uniquitin ligase machinery\u0026nbsp;(Williams et al. 2015; Sun et al. 2018). In addition,\u0026nbsp;its\u0026nbsp;stylar factors such as 120K, NaTrxh, NaStEP, and HT-B have been reported to contribute to S-RNase activity and intracellular dynamics, including compartmentalization and pollen tube rejection outcomes\u0026nbsp;(Cruz-Garcia et al. 2005; Hancock et al. 2005; Goldraij et al. 2006; Jim\u0026eacute;nez-Dur\u0026aacute;n et al. 2013; Torres-Rodr\u0026iacute;guez et al. 2020; Cruz-Zamora et al. 2020). In Maleae, several non-\u003cem\u003eS\u003c/em\u003e components have been associated with SCF complex assembly or with S-RNase regulation within pollen tubes\u0026nbsp;(Xu et al. 2013; Minamikawa et al. 2014; Meng et al. 2014; Li et al. 2018). By comparison, the network of accessory factors in \u003cem\u003ePrunus\u003c/em\u003e remains less comprehensively defined, despite the availability of core SCF-related components such as SSK1, SBP1, and CUL1 that interact with pollen F-box proteins\u0026nbsp;(Zeng et al. 2019; Matsumoto et al. 2012, 2016). Moreover, the \u003cem\u003ePrunus\u003c/em\u003e \u003cem\u003eS\u003c/em\u003e locus\u0026nbsp;is structurally simpler than the corresponding loci in\u0026nbsp;the\u0026nbsp;Solanaceae and Maleae, comprising a compact region with \u003cem\u003eS-RNase\u003c/em\u003e and single \u003cem\u003eSFB\u003c/em\u003e genes rather than multiple \u003cem\u003eF-boxes\u003c/em\u003e (Matsumoto and Tao 2016; Claessen et al. 2019; Wu et al. 2020). Its\u0026nbsp;flanking \u003cem\u003eSLFL\u003c/em\u003e genes, which are orthologous to the pollen determinant F-box genes in Solanaceae and Maleae, are located near but outside the \u003cem\u003eS\u003c/em\u003e locus and have been proposed\u0026nbsp;to function as general detoxification\u0026nbsp;factors rather than haplotype-specific determinants\u0026nbsp;(Akagi et al., 2016; Matsumoto and Tao 2016). Thus,\u0026nbsp;the compact \u003cem\u003eS\u003c/em\u003e locus architecture of \u003cem\u003ePrunus\u003c/em\u003e may mainly dedicate to the allele-specific recognition, whereas\u0026nbsp;the downstream\u0026nbsp;processes mediated by\u0026nbsp;S-RNase likely\u0026nbsp;may\u0026nbsp;require\u0026nbsp;additional non-\u003cem\u003eS\u003c/em\u003e locus proteins for their assembly, stabilization or regulation. Among the candidate pollen-side factors, SLFL-related proteins have also been proposed to participate in S-RNase-associated processes, although the roles of individual members remain unresolved in \u003cem\u003ePrunus\u003c/em\u003e.\u0026nbsp;Consistent with this view,\u0026nbsp;a \u003cem\u003ePrunus\u003c/em\u003e-specific \u003cem\u003eM\u003c/em\u003e locus factor, encoded by \u003cem\u003eMGST\u003c/em\u003e at chromosome 3, distinct from the \u003cem\u003eS\u003c/em\u003e locus on chromosome 6,\u0026nbsp;had been reported as an essential factor to SI behavior in multiple \u003cem\u003ePrunus\u003c/em\u003e species\u0026nbsp;(Akagi et al. 2016; Ono et al. 2018;\u0026nbsp;Ono et al. 2022). In addition,\u0026nbsp;\u003cem\u003ePavAct1\u003c/em\u003e has been suggested to be related to target protein in S-RNase cytotoxicity\u0026nbsp;(Matsumoto and Tao 2012). Together, these findings suggest\u0026nbsp;that the outcome of SI in \u003cem\u003ePrunus\u003c/em\u003e is influenced by additional factors at loci beyond the \u003cem\u003eS\u003c/em\u003e locus with canonical S determinants.\u003c/p\u003e\n\u003cp\u003eIn our previous work to comprehensively identify pollen proteins that bound to silkworm-expressed recombinant S-RNase in sweet cherry (\u003cem\u003ePrunus avium\u003c/em\u003e), pollen-expressed DnaJ-like protein was isolated (S-RNase\u0026ndash;binding DnaJ-like protein, SDJ; Matsumoto and Tao 2019). This finding suggested a potentially new connection between S-RNase regulation and molecular chaperone-like modules. SDJ belongs to the large and diversified family of J-domain proteins (DnaJ/Hsp40), in which members typically function as regulators of protein interaction, folding, or complex assembly (Craig et al. 2006;\u0026nbsp;Kampinga and Craig 2010).\u003c/p\u003e\n\u003cp\u003eCanonical DnaJ-like proteins are commonly classified into three types ( Kampinga and Craig 2010), the type III J-proteins are the most structurally diverse, sharing only the J-domain with Types I and II J-proteins, and in many cases, this domain is also relatively divergent (Hennessy et al. 2000; Ajit Tamadaddi and Sahi 2016). Accordingly,\u0026nbsp;they have frequently evolved lineage- and pathway-specific roles than the canonical housekeeping roles of modulating Hsp70 chaperone function\u0026nbsp;(Hennessy et al. 2000; Ajit Tamadaddi and Sahi 2016). These facts raise the possibility that SDJ may act as a pathway-specialized, adaptor- or chaperone-like factor that contributes to the assembly or stabilization of an S-RNase\u0026ndash;associated protein complex in \u003cem\u003ePrunus\u003c/em\u003e pollen.\u003c/p\u003e\n\u003cp\u003eIn this study, we characterized PavSDJ from sweet cherry using sequence/phylogenetic analyses, expression profiling, and subcellular localization. We also examined its biochemical interactions with S-RNase and pollen proteins, and discussed its role in an S-RNase\u0026ndash;associated protein complex in \u003cem\u003ePrunus\u003c/em\u003e pollen.