Biology subjects

Zuccaro, A.

Publications and source records attributed to Zuccaro, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A fungal effector hijacks conserved plant GSK3-like kinases to reprogram host cell identity

Stable developmental programs normally resist reprogramming, yet pathogens can override these constraints to induce profound tissue and cellular remodeling. How microbial pathogens access conserved developmental signaling networks to reprogram host growth and differentiation remains poorly understood. Here, we show that the maize smut fungus Ustilago maydis deploys the effector Iag1 to reprogram host cell fate by targeting the conserved GSK3-like kinase BIN2. Iag1 contains a PPNT short interaction motif found also in plant viral effectors that mediates its association with BIN2 and related maize GSK3-like kinases and is essential for effector activity and full fungal virulence. This interaction attenuates BIN2-dependent signaling, reduces phosphorylation of the BIN2 substrate BES1, and triggers extensive transcriptional reprogramming consistent with coordinated perturbation of brassinosteroid, auxin, and other BIN2-regulated developmental pathways. These changes result in extensive epidermal developmental reprogramming, including loss of stomatal identity, altered cell division orientation, and aberrant cell expansion. Together, our findings identify GSK3-like kinases as conserved developmental signaling hubs that pathogens can exploit to unlock host developmental plasticity and suggest that short motifs provide an evolutionarily flexible strategy for targeting these central regulatory nodes.

plant biology↗

bioRxiv2026

Low intra-microbiota antagonism underlies stable and protective barley root microbiota

Plants rely on root-associated microbiota for growth and pathogen protection, yet the community properties underlying stable beneficial functions remain unclear. Here, we show that low intra-microbiota antagonism is associated with stability and pathogen protection in the barley root microbiota. Using a comprehensive culture collection of barley root endophytes, we reconstructed bacterial synthetic communities (SynComs) and compared a core SynCom resembling the natural microbiota with a trait-prioritized SynCom assembled from strains selected for predicted host benefits. Although strains from both communities exhibited broad pathogen-inhibitory potential, the communities behaved differently in planta. The core SynCom showed low internal antagonism, remained stable, and protected barley roots against fungal pathogens without impairing growth. In contrast, the trait-prioritized SynCom exhibited strong internal antagonism, unstable assembly, loss of protection, and reduced root growth. Together, our findings indicate that microbiota function cannot be predicted from individual strain traits alone. Rather, low intra-microbiota antagonism promotes community stability and enables protective functions to emerge in barley roots. Significance statementPlants rely on root microbiota for disease protection, yet the principles governing community stability and protective function remain poorly understood. Here, we show that low intra-microbiota antagonism is associated with a stable and protective barley root microbiota, whereas strong internal antagonism destabilizes communities, impairs root growth, and abolishes pathogen protection. These findings show that microbiota function depends not only on beneficial traits of individual strains, but also on compatibility among community members. HighlightsO_LILow intra-microbiota antagonism is associated with stable and protective barley root microbiota. C_LIO_LITrait-prioritized SynCom assembly with higher intra-microbiota antagonism can lead to dysbiosis. C_LIO_LIA highly antagonistic Pseudomonas strain is buffered by the core SynCom but detrimental in an unstable community. C_LIO_LICommunity compatibility better explains protection than individual strain traits. C_LIO_LIStable bacterial communities support fungal endophyte-mediated protection and maintain pathogen protection across barley genotypes. C_LI

plant biology↗

bioRxiv2026

Prion-like domains control plastid targeting of PEPSI effectors and PEPSI6-mediated modulation of DXR during root symbiosis

Microbial effectors often act in the apoplast or cytosol, but how they reach host organelles during beneficial plant-fungal interactions remains poorly understood. Here, we identify a class of secreted prion-like effector proteins from the mutualistic root endophyte Serendipita indica, termed PEPSIs. These proteins contain prion-like domains (PrLDs) embedded within intrinsically disordered regions, and three tested PEPSIs require these domains for plastid localization. Focusing on PEPSI6, we show that plastid targeting depends on its PrLD and is associated with engagement of plastid import and proteostasis machinery. In plastids, PEPSI6 associates with 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), promotes DXR accumulation, alters methylerythritol phosphate pathway metabolites, and enhances tolerance to the DXR inhibitor fosmidomycin. PEPSI6 also undergoes apoplastic C-terminal CAP-domain processing, and its colonization-promoting activity persists after PrLD deletion, indicating a plastid-targeting-independent function. Overall, this work identifies PrLDs as noncanonical plastid-targeting elements in PEPSI effectors and reveals DXR as a target of fungal manipulation during root symbiosis. HighlightsO_LIPrion-like domains control plastid targeting of selected PEPSI effectors. C_LIO_LIPEPSI6 associates with DXR and promotes its accumulation in plastids. C_LIO_LIPEPSI6 alters MEP-pathway metabolites and increases tolerance to fosmidomycin. C_LIO_LIApoplastic processing reveals a plastid-independent PEPSI6 activity during root symbiosis. C_LI

