The Plant Cell

3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) is an essential component of rhamnogalacturonan II (RG-II), a complex pectic polysaccharide required for plant growth and development. While most steps of the KDO biosynthetic pathway have been characterized in plants, KDO-8-phosphatase (KDO8Pase), the phosphatase responsible for converting KDO 8-phosphate (KDO8P) to KDO, remained unidentified. To identify this missing component, we performed gene co-expression analysis and identified At5g57440 (GPP2) as the primary candidate in Arabidopsis (Arabidopsis thaliana L.). Recombinant GPP2 protein exhibited KDO8P-specific phosphohydrolase activity in vitro. A GFP-tagged GPP2 protein was predominantly localized to mitochondria, consistent with the compartmentation of the subsequent step in KDO biosynthesis. Null mutants of GPP2 exhibited significant growth retardation under boron-limited conditions, in which expression of GPP2 and other KDO biosynthetic genes was up-regulated. The growth retardation was also observed in liquid culture in normal media, a condition that induces rapid growth and thus likely increases metabolic demand for KDO. Despite this growth defect, the KDO content per unit cell wall in gpp2 remained equivalent to that in wild-type plants. These results are consistent with the identification of GPP2 as the elusive plant KDO8Pase and suggest a model where KDO availability becomes the rate-limiting factor for cell wall production. Our findings complete the plant KDO biosynthetic pathway and provide new insights into the physiological significance of RG-II in cell wall biosynthesis. Significance statementThis study identifies the previously unknown plant KDO-8-phosphatase, thereby completing the biosynthetic pathway for KDO in rhamnogalacturonan II. Our findings demonstrate that KDO synthesis is up-regulated under boron deficiency, and its supply becomes a rate-limiting factor for cell wall formation.

Diversification of plant development has largely enabled land colonization and establishment of different ecosystems. This diversification relies on the gain/loss of cell-types and tissues, such as stomata, vascular tissue and functional roots. Likewise, diversification of immunity is thought to rely on expansion/contraction, followed by functional specification, of the different components of plant defense mechanisms. Although anatomical changes might result in altering the infection routes of pathogens and the cells and tissues they interact with, very little is known about the co-evolution of plant development and immunity. We have recently described that RNAi-dependent antiviral responses observed in the non-vascular Marchantia polymorpha are confined to leaf vasculature in Nicotiana benthamiana plants, suggesting repatterning of antiviral responses as result of the acquisition of developmental innovations. Here, we explored the genetic basis of that repatterning by establishing the basal immunity toolkit across different cell-types in the non-vascular Marchantia polymorpha and the vascular Arabidopsis thaliana. The results from our comparative transcriptomic studies show that while RNAi is the major antiviral defense across Marchantia cell-types, that configuration is only maintained in phloem companion cells in Arabidopsis leaves, suggesting that plant immunity might co-evolve with developmental diversification. Additionally, differential levels of RNAi expression in different cell-types correlate with their vulnerability to viral countermeasures, with companion cells been the most resilient to the presence of viral silencing suppressors.

Plant receptor-like kinases (RLKs) are involved in diverse processes, ranging from growth and reproduction to interactions with microbes. Variation in the extracellular domains delineates several RLKs subfamilies, including the malectin-like domain leucine-rich repeat receptor-like kinases (MLD-LRR-RLKs). Symbiosis Receptor-like Kinase (SymRK) is the prototypical member of MLD-LRR-RLKs and is required for microbial accommodation in host roots during root endosymbiosis. Yet, comparative phylogenetic analysis of SymRK orthologs in the broader context of MLD-LRR-RLK subfamily evolution remains limited. In this study, we examined the inventory, phylogeny and clade-specific evolutionary and transcriptional characteristics of this receptor group. SymRK and its closest homologs are present in most land plant lineages and group into four major clades and six additional species-specific clades. These clades can be distinguished by their evolutionary characteristics as either conserved with reduced gene copy number changes (including SymRK) or expanded and diversified, as observed in clade IV. Clade IV dynamics are largely driven by tandem gene duplications, which often arise within gene clusters. We further analysed the evolutionary characteristics of MLD-LRR-RLKs at the population level in Arabidopsis thaliana accessions. We found that some genes are conserved across accessions and are therefore likely to be functionally important, whereas a subset of genes, often located within tandem clusters, are highly diverse and likely contribute to accession-specific adaptations. Finally, most MLD-LRR-RLKs in the A. thaliana Col-0 accession are expressed in roots and respond broadly to biotic stimuli at the transcriptional level. Notably, clustered genes frequently exhibited divergent expression profiles, suggesting transcriptional diversification. Together, we revealed two contrasting evolutionary characteristics among members of the MLD-LRR-RLK subfamily, potentially associated with their functions in plants.