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSweet cherry\u0026nbsp;(\u003cem\u003ePrunus avium\u003c/em\u003e)\u0026nbsp;\u0026lsquo;Satonishiki\u0026rsquo; (\u003cem\u003eS\u003c/em\u003e haplotypes: \u003cem\u003eS\u003csup\u003e3\u003c/sup\u003eS\u003csup\u003e6\u003c/sup\u003e\u003c/em\u003e) was used in this study. Anthers were\u0026nbsp;collected from flower buds just before anthesis\u0026nbsp;and then\u0026nbsp;dehisced by overnight incubation\u0026nbsp;in the presence of\u0026nbsp;silica gel desiccant. Pollen was collected by filtering through a\u0026nbsp;100-\u0026mu;m nylon mesh\u0026nbsp;and stored with silica gel desiccant at\u0026nbsp;\u0026minus;80\u0026deg;C. Other floral organs from flower buds and young leaves were also collected during spring, frozen in liquid nitrogen and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted by the cold phenol method as described previously (Tao et al. 1999). First-strand cDNA was synthesized from total RNA using a PrimeScript\u003csup\u003e\u0026reg;\u003c/sup\u003e RT reagent kit with gDNA Eraser (Takara Bio, Otsu, Japan). Quantitative RT-PCR was conducted\u0026nbsp;using\u0026nbsp;gene-specific primers for \u003cem\u003ePavSDJ\u003c/em\u003e (XM_021972310.1; Fw: 5\u0026rsquo;-CGAAGGCCAGACAAGAGCTT-3\u0026rsquo;, Rv: 5\u0026rsquo;-AAAGTCCTCAGGCTCCATCATG-3\u0026rsquo;)\u0026nbsp;and\u0026nbsp;\u003cem\u003ePavSDJ-sister\u0026nbsp;\u003c/em\u003e(XM_021972277.1; Fw: 5\u0026rsquo;-TCACGTGTCTGCCTAGCTG-3\u0026rsquo;, Rv: 5\u0026rsquo;-ACGCAGAATCTATCTTGGTGTCA-3\u0026rsquo;).\u0026nbsp;Reactions were carried out using SYBR\u0026reg; Premix Ex Taq\u0026trade; (Takara Bio) on a LightCycler\u0026reg; Nano system (Roche Diagnostics, Basel, Switzerland) under the following cycling conditions:\u0026nbsp;a denaturation step at 95\u0026deg;C for 30 s, followed by 45 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 20 s. Gene-specific amplifications were\u0026nbsp;verified\u0026nbsp;by melting-curve analysis.\u0026nbsp;A sweet cherry ubiquitin homolog,\u0026nbsp;\u003cem\u003ePavUBQ\u003c/em\u003e (Fw: 5\u0026rsquo;-\u0026nbsp;TGATCCTTGTGGTTCCATCC-3\u0026rsquo;, Rv: 5\u0026rsquo;-\u0026nbsp;CATCCATCAGCCAAGTACGA-3\u0026rsquo;) was used as an internal control.\u0026nbsp;Two technical replicates were analyzed for each of three biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence analysis of SDJ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein sequence of SDJ was obtained from National Center for Biotechnology Information (NCBI) database (RefSeq accession\u0026nbsp;XP_021828002.1; corresponding UniProtKB accession A0A6P5TLJ7). Conserved domains were annotated using InterProScan and NCBI-CD-search. The presence and integrity of the J-domain were\u0026nbsp;assessed by identifying the canonical HPD motif\u0026nbsp;and surrounding conserved residues within the predicted J-domain region.\u0026nbsp;Additional\u0026nbsp;sequence features\u0026nbsp;were analyzed based on\u0026nbsp;the primary amino acid sequence. The hydropathy profile of PavSDJ was calculated using the Kyte\u0026ndash;Doolittle algorithm with a sliding window of 9 residues, in order to assess the distribution of hydrophobic regions along the protein sequence.\u0026nbsp;The predicted three-dimensional structural model of SDJ was\u0026nbsp;retrieved from the AlphaFold Protein Structure Database (AlphaFold DB)\u0026nbsp;based on its sequence.\u0026nbsp;Model confidence was evaluated using the predicted local distance difference test (pLDDT) score, and regions with pLDDT values \u0026gt;70 were considered to be predicted with relatively high confidence (Jumper et al. 2021; Varadi et al. 2022). Structural visualization and figure preparation were performed using PyMOL\u0026nbsp;(Version 2.1, Schr\u0026ouml;dinger, LLC).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the family assignment of PavSDJ, representative type I, type II, and type III J-proteins from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (listed in Supplementary Table 1) were analyzed together with PavSDJ (Rajan and Silva 2009; Pulido and Leister 2018). To further examine the evolutionary relationships of PavSDJ-like proteins, homologous sequences were retrieved by BLASTP search using PavSDJ as the query against the Phytozome v13 database (https://phytozome-next.jgi.doe.gov/), with an E-value cutoff of 1e\u003csup\u003e\u0026minus;25\u003c/sup\u003e. Retrieved sequences included homologs from peach (\u003cem\u003ePrunus persica\u003c/em\u003e), strawberry (\u003cem\u003eFragaria vesca\u003c/em\u003e), apple (\u003cem\u003eMalus domestica\u003c/em\u003e), soybean (\u003cem\u003eGlycine max\u003c/em\u003e), poplar (\u003cem\u003ePopulus trichocarpa\u003c/em\u003e), grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e), cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eAquilegia coerulea\u003c/em\u003e, maize (\u003cem\u003eZea mays\u003c/em\u003e), and rice (\u003cem\u003eOryza sativa\u003c/em\u003e). Protein sequences were aligned using MUSCLE implemented in MEGA version 6.0 (Tamura et al. 2013), and phylogenetic trees were constructed using the Maximum Likelihood (ML) method or Neighbor-Joining (NJ) method in IQ-TREE and MEGA with 1,000 bootstrap replicates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePollen protein extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePollen was cultured in a pollen germination medium [PGM; 50 mM MES-NaOH (pH 6.5), 3 mM Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, 0.8 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1 mM KNO\u003csub\u003e3\u003c/sub\u003e, 1.6 mM H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 15% polyethylene glycol 4000, and 300 mM sucrose] for 3 h at\u0026nbsp;20\u0026deg;C\u0026nbsp;(Hiratsuka et al. 2001). Germinated pollen was collected by centrifugation at 500 \u0026times; \u003cem\u003eg\u003c/em\u003efor 5 min, and homogenized in\u0026nbsp;the immunoprecipitation (IP) buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.3% (v/v) Triton X-100] containing 1 \u0026times; cOmplete\u0026trade; Protease Inhibitor Cocktail (Roche Diagnosis,\u0026nbsp;Mannheim, Germany). After centrifugation at 18,000 \u0026times; \u003cem\u003eg\u003c/em\u003efor 15 min, the supernatant was filtered through a\u0026nbsp;0.45-\u0026mu;m\u0026nbsp;cellulose acetate membrane (Advantec, Tokyo, Japan).\u0026nbsp;Protein concentration\u0026nbsp;was determined using a\u0026nbsp;Bio-Rad Protein Assay Kit\u0026nbsp;(Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation of PavSDJ from pollen extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour microliters of Pierce\u0026trade; Protein A/G Magnetic Beads (Thermo Fisher, Waltham, MA, USA) were incubated with 6\u0026nbsp;\u0026mu;g of anti-PavSDJ antibody for 30 min at room temperature in IP buffer. The anti-PavSDJ antibody was raised against the synthetic peptide SQDYWIHTDTT (amino acids 93\u0026ndash;103) and affinity-purified using the antigen peptide.