Abstract Intracellular accommodation of beneficial fungi requires controlled remodeling of host cell walls while avoiding activation of plant immune responses. During colonization of monocot roots, the endophytic fungus Serendipita indica induces a suite of carbohydrate-active enzymes (CAZymes) targeting xylan and cellulose. Weighted gene co-expression network analysis identified the basidiomycete-specific transcription factor Si ROCX as a key regulator of this monocot-adapted program. DNA affinity purification sequencing (DAP-seq) defined the Si ROCX binding motif and revealed a CAZyme-enriched target regulon. Inducible Si ROCX overexpression selectively activated motif-containing CAZyme genes and markedly enhanced xylanase and cellulase activities on xylan and native barley root cell walls. Premature activation of this program triggered expression of a plant immune marker despite comparable fungal biomass, indicating that precise temporal control of CAZyme deployment is required to maintain symbiotic compatibility. Together, these findings identify Si ROCX as a central regulator of a monocot-adapted cell wall–degrading secretome and reveal a basidiomycete-specific regulatory module that coordinates host cell wall remodeling with immune-compatible symbiotic colonization. Significance Beneficial fungi colonize living plant cells, requiring host cell wall remodeling while avoiding immune activation. However, the key transcriptional regulators coordinating this process in basidiomycetes during symbiotic root colonization remain unknown. We identify Si ROCX, a conserved basidiomycete-specific transcription factor, as a master activator of a monocot-adapted xylan/cellulose degradation program in the root endophyte Serendipita indica . By integrating in planta co-expression networks, DAP-seq, secretome proteomics, and enzymatic assays, we show that Si ROCX overexpression markedly enhances secretion and activity of xylan- and cellulose-degrading enzymes, boosting sugar release from monocot cell walls. These findings reveal a basidiomycete-specific regulatory module for immune-compatible host cell wall remodeling, providing a framework to engineer fungal CAZyme programs for crop symbiosis and biomass conversion.

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.

Hemicelluloses are a group of plant cell wall polysaccharides characterized by their high structural complexity. These glycans are part of an intricate composite polymer network that contribute to the mechanical strength and flexibility of plant cell walls. Hemicellulose structural and functional diversity is further enhanced by the presence of chemical modifications, such as O-acetylation, altering the polysaccharide’s physicochemical properties and the overall functionality. Plant-derived hemicellulose glycans hold great promise for a range of biotechnological applications in a bioeconomy including biomaterials and pharmaceuticals. Synthetic biology approaches have the potential to produce hemicellulose polymers in microbial factories replicating the biosynthetic pathways observed in plants. In this study, we successfully reconstructed in the yeast Yarrowia lipolytica the biosynthesis of two hemicellulose backbone structures i.e., β-glucomannan (GM) and β-glucan, by the expression of glycosyltransferases of diverse plant origins. Oligosaccharide mass profiling combined with compositional and glycosidic linkage analysis confirmed the production of hemicellulose structures analogous to those found in the original plant systems. Furthermore, the additional expression of plant hemicellulose-specific O-acetyltransferases resulted in the biosynthesis of O-acetylated GM and O-acetylated glucan polymers, expanding the repertoire of hemicellulose structures produced in this yeast. These findings demonstrate the feasibility of generating not only compositionally diverse plant-like hemicellulose backbone polymers in microbial systems, but also more structurally complex O-acetylated variants beyond what is found in nature. The use of Y. lipolytica as a biofactory for designer glycans expands the potential of microbial glycoengineering and provides a platform for sustainable production of functionalized polysaccharides with tailored physicochemical properties optimized for specific biotechnological applications.

Intracellular accommodation of beneficial fungi requires host cell-wall remodeling that avoids excessive immune activation. The root endophyte Serendipita indica, a generalist mutualist capable of colonizing both monocot and dicot plants, employs a monocot-specific enzymatic module to deconstruct acetyl-xylan, the dominant hemicellulose of grasses. Central to this module are the glycoside hydrolase SiGH11, which releases acetylated xylooligosaccharides, and SiAXE, a previously uncharacterized SGNH-like acetyl-xylan esterase that sequentially removes acetyl groups from soluble xylooligosaccharides. Both enzymes are co-expressed within a monocot-enriched transcriptional program that also includes sugar transporters and metabolic regulators. Their coordinated activity, together with co-expressed exo-enzymes, promotes efficient xylan hydrolysis while limiting the prolonged accumulation of immunogenic damage-associated molecular patterns. Functional genetics demonstrated that SiAXE is required for sustained intracellular growth in monocot roots: its deletion impaired colonization, whereas overexpression transiently accelerated entry but provoked immune responses, underscoring the importance of temporal regulation and enzyme coordination for immune-compatible colonization. These findings provide mechanistic insights into an immune-compatible fungal strategy for host cell-wall remodeling and reveal how a broadly colonizing mutualist has repurposed ancestral saprotrophic enzymes into specialized host-adapted modules that balance nutrient acquisition with immune modulation.