plant biology↗

bioRxiv2026

The transcription factor SiROCX controls a monocot-specific CAZyme program for host cell wall remodeling during symbiotic root colonization

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 SiROCX as a key regulator of this monocot-adapted program. DNA affinity purification sequencing (DAP-seq) defined the SiROCX binding motif and revealed a CAZyme-enriched target regulon. Inducible SiROCX 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 SiROCX 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. SignificanceBeneficial 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 SiROCX, 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 SiROCX 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.

microbiology↗

bioRxiv2025

A fungal root endophyte functionally complements host immunity and mitigates natural immune variation in Arabidopsis

Beneficial root-associated microbes can enhance plant resilience by complementing aspects of host immunity. The fungal root endophyte Serendipita indica (Si) is known to promote plant growth and confer broad stress tolerance. To assess how natural host genetic variation influences Si-mediated protection, we screened 47 Arabidopsis thaliana accessions for susceptibility to the fungal pathogen Bipolaris sorokiniana (Bs) with and without Si colonization. All accessions benefited from Si, indicating that endophyte-mediated disease mitigation occurs broadly across diverse host genotypes. A focused comparison of two genetically and geographically proximate Swedish accessions, T510 and T530, which displayed the most divergent protection scores, revealed substantial differences in Bs susceptibility. Transcriptome profiling under bi- and tripartite colonization showed conserved defense responses in both accessions. Bs infection downregulated growth- and development-related genes, consistent with a growth-immunity trade-off, with T530 exhibiting higher Bs colonization and a stronger transcriptional response than T510. Co-colonization with Si effectively suppressed pathogen growth and disease symptoms in both accessions. Comparative genomic and transcriptomic analyses identified four immune receptor genes, including the TIR-NLR ISI, present in T510 but absent in T530. An isi T-DNA insertion mutant phenocopied the heightened Bs susceptibility of T530, confirming that ISI contributes to root immunity, while Si-mediated protection remained intact despite increased pathogen susceptibility. Together, these findings demonstrate that fungal endophytes can mitigate the functional consequences of natural immune variation and enhance the resilience of genetically diverse plant populations. HighlightsO_LIS. indica confers broad protection against B. sorokiniana largely independent of host genotype or pathogen susceptibility. C_LIO_LIThe TIR-NLR immune receptor ISI contributes to root immunity but is not essential for S. indica-mediated protection. C_LIO_LIBeneficial endophytes can mitigate natural immune variation effects and support overall plant health. C_LI

ecology↗

bioRxiv2025

Targeting Pleiotrophin to mitigate high fat diet-induced liver metabolic disease: Insights into sex-specific metabolic protection.

Obesity is a global health problem linked to the development of metabolic syndrome (MetS) and comorbidities such as metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). These diseases are characterised by systemic inflammation, lipid accumulation and tissue damage, which contribute to liver fibrosis and dysfunction. Pleiotrophin (PTN), a cytokine known for its role in tissue regeneration and energy metabolism, has emerged as a potential regulator of liver homeostasis. Herein, we demonstrate that Ptn deletion protects against body weight gain, metainflammation and high-fat diet (HFD)-induced MASLD and MASH development. Furthermore, our work uncovers the molecular mechanisms by which PTN may promote lipid synthesis and hepatic extracellular matrix remodelling. Results highlight PTN as a critical modulator of liver metabolism and systemic inflammation in the context of obesity, identifying it as a promising therapeutic target for the treatment of MASLD, MASH and related metabolic disorders, and point to a sexual dimorphism in adaptive metabolic strategies, with females demonstrating a greater degree of protection against the liver-damaging effects of diet-induced obesity. HIGHLIGHTSO_LIFemales are more protected against the liver-damaging effects of the high-fat diet (HFD), suggesting a sexual dimorphism in adaptive metabolic strategies. C_LIO_LIPTN regulates lipid metabolism by promoting lipogenesis and lipid accumulation in liver cells through the activation of AKT and the inhibition of AMPK; the absence of PTN reverses this process. C_LIO_LIDeletion of Ptn protects against high-fat diet (HFD)-induced weight gain, systemic inflammation, hepatic lipid accumulation, and the development of steatosis and liver fibrosis. C_LIO_LIPTN is a key modulator of metabolic, inflammatory and remodelling processes in the liver, and it is proposed as a promising therapeutic target for MASLD, MASH and other metabolic comorbidities. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/691898v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f8846borg.highwire.dtl.DTLVardef@bdbaf2org.highwire.dtl.DTLVardef@3c8c33org.highwire.dtl.DTLVardef@118a0cf_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO PTN liver signalling pathway. acetyl-CoA carboxylase (ACC); 5 adenosine monophosphate-activated protein kinase (AMPK); protein kinase B (AKT); aquaporin 9 (AQP9); diacylglycerides (DAG); diacylglycerol O-acyltransferase 2 (DGAT2); free fatty acids (FFA); hepatic stellate cell (HSC); insulin receptor (IR); pleiotrophin (PTN); triacylglycerides (TAG). C_FIG