\u0026nbsp;After\u0026nbsp;one wash with IP buffer, 5 mg of crude pollen extract was added to the beads, and the mixture was incubated at 4\u0026deg;C\u0026nbsp;for 2 h.\u0026nbsp;The beads were then\u0026nbsp;washed five times with IP buffer, and used for the recovery of innate PavSDJ complex or\u0026nbsp;mass spectrometric\u0026nbsp;analysis.\u003c/p\u003e\n\u003cp\u003eAs a parallel antibody control, purified anti-PavS6-RNase antibody was used in the same co-immunoprecipitation procedure. Because S-RNase is a pistil-expressed determinant and no specific endogenous PavS6-RNase antigen was expected to be present in the mature pollen extracts under our assay conditions, this control was used to estimate nonspecific proteins recovered through the antibody/IP procedure. For the recovery of innate PavSDJ complex, the beads were incubated with 1\u0026nbsp;mg mL⁻\u0026sup1;\u0026nbsp;antigen epitope in\u0026nbsp;50 mM tris-HCl (pH 7.5) and 150 mM NaCl\u0026nbsp;for 20min for three times, and eluted proteins were diafiltered with Milli-Q water using Amicon Ultra 0.5\u0026nbsp;centrifugal\u0026nbsp;filters (Millipore,\u0026nbsp;Billerica, MA, USA).\u0026nbsp;For proteomic analysis, bead-bound proteins were subjected to nanoLC-MS/MS analysis by Kazusa Genome Technologies (Kisarazu, Japan).\u0026nbsp;Each sample was digested with trypsin and analyzed\u0026nbsp;using an UltiMate 3000 RSLCnano LC system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific).\u0026nbsp;Data were analyzed using Scaffold DIA (Proteome Software Inc., Portland, OR, USA) against the database of \u003cem\u003ePrunus avium\u003c/em\u003e UniProtKB/Swiss-Prot database (UP000515124).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient recombinant protein expression in tobacco leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecMyc-tagged PavSDJ, cMyc-tagged PavSDJ-sister and HA-tagged S-locus F-box like 6 (PavSLFL6) was expressed in\u0026nbsp;\u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves using the agroinfiltration method as described previously, with slight modification (Matsumoto and Tao 2016). The coding sequences (CDSs) of\u003cem\u003e\u0026nbsp;PavSDJ\u003c/em\u003e and \u003cem\u003ePavSDJ-sister\u0026nbsp;\u003c/em\u003ewere amplified from \u0026lsquo;Satonishiki\u0026rsquo; pollen cDNA using\u0026nbsp;PrimeSTAR\u003csup\u003e\u0026reg;\u003c/sup\u003e HS DNA Polymerase (Takara Bio)\u0026nbsp;and further fused with cMyc tag sequence at their N-terminus by PCR.\u0026nbsp;The resulting PCR\u0026nbsp;products were cloned into the pGWB2 vector (Nakagawa et al. 2007) using an In-Fusion HD cloning kit (Takara Bio). The previously constructed pGWB2 constructs cloning \u003cem\u003eHA-PavSLFL6\u003c/em\u003e and \u003cem\u003eSFB-interacting Skp1 like\u003c/em\u003e \u003cem\u003e1\u003c/em\u003e (PavSSK1), whose co-expression enhances accumulation of PavSLFL protein, were also used in this study (Matsumoto and Tao 2016, 2019). \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain LBA4404 (Takara Bio) was transformed with either of the pGWB2 constructs or pBIN61 \u003cem\u003eturnip crinkle virus coat protein\u003c/em\u003e (\u003cem\u003eTCVCP\u003c/em\u003e). Transformed \u003cem\u003eAgrobacterium\u003c/em\u003e strains were cultured separately in LB medium to OD\u003csub\u003e600\u003c/sub\u003e 0.3\u0026ndash;0.5 and resuspended in an MS medium containing 100 \u0026mu;M acetosyrigone to a final OD\u003csub\u003e600\u003c/sub\u003e of 2.0. After an \u003cem\u003eAgrobacterium\u003c/em\u003e strain transformed with pGWB2 construct and that transformed with pBIN61 were mixed equally, two to three leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e plants at the six-leaf stage were agroinfiltrated. Plants were then grown for 4\u0026ndash;5 days at 18\u0026deg;C. Agroinfiltrated leaves were homogenized with a pestle in the immunoprecipitation (IP) buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM 2-mercaptoethanol, 0.3% (v/v) Triton X-100] containing 1 \u0026times; cOmplete\u0026trade; Protease Inhibitor Cocktail (Roche Diagnosis,\u0026nbsp;Mannheim, Germany).\u0026nbsp;The protein concentration was determined as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation using recombinant PavS-RNases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant PavS\u003csup\u003e3\u003c/sup\u003e-, S\u003csup\u003e4\u003c/sup\u003e-, S\u003csup\u003e6\u003c/sup\u003e-RNase tagged with DDDDK at the N-terminus were expressed with the silkworm-baculovirus system (Sysmex, Kobe, Japan) and purified using a DDDDK-tagged protein purification kit (MBL \u0026amp; Biological Laboratories, Nagoya, Japan), as described previously (Matsumoto and Tao 2019).\u0026nbsp;For pull-down assays,\u0026nbsp;1 mg crude protein extract from pollen or agroinfiltrated tobacco leaves was incubated\u0026nbsp;with\u0026nbsp;1 \u0026mu;g recombinant S-RNase\u0026nbsp;and 10\u0026nbsp;\u0026mu;l DDDDK-tagged protein purification gel\u0026nbsp;for 2 h at 4\u0026deg;C. After four washes with IP buffer,\u0026nbsp;bead-bound proteins were eluted\u0026nbsp;with a Laemmli SDS sample buffer [0.2 M tris-HCl (pH 6.8), 2% (w/v) SDS, 0.85 M 2-mercaptoethanol, and 10% (v/v) glycerol].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE and protein detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were visualized by silver staining using Sil-Best Stain One (Nacalai Tesque, Kyoto, Japan) or blotted onto PVDF membranes (Millipore) for immunoblot analysis. Anti-DDDDK mouse monoclonal antibodies (MBL\u0026nbsp;\u0026amp; Biological Laboratories), anti-HA mouse monoclonal antibodies (TANA2, MBL\u0026nbsp;\u0026amp; Biological Laboratories), anti-Myc rabbit ployclonal antibodies (MBL\u0026nbsp;\u0026amp; Biological Laboratories) and antiserum against PavSDJ were used as the primary antibodies. Immune complexes were detected using an ECL Advance Western Blotting Detection Kit (GE Healthcare) and the LAS-500 system (Cytiva, Tokyo, Japan). Exposure time in detection was adjusted according to the signal intensity of the target protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular localization analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u003cem\u003ePavSDJ\u003c/em\u003e PCR fragment was fused \u003cem\u003eGFP\u003c/em\u003e at its C-terminus and cloned into the pGWB2 vector using an In-Fusion HD cloning kit (Takara Bio).