Journal of Integrative Plant Biology

Protein-protein interactions (PPIs) play a crucial role in enabling proteins to carry out their functions within various biological processes (Hui et al., 2003). Since the introduction of the yeast two-hybrid (Y2H) method for PPI detection in 1989 (Fields and Song, 1989), the identification of PPIs has become a significant focus in modern biological research. PPI goes beyond examining individual proteins, allowing researchers to establish a comprehensive network that regulates biological processes. Rice, as a key model organism in plant biological studies, has been at the forefront of PPI research. In 2008, prominent rice scientists in China called for concerted efforts to define a comprehensive protein-protein interaction network experimentally, which aimed to facilitate the prediction of the functional mechanisms operating throughout a plants lifecycle (Zhang et al., 2008). With efforts for 2 decades, the experimentally identified rice PPIs have reached over ten thousand. Several public databases have been established to systematically collate and store PPIs, including STRING (Szklarczyk et al., 2019), BioGRID (Oughtred et al., 2020), IntAct (del Toro et al., 2022), PRIN (Gu et al., 2011), RicePPINet (Liu et al., 2017) and RiceNet v2 (Lee et al., 2015). However, most PPI datasets in rice stem from computational predictions, while experiment-based rice PPI datasets are fragmented due to the lack of systematic profiling at the rice PPIome level, which largely hinders information sharing in the rice research community. To bridge this gap, we constructed the Port of Protein-Protein Interactomes (POPPIN; https://riceome.hzau.edu.cn/poppin/), an integrated database dedicated to sharing experimentally verified PPIs and functional clues in rice. Empowered by high-throughput PPIome profiling technologies and text mining assisted by a large language model (Huang et al., 2025; Liu et al., 2025), POPPIN currently has deposited over 150,451 pieces of rice PPI-related information. Additionally, POPPIN provides detailed protein information, including GO annotations, subcellular localizations, domains, trait ontology (TO) information, and hyperlinks to external biological databases. Through offering a user-friendly web interface for search and dynamic network visualization, POPPIN serves as the first large-scale, experiment-based database for searchable PPIs in rice, and has the potential to be extended to other species under this structural framework.

Cellulose, a central structural component of plant cell walls, is produced by cellulose synthase complexes (CSCs) at the plasma membrane. Salinity stress is particularly damaging to cellulose biosynthesis, and therefore, plants have developed adaptive mechanisms to cope with these conditions. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are essential for growth under salt stress and show a salt-dependent association with CSCs through an as-yet unknown mechanism. Here, we identify a phosphorylation-dependent regulatory mechanism linking salt stress signaling to cellulose biosynthesis through the coordinated action of TTL3 and the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1). Phosphorylation of Serine 93 in the N-terminal intrinsically disordered region of TTL3 controls its localization, retaining it in the cytosol, while dephosphorylation promotes association with CSCs at the plasma membrane. Biochemical and genetic analysis identified BIK1 as the kinase responsible for TTL3-S93 phosphorylation, with bik1 mutants phenocopying the phosphoablative TTL3S93A in vivo. Transcriptomic analyses reveal a strong overlap of differentially expressed genes between bik1 and a cellulose-deficient mutant, supporting a broader role for BIK1 in cell wall regulation. Notably, TTL proteins do not appear to be involved in the assayed canonical immune responses, suggesting pathway specificity downstream of BIK1. Together, these findings define a signaling module that connects salt stress perception to CSCs regulation and establish BIK1-dependent TTL3 phosphorylation as a molecular switch for maintaining cell wall integrity under abiotic stress.