biochemistry↗

bioRxiv2025

Host-adapted enzymatic deconstruction of acetylated xylan enables mutualistic colonization of monocot roots

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 SiGH11 cleaves the xylan backbone and releases O-acetylated oligosaccharides, which are subsequently deacetylated by the XynE-like esterase SiAXE. 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 SiAXE 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 BriefSerendipita 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. HighlightsO_LIMutualistic fungal endophyte repurposes saprotrophic enzymes to enable monocot-specific intracellular root colonization C_LIO_LICoordinated xylanase and esterase activity remodels host cell walls and dampens immune responses C_LIO_LIExpression of cell wall degrading enzymes is regulated by host species and colonization stage C_LIO_LIResults reveal fungal adaptation to monocot roots along the saprotrophy to symbiosis transition C_LI

plant biology↗

bioRxiv2025

Domain gain or loss in fungal chitinases drives ecological specialization toward antagonism or immune suppression

The evolutionary origins of fungal effector proteins remain poorly understood, particularly how structural changes reprogram antimicrobial enzymes into host-adapted immune suppressors. Here, we show that domain modularity drives ecological specialization in chitinase effectors of the beneficial root endophyte Serendipita indica. The GH18 chitinase SiCHIT, which carries a C-terminal carbohydrate-binding module (CBM5), is expressed during fungal competition and antagonizes the fungal pathogen Bipolaris sorokiniana in the rhizosphere, thereby protecting plant roots. Deletion of the CBM5 abolishes this antifungal activity, while fusion of CBM5 to the CBM5-lacking paralog SiCHIT2 restores pathogen inhibition. In contrast, SiCHIT2 is induced during root colonization and suppresses chitin-triggered reactive oxygen species production, promoting immune evasion and host compatibility. These results identify CBM5 as a modular determinant of effector function, switching chitinase activity between microbial antagonism and host immune suppression. Our findings support an evolutionary scenario in which effector function in planta arises through domain loss and transcriptional divergence from an antimicrobial precursor, consistent with transitions along the saprotrophy-to-symbiosis continuum. Significance StatementEffector proteins play key roles in shaping fungal interactions with both plant hosts and microbial competitors, yet how these functions evolve remains unclear. Here, we show that structural domain modularity enables ecological specialization of two paralogous chitinases arising from gene duplication in the root endophyte Serendipita indica. Through domain deletion and fusion, we demonstrate that a carbohydrate-binding module (CBM5) determines whether a chitinase functions in microbial antagonism or immune evasion. Our findings provide mechanistic evidence for effector diversification via domain loss and transcriptional divergence, supporting an evolutionary trajectory from antimicrobial activity to host adaptation. This work advances our understanding of how modular architecture drives effector evolution and niche specialization in symbiotic fungi. HighlightsO_LIGain or loss of a CBM5 binding domain drives effector specialization between fungal antagonism and immune suppression C_LIO_LICBM5 acts as a modular determinant enabling antifungal activity in the GH18 chitinase SiCHIT C_LIO_LIThe CBM5-lacking paralog SiCHIT2 suppresses host immunity and promotes root colonization C_LIO_LIFunctional divergence following gene duplication illustrates evolutionary repurposing of an antimicrobial enzyme into an immune-suppressive effector C_LI

plant biology↗

bioRxiv2024

Pleiotrophin deletion prevents high-fat diet-induced cognitive impairment, glial responses, and alterations of the perineuronal nets in the hippocampus