\u0026nbsp;The resulting\u0026nbsp;pGWB2-PavSDJ-GFP was introduced into \u003cem\u003eAgorbacterium\u003c/em\u003e strain EHA105. Agroinfiltration against\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e leaves was conducted as described above. Three days after inoculation, leaf discs (approximately 5 mm square) were immersed in 1 mg L⁻\u0026sup1; Propidium Iodide (PI) solution for 10 min. Subcellular localization (GFP fluorescence) of PavSDJ-GFP was observed using a FLUOVIEW FV3000 confocal laser microscope system (Olympus, Japan). The transient expression assay was repeated more than three times to confirm the protein localization.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSequence features and structural characteristics of PavSDJ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PavSDJ protein from \u003cem\u003ePrunus avium\u003c/em\u003e (XP_021828002.1) is predicted to be a 355-amino-acid protein with a molecular mass of approximately 42 kDa. Its encoding gene is located on chromosome 8, distinct from the \u003cem\u003eS\u003c/em\u003e locus on chromosome 6 and from \u003cem\u003eMGST\u0026nbsp;\u003c/em\u003eon chromosome 3. Domain annotation identified a conserved J-domain at the N-terminus (residues 24\u0026ndash;84), including the canonical HPD (His-Pro-Asp) motif at positions 51\u0026ndash;53, a hallmark of J-domain proteins that can stimulate Hsp70 ATPase activity (Fig 1a; Hennessy et al. 2000; Kampinga and Craig 2010). PavSDJ lacks the glycine/phenylalanine (G/F)-rich region and the cysteine-rich zinc-finger motifs typical of type I/II J-proteins, supporting its classification as a type III J-protein with a canonical J-domain. Structural analysis of the C-terminal region revealed features consistent with a potential client-interaction surface. Two clusters of hydrophobic residues were identified between positions 286 and 328 (Fig 1b). Secondary structure predictions combined with AlphaFold suggested the N-terminal region contains a canonical J-domain with the conserved HPD motif, consistent with a four-helix bundle, and the C-terminal region shows a structurally distinct segment with an extended terminal segment, which may contribute to an interaction-related surface (Fig 1c) (Dyson et al. 2006; Marinko et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvolutionary p\u003c/strong\u003e\u003cstrong\u003eosition\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of PavSDJ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo place PavSDJ within the plant J-protein repertoire, we first compared PavSDJ with representative \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003etype I, type II, and type III J-proteins. In this broader classification tree (Supplementary Fig 1), PavSDJ clustered within the type III J-protein assemblage, in proximity to the \u003cem\u003eArabidopsis\u003c/em\u003e type III reference DNAJC75 (NP_192673.1), supporting its assignment as a type III J-protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined the relationships among PavSDJ-like proteins in a focused phylogenetic analysis. Notably, the retrieved SDJ-like sequences were resolved into three major clades (Clades I\u0026ndash;III), all of which belong to the type III J-protein/SDJ-like group rather than corresponding to the three canonical J-protein families (Fig 2; Supplementary Fig 2). Within this framework, PavSDJ was placed in Clade III, where it grouped with the peach homolog Prupe.8G091400.1 and the strawberry homolog Fvesca mrna21834.1. A closely related but distinct neighboring branch within the same clade contained Prupe.8G091300.1, Fvesca mrna21835.1, and apple homologs, and was therefore designated as the SDJ sister gene group. Thus, SDJ occupies a distinct sublineage within Clade III, whereas \u003cem\u003eMalus/Pyrus\u003c/em\u003e-related homologs were associated more closely with the sister branch than with the PavSDJ branch itself. Together, these phylogenetic patterns are consistent with divergence between PavSDJ and its sister lineage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression patterns and divergence from the SDJ sister gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine whether \u003cem\u003ePavSDJ\u003c/em\u003e and its sister gene differ in expression and biochemical behavior, we compared their transcript abundance across tissues and tested their interaction with recombinant S-RNase. qRT-PCR showed that \u003cem\u003ePavSDJ\u003c/em\u003e transcripts accumulated predominantly in anther and pollen, with little to no detectable expression in vegetative organs (leaf) and most floral tissues (sepal, petal, ovary, pistil) (Fig 3a). In contrast, the \u003cem\u003ePavSDJ\u003c/em\u003e sister gene displayed broad expression across organs, with relatively higher transcript levels in non-reproductive tissues (Fig 3a). Consistent with these divergent expression profiles, \u003cem\u003ePavSDJ\u003c/em\u003e and its sister gene differed in S-RNase association in a recombinant co-immunoprecipitation assay. DDDDK-tagged PavS6-RNase precipitated cMyc-tagged PavSDJ, whereas the cMyc-tagged sister protein was not detectably co-precipitated under the same conditions (Fig 3b). Recovery of the bait protein was confirmed by anti-DDDDK immunoblotting. These results support functional divergence between\u003cem\u003e\u0026nbsp;PavSDJ\u003c/em\u003e and its sister gene, with \u003cem\u003ePavSDJ\u003c/em\u003e displaying a pollen-enriched expression pattern and S-RNase association.