Programmable DNA integration is a major challenge in plant genome engineering. CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants. Here, we reconstituted and engineered a Type I-F CAST for programmable DNA integration in plant cells. We validated expression of the wild-type Pseudoalteromonas CAST (PseCAST) machinery in plants and established targeted episomal integration in Arabidopsis thaliana protoplasts and chromosomal integration at a transgenic locus in Nicotiana benthamiana. The evolved PseCAST system, evoCAST, showed chromosomal integration efficiencies of 2.7%, representing a 6-fold improvement over wild-type PseCAST. evoCAST also enabled the insertion of cis-regulatory elements into a synthetic landing pad with 8% efficiency. evoCAST was subsequently retargeted to six endogenous genomic loci, demonstrating programmable integration across diverse chromosomal contexts. Finally, a cofactor screen identified the chromatin-associated factor AtHMGB2 as an enhancer of evoCAST-mediated integration activity in plants. These results establish CAST as a functional platform for programmable DNA insertion in plants and provide a foundation for developing targeted genome-engineering technologies for crop biotechnology.

O_LIViroids are small, non-coding RNAs that rely on host factors for replication, intracellular trafficking and systemic movement. VIRP1, a Bromodomain and Extra-terminal domain (BET) protein, has previously been implicated in Potato spindle tuber viroid (PSTVd) infection, yet its precise role and mode of action remain unresolved. C_LIO_LIIn this work, we highlight VIRP1 as the only Solanaceae BET protein containing a proline-rich domain, which overlaps with the PSTVd-binding site. VIRP1-deficient plants exhibit delayed flowering and increased ABA sensitivity, with differentially expressed genes enriched in stress-related pathways. C_LIO_LIVIRP1 forms condensates in planta and in vitro, consistent with phase-separation behaviour. Condensate morphology is altered by PSTVd RNA, deletion of the intrinsically disordered CTD and bromodomain mutations. C_LIO_LIVIRP1 is particularly important for the early establishment of PSTVd infection, while nuclear localization and bromodomain integrity are required for efficient viroid accumulation. By contrast, the disordered CTD region is dispensable for complementation of PSTVd accumulation. C_LIO_LIOur results support a model in which VIRP1 acts as a host nuclear factor that links stress-related functions, nuclear condensate formation and early viroid infection. C_LI

Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.

O_LIThe control of amount of nuclear proteins is fundamental in regulating plant gene expression, but the mechanisms of quantitative dynamics of the nuclear proteome are largely unstudied during adaptive responses to pathogens. C_LIO_LIHighly specific labeling, enrichment and measurement of the nuclear proteome was performed using TurboID LC-MS of Arabidopsis thaliana leaves treated with the pathogen-associated molecular pattern (PAMP), flg22, and/or cycloheximide. The chosen experimental approach allowed discrimination of the effects of translation, nuclear protein import, trafficking of preexisting proteins, derepression, and nuclear protein turn-over upon elicitation of basal immunity. C_LIO_LIThe highly specific, deep coverage of proteins in the nucleus makes this study a resource for anyone interested in plant nuclear proteome dynamics and defense. C_LIO_LIAround 2,000 nuclear proteins were repeatedly quantified, including more than 300 transcription factors or other proteins related to transcription. Several proteins with documented activity in endosomes were newly synthesized and imported into nuclei upon PAMP challenge, suggesting alternative nuclear functions in PAMP-triggered immunity (PTI). Circadian clock components, including the transcription factor, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-HIKING EXPEDITION (CHE), were depleted upon PAMP challenge, suggesting a safeguard against untimely induction of systemic acquired resistance (SAR). C_LIO_LIBased on proteomic patterns, proteins moonlighting in the nucleus as well as trafficking and turn-over regulation of the proteome are common elements during plant immunity. C_LI