Abstract Explosive seed dispersal distinguishes Cardamine species from Arabidopsis and depends on polarized secondary cell wall (SCW) deposition in fruit endocarp b (end b ) cells. How this SCW pattern is specified and environmentally modulated remains unclear. The polyploid Cardamine chenopodiifolia produces explosive aerial fruit and non-explosive subterranean fruit, creating a tractable system to address this problem. We show light triggers underground fruit to explode by reprogramming end b SCW patterning from uniform to polar. We identify the HD-ZIPIII transcription factor REVOLUTA as a central regulator of end b cell fate, SCW formation, and organ polarity in Arabidopsis and Cardamine hirsuta . In C. hirsuta , duplicated REVOLUTA paralogs are required for end b SCW deposition, while other HD-ZIPIII genes contribute redundantly to cell fate and organ polarity. REVOLUTA over-expression converts polar end b SCWs to uniform, producing non-explosive fruit. Together, these findings reveal a tunable developmental module underlying evolutionary transitions between explosive and non-explosive seed dispersal strategies.

SUMMARY O- Acetylation is the most abundant xylan decoration in eudicot plants and plays a critical role in determining xylan conformation and its interactions with cellulose and lignin, thereby contributing to secondary cell wall (SCW) integrity. In Arabidopsis , loss of the xylan O -acetyltransferase TBL29/ESK1 causes collapsed xylem and growth defects that can be suppressed by mutations in strigolactone (SL) biosynthesis genes such as MAX3 . However, the molecular basis of this suppression remains unknown. Hypoacetylated xylan in tbl29 has a higher frequency of methyl glucuronic acid (MeGlcA) substituents, while the ratio of GlcA/MeGlcA is recovered in tbl29 max3 . Furthermore, gene expression analyses reveal that the three xylan glucuronoxylan methyltransferases (GXM1/2/3) involved in xylan MeGlcA modification are upregulated in tbl29 SCWs but downregulated in tbl29 max3 . Genetic analysis shows that the transcription factor MYC2 is required for max3 -mediated suppression: the loss of MYC2 in tbl29 max3 prevents growth recovery and reverts GXM genes expression and xylan MeGlcA substitution levels. We propose a model where SL deficiency enhances MYC2 transcription, which in turn represses GXMs , thereby fine-tuning xylan methylation and re-establishing the MeGlcA/GlcA substitution balance under conditions of reduced O -acetylation. Our findings identify a MYC2-dependent regulatory module linking SL signalling to xylan methylation and reveal a genetically encoded compensatory mechanism that mitigates the consequences of defective xylan O -acetylation. More broadly, this work demonstrates that plants can preserve SCW function through adaptive remodelling of polysaccharide substitution patterns, highlighting an unexpected plasticity in SCW biosynthesis. Significance Statement Secondary cell wall integrity depends on the coordinated modification of xylan. We show that defects caused by reduced xylan O -acetylation can be alleviated through a strigolactone- and MYC2-dependent pathway that alters xylan methylglucuronidation. Rather than restoring the original wall composition, this mechanism appears to compensate for the loss of O -acetyl groups by remodelling polysaccharide substitution patterns to maintain cell wall function, revealing a new layer of plasticity in secondary wall biosynthesis.

Brazilwood (Paubrasilia echinata) is an endangered species endemic to the Brazilian Atlantic Forest. It was historically overexploited for red dye extraction and for crafting bows for stringed instruments. Its distinctive extractives and mechanical properties confer high tonal quality, durability, and resistance to fungal and termite decay. In this study, the cell wall chemical composition of brazilwood was compared with that of eucalyptus, angico-preto (Anadenanthera macrocarpa), and pine to identify traits that may be related to its quality and resistance. Analyses include fractionation, Maule staining, GC/MS, and HPAEC/PAD. Brazilwood sapwood shows a lower syringyl-to-guaiacyl (S/G) ratio than heartwood. It also has higher lignin content and lower polysaccharide extractability, indicating a highly cross-linked lignin-carbohydrate complex. Heartwood lignin composition resembles that of angico-preto, a decay-resistant species. However, brazilwood hemicelluloses, mainly arabinoxylans, are less tightly associated with cellulose. The lack of detectable mannose indicates that glucomannans are a very minor component, distinguishing brazilwood from typical hardwoods and softwoods. Pectic fractions contain lower rhamnose and galactose levels. Xylose dominates the alkali-extracted fractions, indicating poorly branched xylans. Although brazilwood shares traits with angico-preto, differences in cell wall composition may be associated with enhanced mechanical performance and decay resistance, potentially contributing to its distinctive properties.