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular localization of PavSDJ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransient expression of PavSDJ\u0026ndash;GFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e epidermal cells revealed GFP fluorescence distributed throughout the cytoplasm, with no clear enrichment at the cell wall (Fig 4). The cytosolic distribution is consistent with the expectation that PavSDJ could access cytoplasmic protein interaction partners. While this heterologous system does not directly report localization in pollen tubes, the observed cytosolic distribution complements the pollen-enriched expression pattern and is compatible with a role in intracellular S-RNase-associated processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic profiling of PavSDJ co-immunoprecipitates identifies an SLFL6/SSK1-associated module\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify proteins associated with endogenous PavSDJ in pollen, we performed co-immunoprecipitation (co-IP) from \u0026lsquo;Satonishiki\u0026rsquo; pollen extracts using anti-PavSDJ polyclonal antibody,\u0026nbsp;in parallel,\u0026nbsp;purified anti-PavS6-RNase antibody was used as a antibody control, as no specific endogenous S-RNase target was expected in the pollen extracts under our assay conditions. Silver staining of the co-IP eluates revealed a prominent band at ~42 kDa in the anti-PavSDJ pull-down, consistent with the predicted molecular weight of PavSDJ,\u0026nbsp;together with additional co-precipitated or non-specific bands\u0026nbsp;(Fig 5). The control pull-down showed a distinct banding pattern, indicating that the two antibodies recovered different protein populations under the tested conditions.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"557\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"bottom\" style=\"width: 557px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eTop 15 proteins identified in MS/MS analysis of the co-IP using anti-SDJ pAb against the pollen extract of \u0026apos;Satonishiki\u0026apos;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eProteins preferentially identidfied in co-IP\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eAccessions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eMolecular mass\u003cbr\u003e\u0026nbsp;(kDa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003ePeak\u0026nbsp;\u003cbr\u003e\u0026nbsp;intensities\u0026nbsp;\u003cbr\u003e\u0026nbsp;in co-IP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003ePeak\u0026nbsp;\u003cbr\u003e\u0026nbsp;intensities\u0026nbsp;\u003cbr\u003ein control\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eEnrichment\u003cbr\u003e\u0026nbsp;ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003ePavSDJ1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5TLJ7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e2.3.E+10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6.6.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e342.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003ePavSLFL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5SY21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6.9.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6.9.E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1004.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003ePavSSK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RK47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e5.5.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e7.6.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e71.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S acidic ribosomal protein P2-2-like isoform X1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5S8S2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e2.4.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.6.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e15.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L13a-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RWM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.5.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.4.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5TU86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.3.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.1.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e12.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L10-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RWC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.2.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.2.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L7-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RV38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.2.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e8.4.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e14.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S acidic ribosomal protein P0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RIH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.2.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e8.3.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RYC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.1.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e9.3.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e11.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L22-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RJF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.0.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6.2.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e16.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L34-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5SV33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.0.E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.0.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e50S ribosomal protein L23, chloroplastic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5SJW1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e9.0.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1.3.