Ulla Neumann

Meiotic recombination ensures accurate chromosome segregation and promotes genetic diversity by generating crossovers between homologous chromosomes ¹ . Although essential in most sexually reproducing organisms, recombination is variably regulated and can be absent in some lineages, a condition known as achiasmy ² . However, obligate achiasmy in both sexes of a sexual species has not been documented. Here we investigate Rhynchospora tenuis , a flowering plant with the lowest known chromosome number and inverted meiosis ³ . Combining genomics with molecular experiments, we show that R. tenuis undergoes obligate, genome-wide achiasmy in both male and female meiosis. Despite normal early meiotic axis formation, synapsis fails, crossovers are undetectable cytologically and genetically, and univalents persist at metaphase I. Haplotype-specific accumulation of transposable elements generates segregation distortion favouring the transmission of larger, repeat-rich chromosomes. Sexual reproduction is nevertheless retained: fertilization yields viable seeds only when translocation-compatible gametes meet, indicating strong post-meiotic selection against incompatible homozygous combinations. As a result, all surviving offspring are genetically identical, effectively maintaining heterozygosity by sexual reproduction with parental genotype restitution mimicking clonal reproduction. We propose that recombination loss, a low chromosome number, inverted meiosis and selection for compatible gamete combinations together enable faithful segregation and clonal-like inheritance despite sexual reproduction. These findings blur the boundary between sex and clonality, linking genome architecture, recombination loss and transmission bias.

Pseudomonas species, spanning both beneficial and pathogenic lifestyles, possess conserved mechanisms to modulate plant immunity. Nevertheless, the mechanisms by which commensal bacteria establish and maintain host colonization remain poorly understood. Here, we report the characterization of a Pseudomonas chagasin-like protease inhibitor (Cpi1), conserved across pseudomonads representing a novel class of membrane-anchored PLCP inhibitor. Unlike previously described secreted protease inhibitors, P. putida Cpi1 is a lipoprotein localized to the bacterial surface and outer membrane vesicles (OMVs), positioning it to selectively inhibit immune-related papain-like cysteine proteases (PLCPs) during host interactions. Functional assays demonstrated inhibition of maize PLCP activity in the nanomolar range, while cpi1 deletion and chagasin motif mutants exhibited significantly impaired early root colonization, particularly in the meristematic and elongation zones. Besides, lack of cpi1 resulted in an altered structure of a maize root-associated synthetic community. We hypothesize that, Cpi1 may protect critical bacterial surface proteins from cleavage by inhibiting plant proteases and thereby modulate the release of MAMPs, dampening host immune responses. Moreover, the release of Cpi1 via OMVs could further extend its function within the root periphery and the apoplast. Together, our results uncover a conserved, membrane-anchored mechanism among pseudomonads for subverting plant immunity and establishing host-microbe interactions.

Secondary cell walls (SCW) constitute the most abundant form of renewable plant biomass and are major sinks for atmospheric carbon. Their highly ordered patterns underpin specialized cell functions. In Cardamine hirsuta, the geometry of a polarly localized SCW in fruit endocarp b (endb) cells determines the mechanics of explosive seed dispersal. Yet, the genetic control of SCW synthesis and patterning in these specialized cells remains poorly understood. Here we show that CELLULOSE SYNTHASE 7 (CESA7) is required to synthesize SCW cellulose in endb cells. While lignin and xylan deposition occurs independently of cellulose patterning in cesa7 endb SCWs, the final geometry and layered organization of wild-type endb SCWs depend on CESA7. Cellulose serves as a scaffold for the organized assembly of SCW polymers, thereby maintaining the precise SCW patterns observed in endb cells of fruits and metaxylem cells in roots. Cortical microtubules guide the patterned deposition of cellulose, lignin and xylan in endb cells, creating SCW-depleted domains along cell edges that produce the specific hinged SCW geometry. Disrupting microtubules abolished this pattern and prevented explosive coiling of the fruit valves. Our findings show that microtubules and CESA7 shape the form and function of endb SCWs in exploding seed pods.

Secondary cell walls (SCW) constitute the most abundant form of renewable plant biomass and are major sinks for atmospheric carbon. Their highly ordered patterns underpin specialized cell functions. In Cardamine hirsuta , the geometry of a polarly localized SCW in fruit endocarp b (end b ) cells determines the mechanics of explosive seed dispersal. Yet, the genetic control of SCW synthesis and patterning in these specialized cells remains poorly understood. Here we show that CELLULOSE SYNTHASE 7 ( CESA7 ) is required to synthesize SCW cellulose in end b cells. While lignin and xylan deposition occurs independently of cellulose patterning in cesa7 end b SCWs, the final geometry and layered organization of wild-type end b SCWs depend on CESA7 . Cellulose serves as a scaffold for the organized assembly of SCW polymers, thereby maintaining the precise SCW patterns observed in end b cells of fruits and metaxylem cells in roots. Cortical microtubules guide the patterned deposition of cellulose, lignin and xylan in end b cells, creating SCW-depleted domains along cell edges that produce the specific hinged SCW geometry. Disrupting microtubules abolished this pattern and prevented explosive coiling of the fruit valves. Our findings show that microtubules and CESA7 shape the form and function of end b SCWs in exploding seed pods.