Plant architecture is a crucial component of maize productivity. Tailoring architectural component traits like leaf area and angle can increase productivity by promoting deeper light penetration into the canopy and better resource utilization. Novel genetic variants can increase the rate of gain for optimized plant architecture. Here, we map a moderate-effect mutation denoted reduced leaf area1 (rdla1) to the RAGGED5 (RGD5) locus and characterize it as a transposon insertion allele. Mutant leaf area reductions were most extreme in mid-upper canopy positions. Photosynthetic gas exchange rates were not significantly impacted in rdla1 relative to wild-type, indicating that mutant leaf structure, but not function, is altered. Functional annotations of RDLA1 were supported by metabolite profiles suggesting a role in cuticular wax biosynthesis. Introgression of the rdla1 allele into 27 commercially relevant genetic backgrounds identified differences in effect size across genotypes, revealing modifier effects that could serve as targets for modulating plant architecture.

In plant cytokinesis, the partitioning membrane is made by homotypic fusion of secretory vesicles, progressing in a centre-to-periphery direction. In Arabidopsis, this process is mediated by a cytokinesis-specific fusion machinery involving Qa-SNARE KNOLLE which is made during G2/M phase and degraded at the end of cytokinesis. Here we analyse how the turnover of KNOLLE protein is regulated. KNOLLE is ubiquitinated, which is best detected after combined treatment with inhibitors of endocytosis and de-ubiquitination. Site-directed mutagenesis of three clustered lysine residues prevented ubiquitination and internalisation, resulting in stable accumulation of KNOLLE at the plasma membrane in all cells of the seedling root. This is in stark contrast to the transient accumulation of wild-type KNOLLE in dividing cells only. Partial-substitution mutant lines revealed redundancy of lysine residues in both KNOLLE ubiquitination and turnover. KNOLLE ubiquitination resulted in K63-linked ubiquitin chains known to be involved in endocytosis whereas K48-linked chains were not detected. To explore the spatio-temporal conditions, we analysed KNOLLE ubiquitination in cis-SNARE and trans-SNARE complexes during membrane traffic and cell-plate formation. Our findings suggest that KNOLLE protein turnover is caused by a ubiquitination process that depends on successful membrane fusion generating the cell plate.

Aldoximes are amino acid-derived metabolites that serve as precursors of auxins and modulate phenylpropanoid production in Arabidopsis. However, the enzymes responsible for aldoxime production in Solanaceae remain unknown. Here, we report the identification of aldoxime-producing enzymes in tomato (Solanum lycopersicum) and examine how altered aldoxime production affects auxin production and phenylpropanoid metabolism. Through homology-based analysis, we identified five putative CYP79 homologs in tomato, among which SlCYP79DB32 and SlCYP79DB52 exhibited aldoxime-producing activity toward multiple amino acids, including phenylalanine and tryptophan. SlCYP79DB32 and SlCYP79DB52 converted phenylalanine into phenylacetaldoxime (PAOx), whereas only SlCYP79DB52 converted tryptophan into indole-3-acetaldoxime (IAOx). Stable isotope-labeled feeding experiments revealed that IAOx and PAOx can be converted to the auxins indole-3-acetic acid (IAA) and phenylacetic acid (PAA), respectively. Consistently, tomato plants engineered to overproduce IAOx and PAOx accumulated elevated levels of IAA and PAA. These plants also accumulated lower levels of phenylpropanoids. In Brassicaceae plants such as Arabidopsis and Camelina, aldoxime accumulation represses phenylpropanoid production by promoting degradation of phenylalanine ammonia-lyase (PAL). However, aldoxime accumulation did not reduce PAL activity in tomato, suggesting an alternative mechanism in this species. Transcriptome analysis revealed extensive transcriptional reprogramming in aldoxime-overaccumulating tomato plants, including upregulation of stress- and defense-related genes. Despite the observed reduction in phenylpropanoid levels, transcript levels of most phenylpropanoid biosynthetic genes were not decreased, suggesting possible post-transcriptional regulation of this repression. Together, our findings demonstrate that aldoximes can serve as intermediates in auxin biosynthesis in tomato and reveal that aldoxime-mediated repression of phenylpropanoid metabolism extends beyond Brassicaceae.