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L4-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5REB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e8.8.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6.9.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e12.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60S ribosomal protein L14-2-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eA0A6P5RNY9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e8.5.E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e8.1.E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTotal intensities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e9.7.E+10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e1.2.E+11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"bottom\" style=\"width: 557px;\"\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Proteins with a 4-fold or greater increase in signal intensity by co-IP using anti-PavSDJ antibody are shown.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 362px;\"\u003e\n \u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Purified anti-PavS6-RNase rabbit antibody was used in control.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePrecipitated proteins detected by LC\u0026ndash;MS/MS were quantified by protein peak intensities, and enrichment ratios were calculated as the ratio of peak intensity in the anti-PavSDJ co-IP to that in the control IP (Table 1). Proteins showing a \u0026ge;4-fold higher peak intensity in the anti-PavSDJ co-IP relative to the control are listed in Table 1. Among these proteins, PavSDJ was strongly enriched (enrichment ratio 342.8), supporting efficient recovery of the bait protein. Notably, PavSLFL6 and PavSSK1 were among the most enriched non-bait proteins (enrichment ratios of 1004.5 and 71.9, respectively), and no other SLFL family members were detected other than PavSLFL6 under our experimental conditions. In addition, multiple ribosomal proteins displayed moderate enrichment (enrichment ratios ~7.1\u0026ndash;16.7) (Table 1). These results support the view that PavSDJ is associated with an SLFL6/SSK1-containing module in pollen, suggesting a possible role in the organization of S-RNase-associated protein complexes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePavSDJ associates with recombinant S-RNases in pollen extracts and promotes co-precipitation of PavSLFL6 with S-RNase\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether PavSDJ associates with S-RNase in the non-allelic specific manner, pollen protein extracts were incubated with recombinant DDDDK-tagged PavS3-, PavS4-, or PavS6-RNase, followed by pull-down with anti-DDDDK beads and immunoblot detection. PavSDJ was recovered in the DDDDK pull-downs for all three S-RNase alleles (Fig 6a, anti-PavSDJ), signal bands are of the same size with the pollen extract input control. These data support that PavSDJ can associate with multiple S-RNase alleles.\u003c/p\u003e\n\u003cp\u003eTo further confirm the PavSDJ containing protein complex binds to S-RNase directly, we prepared PavSDJ-containing eluates by anti-PavSDJ immunoprecipitation from pollen extracts and incubated them with recombinant DDDDK-PavS6-RNase prior to anti-DDDDK pull-down. PavSDJ was detected in the DDDDK pull-down when DDDDK-PavS6-RNase was included (Fig 6b), supporting that PavSDJ present in the PavSDJ-enriched fraction remains competent for S-RNase association. Together, the pollen-extract and PavSDJ-enriched assays support that PavSDJ associates with S-RNase in a pollen-derived biochemical context.\u003c/p\u003e\n\u003cp\u003eWe next examined whether PavSDJ influences the association of PavSLFL6 with S-RNase in a reconstituted pull-down assay. DDDDK-PavS6-RNase was used as a bait, cMyc-tagged PavSDJ was stably recovered in the pull-down both in the presence and absence of HA-tagged PavSLFL6 (Fig 6c, anti-cMyc). In contrast, HA-PavSLFL6 alone showed little to no recovery with PavS6-RNase, whereas clear co-precipitation of HA-PavSLFL6 was observed when PavSDJ was included (Fig 6c, anti-HA). Bait recovery was confirmed by anti-DDDDK immunoblotting. These results indicate that PavSDJ is sufficient for detectable association with PavS6-RNase in this assay and that PavSDJ promotes the co-precipitation of PavSLFL6 with S-RNase.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we characterize PavSDJ as a pollen-expressed cytosolic type III J-protein in sweet cherry and provide biochemical evidence linking PavSDJ to S-RNase association and to an SLFL6/SSK1-containing pollen interaction module. By integrating sequence/phylogenetic analyses, expression profiling, co-immunoprecipitation coupled with mass spectrometry, and recombinant pull-down assays, we establish a framework in which PavSDJ serves as an S-RNase\u0026ndash;binding factor that can facilitate recruitment of PavSLFL6 to an S-RNase\u0026ndash;containing complex. While the present data do not directly demonstrate a causal role in SI outcomes, they nominate PavSDJ as a candidate pollen-side component relevant to S-RNase regulation and provide a mechanistic hypothesis that can guide future functional tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1 PavSDJ combines a conserved J-domain with a divergent C-terminal region that may support specialized interactions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePavSDJ was appeared to contain a conserved N-terminal J-domain with the canonical HPD motif, consistent with type III J-protein classification. Unlike type I/II J-proteins, PavSDJ lacks the G/F-rich and zinc-finger regions, suggesting a streamlined architecture that may favor pathway-specific protein interactions. The C-terminal region of PavSDJ contains hydrophobic clusters and a short C-terminal motif that may contribute to client binding or partner recruitment, although these hypotheses require further experimental validation. Such features are consistent with the broader theme that type III J-proteins frequently evolve specialized interaction modules beyond canonical Hsp70 