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

The plant immune system comprises a complex signaling network that is activated upon perceiving molecules derived from invading pathogens. The first line of defense at the plant cell surface is mediated by receptor-like proteins (RLPs) and receptor-like kinases (RLKs). RLPs, which lack a cytoplasmic signalling domain themselves, constitutively interact with the RLK SUPRESSOR OF BIR1-1 (SOBIR1), which is a key component initiating immune signal transduction upon pathogen perception. Therefore, elucidating the composition of the SOBIR1 protein complex will contribute to understanding the basic molecular mechanisms of plant disease resistance. Most of the studies focused on the identification of SOBIR1-interacting proteins are limited to model plants, due to technical challenges and lack of reliable genome and proteome databases in crop plants. Here, we evaluate the application of the biotin ligase TurboID (TbID)-based proximity-dependent labeling (PL) approach by transiently expressing SOBIR1 from Nicotiana benthamiana (NbSOBIR1), fused to TbID in leaves of the wild potato Solanum microdontum. We show that NbSOBIR1-YFP-TbID properly accumulates in potato and that proximal proteins are biotinylated. Quantitative proteomic analysis yielded over 130 candidate proteins to be in the proximity of the cytoplasmic kinase domain of NbSOBIR1, of which some could be linked to disease resistance by KEGG pathway and gene ontology (GO) molecular function analysis. We also studied the dynamics of the proteome in proximity of NbSOBIR1 upon perception of the INF1 elicitin of Phytophthora infestans that was co-expressed in potato with the elicitin receptor ELR, which is an RLP that constitutively interacts with SOBIR1. We found more than 80 proteins, including the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH 1 (NRC1), putatively interacting with NbSOBIR1. In conclusion, we were able to successfully apply PL in potato and a future roadmap for further research on deciphering the composition of protein complexes involved in immune signaling has been established.

The Arabidopsis PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) and Telomere Repeat-Binding Proteins 1-3 (TRB1-3, TRBs) associate with distinct and shared protein complexes involved in epigenetic regulation, yet their cooperative roles in chromatin control and plant development remain largely unexplored. Here, we show that the interaction between PWO1 and TRBs is evolutionarily conserved. Both PWO1 and TRBs associate with plant telomeres, interact at these regions, and are co-enriched at subsets of interspersed telo-box motifs across regulatory regions genome-wide. TRBs facilitate PWO1 binding at shared genomic regions, including telo-box motifs. PWO1 and TRBs share a substantial number of genomic targets and preferentially bind chromatin regions associated with transcriptionally active states, whereas TRBs alone associate with repressive marks at thousands of loci. Genetic analyses show that the pwo1 trb1 trb3 triple mutant displays severe developmental defects, including main stem arrest and early maturation associated with aberrant lignin deposition in interfascicular tissues. In the triple mutant, key enzymes in the lignin biosynthesis pathway are upregulated, indicating that PWO1, TRB1, and TRB3 cooperatively regulate secondary cell wall formation. Together, our findings provide new insights into how PWO1 and TRBs cooperate to regulate chromatin states and orchestrate plant development, highlighting their central role in controlling gene expression programs. Significance statementThis study shows that PWO1 and TRB proteins co-occupy telomeres, including interspersed telo-box motifs, to regulate chromatin organization and plant development, particularly ectopic lignin deposition. Our findings reveal how these nuclear protein factors coordinate epigenetic states in Arabidopsis thaliana, providing a framework for understanding the control of developmental programs. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740627v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1370171org.highwire.dtl.DTLVardef@3faea5org.highwire.dtl.DTLVardef@e30921org.highwire.dtl.DTLVardef@16c948e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Evolutionarily conserved PWO-TRB interactions and their shared roles in chromatin regulation and plant development. Created with BioRender.com. C_FIG

Polycomb Repressive Complex 2 (PRC2) is a histone H3K27 methyltransferase that represses gene expression. Arabidopsis thaliana (A. thaliana) has several different PRC2 isoforms that are functionally distinct during the life cycle of the plants. However, their biochemical and structural characteristics have not been investigated. Here, we biochemically characterized PRC2 isoforms having different catalytic subunits: SWNINGER (SWN; PRC2 SWN ) and CURLY LEAF (CLF; PRC2 CLF ). Interestingly, PRC2 SWN showed much lower activity than PRC2 CLF . In addition, PRC2 SWN methylates histone H3K27 in mono and di-methylation, while PRC2 CLF shows robust tri-methylase activity. We also determined the cryo-electron microscopy (cryo-EM) structures of PRC2 SWN and PRC2 CLF , revealing that the substrate binding pocket of the SWN SET domain is blocked by a loop in the pre-SET domain, functioning as an auto-inhibitory loop, while that of the CLF SET domain is freely accessible. Introduction of CLF-like mutations in the auto-inhibitory loop in SWN enhances PRC2 SWN activity. Furthermore, structure-guided in planta analysis shows that a CLF-mimetic SWN mutant rescues the CLF knockout phenotype. Our work provides structural and molecular insights into the isoform-specific regulatory mechanism of plant PRC2.