Plants, as sessile organisms, must continually adapt to fluctuating temperatures to ensure survival. The plasma membrane-localized receptor-like kinase FERONIA (FER) coordinates diverse physiological processes and responses to various biotic and abiotic stresses. However, the role of FER in plant adaptation to elevated temperatures remains largely unexplored. Here, we report that FER is indispensable for plant thermotolerance. We found that fer loss-of-function mutants exhibit impaired thermomorphogenic growth and are hypersensitive to mild heat stress, displaying extensive oxidative stress-mediated cell death at elevated temperatures. Combined genetic and molecular analyses revealed that these temperature-sensitive defects in fer mutants are caused by an overaccumulation of jasmonic acid (JA), which subsequently triggers excessive production of reactive oxygen species. Furthermore, we show that this aberrant JA accumulation and oxidative stress are attributable to impaired FER-mediated regulation of turgor-dependent cell wall tensile stress. Taken together, our results suggest that FER-mediated cell wall tensile stress regulation serves as a critical mechanism to prevent aberrant JA accumulation and oxidative stress at elevated temperatures, thereby enabling plants to adapt to and survive under high-temperature conditions.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pentose phosphate pathway, generating NADPH to sustain redox metabolism and signaling. However, whether individual G6PDH isoforms directly regulate oxidative stress signaling remains unclear. To determine the contribution of the different Arabidopsis G6PDH isoforms to oxidative stress signaling, we introduced single T-DNA mutants into the catalase-deficient cat2 background, a genetic system in which intracellular H2O2 production activates salicylic acid (SA)-dependent cell death and defense pathways. Interestingly, impairment of cytosolic, but not chloroplastic G6PDH activity suppressed cat2-triggered phenotypes, with loss of G6PD5 function fully abolishing lesion formation. The cat2 g6pd5 double mutant phenocopied the SA biosynthesis-deficient mutant cat2 sid2 and showed reversion of defense responses as well as metabolomic and transcriptomic profiles to the wild-type state. Strikingly, despite the suppression of SA-dependent lesions, loss of G6PD5 activity does not appear to reduce stress intensity. On the contrary, cat2 g6pd5 plants exhibit increased glutathione synthesis and oxidation, elevated expression of oxidative stress marker genes, and enhanced accumulation of reactive nitrogen species relative to cat2. Protein-protein interaction analyses revealed that G6PD5 associates with several redox and defense-related proteins. In particular, we confirmed a physical interaction between G6PD5 and thioredoxin h5, a key component of redox-dependent SA signaling. However, analysis of cat2 trxh5 and cat2 npr1 lines indicated that this interaction alone cannot explain the G6PD5-dependent control of SA responses. Our work reveals that cytosolic G6PD5 integrates redox metabolism with immune signaling to control plant responses to oxidative stress.

In tomato plants, the potassium (K) transporter SlHAK5 is integral to root K uptake and overall plant fertility. Under K deficiency, SlHAK5 expression is induced in roots and the encoded transporter is activated via the Ca{superscript 2}-sensing CIPK/CBL complex SlCIPK23/SlCBL1-9. In Arabidopsis, multiple CIPK/CBL complexes can activate AtHAK5, providing alternative regulatory pathways that enhance K uptake. However, the architecture of CIPK/CBL signaling networks has diverged among plant species, necessitating species-specific identification of novel regulatory components. Accordingly, we screened additional tomato CIPK proteins for their capacity to modulate SlHAK5 activity in yeast. SlCIPK15 and SlCIPK26 emerged as potent activators of SlHAK5, acting in concert with SlCBL9. Functional characterization of slcipk15 and slcipk26 mutants revealed that neither contributed significantly to SlHAK5-mediated K uptake in roots. Conversely, both mutants exhibited impaired pollen tube elongation, correlating with reduced K content in pollen relative to wild type. Notably, slcipk26 mutants displayed more severe pollen defects, phenocopying the slhak5 mutant. Further analyses demonstrated that slcipk26 plants suffered compromised seed set and pistil function, paralleling the reproductive deficiencies observed in slhak5 mutants. These findings implicate SlCIPK26 as the principal regulator of SlHAK5 in reproductive tissues. Collectively, our data underscore the role of CIPK paralogs in orchestrating tissue-specific regulation of target proteins, thereby enabling fine-tuned modulation of K transport essential for both vegetative and reproductive development.