co-chaperone functions (Ajit Tamadaddi and Sahi 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 PavSDJ associates with multiple PavS-RNase alleles without strong allele preference, consistent with a general S-RNase\u0026ndash;binding factor in pollen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the key findings of this study is that PavSDJ was repeatedly consistently recovered in pull-down assays from pollen extracts of sweet cherry \u0026lsquo;Satonishiki\u0026rsquo;, using multiple recombinant S-RNase alleles (\u003cem\u003eS\u003csup\u003e3\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eS\u003csup\u003e4\u003c/sup\u003e\u003c/em\u003e, and \u003cem\u003eS\u003csup\u003e6\u003c/sup\u003e\u003c/em\u003e) as baits (Fig 6a). Notably,\u003cem\u003e\u0026nbsp;S\u003csup\u003e3\u003c/sup\u003e\u003c/em\u003e and\u003cem\u003e\u0026nbsp;S\u003csup\u003e6\u003c/sup\u003e\u003c/em\u003e correspond to the \u003cem\u003eS\u003c/em\u003e haplotypes of \u0026lsquo;Satonishiki\u0026rsquo;, whereas \u003cem\u003eS\u003csup\u003e4\u003c/sup\u003e\u003c/em\u003e represents a non-self (foreign) \u003cem\u003eS\u003c/em\u003e haplotype. Under our assay conditions, PavSDJ co-precipitated with all three S-RNases in an \u003cem\u003eS\u003c/em\u003e haplotype-independet manner. This interpretation is further supported by the reconstitution assays using recombinant proteins Specifically, cMyc\u0026ndash;PavSDJ was robustly pulled down by recombinant PavS6-RNase, whereas its recombinant sister protein (cMyc\u0026ndash;PavSDJ-sister) was not detectably recovered under the same conditions (Fig 3b, 6c). Together, the lack of a strong allele-specific preference, combined with the specificity difference between PavSDJ and its sister protein suggest that PavSDJ is likely to be a general factor that engages S-RNase after its entry into the pollen tube. This is consistent with its pollen-enriched expression and cytosolic localization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 PavSDJ promotes recruitment of PavSLFL6 to S-RNase and links to an SLFL6/SSK1 module\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA central finding of this study is that PavSDJ promoted co-precipitation of PavSLFL6 with S-RNase in a reconstituted assay: SLFL6 was not recovered with S-RNase alone but was robustly recovered when PavSDJ was included. Together with the co-IP/MS enrichment of SLFL6 and SSK1 in anti-PavSDJ co-IP/MS analyses, these results support the association of PavSDJ with an SLFL6/SSK1-containing module. Because SSK1 is a Skp1-like component that interacts with pollen F-box proteins in \u003cem\u003ePrunus\u0026nbsp;\u003c/em\u003e (Zeng et al. 2019; Matsumoto et al. 2016), this network is compatible with a model in which PavSDJ facilitates the assembly or stabilization of S-RNase\u0026ndash;associated complexes that include F-box\u0026ndash;related partners. In the broader context of S-RNase-based GSI, pollen tubes are likely to require accessory factors that influence S-RNase stability, accessibility, localization, or interaction competence after S-RNase entry. From this perspective, PavSDJ may act as an adaptor- or chaperone-like general binder that stabilizes S-RNase and/or facilitates its productive association with downstream pollen proteins, rather than conferring allele specificity per se.\u003c/p\u003e\n\u003cp\u003eIn addition, multiple ribosomal proteins showed moderate enrichment in the anti-PavSDJ co-IP. Given their high cellular abundance and frequent recovery in proteomic datasets, these proteins may in part represent common co-purifying components under the present experimental conditions. However, their detection is also noteworthy from a broader perspective of the J protein concept. For example, specialized cytosolic J-proteins such as Zuo1 and Jjj1 associate with 60S ribosomal particles and function in nascent polypeptide regulation or ribosome biogenesis (Kaschner et al. 2015; Hong et al. 2014). The enrichment of ribosomal proteins raises the possibility that PavSDJ operates in a ribosome-proximal chaperone environment.\u003c/p\u003e\n\u003cp\u003ePrevious studies proposed \u003cem\u003ePrunus\u003c/em\u003e SLFL proteins as candidate general inhibitors because of their interactions with tested S-RNases (Matsumoto et al. 2012, 2019), however, their S-RNase-binding behavior may not be uniformly aligned. Matsumoto et al (2016) proposed that, PavSLFL1 bound only PavS3-RNase, PavSLFL2 bound all four tested PavS-RNases (PavS1-, PavS3-, PavS4-, and PavS6-RNase), and PavSLFL3 showed no detectable binding to any of the tested S-RNases, and in the subsequent study of Matsumoto et al (2019), PavSLFL6 was further identified but did not show detectable binding to the four tested PavS-RNases. This study demonstrated that PavSDJ promoted the binding of PavSLFL6 to S-RNase. Against this background, rather than indicating that PavSLFL6 is itself a strong direct binder of S-RNase, our results suggest that PavSLFL6 may not be a strong direct binder of S-RNase on its own. Instead, they are more consistent with a model in which PavSDJ facilitates or stabilizes the assembly of PavSLFL6 into an S-RNase-associated complex. \u0026nbsp;Consistent with this view, we observed that PavSDJ exhibits broad binding affinity toward various S-RNases, rather than showing a preference for specific alleles. However, our current data are not yet sufficient to elucidate the specific biochemical events following S-RNase binding mediated by PavSDJ. Mechanistically, PavSDJ may provide an additional interaction surface for SLFL6, alter the conformation or accessibility of S-RNase, or stabilize protein assemblies by preventing protein aggregation. These possibilities are consistent with known modes of action for specialized J-proteins (Kampinga and Craig 2010), but remain to be tested in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Divergence from the SDJ sister gene supports pollen-enriched specialization of PavSDJ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetically, PavSDJ and its sister