Topologically associating domains (TADs) are essential structural and functional modules of the genome that play a crucial role in regulating gene expression. In this study, we systematically investigated the conservation and evolution of TADs in five closely related crops, including maize, sorghum, coix, foxtail millet and broomcorn millet. Our results show that 74% of TAD boundaries are conserved between two inbred maize lines, B73 and Mo17, and that approximately 50% or more of TAD boundaries are conserved across different crop species. TAD number remains relatively stable in the face of changes in genome size. However, the length of TADs varies depending on genome size. Furthermore, we found that large-scale transposable element expansion leads to TAD expansion, while chromosomal inversions lead to TAD fusion and the formation of new TAD boundaries. Frequent chromatin interactions between subgenome chromosomes occur after whole-genome duplication. Moreover, we also found that crossovers are enriched at TAD boundaries in maize, indicating the importance of TADs as a fundamental unit during species evolution. Overall, our study provides insights into the conservation and evolution of TADs in crop genomes and their roles in genome organization and function.

Brown spot (BS), caused by the fungal pathogen Bipolaris oryzae, is a major disease threatening global rice production. However, the genetic basis of host BS resistance remains unclear. Here, we identified brown spot resistance 1 (bsr1), a quantitative trait locus conferring BS resistance, by map-based cloning. We show that bsr1 encodes a sucrose transporter and that a near-isogenic line carrying bsr1 (bsr1-NIL) in the susceptible Koshihikari genetic background exhibited resistance to BS by suppressing sucrose efflux into the apoplast after pathogen attack. Furthermore, bsr1-NIL also showed strain-specific resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae through the same mechanism. These findings demonstrate that bsr1 confers dual resistance to fungal and bacterial diseases by regulating sucrose efflux. Our study identifies a previously unrecognized mechanism underlying resistance to both BS and bacterial blight and highlights bsr1 as a promising target for breeding disease-resistance rice cultivars. Rice (Oryza sativa L.) is a staple food for more than half of the worlds population1. Brown spot (BS), caused by the fungus Bipolaris oryzae, is one of the most prevalent fungal diseases of rice, and its incidence has increased under global warming2. BS infects coleoptiles, leaves, leaf sheaths, panicle branches, glumes, and spikelets, and severe infection can substantially reduce grain yield.

Exocyst was initially uncovered in yeast genetic sec-screen as a tethering complex for exocytotic vesicles and this function was later found to be evolutionarily conserved in other eukaryotes including plants. Later however, a surprising engagement of the exocyst complex in autophagy was observed in animals, plants and recently also in yeast. Using the genetic approach we observed EXO70E2 exocyst complex subunit engagement in the defence response to Pseudomonas syringae attack linked to the autophagy pathway. CRISPR/CAS LOF mutant of EXO70E2 is more sensitive to Pseudomonas infection (both virulent as well as T3SS mutant) and autophagy flux monitored by NBR1 antibody is compromised in comparison to WT. We conclude that the plant exocyst complex linked to the EXO70E2 subunit participates in defence against Pseudomonas bacteria in conjunction with the autophagy pathway. HighlightArabidopsis exocyst subunit EXO70E2 affects selective autophagic flux monitored by NBR1 and is participating in defense against Pseudomonas syringae infection.

