Molecular Plant Pathology

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

Accessory genes are thought to contribute to fungal adaptation and pathogenicity by modulating host immunity, while core genes play crucial roles in maintaining fundamental biological processes. Rust fungi (order Pucciniales) are obligate biotrophic plant-pathogens and infect economically relevant crops. Here, we characterize core and accessory gene repertoires across rust fungi, with a particular focus on Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Across Pucciniales genomes, accessory genes represented the largest fraction of gene content (~44.6% on average), whereas core genes accounted for a smaller proportion (~18-35%). Notably, variations in accessory gene content among rust fungi are perhaps attributed to lineage-specific gene expansions and losses. Core gene content was positively correlated with total gene number across Pucciniales genomes, suggesting retention after gene duplication events, consistent with their essential biological functions. Among P. pachyrhizi genes expressed during soybean infection, core effectors were associated with cysteine-rich proteins, pectin-degrading enzymes, and SPFH/Band 7 family, while accessory effectors included phosphatidylethanolamine-binding proteins, trehalose phosphatases, and CFEM domain-containing proteins. The in-plant induced core and accessory genes in P. pachyrhizi also comprised multiple families of CAZymes (GH5/GH7 cellulases, CE5 cutinases, CE8 pectinesterases, CE4/GH18 chitin-modifying enzymes); proteases (aspartyl proteases, serine carboxypeptidases, alpha/beta hydrolases); transporters (amino acid permeases, ferric reductase-like transmembrane proteins, and OPT oligopeptide transporter), and transcription factors (bZIP, GATA zinc finger, STE-like, and homeobox KN). Our study highlights that core and accessory gene families have shaped P. pachyrhizi-soybean interactions, identifying promising targets for functional studies aimed at elucidating host-adaptation mechanisms in rust fungi.

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the ~65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

The receptor kinase FERONIA (FER) perceives endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides and regulates a plethora of plant physiological processes, including immunity. RALF peptides also bind to LEUCINE-RICH REPEAT EXTENSIN (LRX) proteins as structural components of the cell wall. We recently showed that the FER-RALF-LRX pathway supports colonization by the obligate biotrophic fungal pathogen Erysiphe cruciferarum (Ecr), a member of the powdery mildew species complex that infects Arabidopsis. Genetic disruption of the pathway primarily affects conidiation of the fungus, raising the question of effects on fungal nutrition. To get further insight into the underlying mechanisms, we performed RNA sequencing (RNAseq) to identify differential transcriptional responses of FER-RALF-LRX pathway mutants upon Ecr infection. Surprisingly, our results revealed that pathway disruption has a limited impact on the overall transcriptional changes upon fungal infection. However, consistent with previous reports, FER-RALF-LRX pathway mutants show changes in basal expression of a plethora of genes, mainly associated with cell wall metabolism, jasmonic acid signalling, amino acid biosynthesis and secondary metabolism. Many of these genes are regulated by Ecr infection across genotypes, too. This raises the question whether these are relevant pathway components for powdery mildew host establishment downstream of the FER-RALF-LRX module. In summary, our data reveals new insights into FER-RALF-LRX-dependent responses that may support host susceptibility to biotrophic plant pathogens.

Light leaf spot (LLS), caused by subcuticular hemibiotrophic ascomycete fungus Pyrenopeziza brassicae, is a major constraint on oilseed rape (Brassica napus) production, yet the genetic and biochemical mechanisms of quantitative disease resistance (QDR) remain poorly defined. Here, disease phenotyping, pathogen quantification, microscopy, gene expression profiling and glucosinolate (GSL) analysis were integrated to dissect resistance mechanisms in B. napus. Disease assays of 19 diverse lines revealed clear contrasts between susceptible and resistant genotypes, with the commercial cultivar Ambassador showing a phenotype inconsistent with the UK Recommended List rating. Microscopy demonstrated that resistance within doubled haploid line Cubs Root does not inhibit spore germination or penetration but restricts hyphal branching and subcuticular colonisation from 4 to 8 days post-inoculation. Expression profiling of seven candidate gene expression markers (GEMs) and pathogenesis-related PR1 showed that cinnamate-4-hydroxylase, phospholipase C4, {beta}-adaptin, universal stress protein and the 40S ribosomal subunit protein S24 were strongly pathogen-induced in resistant lines, whereas a BAHD acyltransferase, a putative susceptibility factor, was induced only in susceptible cultivars. GSL profiling identified negative correlations between disease severity and total GSLs, particularly aliphatic and aromatic GSLs, with 2{square}phenylethyl and 7-methylsulfinyl heptyl GSLs showing the strongest associations with resistance. Together, these results highlight coordinated transcriptional and metabolic responses that limit pathogen proliferation and provide targets for breeding durable LLS resistance in B. napus.