The uptake of nitrogen-fixing bacteria into living plant cells and the intracellular accommodation of arbuscular mycorrhiza (AM) fungi requires the plasma membrane-localised Symbiosis Receptor-like Kinase (SymRK). AM is widespread across terrestrial vascular plant lineages, while the nitrogen-fixing root nodule symbiosis (RNS) is restricted to one clade within the eurosids. This distribution led to the concept that SymRK was adopted during evolution to mediate RNS. Comparative analyses revealed that SymRK orthologs from the eurosid clade support RNS while SymRK from the phylogenetically distant species Solanum lycopersicum (tomato) does not. To dissect the molecular basis for this different functionality, we carried out complementation analyses of the Lotus japonicus symrk-3 mutant which is unable to form AM or RNS. Domains swap chimera from the tomato and L. japonicus SymRK orthologs revealed that the intracellular domain of L. japonicus SymRK is necessary and for cortical infection thread (IT) and symbiosome development at 21 days post inoculation. Notably, this signalling specificity could be overcome by ectopic expression of tomato SymRK, pointing to altered protein dosage as a potential determinant of function. Consistent with this idea, SINA family E3 ubiquitin ligases interacted with and ubiquitinylated L. japonicus SymRK, but not tomato SymRK. In yeast two hybrid analysis, the interaction of SymRK with SINA2 and SINA4 depended on the C-terminal intrinsically disordered tail region of L. japonicus SymRK. We conclude that the SymRK intracellular domain evolved interaction capabilities with SINA E3 ligases which correlates with its ability to support RNS.

Diurnal changes in light availability are a defining feature of life on Earth. Photoautotrophic organisms therefore store reduced carbon during the day to sustain energy metabolism at night. In cyanobacteria, glycogen is the primary carbon storage compound and supports both energy homeostasis and stress responses. Although glycogen-deficient Synechocystis strains have been studied previously, how these mutants cope with the loss of the major daytime carbon sink and can sustain themselves during the night remains unclear. Using single-cell microfluidics, transcriptomics, and metabolomics, we show that {Delta}glgC mutants exhibit pronounced light sensitivity. At sub-lethal light intensities, daytime transcriptional responses are dominated by downregulation of photosynthesis-related genes, likely preventing NADPH overaccumulation in the absence of a carbon sink. During the night, mutants display severe energy limitation, characterized by reduced ATP levels, altered redox balance, and depletion of central carbon intermediates. In contrast, fumarate and malate accumulate, indicating enhanced respiratory flux through succinate dehydrogenase. These metabolic constraints lead to extended lag phases and delayed cell divisions after the onset of light, demonstrating that glycogen-deficient cells fail to efficiently reinitiate growth after dawn. Overall, our results as a snapshot of the initial response to diurnal regimes highlight glycogen as a central integrator of diurnal physiology in Synechocystis, coordinating energy metabolism, redox balance, and cell division, with implications for metabolic robustness and the evolutionary constraints shaping (endo)symbiosis. Short summaryGlycogen deficiency disrupts day-night energy and redox homeostasis in Synechocystis, revealing constraints on growth, division, and symbiotic potential.

Expression of the chloroplast AAA+ chaperone CLPD gene increases during senescence and drought, but its functional role in chloroplast proteostasis is poorly understood. This study provides a comprehensive analysis of Arabidopsis CLPD protein accumulation across development from early seedlings to senescence, and compares results to its homologs CLPC1,2, as well as CLPB3 and cpHSP90. The developmental consequences of complete loss of CLPD expression (clpd-1), as well as overexpression of functional CLPD or CLPD impaired in ATP hydrolysis (CLPD-TRAP), were determined in Arabidopsis. clpd-1 has accelerated seedling development while functional CLPD overexpression lines, but not CLPD-TRAP, have delayed development. To determine if CLPD is a bona fide CLP chaperone associating with the CLPPRT protease and to identify in vivo candidate substrates, we employed the CLPD-TRAP line during the vegetative and flowering (senescent) growth stages. Affinity purification of CLPD-TRAP followed by mass spectrometry showed high enrichment of the CLP protease complex, thus providing direct support for the role of CLPD in substrate delivery to the CLP protease. CLPC1,2 were also highly enriched in the CLPD-TRAP interactome, suggesting hetero-oligomerization and cooperation between the three chaperones is likely. Nine chloroplast candidate substrates were identified in the CLPD-interactomes, including: FHY2 involved in riboflavin synthesis, THI1 and THIC involved in thiamin metabolism, and four proteins of unknown function. Several of these have been previously identified as potential CLPC1 substrates; however, others appear to be specific to CLPD. CLPD acts in substrate selection within a heteromeric CLPC-CLPD hexamer, likely to make unique contributions through its divergent N-terminus.