Fungi engage in associations with other organisms across a continuum from pathogenic to mutualistic lifestyles. Hence, they require a compendium of molecular capacities, including partner recognition, extracellular signaling, nutrient exchange, immune modulation, and control of microbial competitors. In filamentous pathogens such traits are frequently associated with compartmentalized genomes, including rapidly evolving secreted proteins known as effectors and expanded receptor families, but it remains unclear whether similar genomic principles shape mutualistic fungal symbioses. Here, we generated a chromosome-scale super-pangenome for the lichen-forming genus Peltigera, comprising 41 mycobiont assemblies representing eleven species, together with genomes of associated Nostoc and, in tripartite species, Coccomyxa photobionts. The mycobiont genomes revealed extensive variation in genome size, transposable element content, biosynthetic gene clusters, and lineage-specific gene content, with pronounced expansions in tripartite species. Across Peltigera, secreted protein encoding genes were preferentially located in TE-rich regions. We further identified Starship-like transposon elements, expanded antimicrobial protein repertoires, and a large, previously underestimated repertoire of fungal GPCRs dominated by Pth11-like receptors. Layer-specific transcriptomics of a P. rufescens thallus showed differential expression of several interaction-associated gene families, e.g. lectins, antimicrobial proteins and Pth11-like GPCRs. These data indicate that pathogenic and mutualistic fungi might exhibit shared genomic principles, including genome compartmentalization, mobile-element-associated diversification, and the expansion of molecular repertoires involved in recognition, extracellular control and signaling.

The interactions between hosts and their microbial symbionts play a crucial role in shaping biological diversity and ecosystem function. Bacteria can adapt to specific host environments over evolutionary timescales, leading to co-speciation and the formation of specialized host-microbe relationships. Understanding the molecular mechanisms underlying these adaptations provides key insights into the evolution of symbiosis and the stability of microbial communities. This study investigates the co-speciation and molecular adaptations of Curvibacter to its host Hydra , a well-established model for the study of host-microbe interactions. We provide strong evidence of co-speciation, as demonstrated by phylogenetic congruence between different Hydra species and their corresponding Curvibacter symbionts, along with preferential recolonization of germ-free Hydra by their native Curvibacter strains. Comparative genomic analyses reveal that host-associated Curvibacter strains exhibit distinct metabolic and biosynthetic adaptations compared to their free-living relatives. Specifically, the enrichment of proteins involved in sugar metabolism and transport, as well as the selective purification of proteins linked to macromolecule biosynthesis, highlights the specialization of Curvibacter symbionts within the Hydra glycocalyx. Functional experiments identify a symbiont-specific extracellular polymeric substances (EPS) operon as key factor for microbial adhesion and host colonization, underscoring its role in facilitating symbiont specificity and stability. These findings provide insights into the molecular mechanisms driving host-microbe co-evolution and highlight the evolutionary forces shaping microbial specialization within host-symbiont relationships. Significant Statement The vast majority of animal and plant species are associated with microbial organisms that offer a wide range of benefits for development, physiology, and health. Understanding the evolutionary forces that shape these symbiotic interactions is crucial for elucidating the mechanisms through which organisms mitigate environmental stress and enhance their resilience, particularly in response to rapidly changing environmental conditions. Using advanced bioinformatic, bioanalytical, and genetic approaches, this study demonstrates that Curvibacter symbionts have evolved specific extracellular polymeric substances to enhance their ability to form a specific symbiosis with Hydra . ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, FR 3041/3-1, 517563163, 390686111