Septoria nodorum blotch (SNB) is an economically important fungal disease of wheat caused by Parastagonospora nodorum. It is primarily controlled by the breeding of resistant wheat cultivars, but experience over the last 50 years shows that new pathogen populations soon evolve that are more virulent on the current popular cultivars. In this study, we assembled a panel of 360 P. nodorum isolates. The collection resolved into eight subpopulations. One core and seven transient populations were found possessing contrasting characters in term of spatial and temporal distribution, mating-type, effector haplotypes and patterns of intact and degraded copies of a Tc-1 mariner transposon, called Molly. Molly can proliferate and randomly insert throughout the fungal genome. Its multiplication in sexual population likely triggered RIP which partially explains the extensive genetic diversity and explains the ability to form new adapted lineages and the observed population structure of this important pathogen of wheat. When tested on wheat, the recently emerged groups exhibited greater pathogenicity on modern elite cultivars consistent with the low-amplitude boom-and-bust cycle observed previously. It is possible that active copies of Molly transpose and contribute to both the birth and death of the transient groups. This study identified and characterised a fungal specific transposable element (TE) which plays a vital role in shaping Australian P. nodorum population structure and creating extensive genetic diversity which potentially leads to better adaptation of the pathogen. The study suggests practical measures to improve the efficiency and longevity of resistance breeding for SNB.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Potato leafroll virus (PLRV) is an economically important viral disease of potato (S. tuberosum). Genetic resistance to this phloem-limited virus is rare, and no cloned resistance (R) genes have been reported. Rladg confers resistance to PLRV in an Andean potato landrace, LOP-868 (Velasquez et al. 2007). We identified the functional Rladg gene as a homolog of the tomato TIR-NLR-encoding Bs4. Rladg interacts with the serine protease domain of the PLRV protein P1, which is essential for virus replication. This recognition is independent of the proteases enzymatic activity, and the Rladg immune receptor oligomerizes upon direct association with the protease. Like PLRV, many poleroviruses contain a serine protease. Despite their diverse amino acid sequences, these proteases are predicted to share similar structures. Rladg recognizes all ten tested polerovirus proteases, suggesting a conserved structural recognition mechanism. We propose that Rladgs broad recognition capacity could enable resistance to poleroviruses in many crop species. Rladg is the first R-gene reported to confer resistance to a phloem-limited pathogen and could provide enhanced resistance to many economically important poleroviruses.

Symbiotic interactions between plants and nitrogen-fixing microorganisms are essential for sustainable agriculture, yet the molecular mechanisms underlying plant-cyanobacterium symbiosis remain poorly understood. In particular, the nature of the signalling mechanisms mediating partner recognition in associations involving Nostoc species is largely unknown. Recent proteomic analyses have identified proteins homologous to rhizobial Nod factors biosynthetic enzymes in Nostoc punctiforme, suggesting the existence of a Nod-like signalling system. However, the functional role of these components has not been experimentally validated. Here, we investigate the contribution of nod-like biosynthetic and regulatory genes to symbiosis by analysing mutants of N. punctiforme affected in genes with homology to nodB and nodD. Phenotypic characterization revealed that disruption of nodB-like genes does not impair free-living growth but affects early stages of plant association and colonization. Specifically, the nodB1 mutant is impaired in plant association and shows a mild defect in colonization, whereas the nodB3 mutant exhibits a severe defect in colonization. In contrast, nodD-like mutants exhibited altered symbiotic phenotypes, with specific regulators differentially affecting interaction and colonization efficiency in rice (Oryza sativa). In particular, mutation of nodD2 and nodD3 reduced plant association and severely compromised colonization in Oryza sativa, with a more pronounced phenotype in nodD3 mutant. Altogether, our results provide genetic evidence supporting the involvement of Nod-like components in cyanobacterial symbiosis and suggest the existence of a regulatory and biosynthetic module contributing to plant colonization. These findings shed new light on the evolution and diversity of symbiotic signalling mechanisms across plant-microbe interactions.

