Plants encounter diverse pathogens and have evolved a two-layered innate immune system to detect pathogen molecules and activate defense mechanisms that restrict infection. Most cloned plant Resistance (R) genes encode NLR immune receptors. NLR genes are often found in clusters of paralogs with sequence and copy number variation; whether these NLR clusters evolve in response to single or multiple pathogens has been unclear. We report here the isolation of a Phytophthora capsici resistance gene, Rpc2, along with a novel P. infestans resistance gene, Rpi-amr5, from two Solanum americanum accessions. These orthologous genes reside in the Rpi-amr1 cluster, which has previously been associated with resistance to P. infestans. By screening RXLR effector libraries of P. infestans and P. capsici, we identified multiple effectors recognised by both NLRs. Our findings highlight the complexity of NLR clusters and evolution driven by interactions with multiple pathogens. This work will underpin efforts to elevate resistance against Phytophthora pathogens and enhances our understanding of NLR evolution.
Plant germ cells are specified de novo from somatic tissues, yet how early anther progenitors initiate divergent germline and somatic fates remains unclear. Here, we generated a high-resolution single-cell transcriptomic atlas of Arabidopsis anther development, profiling 66,864 cells from early lineage specification through pollen maturation. We resolved major anther cell types and identified an early L2-derived intermediate population, termed the archesporial-derived state (Ar-d), marked by REM22, ER/ERL1/2, SPL/NZZ, and BAM1. Although classical anatomical studies define archesporial cells, primary sporogenous cells, primary parietal cells, and secondary parietal derivatives based on developmental position and inferred lineage relationships, our analysis showed that these early L2-derived intermediates remain globally similar, forming a shared progenitor-like transcriptional state before lineage stabilization. Trajectory reconstruction, RNA velocity, and module analyses revealed that germline and somatic trajectories first emerge through restricted branch-biased transcriptional programs, which are later amplified into robust lineage-specific identities. Mutant single-cell analyses defined two regulatory steps: SPL/NZZ establishes early branch-associated transcriptional programs within the Ar-d state, whereas EMS1 stabilizes the somatic/tapetal trajectory and restricts inappropriate enrichment of germline-associated states. Lineage-resolved analyses further defined maturation programs in germline, tapetum, middle layer, and endothecium cells, and identified LBD transcription factors required for normal pollen development. Together, our study reveals a cryptic mode of anther fate priming in which shared early L2-derived intermediates progressively transform limited branch-biased transcriptional programs into distinct reproductive and somatic cell identities through SPL-dependent priming and EMS1-dependent somatic/tapetal stabilization.
The endoplasmic reticulum (ER) is the largest intracellular membrane-bound organelle in eukaryotic cells, comprising an interconnected network of tubules and sheets. Lunapark (LNP) functions as an E3 ubiquitin ligase that targets RHD3, a dynamin-like GTPase essential for homotypic fusion of ER tubules, for proteasomal degradation, thereby modulating ER tubule stability. However, the regulatory mechanism governing LNP activity remains largely unknown. Here, we report the characterization of the rice oskish mutant, which exhibits aberrant aggregation of ER tubules and defective ER exit of seed storage proteins. OsKish is an ER-localized small protein that physically interacts with both OsLNPs and RHD3-like (RHD3L) protein. OsKish stabilizes OsLNPs by suppressing their auto-ubiquitination; concomitantly, OsKish attenuates the membrane fusion activity of RHD3L possibly by inhibiting its oligomerization. Altogether, our studies propose an OsKish-OsLNPs-RHD3L framework for fine-tuning homotypic ER tubule fusion in rice, providing mechanistic insights into the maintenance of ER architecture in plants.
Seed physical dormancy is a crucial adaptive trait conferred by the water-impermeable seed coat, particularly its specialized palisade layer. Although the structural components of this barrier, including the cuticle and cell wall polysaccharides, are well characterized, the molecular mechanisms coordinating their formation and remodeling remain poorly understood. Here, using single-nucleus RNA sequencing of Medicago truncatula, we identify a regulatory module essential for palisade layer formation and the establishment of physical dormancy. We show that MtKNOX4 interacts with cytoplasmic MtSAW1/2 proteins and facilitates their nuclear recruitment, thereby enabling the formation of functional nuclear MtKNOX4-MtSAW1/2 complexes. These complexes activate the expression of downstream target genes, including MtATT1 (encoding a fatty acid ω-hydroxylase belonging to the CYP86A subfamily of cytochrome P450 enzymes) and MtPAE8 (encoding a pectin acetylesterase). Thus, the MtKNOX4-MtSAW1/2 module coordinates two parallel barrier-forming pathways involving lipid polyester remodeling and pectin deacetylation, thereby reinforcing seed coat impermeability. Loss-of-function mutations in these regulators disrupt palisade layer integrity and impair physical dormancy. Collectively, our results reveal a regulatory module in which MtKNOX4-mediated nuclear recruitment of MtSAW1/2 links transcriptional regulation of lipid metabolism and cell wall remodeling to seed coat barrier formation, providing potential targets for tuning hard-seededness in legume crops.