Toll/interleukin-1 receptor (TIR) enzymes are prominent immune components in diverse organisms across the tree of life. In flowering plants, TIRs are often integrated into nucleotide binding leucine-rich repeat (NLR) receptors whose oligomerization-dependent biochemical activities create second messengers perceived by ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-family signaling complexes. TIR-NLRs and TIR proteins are present across the full spectrum of plant evolution, yet EDS1 signaling is a derived trait in seed plants. Here, we examined the functional dependency of diverse plant TIRs on the EDS1 pathway in the angiosperms Nicotiana benthamiana and Nicotiana tabacum. While the isolated TIR domains from non-seed plants generally required EDS1 for immune cell death activation, we also identified TIRs that functioned independent of EDS1. However, chimeric TIR-NLRs incorporating these diverse TIR domains onto the AtWRR4a receptor chassis showed a full reversion to EDS1-dependency. Extending this phenomenon further, we demonstrated that the AtWRR4a architecture enforces EDS1-dependence onto a bacterial TIR domain that is otherwise EDS1-independent. Collectively, our work demonstrates that NLR immune receptor architecture influences TIR-related immunity and provides further context to their ancient acquisition into plant immune systems.

Salicylic acid (SA), a central hormone in plant immunity, is biosynthesized via a recently elucidated phenylalanine-derived pathway in most seed plants. This pathway requires benzyl alcohol as a key substrate for the formation of the SA precursor benzyl benzoate. However, how benzyl alcohol is produced in plants was unclear. Here, we identify a two-step conversion of benzoyl-CoA to benzyl alcohol via benzaldehyde in Nicotiana (N.) benthamiana. From a forward genetic screen for SA-deficient mutants, the and {beta} subunits of heterodimeric benzaldehyde synthase (BalS) involved in the conversion of benzoyl-CoA to benzaldehyde were found to be required for SA biosynthesis in N. benthamiana. Further reverse genetic analysis revealed that the NADPH-dependent benzaldehyde reductase (BalR1) acts downstream of BalS to convert benzaldehyde to benzyl alcohol. Interestingly, OsBalR1, but not OsBalS or OsBalS{beta}, is required for maintaining high basal SA levels in rice, suggesting the presence of redundant benzoyl-CoA-reducing activities or alternative biosynthesis routes for benzyl alcohol production. Together, this work defines the missing enzymatic steps in phenylalanine-derived SA pathway and provides insights into the evolutionary diversification of SA production strategies in plants.

The COP1/SPA ubiquitin ligase is a key repressor of photomorphogenesis that is inactivated by photoreceptors to initiate light signalling. The four SPA proteins (SPA1-SPA4) confer functional specificity to COP1 during plant growth, yet the underlying molecular mechanisms remain unclear. Here, we used a domain-swap approach in transgenic seedlings to address the functional divergence of SPA2 and SPA3. We show that the respective N-terminal kinase domain determines the contrasting protein stabilities of SPA2 and SPA3 in light-grown seedlings. The instability of SPA2 correlates with a specific ability of the SPA2 N-terminal domain to bind phytochrome A in the light, suggesting that phytochrome A promotes the CUL4DET1/COP1-dependent degradation of SPA2 but not of SPA3. We uncover that the coiled-coiled and WD-repeat domains of SPA2 and SPA3 substantially differ in their activity in repression of photomorphogenesis, with those of SPA2 being more active repressors than those of SPA3. Thus, SPA2 combines a potent repressor activity with light-induced instability. We conclude that the evolution of SPA2 instability in response to light counterbalances its inherent strong repressor activity, thereby allowing seedling etiolation in darkness followed by rapid reduction in COP1 activity through SPA2 degradation upon light-exposure as seedlings emerge from soil to initiate photosynthetic growth. HighlightThe repressor of light signaling SPA2 combines a phytochrome A-interacting instability domain with a potent repressor domain to allow greatly contrasting activities of COP1 in skoto- and photomorphogenesis.

Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeqcyt/pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

Grain legumes such as pea (Pisum sativum L.) accumulate large amounts of seed storage proteins without nitrogen fertilization due to their symbiosis with nitrogen-fixing bacteria, making them a key source of plant-based proteins. Seed growth and the accumulation of seed storage proteins are tightly regulated by complex gene networks; however, the mechanisms governing these processes in pea remain poorly understood. In this study, we generated a comprehensive seed expression atlas covering six developmental stages in pea (cv Cameor), including the key transition stage from embryogenesis to early seed filling, providing a detailed temporal resolution of transcriptional dynamics throughout seed development in this species. Co-expression network analysis highlighted several candidate transcription factors potentially involved in the transition towards seed filling. Among them, we characterized the seed-specific NF-YB transcription factor PsLEC1-like (PsL1L), the major LEC1-type factor expressed during early pea seed development. Functional analyses using TILLING mutants demonstrated that loss of PsL1L function reduces seed size and seed nitrogen content and impairs early embryo growth from the end of embryogenesis. Finally, we show that the expression of the B3-domain transcription factor PsFUS3, but not that of PsLEC2 or PsABI3, is reduced in the loss-of-function l1l mutant, suggesting that PsL1L acts upstream of PsFUS3 to control seed size.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

Plant immunity can be activated by membrane-localized pattern recognition receptors or by cytosolic nucleotide-binding leucine-rich repeat (NLR) receptors, while it can be counteracted by pathogen secreted effectors. Manifestation of immunity often involves endomembrane traffic. However, limited evidence is available for such an involvement in NLR-mediated immunity, which typically includes a hypersensitive reaction (HR)-programmed cell death response. Here we show that the barley powdery mildew fungus uses several effectors to target and inhibit the vacuolar trafficking pathway, which causes endomembrane markers to stall in the endoplasmic reticulum (ER). We used this ER-stalling phenotype as a proxy to track fungal manipulation of the vacuolar pathway during different stages of the actual infection. Our data indicate that powdery mildew fungi interfere with the vacuolar pathway, but only temporarily, as the stalling is lifted once the fungal haustorial feeding structures are well-developed for nutrient uptake in epidermal cells. Notably, we show evidence that blocking the vacuolar pathway causes a general inhibition of NLR-mediated HR, and that this mechanism is taken advantage of by the pathogen. Finally, we provide an example that the fungus can secrete a different set of effectors at the haustorial stage that result in the re-opening of the vacuolar pathway by inhibiting the initial vacuolar traffic-suppressing effectors.

Development of arbuscular mycorrhiza (AM), a symbiosis between plants and beneficial Glomeromycotan fungi, is largely under plant control. Several genes, required for AM development, are proposed to be regulated by the karrikin signalling module, comprising the alpha/beta hydrolase receptor KARRIKIN INSENSITIVE 2 (KAI2), the F-box protein MORE AXILLARY GROWTH2 (MAX2) and the transcriptional repressor SUPPRESSOR OF MAX2 1 (SMAX1), which is ubiquitylated for proteasomal degradation upon KAI2-ligand-induced binding to the KAI2-MAX2 complex. Rice and Brachypodium distachyon kai2 mutants are incapable of forming AM. Here, we show that in Lotus japonicus, Pisum sativum, and Nicotiana benthamiana, KAI2 only quantitatively affects AM development, indicating angiosperms vary in their requirement for KAI2-signalling to support AM. Comparative transcriptomics of L. japonicus and B. distachyon roots after treatment with fungal signalling molecules revealed some AM-relevant genes respond KAI2-independently in L. japonicus but not in B. distachyon. Consistently we obtained evidence for low-level degradation of SMAX1 in Ljkai2a,b observed through a ratiometric reporter for the SMAX1 degron (SMAX1D2). Further, we found an unexpected accumulation of SMAX1D2 in in response to AM even in wild type. Together, this suggests an unexpected role of SMAX1 accumulation in AM roots and that in AM symbiosis of L. japonicus, redundant mechanisms drive SMAX1 degradation and gene activation independently of KAI2.