Endophytic biocontrol agents may contribute to grapevine health, but their ability to establish, persist, and move within woody tissues remains poorly understood. In this study, a stem injection method was developed to introduce Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into rooted and grafted grapevine plants, and their spatial and temporal colonization patterns were compared with the movement of a dye tracer. The dye tracer moved rapidly through xylem tissues, whereas both microorganisms showed more restricted early distribution. Over time, M71 and SC1 displayed distinct colonization patterns. M71 persisted after injection, but remained localized near the inoculation site, with limited movement toward roots or distal aerial tissues. In grafted plants, M71 recovery depended on the injection site and declined more markedly after rootstock injection than after scion injection. In contrast, SC1 showed broader and more persistent colonization. In rooted cuttings, SC1 was recovered from stem and root tissues up to 56 days post-injection, and in grafted plants it was recovered across the graft union, particularly after scion injection. Microscopy supported internal localization of both microorganisms. GFP-labelled M71 and SC1 hyphae were observed mainly within xylem vessels, and viable microorganisms were recovered from corresponding wood tissues. No contamination was observed in control plants. These results show that beneficial microorganisms can be introduced into grapevine tissues by stem injection and that bacterial and fungal biocontrol agents differ markedly in their internal movement and persistence. IMPORTANCEIntroducing beneficial microorganisms directly into plant tissues could help in establishing protective endophytic populations, but little is known about how such microorganisms move and persist inside grapevine. This study shows that stem injection can deliver Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into grapevine tissues without visible phytotoxicity. The two microorganisms followed different colonization patterns. M71 remained mostly localized near the injection site, whereas SC1 spread more broadly and persisted in both rooted and grafted plants. These findings provide a basis for developing targeted endophytic biocontrol strategies in grapevine propagation and early plant establishment. This approach may be particularly relevant for grapevine trunk diseases and other vascular disorders, in which pathogens colonize internal woody tissues.

The pathogenic fungus Trichosporon asahii causes severe mycoses in immunocompromised hosts, such as neutropenic patients. In Cryptococcus neoformans, the unfolded protein response (UPR) sensor Ire1 induces hxl1 mRNA splicing and contributes to stress responses and virulence. The function of Ire1-triggered hxl1 mRNA splicing in stress tolerance and virulence of T. asahii, however, remains unclear. Here, we demonstrated that ire1- and hxl1 gene-deficient T. asahii mutants are sensitive to dithiothreitol (DTT), an inducer of endoplasmic reticulum stress, and exhibit reduced virulence in a silkworm infection model. DTT treatment induced hxl1 mRNA splicing in the wild-type strain, whereas ire1 gene-deficient mutants did not undergo hxl1 mRNA splicing. The ire1 gene-deficient mutants were more sensitive than the parent strain to DTT, H2O2, Congo red, and SDS, and showed reduced virulence in silkworms. Similarly, hxl1 gene-deficient mutants exhibited increased sensitivity to these stressors and reduced virulence. Both the ire1 gene-deficient and hxl1 gene-deficient mutants showed decreased expression of reactive oxygen species-detoxifying related genes CAT2, SOD1, and SOD2, compared with the parent strain. Together, these findings suggest that Ire1-triggered hxl1 mRNA splicing contributes to stress resistance and virulence in T. asahii.