High soil salinity poses a major threat to global crop yields. Although cytoplasmic stress granules are known to contribute to plant responses to salt stress, the function and regulatory mechanisms of nuclear condensates in rice during stress response have remained elusive. Here, we demonstrate that the RNA-binding protein OsGRP3 assembles into nuclear condensates in response to salt stress. OsGRP3 undergoes deacetylation, which promotes its liquid-liquid phase separation (LLPS) and the formation of nuclear condensates. These condensates specifically recruit the OsZHD1.1 splice variant of the transcription factor OsZHD1, but not OsZHD1.2. Mechanistically, OsGRP3 enhances the transcriptional activity of OsZHD1.1, leading to the upregulation of key salt stress-responsive genes, including OsHKT1;4 and OsSOS1, ultimately limiting shoot sodium accumulation and enhancing root sodium extrusion. Our findings identify deacetylation-driven OsGRP3 condensation as a mechanism that promotes OsZHD1.1 recruitment and transcriptional activation of salt-responsive genes, thereby contributing to Na+ homeostasis and salt tolerance in rice. This study provides a mechanistic framework for understanding how stress-responsive nuclear condensates coordinate transcriptional reprogramming in plants.
Land plant chloroplasts are characterized by a prominent morphological feature: the stacking of thylakoid membranes into grana, i.e. defining a continuous membranous structure surrounded by the stroma and folded into appressed grana and non-appressed domains, enclosing the thylakoid lumen. Photosystem II and its antenna system are concentrated, whereas Photosystem I and ATPase are located in the grana end membranes and, in the stroma, membranes connecting grana. Thylakoid stacking promotes the physical separation of Photosystems I and II, thereby ensuring a balanced flow of excitation energy between them. Through a genetic dissection of the light-harvesting complex subfamily Lhcb, we showed that removal of the antenna system serving Photosystem II completely abolished grana formation, whereas deletion of specific Lhcb subgroups reduced stacking by up to 40%. Lhcb5 alone was sufficient to partially restore stacking, whereas exclusive expression of Lhcb2 resulted in grana with an unusually large size, which disassembled upon illumination. Time-resolved fluorescence measurements revealed that the extent of excitation energy spillover from PSII to Photosystem I correlated with the level of Lhcb depletion, rather than with granal architecture. These findings provide genetic evidence for a functional link between Photosystem II antenna composition, lateral heterogeneity and excitation energy partition between photosystems.
Proximity labeling (PL) proteomics, primarily powered by engineered biotin ligases such as BioID and TurboID, has emerged as a transformative approach for mapping protein association networks and subcellular proteomes in living cells. By covalently biotinylating proteins within a nanometer-scale radius of a bait protein, PL captures transient, weak, and spatially restricted associations that often escape conventional affinity-based methods. However, applying PL in plants introduces distinctive challenges, including rigid cell walls that can limit biotin penetration, tissue-specific variation in substrate accessibility, enzyme temperature sensitivity, and the difficulty of removing excess free biotin after labeling. Here, we outline best practices for designing and implementing biotin ligase-based PL experiments in plant systems, drawing on experience across multiple species. We discuss key considerations, including enzyme selection, expression system design, fusion protein validation, biotin delivery strategies, protein extraction and enrichment, and mass spectrometry-based analysis. We also highlight emerging quantitative and conditional PL approaches that enable dynamic comparison of proxiomes across developmental stages, environmental conditions, and genetic backgrounds. Throughout, we emphasize the importance of rigorous controls, careful terminology, and orthogonal validation to ensure biologically meaningful interpretation of PL datasets. These guidelines aim to standardize experimental design and interpretation, facilitating reproducible and biologically meaningful PL studies in plant systems.