Obesity and metabolic disorders, such as metabolic syndrome (MetS) facilitate the development of neurodegenerative diseases and cognitive decline. Persistent neuroinflammation plays an important role in this process. Pleiotrophin (PTN) is a cytokine that regulates energy metabolism and high-fat diet (HFD)-induced neuroinflammation, suggesting that PTN could play an important role in the connection between obesity and brain alterations, including cognitive decline. To test this hypothesis, we used an HFD-induced obesity model in Ptn genetically deficient mice (Ptn-/-). First, we confirmed that Ptn deletion prevents HFD-induced obesity. Our findings demonstrate that feeding wild-type (Ptn+/+) mice with HFD for 6 months results in short- and long-term memory loss in the novel object recognition task. Surprisingly, we did not observe any sign of cognitive impairment in Ptn-/- mice fed with HFD. In addition, we observed that HFD induced microglial responses, astrocyte depletion, and perineuronal nets (PNNs) alterations in Ptn+/+ mice, while these effects of HFD were mostly prevented in Ptn-/- mice. These results show a crucial role of PTN in metabolic responses and brain alterations induced by HFD and suggest the PTN signalling pathway as a promising therapeutic target for brain disorders associated with MetS.

neuroscience↗

bioRxiv2023

Time-resolved transcriptomics reveal a mechanism of host niche defense: beneficial root endophytes deploy a host-protective antimicrobial GH18-CBM5 chitinase

Associations between plants and beneficial root-endophytic fungi enhance plant performance by improving nutrient uptake, abiotic stress tolerance and disease resistance. To successfully colonize different host plants and defend their host niche against competing microbes, but also to cooperate with beneficial bacterial members of the microbiota, root endophytes such as Sebacinales secrete a multitude of tightly regulated effector-proteins and carbohydrate-active enzymes. However, the functions, specificity, and regulation of these proteins remain poorly understood. In this study, we employ time-resolved transcriptomics to analyse the gene expression profiles of two Sebacinales members interacting with organisms from different kingdoms of life. We identified crucial genes for plant colonization and intermicrobial competition, including a fungal GH18-CBM5 chitinase specifically upregulated in response to the phytopathogenic fungus Bipolaris sorokiniana. This chitinase protects the plant hosts against the pathogen, reducing fungal biomass and disease symptoms in barley and Arabidopsis thaliana. Our findings shed light on interaction partner specific gene expression in Sebacinales endophytes, with potential applications in enhancing plant health and resilience. Bullet pointsO_LIBoth Serendipita indica (Si) and Serendipita vermifera (Sv) show similar transcriptional responses to three host species and the phytopathogen Bipolaris sorokiniana (Bs), indicating common interaction principles between Sebacinales and plant hosts or fungi. C_LIO_LIThese shared mechanisms involve the activation of effector genes like small secreted proteins and carbohydrate-active enzymes. C_LIO_LICooperation with beneficial bacteria elicits only minimal transcriptomic alterations in Sebacinales compared to plants and Bs. C_LIO_LISebacinales respond to Bs by upregulating a specific GH18-CBM5 chitinase unique to Basidiomycota within the fungal kingdom, inhibiting Bs growth and reducing disease symptoms in Arabidopsis thaliana and barley. C_LI

microbiology↗

bioRxiv2023

Metabolic arms race between a plant and a fungal pathogen

In this work, we uncover a metabolite interaction between barley and the fungal pathogen Bipolaris sorokiniana (Bs), involving hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties. Bs infection of barley roots elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the synthesis pathway in yeast produced several hordedanes, including one of the most advanced products 19-b-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of the cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, while it inhibits other fungi, and Bs chemically modifies 19-OH-HTA. Thus, plant and pathogen molecular interactions extend beyond protein-protein recognition and the simple detoxification of plant antimicrobial metabolites. One-Sentence SummaryA fungal pathogen uses barley diterpenoid phytoalexins to facilitate root colonization.