protein occupy adjacent but distinct branches within the type III J-protein group. Homologs from \u003cem\u003ePrunus\u0026nbsp;\u003c/em\u003eand Fragaria are present in both branches, and their separation is consistent with a duplication event predating the divergence of these rosaceous lineages, followed by subsequent sequence divergence (Lallemand et al. 2020). The closer association of \u003cem\u003eMalus/Pyrus\u003c/em\u003e-like homologs with the sister branch, rather than with the PavSDJ branch itself, suggests differential retention, gene loss, and/or divergence among rosaceous lineages (Panchy, Lehti-shiu, and Shiu 2016), even though further support from synteny and broader taxon sampling is still required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePavSDJ\u003c/em\u003e and its sister gene display strikingly distinct expression patterns: \u003cem\u003ePavSDJ\u003c/em\u003e was enriched in pollen/anther, whereas the sister gene was broadly expressed across organs. Combined with their contrasting S-RNase association in recombinant assays, these patterns support functional divergence following gene duplication. This is consistent with pollen-lineage specialization, similar to that previously observed for the non-\u003cem\u003eS\u003c/em\u003e modifier MGST (Ono et al. 2018). Such neofunctionalization is plausible within the context of pollen\u0026ndash;pistil interactions, where selective pressures can drive rapid evolution of reproductive proteins and their interaction interfaces (Swanson and Vacquier 2002; Assis 2018).\u003c/p\u003e\n\u003cp\u003eCollectively, our results support a working model in which PavSDJ is a cytosolic, pollen-expressed type III J-protein that associates with S-RNase in an allele-independent manner. It facilitates the recruitment of PavSLFL6, likely within an SSK1-containing SCF-related module, into an S-RNase-associated complex. These findings suggest that PavSDJ may regulate S-RNase in pollen by facilitating its interaction with partner proteins. Although this study primarily relies on biochemical and interaction assays, it provides a foundational framework for future studies to elucidate the underlying mechanism in vivo. Furthermore, the evolutionary divergence between PavSDJ and its sister lineage, together with their contrasting expression and biochemical profiles, suggests that specialized type III J-proteins have been co-opted into reproductive protein-processing pathways in the Rosaceae.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDM and XD conceived the study, performed experiments, analyzed data, data interpretation and wrote the manuscript. DM contributed to experimental design, supervision of the study and served as corresponding author. RT, DM and SN contributed to study design, discussion, and manuscript revision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Grant-in-Aid for Scientific Research (A) with JSPS KAKENHI Grant Number 24H00510 of RT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjit Tamadaddi C, Sahi C (2016) J domain independent functions of J proteins. 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Plant Commun 5:100719. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.xplc.2023.100719\u003c/span\u003e\u003cspan address=\"10.1016/j.xplc.2023.100719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"J-protein, Prunus avium, self-incompatibility, SLFL6, S-RNase, pollen","lastPublishedDoi":"10.21203/rs.3.rs-9513049/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9513049/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn \u003cem\u003ePrunus\u003c/em\u003e, self-incompatibility (SI) is controlled by S-RNases and pollen-expressed F-box proteins, whereas the molecular processes governing S-RNase regulation in pollen remain incompletely understood. Here, we characterized PavSDJ, a novel pollen protein from sweet cherry (\u003cem\u003ePrunus avium\u003c/em\u003e), as a candidate factor involved in pollen-side S-RNase-associated processes. Sequence and structural analyses identified PavSDJ as a type III J-protein. Phylogenetic analyses placed PavSDJ within a distinct SDJ-like sublineage of the type III J-protein group, separate from a closely related sister lineage. Consistent with this divergence, PavSDJ was strongly expressed in anthers and pollen, whereas its sister gene was broadly expressed across organs. Transient expression assays indicated that PavSDJ localized predominantly to the cytosol. Biochemical analyses showed that PavSDJ associated with recombinant PavS-RNases in pollen extracts and in reconstituted pull-down assays, without obvious allele preference. Proteomic analysis of PavSDJ co-immunoprecipitates from pollen extracts identified a complex including PavSLFL6 and PavSSK1. Reconstitution assays further showed that PavSDJ promoted the co-precipitation of PavSLFL6 with S-RNase. These findings identify PavSDJ as a candidate pollen-side factor in the \u003cem\u003ePrunus\u003c/em\u003e SI pathway and provide evidence that a specialized J-protein may contribute to SI-related protein complex assembly.\u003c/p\u003e","manuscriptTitle":"SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 05:29:11","doi":"10.21203/rs.3.rs-9513049/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ed4254b-3626-4ac6-afe1-1f4e41017ebe","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"145037609941581291106701911986159169765","date":"2026-05-21T14:48:15+00:00","index":13,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T12:39:39+00:00","index":12,"fulltext":""},{"type":"reviewerAgreed","content":"188503124748644607849179760733326291794","date":"2026-05-08T00:03:06+00:00","index":10,"fulltext":""},{"type":"reviewersInvited","content":"3","date":"2026-05-07T21:21:30+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T05:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 05:29:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9513049","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9513049","identity":"rs-9513049","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}