Ribosome biogenesis is critical for plant development and environmental responses. A large number of ribosomal proteins (RPs) and ribosomal biogenesis factors (RBFs) are required for ribosome biogenesis, many of which remain uncharacterized in plants. We report here the identification of Arabidopsis RBF FAN and its interacting partner FAN-INTERACTING PROTEIN 1 (FIP1). As their human and yeast orthologues, FAN-FIP1 interact. Both FAN and FIP1 participate in the processing of pre-rRNAs. Functional loss of FAN or FIP1 knock-down results in developmental retardation and hypersensitivity to heat stresses. We demonstrate that FAN-FIP1 positively mediates brassinosteroid (BR) signaling by ensuring the translation efficiency of the BR receptor-coding gene BRASSINOSTEROID INSENSITIVE 1 (BRI1) through the presence of its upstream open reading frame (uORF). Importantly, BR signaling positively mediates the processing of pre-rRNAs, which may be critical not only for development but also for heat tolerance.

Transcription factors (TFs) orchestrate environmental responses by activating target genes, yet how they reshape epigenome architecture to coordinate gene expression remains poorly understood. We previously identified SIENA (Stimulus-Induced ENhancer Acetylation) domains as large regions of jasmonic acid (JA)-induced H3K9 hyperacetylation surrounding MYC2 TF binding sites in Arabidopsis and tomato. However, the mechanisms underlying the formation of SIENA domains (SIENAs) and their functional significance remained unknown. Here, we show that SIENAs also form at major JA-responsive genes and gene clusters in soybean, extending this phenomenon to an evolutionarily distant crop species. Comprehensive chromatin profiling revealed that SIENAs accumulate multiple histone acetylation marks, including H3K9ac, H3K27ac, H3K56ac, H2BK20ac, and H2A.Zac, establishing them as regions of broad histone hyperacetylation. Pharmacological disruption of proteasomal turnover and histone acetylation dynamics compromised SIENA formation. Chromatin accessibility analyses further showed that inducible accessibility within SIENAs is tightly associated with MYC2 binding sites, supporting a model in which MYCs nucleate localized chromatin reprogramming events. Together, our findings establish SIENAs as MYC2-dependent chromatin-organizing domains and identify histone hyperacetylation as a central feature of MYC2-mediated gene activation.

Nucleotide-binding leucine-rich repeat (NLR) immune receptor genes are of significant value in disease resistance breeding and the control of viral diseases. Soybean mosaic virus (SMV) poses a serious threat to soybean production and the Rsv1 locus in soybean cultivar Suweon 97 confers broad-spectrum resistance against SMV strains G1 to G7; however, this locus harbors no fewer than 18 NLR genes, and thus the broad-spectrum antiviral mechanisms underlying the Rsv1 locus remain poorly understood to date. Here, we established a rapid and highly efficient screening system for cloning NLR genes from soybean Rsv1 locus and identified a broad-spectrum antiviral NLR gene 13g184900 from this highly complicated locus. The NLR encoded by 13g184900 can recognize viral P3 protein from all SMV strains (G1-G7) and another potyvirus Bean common mosaic virus (BCMV). The coiled-coil (CC) domain of this NLR directly interacts with viral P3 protein. Additionally, we showed that this NLR originated from wild soybean accession in East China and has been introduced into several soybean cultivars during domestication. Collectively, we developed a high-throughput screening system for identifying NLR genes in soybean and our study provides new mechanistic perspective on how the Rsv1 locus mediates the broad-spectrum resistance to all SMV G1-G7 strains.

In angiosperms, ovule development requires the activity of the C, D and E classes of MADS genes, which encode key transcriptional regulators of reproductive development. The SEPALLATA (SEP) MADS transcription factors (MTFs), which belong to the E class, act as organizing hubs of MADS heterotetrameric complexes and play an essential role in the development of flower organs. However, the role of the SEP genes in ovule and seed development has been difficult to determine due to redundancy in the subclade, the lack of observable phenotypes in single and double sep1 sep2 mutants and the homeotic conversion of the carpel into sepal or leaf in higher order sep mutants. Here, we engineered a version of SEP3 (SEP3{Delta}M) that encodes a protein lacking the DNA-binding MADS-domain but retains the oligomerization domains needed for MADS protein heterotetramerization. In vitro experiments demonstrated the ability of SEP3{Delta}M to interact with the C and D classes of MTF, reducing the capability of such MADS complex to efficiently bind DNA. sep3{Delta}M plants showed a delay in flower opening and organ maturation and a reduced fertility. The ovules exhibited reduced outer integument growth, and the few seeds that developed showed impaired mucilage secretion upon imbibition. RNA-seq analysis of sep3{Delta}M demonstrated misregulation of genes involved in outer integument and seed coat development. Taken together, these data indicate the key role of SEP3-containing MADS complexes in proper ovule outer integument growth and seed coat development.