plant biology↗

bioRxiv2023

Autophagy restricts fungal accommodation in the roots ofArabidopsis thaliana

Endophytic colonization of Arabidopsis thaliana by the beneficial root endophyte Serendipita indica is characterized by an initial biotrophic phase followed by a restricted host cell death-associated phase. This latter phase involves regulated cell death (RCD) for fungal accommodation. However, the host molecular pathways that limit S. indica colonization and govern symbiosis remain largely unknown. Our study demonstrates that autophagy, a major cellular degradation pathway, is activated during S. indica colonization and is required to restrict fungal colonization in Arabidopsis. Independent Arabidopsis knockout (KO) mutants deficient in autophagosome formation are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by two secreted S. indica effectors at the onset of the cell death-associated phase. In the atg5 autophagy mutant background, impaired dAdo uptake prevents dAdo-induced and symbiosis-mediated cell death. Based on our data, we propose that autophagy-mediated pro-survival responses in the host are crucial for maintaining a balanced symbiotic interaction between S. indica and Arabidopsis. In a NutshellOur study reveals that during colonization of Arabidopsis thaliana roots by the beneficial root endophyte Serendipita indica, autophagy, a key cellular degradation pathway, is activated to limit fungal colonization. Autophagy-deficient Arabidopsis mutants are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by S. indica. We propose that autophagy-mediated pro-survival responses are essential for maintaining a balanced symbiotic interaction between S. indica and Arabidopsis.

microbiology↗

bioRxiv2023

A GH81-type β-glucan-binding protein facilitates colonization by mutualistic fungi in barley

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 others 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 {beta}-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 {beta}-glucan laminarin. Thereby, we identified a glycoside hydrolase family 81-type glucan-binding protein (GBP) as the major {beta}-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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/536646v1_figu1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@c47957org.highwire.dtl.DTLVardef@fa6727org.highwire.dtl.DTLVardef@18a54d2org.highwire.dtl.DTLVardef@c6b103_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefGBP1, a family 81 glycoside hydrolase, is an important {beta}-glucan interactor in barley. Mutation of GBP1 and its sole paralogue GBP2 leads to reduced colonization by beneficial root endophytes, AM fungi and pathogens, accompanied by enhanced responses at the plant cell wall. This indicates that GBP1 and {beta}-glucans are compatibility factors involved in the establishment of symbiotic associations with beneficial fungi, a role possibly hijacked by pathogens.

plant biology↗

bioRxiv2023

Root cap cell corpse clearance limits microbial colonization

Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica, primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica-induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting a transcriptional control mechanism that facilitates the accommodation of beneficial microbes in the roots. Key findings in bullet pointsO_LIThe process of programmed cell death in root development (dPCD) influences the extent and outcomes of fungal symbiosis C_LIO_LIFungal colonization of the root tip and differentiation zone is restricted by SMB-mediated clearance of dead cells, which preserves the meristem and regulates symbiosis C_LIO_LIExpression of plant nuclease BFN1, which is associated with senescence, is modulated to facilitate root accommodation of beneficial microbes C_LI

plant biology↗

bioRxiv2022

Chloroplast protein import determines plant proteostasis and retrograde signaling

Proteins containing polyglutamine (polyQ) repeats are prone to aggregation and can lead to distinct human pathologies. For instance, Huntingtons disease is caused by an abnormal expansion of the polyQ stretch (> Q35) of Huntingtin (HTT) protein. However, plants express hundreds of proteins containing polyQ regions, but no pathologies arising from these factors have been reported to date. Here, we ask how plants maintain the proteostasis of polyQ-containing proteins, which are intrinsically enriched in the plant proteomes. To this end, we overexpressed an aggregation-prone fragment of human HTT (Q69) in plant cells. In contrast to invertebrate and mammalian transgenic models, we find that Arabidopsis thaliana plants suppress Q69 aggregation. This elevated proteostasis ability is mediated through the import and degradation of Q69 in chloroplasts. Conversely, inhibition of chloroplast protein import either genetically or pharmacologically reduces the capacity of plant cells to prevent Q69 aggregation. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Notably, expression of synthetic Arabidopsis SPP is sufficient to suppress aggregation of polyQ-expanded HTT in human cells. Beyond ectopically expressed Q69-HTT, endogenous polyQ-containing proteins also aggregate in Arabidopsis upon inhibition of chloroplast import. Among them, the plastid casein kinase 2 (pCK2), which contains a polyQ region next to the chloroplast targeting sequence motif, can also be localized into the nucleus. Upon inhibition of chloroplast import, pCK2 accumulates at higher levels in the nucleus and forms diamond-shaped amyloid-like fibrils surrounding the chloroplasts. These results indicate that the differential conformation and redistribution of pCK2 to the nucleus depends on chloroplast import efficiency, providing a role of polyQ repeats in chloroplast to nucleus communication (i.e. retrograde signaling). Together, our findings establish chloroplast protein import and proteases as determinants of polyQ proteostasis, with important implications for plant biology that can also lead to therapeutic approaches for human diseases that involve protein aggregation.