Florigen, encoded by FT genes, is synthesized in leaves and transported to the shoot apical meristem (SAM) to induce flower development ^1–3 . At the SAM, 14-3-3 proteins are proposed to act as receptors for FT protein and to mediate the indirect interaction between FT and the basic leucine zipper (bZIP) transcription factor FD to form the florigen activation complex (FAC) that activates transcription of flowering genes ^4–6 . Here we demonstrate a different mechanism of FAC assembly, diverse functions for the 14-3-3 proteins within the complex, and an unexpected spatiotemporal distribution of the FAC. We show that FT is not recruited by 14-3-3 alone, but that it interacts with the DNA–FD–14-3-3 complex through two interfaces, one of which binds DNA via the unstructured C terminus of FT. We also find that interaction of 14-3-3 proteins with the C terminus of phosphorylated FD reduces liquid phase condensation of the intrinsically disordered FD protein, allowing it to bind DNA, and that the 14-3-3 proteins strengthen DNA binding of FD by promoting dimerization, which ultimately results in the recruitment of FT. Unexpectedly, we also find that after FT movement to the shoot apex, FT and FD are co-transcribed in young floral primordia, forming a boundary with the suppressed bract and allowing formation of the FAC during the first stages of floral development. Our studies propose a new mechanism by which the florigen FT transcriptional complex is formed, and indicate distinct functions for the complex during SAM and floral primordium development.

Structural biology is fundamental to understanding the molecular basis of biological processes. While machine learning-based protein structure prediction has advanced considerably, experimentally determined structures remain indispensable for guiding structure-function analyses and for improving predictive modeling. However, experimental studies of protein complexes continue to pose challenges, particularly due to the necessity of high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy. Transient transformation of Nicotiana benthamiana has emerged as a promising expression system for recombinant protein production, offering advantages such as low operating costs, rapid cultivation, short experimental turnaround and scalability compared with other established platforms such as insect or human cell culture systems. Here we present a versatile protocol leveraging N. benthamiana for the purification and structural analysis of protein complexes of diverse origin and composition, exemplified by six oligomeric complexes ranging from ~140 to ~660 kDa, originating from plant, vertebrate, fungal and bacterial species. In most cases, purification only requires a single epitope tag, simplifying workflows and reducing complications that come with multitag and sequential affinity purifications. The protocol enables rapid application, allowing protein sample production in fewer than 7 days. Critical parameters influencing expression and purification efficiency include codon alteration, epitope tag selection and detergent supplementation.

Establishment of the apoplastic root barrier known as the Casparian strip occurs early in root development. In legumes, this area overlaps with nitrogen-fixing nodule formation, which raises the possibility that nodulation and barrier formation are connected. Nodules also contain Casparian strips, yet, in this case, their role is unknown. We established mutants with defective barriers in Lotus japonicus . This revealed that effective apoplastic blockage in the endodermis is important for root-to-shoot signals underlying nodulation. Our findings further revealed that in nodules, the genetic machinery for Casparian strip formation is shared with roots. Apoplastic blockage controls the metabolic source-sink status required for nitrogen fixation. This identifies Casparian strips as a model system to study spatially constrained symbiotic plant-microbe relationships.

Structural biology is an essential tool for understanding the molecular basis of biological processes. Although predicting protein structures by fold recognition algorithms has become increasingly powerful, especially with the integration of deep-learning approaches, experimentally resolved structures are indispensable for guiding structure-function studies and for improving modelling. However, experimental structural studies of protein complexes are still challenging, owing to, for example, the necessity for high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy (cryo-EM). The use of Nicotiana benthamiana leaves as a transient expression system for recombinant proteins has become an increasingly attractive approach as the plant is inexpensive to cultivate, grows rapidly, allows fast experimental turnaround and is easily scalable compared to other established systems such as insect cell cultures. Using N. benthamiana as an expression system, we present here a robust and versatile protocol for the purification of five heterocomplexes with sizes ranging from ∼140 kDa to ∼660 kDa consisting of immunoreceptors and their associated pathogen effectors, followed by electron microscopy. The plant-based protocol was applied to verify the structure of the insect cell-derived wheat Sr35 resistosome and to co-purify and co-resolve a ∼140 kDa homodimer of the AvrSr35 effector from the fungus Puccinia graminis f sp tritici ( Pgt ). In several cases, only a single epitope tag is needed for complex purification, reducing complications that come with multiple epitope tags and two-step affinity purifications. We identify codon usage, signal peptide fusion, epitope tag choice and detergents as critical factors for expression and purification of recombinant protein from N. benthamiana leaves.