The ubiquitin-proteasome system (UPS) serves as the primary proteolytic machinery in eukaryotes, governing intracellular protein turnover to maintain proteome homeostasis. In plants, the HECT-type UPL3/4 ubiquitin ligases play vital roles in developmental and immune signaling. After ubiquitination by pathway-specific E3 ligases, substrates are physically relayed to proteasome-associated UPL3/4 ligases for further modification, which is necessary for their proteasome-mediated degradation. In this study, we investigated if the cellular influence of UPL3/4 extends beyond their direct role in substrate degradation. We discovered that UPL3/4 govern the ubiquitination not only of a broad array of immune-related substrates, but also of many UPS components, including E3 ligases. UPL3 physically interacts with PUB22, a pathway-specific U-box E3 ligase that negatively regulates immunity. PUB22 is controlled by a phospho-switch that converts it from an instable autoubiquitinated state to a stable phosphorylated E3 ligase that marks substrates for degradation. Remarkably, UPL3 only interacted with unphosphorylated PUB22 and facilitated its autoubiquitination-mediated degradation, thereby promoting the accumulation of PUB22 substrates. Moreover, the compromised immune phenotypes of upl3 upl4 mutant plants were largely dependent on PUB22 and its close paralogues. Thus, UPL3/4 control the stability of immune-related substrates not only through direct ubiquitination, but also indirectly by promoting autoubiquitination of PUB22 ligase and its paralogues. Controlling the stability of autoubiquitinating E3 ligases may be a universal mechanism whereby HECT-type ligases and the proteasomes they associated with, orchestrate cellular proteostasis in eukaryotes. Significance StatementThe ubiquitin-proteasome system (UPS) governs intracellular protein turnover to maintain proteome homeostasis in eukaryotes. Proteasome-associate HECT-type ubiquitin ligases play an important role in processing and degrading substrates delivered to the proteasome by pathway-specific E3 ligases. Here, we discover that in plants, HECT-type ligases not only promote the degradation of substrates, they also modify the E3 ligases that target these substrates to the proteasome. Specifically, HECT-type ligases facilitated or expanded the autoubiquitination of immune-suppressive E3 ligases, resulting in their proteasome-mediated degradation and onset of immunity. Our discoveries suggest that during plant immunity, HECT-type ligases and the proteasomes they associate with, control cellular proteostasis by governing the stabilities of both E3 ligases and their substrates.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

Bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is a devastating disease of tomato that severely limits global tomato productivity. Understanding the molecular mechanisms underlying Pst and tomato is essential for developing disease resistant varieties. Here, we demonstrate that Pto, the first disease-resistance gene conferring recognition of a specific pathogen, phosphorylates Pst type III effector AvrPtoB at serine 335 site. This post-translational modification triggers the dissociation of the Prf immune complex, enhancing immune signaling and reducing bacterial pathogenicity. Furthermore, evolutionary analyses indicate that Pto-associated proteins originated from malectin-like receptor kinases (MLRs) through loss of the extracellular domain. Crucially, we identified two key amino acid substitutions, Arg158 and Glu258 in Pto, which replace the ancestral lysine residues in MLRs (SpHREK1-1, SpHERK1-2 and SpHERK1-3). These substitutions stabilize Pto by preventing degradation mediated by AvrPtoB's E3 ubiquitin ligase activity. Our findings reveal a novel mechanism, by which Pto phosphorylates a bacterial effector to trigger enhanced immunity and elucidate the key evolutionary adaptations that have shaped Pto into a stable resistance protein.