plant biology↗

bioRxiv2022

A fungal endophyte-generated nucleoside signal regulates host cell death and promotes root colonization

The intracellular colonization of plant roots by the beneficial fungal endophyte Serendipita indica follows a biphasic strategy. After an early biotrophic phase, the interaction transitions to a host cell death phase restricted to the epidermal and cortex layers of the root. Host cell death contributes to the successful accommodation of the fungus during the beneficial interaction in Arabidopsis thaliana. How host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the plant apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. The uptake of extracellular dAdo, but not the structurally related adenosine (Ado), activates a previously undescribed cell death mechanism in A. thaliana. Mutation of the equilibrative nucleoside transporter ENT3 in A. thaliana results in resistance to cell death triggered by extracellular dAdo and reduced fungal-mediated cell death during root colonization. A library screen of A. thaliana T-DNA insertion lines identified a toll/interleukin-1 receptor nucleotide-binding leucine-rich repeat (TIR-NLR) protein as an additional intracellular component in dAdo-triggered cell death. Mutation of this previously uncharacterised TIR-NLR, which we have named ISI (induced by S. indica), affects host cell death, fungal colonization and growth promotion, suggesting a key role in the regulation of root cell death and plant-microbe interaction. Our data show that the combined activity of two fungal apoplastic enzymes leads to the production of a metabolite that, upon uptake, triggers TIR-NLR-modulated plant cell death, providing a link to immunometabolism in plants. Short summaryEfficient intraradical colonization by the beneficial fungal endophyte Serendipita indica requires restricted host cell death. How this symbiotic host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. Uptake of extracellular dAdo activates a previously undescribed cell death mechanism in plants. Mutation of the A. thaliana equilibrative nucleoside transporter ENT3 leads to resistance to cell death triggered by uptake of extracellular dAdo and to reduced fungal-mediated cell death during colonization. A library screen of A. thaliana T-DNA insertion lines identified a TIR-NLR protein as an additional intracellular component in dAdo-triggered cell death, providing a link to immunometabolism in plants. In a nutshellRegulated host cell death is part of the plant defense strategy against pathogens, but it is also involved in the accommodation of certain beneficial microbes in the roots. We have identified extracellular metabolites and intracellular metabolic signals that contribute to colonization by beneficial root fungal endophytes and uncovered a conserved cell death mechanism likely co-opted for establishing plant-endophyte symbiosis.

plant biology↗

bioRxiv2021

A barley gene cluster for the biosynthesis of diterpenoid phytoalexins

Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley (Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana. The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana, validating the function of these genes in planta. Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome.

plant biology↗

bioRxiv2021

Fungi hijack a plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall (CW) layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different biological functions. The barley (Hordeum vulgare) {beta}-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved {beta}-1,3;1,6-glucan decasaccharide ({beta}-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight {beta}-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by {beta}-1,3-endoglucanases due to the presence of three {beta}-1,6-linked glucose branches and can scavenge reactive oxygen species. Additionally, exogenous application of {beta}-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface. SignificanceHere we identify and characterize a conserved {beta}-1,3;1,6-glucan decasaccharide with antioxidant activity released from the fungal extracellular polysaccharide (EPS) matrix by the activity of a plant apoplastic endoglucanase. In addition, we provide a quantitative proteomic analysis of the fungal EPS and cell wall (CW) layers. HIGHLIGHTSO_LIThe fungal extracellular polysaccharide (EPS) matrix and the cell wall (CW) are specific layers with distinct protein and carbohydrate signatures C_LIO_LIA conserved {beta}-1,3;1,6-glucan decasaccharide ({beta}-GD) is released from the EPS matrices of different fungi by the activity of the barley {beta}-1,3-endoglucanase BGLUII, a member of the widely distributed apoplastic GH17 family C_LIO_LIThe {beta}-GD efficiently scavenges reactive oxygen species (ROS) and enhances fungal colonization C_LIO_LIThe immunomodulatory potential as microbe-associated molecular pattern (MAMP) as well as the biochemical activity as ROS scavenger of soluble low molecular weight {beta}-glucans are defined by the presence of {beta}-1,6-glucose branches C_LI

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