Although chromatin states are closely associated with epigenetic modifications, how they influence each other remains to be fully elucidated. In this study, we found that mutation of DECREASED IN DNA METHYLATION 1 (DDM1) in soybean leads to genome-wide chromatin homogenization, characterized by a loosening of heterochromatin and a tightening of euchromatin. This remodeling triggers 2 distinct molecular pathways: in heterochromatin, increased release of small RNAs activates the noncanonical RdDM pathway; while in euchromatin, transposable elements (TEs) are enriched with H3K9me2 modifications, which in turn activate the CMT2 pathway. Together, these pathways drive a pronounced burst of CHH methylation at the whole genome level. This epigenetic remodeling correlates with massive transcriptional dysregulation, upregulating stress responses and downregulating photosynthesis, linking local H3K9me2 changes on TEs, ultimately leading to severe phenotypes. Our findings reveal a unique, bidirectional pathway for CHH regulation in plants with complex chromatin states remodeling.
Plant immunity relies on dynamic modulation of phytohormone signaling to balance growth and defense, with auxin contributing to resistance against the necrotrophic pathogen Botrytis cinerea (B. cinerea). However, how plants rapidly and precisely regulate auxin biosynthesis during pathogen infection remains unclear. Here, we identified the nitrilases NIT1, NIT2, and NIT3, as key components of auxin biosynthesis during infection. Upon chitin perception, CERK1 associates with and phosphorylates nitrilases to stimulate the conversion of indole-3-acetonitrile (IAN) into indole-3-acetic acid (IAA), thereby activating auxin signaling and promoting resistance to B. cinerea. CRISPR/Cas9-generated nit1, nit2, and nit3 triple mutants (ntm) exhibit increased susceptibility to B. cinerea, whereas reintroduction of individual nitrilases restores resistance. IAN, similar to IAA, enhances resistance to B. cinerea in WT plants in a nitrilase-dependent manner. Furthermore, CERK1 phosphorylates NIT2 at a conserved threonine residue (T149), thereby enhancing nitrilase activity and stimulating IAA biosynthesis and auxin signaling during infection. Notably, nitrilase-dependent defense is likely conserved, as RNAi-silencing of the tomato nitrilase SlNIT impairs IAN-mediated resistance to B. cinerea. Collectively, our findings demonstrate that CERK1-mediated chitin signaling activates nitrilase-dependent IAA biosynthesis, directly linking pathogen perception to auxin-regulated defense.
Leaf angle is a key determinant of plant architecture and grain yield in rice, yet the complex genetic regulatory network remains unclear. Here, we reveal that the HD-ZIP III transcription factor LF1 mediates brassinosteroid (BR) signaling to modulate leaf angle. A miRNA165/166-resistant, gain-of-function lf1 mutant displays an enlarged leaf angle driven by elongated adaxial parenchymal cells and diminished abaxial sclerenchyma. At the transcriptional level, the BR-activated transcription factor OsBZR1 directly induces LF1, which in turn upregulates OsOFP8, forming an OsBZR1-LF1-OsOFP8 hierarchical transcriptional cascade that mediates BR signaling to regulate leaf angle development. At the post-translational level, LF1 protein stability is fine-tuned by reciprocal post-translational modifications, wherein OsMAPK6-mediated phosphorylation at threonine residue at position 148 promotes LF1 accumulation and transcriptional activity, directly counteracting its ubiquitination and degradation by the APC/CTAD1 complex. Genetic analyses demonstrate that LF1, OsMAPK6, and TAD1 function in a common pathway to regulate leaf angle development. Overall, our study establishes LF1 as a central hub coupling transcriptional and post-translational mechanisms to modulate BR-mediated leaf angle, providing targets for plant architecture improvement.
The demethylation status of pectin, a major plant cell wall polysaccharide, is regulated by pectin methylesterases (PMEs) and their endogenous inhibitors (PMEIs). While PME-mediated removal of methyl groups increases pectin susceptibility to enzymatic degradation and facilitates wall remodeling, this process is suppressed by PMEIs to strengthen the wall, preventing pathogen invasion. How biotrophic pathogens overcome this inhibitory defense remains unclear. Here, we demonstrate how N-glycosylation serves as a post-translational mechanism that preshapes a conserved smut effector, directing its adaptation toward a host-specific PMEI to facilitate colonization. We identify the Ustilago maydis N-glycosylated effector Nge1, which interacts with 2 host inhibitors, PMEI45 and PMEI46, to suppress their inhibitory function. This release of PME activity leads to highly demethylated pectin in the host cell wall that is prone to degradation, likely driving the cell wall loosening favored by pathogens. Crucially, we show that N-glycosylation of Nge1 is essential for its interaction with PMEI45, whereas the interaction with PMEI46 is glycosylation-independent. Engineering N-glycosylation sites into a naturally nonglycosylated Nge1 ortholog restored its ability to neutralize host PMEIs and functionally complement the U. maydis Δnge1 mutant. Our findings reveal that smut fungi utilize post-translational glycan modifications to fine-tune effector specificity, allowing them to overcome host-adapted PMEIs and manipulate plant cell wall dynamics in an ongoing plant-fungal interplay.