Core members of the fungal root microbiota include pathogens capable of colonizing multiple hosts, yet the underlying genetic determinants remain unknown. We report that Plectosphaerella cucumerina is a core member of the Arabidopsis thaliana root microbiota displaying high pathogenic potential and multi-host colonization capabilities. Establishment of a Plectosphaerella reference culture collection, followed by whole-genome sequencing of 72 strains reveals subtle phenotypic and genotypic variation that associate with fungal phylogeny, but not host plant identity. Transcriptome profiling of a model P. cucumerina isolate in roots of multiple hosts identifies core and host-specific fungal processes linked to carbon catabolism and root cell wall deconstruction of the hosts. A fungal gene encoding a candidate β-1,3-glucanase (GH64) was identified as a key genetic factor driving infection and disease in plants that diverged 110 million years ago. The gene is enriched in plant-colonizing fungi and consistently functions as a disease determinant in the root pathogen Colletotrichum incanum . We conclude that diverse and tunable fungal repertoires of carbohydrate-active enzymes act as disease determinants and drive multi-host compatibility belowground. ### Competing Interest Statement The authors have declared no competing interest. European Research Council consolidator grants, MICRORULES (758003), MICROBIOSIS (101089198) Cluster of Excellence on Plant Sciences (CEPLAS), 390686111 Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)- SPP DECRyPT 2125 Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)- HA 8169/2-2 A.K.B was funded by Polish National Agency for Academic Exchange NAWA J.N. was funded by JST SPRING grant, JPMJSP2108 K.H. was funded by JST grant, JPMJAN23D4 J.G.M-V. is funded by Beatriz Galindo program

The plant cell wall is a crucial structure that ensures plant cell integrity and facilitates environmental adaptation. Cellulose is the primary component of the plant cell wall. Its biosynthesis is orchestrated through the plasma membrane-localized multiprotein cellulose synthase complex, which includes a membrane-anchored endo-1,4-ß-glucanase. Here, we identified a barley (Hordeum vulgare) mutant with short roots resulting from repressed cell division and elongation, which we designated H. vulgare endo-β-1,4-D-glucanase 3-1 (hvglu3-1). HvGLU3 encodes a putative membrane-anchored endo-1,4-ß-glucanase that is highly conserved across plant species. The hvglu3-1 mutant exhibited a 60% reduction in cellulose content, accompanied by changes in hemicellulose and suberin levels and an altered lignin structure in the roots. Subcellular localization analyses and bimolecular fluorescence complementation assays suggested a direct interaction between HvGLU3 and primary cellulose synthases. We investigated the reprogramming of the tissue-specific transcriptome in hvglu3-1 root tips using a combination of laser capture microdissection and RNA sequencing. This approach revealed that 74% of all genes that are actively expressed in the elongation zone are influenced by root cellulose biosynthesis. Gene coexpression analyses highlighted the essential role of cellulose biosynthesis in diverse biological processes, including cell wall organization, phytohormone signaling, and stress responses, to regulate root tissue development. Overall, our study demonstrates the partially conserved role of HvGLU3 in controlling cellulose biosynthesis in roots and provides valuable transcriptomic resources for future studies.

Cellulose, the most abundant polymer on this planet, is widely produced by plants and many bacterial species. Certain cyanobacterial species also synthetize cellulose, though typically at much lower yields compared to other bacteria. Cyanobacteria are particularly intriguing in this context, as they uniquely combine the features of Gram-negative bacteria with plant-like features, such as oxygenic photosynthesis and CO2 fixation. This review highlights the structure and biosynthesis of cellulose in cyanobacteria, and explores the distinctive features compared with those of bacterial and vascular plants. We also discuss current strategies to enhance cellulose production in cyanobacteria through genetic engineering, synthetic redesign and environmental modulation, and propose key knowledge gaps. This review thus provides a foundation for advancing both fundamental understanding and the development of sustainable cellulose-based biotechnologies.

Intracellular accommodation of mutualistic fungi in plant roots depends on selective remodeling of host cell walls while minimizing activation of plant immune responses. In this study, we identify a host-adapted enzymatic module in the root endophyte Serendipita indica that targets acetylated xylan, a major structural component of monocot cell walls. The glycoside hydrolase Si GH11 cleaves the xylan backbone and releases O -acetylated oligosaccharides, which are subsequently deacetylated by the XynE-like esterase Si AXE. These enzymes are co-expressed within a monocot-specific transcriptional program that is enriched in carbohydrate-active enzymes and sugar transporters. Their combined activity enhances enzymatic degradation and facilitates downstream hydrolysis by exo-xylanases, which reduces the production of apoplastic reactive oxygen species triggered by damage-associated molecular patterns. Functional analysis shows that overexpression of Si AXE promotes early root colonization, while deletion of the gene compromises fungal proliferation during later stages. These findings define a coordinated and immune-compatible strategy for host cell wall deconstruction that enables fungal adaptation and endophytic colonization of monocot roots. In Brief Serendipita indica utilizes a transcriptionally coordinated xylanase and esterase module to degrade acetylated xylan in monocot roots. This enzyme cooperation enhances substrate breakdown, suppresses immune responses, and enables endophytic colonization, illustrating how mutualistic fungi adapt saprotrophic enzymes for host-specific intracellular accommodation. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