DELLA proteins are negative regulators of gibberellin (GA) signaling. Under GA-deficient conditions, DELLA accumulates in the nucleus and interacts with numerous transcription factors (TFs) via the GRAS domain. Recent studies have shown that DELLA interacts with TFs via the LHR1 subdomain and histone H2A via the PFYRE subdomain, respectively. It is essential for DELLA-mediated global transcription reprograming. DELLA exhibits strong transcriptional activity and acts as a co-activator of GAI-ASSOCIATED FACTOR1 (GAF1)/IDD2. The N-terminus of DELLA is necessary for strong transcriptional activity, but the regulatory activities and mechanisms of DELLA remain unclear. Therefore, we screened DELLA-interacting factors using the proximity biotinylation enzyme AirID and identified HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY1 (HAC1). HAC1-mediated acetylation of histone H3 at the GAF1 target chromatin was necessary for transcriptional activation by the GAF1-DELLA complex. Additionally, the HAC1-mediated transcriptional activity of DELLA was involved in regulating plant growth. Moreover, ETHYLENE RESPONSE FACTOR2 (ERF2) was identified as a growth repressor gene regulated by the GAF1-DELLA-HAC1 complex. Here, we showed that DELLA proteins regulate plant growth via HAC1-mediated transcriptional regulation of ERF2.
Faithful pollen wall formation requires tight control of both the amplitude and duration of tapetal transcriptional activity. However, how such dynamic restraint is achieved during anther development remains poorly understood. Here, we identify a phosphorylation-dependent regulatory module in which SBI-PP2A-C1/C5 constrains the functional activity window of the tapetal transcription factor bHLH089 to safeguard male fertility. PP2A-C1 and PP2A-C5 physically associate with bHLH089 and limit its transcriptional output by reducing bHLH089 phosphorylation and nuclear retention, thereby restricting its nuclear accumulation and effective activity. Genetic analyses reveal that precise PP2A-C1/C5 dosage is critical for pollen wall integrity, as both reduced PP2A-C function and PP2A-C1/C5 overexpression disrupt tapetal transcriptional homeostasis and compromise male fertility. Mechanistically, Ser28 and Ser58 function as conserved phosphorylation-dependent determinants of bHLH089 nuclear retention and are required for PP2A-C1/C5-sensitive regulation of bHLH089 activity. Together, these findings identify a phosphatase-mediated timing mechanism that integrates transcriptional feedback with post-translational control to constrain bHLH089-driven transcriptional amplification within a self-limiting tapetal activity window required for tapetal homeostasis, pollen wall formation, and male fertility.
Asynchronous floret opening hinders hybrid rice seed production, particularly between indica and japonica subspecies. Although jasmonate (JA) signaling regulates diurnal floret opening time (DFOT), the mechanism underlying structural changes in the lodicule as a swellable organ driving floret opening remains unclear. Here, we identified OsbHLH091 as a lodicule-specific transcription factor mediating JA-induced floret opening. The null mutants of OsbHLH091 exhibit cleistogamy (closed flowers) without defects in floral organ, while OsbHLH091 overexpression accelerates floral opening. Mechanistically, OsbHLH091 activates cell expansion genes, including EXPANSINs and CELLULOSE SYNTHASEs, and such activity is repressed by JAZ proteins, the JA signaling inhibitors. Increasing JA before anthesis degrades JAZs, which releases OsbHLH091 to trigger lodicule expansion. Natural variation in OsbHLH091 contributes to DFOT differences between subspecies: the japonica haplotype interacts more strongly with JAZs, thus delaying opening. Together, as a lodicule-specific regulator, OsbHLH091 not only reveals a previously unknown mechanism controlling floret opening but also provides a precise tool to synchronize DFOT across subspecies, enabling efficient indica-japonica hybrid breeding.