AbstractA key aspect of sustainable bioeconomy is the recirculation of renewable, agricultural waste streams as substrates for microbial production of high-value compounds. One approach is the bioconversion of corn stover, an abundant maize crop byproduct, using the fungal maize pathogen Ustilago maydis. U. maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates. In this study, we demonstrate that U. maydis can grow using untreated corn stover as its sole carbon source. We developed a small-scale bioreactor platform to investigate U. maydis processing of corn stover, combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre- and post-fermentation residues. Our results reveal that U. maydis primarily utilizes soluble sugars i.e., glucose, sucrose and fructose present in corn stover, with only limited exploitation of the abundant lignocellulosic carbohydrates. Thus, we further explored the biotechnological potential of enhancing U. maydis´ lignocellulosic utilization. Additive performance improvements of up to 120 % were achieved when using a maize mutant with increased biomass digestibility, co-fermentation with a commercial cellulolytic enzyme cocktail, and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes. This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U. maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates. Graphical abstract

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava.

Cell walls are important interfaces of plant-fungal interactions. Host cell walls act as robust physical and chemical barriers against fungal invaders, making them an essential line of defense. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to reinforce their walls and prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release a wide range of sugar oligomers into the apoplast, some of which trigger the activation of host immunity via host surface receptors. Recent characterization of cell walls from plant-colonizing fungi have emphasized the abundance of β-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated β-glucan laminarin. Thereby, we identified a glycoside hydrolase family 81-type glucan-binding protein (GBP) as the major β-glucan interactor. Mutation of GBP1 and its only paralogue GBP2 in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of symbiotic colonization was accompanied by enhanced responses at the host cell wall. Moreover, GBP mutation in barley also increased resistance to fungal infections in roots and leaves by the hemibiotrophic pathogen Bipolaris sorokiniana and the obligate biotrophic pathogen Blumeria graminis f. sp. hordei, respectively. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi, a role that has potentially been appropriated by barley-adapted pathogens.

Plant cell walls contain the hemicellulose xyloglucan, whose fine structure may vary depending on cell type, tissue, and/or plant species. Most but not all of the glycosyltransferases involved in the biosynthesis of xyloglucan sidechains have been identified. Here, we report the identification of several functional glycosyltransferases from blueberry (Vaccinium corymbosum bluecrop). Among those transferases is a hitherto elusive Xyloglucan:Beta-xylosylTransferase (XBT). Heterologous expression of VcXBT in the Arabidopsis thaliana double mutant mur3 xlt2, where xyloglucan consists only of an unsubstituted xylosylated glucan core structure, results in the production of the xylopyranose-containing "U" sidechain as characterized by mass spectrometry, glycosidic linkage, and NMR analysis. The introduction of the additional xylopyranosyl residue rescues the dwarfed phenotype of the untransformed Arabidopsis mur3 xlt2 mutant to wild-type height. Structural protein analysis using Alphafold of this and other related xyloglucan glycosyltransferase family 47 proteins not only identifies potential domains that might influence the regioselectivity of these enzymes but also gives hints to specific amino acids that might determine the donor-substrate specificity of these glycosyltransferases.

The hemicelluloses comprise a group of matrix glycans that interact with cellulose microfibrils in plant cell walls and play important roles in establishing wall architecture. The structures of hemicelluloses are determined by carbohydrate-active enzymes (CAZymes) that synthesize, integrate, and break down these polymers. Specifically, endo-glucanase 16 (EG16) enzymes, which are related to the well-known xyloglucan endotransglycosylase/hydrolase (XTH) gene products in Glycoside Hydrolase Family 16 (GH16), have been implicated in the degradation of the β(1,4)-linked backbone of mixed-linkage β(1,3);β(1,4)-glucans (MLG) and xyloglucans. EG16 members are single-copy genes found in most plant clades but are absent from many eudicots, including the model plant Arabidopsis thaliana. Until recently, EG16 members had only been characterized in vitro, establishing their substrate specificity, protein structure, and phylogenetic history, but their biological function was unknown. Here we used a hybrid polar, Populus alba × Populus grandidentata (P39), as a model to examine EG16 expression, subcellular localization, and pheno- and chemotypes of EG16-downregulated P39 plants. Populus EG16 expression is strong in young tissues, but RNAi-mediated downregulation did not impact plant growth nor the fine structure of the hemicellulose xyloglucan, suggesting a restricted or currently unknown role in angiosperm physiology.

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of 0-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated 0-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a 0-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley adapted pathogens.