Biological control strategies are increasingly being explored as sustainable alternatives for managing rice blast disease caused by Magnaporthe oryzae. In this study, we characterized three Bacillus pumilus isolates (DC01, DC09, and DC13) and evaluated their antifungal and plant-beneficial properties against M. oryzae. Whole genome sequencing revealed multiple biosynthetic gene clusters associated with the production of antimicrobial metabolites. All three isolates inhibited fungal growth in dual-culture assays, whereas heat-stable diffusible antifungal activity was primarily associated with the cell-free supernatants of DC09 and DC13. Exposure to bacterial supernatants disrupted fungal development, inducing abnormal hyphal morphology characterized by bulbous swelling, altered polarity, and increased branching in M. oryzae. Volatile organic compound assays further revealed that the DC isolates suppress fungal growth in the absence of physical contact. The isolates additionally inhibited the growth of other phytopathogenic fungi and selected human bacterial pathogens. All strains exhibited plant growth-promoting traits, including indole-3-acetic acid production and osmotic stress tolerance, whereas DC09 also displayed phosphate-solubilizing activity. Importantly, root inoculation with the DC isolates significantly reduced rice blast disease severity and induced expression of defense-associated genes involved in jasmonic acid/ethylene signaling and immune priming. Collectively, these findings identify the DC isolates, particularly DC09 and DC13, as promising multi-mechanistic biological control agents for sustainable rice blast management.

Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), causes yield losses exceeding 50% in affected areas, including the Bagre rice plain in Burkina Faso. Genome-edited (GEd) rice lines have been successful in tackling BB. Modifications in the Xoo virulence protein target site upstream of three SWEET susceptibility genes in two elite rice varieties, IR64 and Ciherang-Sub1, have been demonstrated to confer broad-spectrum resistance to Asian and East African Xoo strains. Here, we evaluate the potential of the GEd lines as a solution for BB management in Burkina Faso. We challenged the GEd lines against five locally collected Burkinabe Xoo strains under controlled green-house conditions and assessed their agro-morphological performance under field conditions representative of local agroecological conditions. Greenhouse pathogen assays demonstrated that GEd IR64 and Ciherang-Sub1 lines were resistant to all tested local Xoo strains across three successive generations. We identified TalC as the primary disease-causing effector in the local Xoo populations. Irrigated field trials conducted over two seasons in the Kou Valley, Burkina Faso, revealed absence of agro-morphological penalties in GEd lines compared to their parental wild-type lines. Observed trait variation was attributable to environmental fluctuations rather than genomic modifications. Collectively, our findings demonstrate that genome editing of the rice lines does not impose growth penalties, and support the suitability of GEd IR64 and Ciherang-Sub1 for large-scale adoption in Burkina Faso, pending multi-location validation and introgression into locally adapted varieties.

Parastagonospora nodorum (Berk.) Quaedvlieg, Verkley & Crousis, a necrotrophic fungal pathogen, is the causal agent for septoria nodorum blotch, a major constraint on global wheat production. Pathogen-produced necrotrophic effectors (NEs) that interact with host-sensitivity genes in an inverse gene-for-gene manner, collectively leading to effector-triggered susceptibility (ETS). Here, we investigated the transcriptional responses of two Triticum aestivum L. genotypes, Mace and Lancer, following infiltration with a novel NE, SnTox8. A total of 12,679 unique differentially expressed genes in Mace and 149 in Lancer were detected from transcriptomic analysis. In the SnTox8-sensitive cultivar, Mace, numerous defence-related genes were induced, including protein phosphorylation cascades, reactive oxygen species bursts, calcium signalling, phytohormone modulation, and suppression of photosynthesis, consistent with findings from other ETS models, in which necrotrophic fungal pathogens hijack host defence systems to proliferate. The interaction also activated genes involved in signal transduction, metabolism, membrane modification, and molecular transport, reflecting a coordinated host reprogramming that promotes cellular dysfunction and cell death, thereby facilitating necrotrophic pathogenesis. In contrast, Lancer, an SnTox8-insensitive cultivar, exhibited minimal transcriptional changes with no evidence of effector recognition or downstream defence-related activities. Overall, this study exhibited that SnTox8 manipulates kinase-mediated immune signalling and metabolic reprogramming to convert defence activation into host cell death, revealing a mechanistic basis for ETS in wheat. The identified SnTox8-Snn8-triggered processes were confirmed through additional transcriptome analysis of Mace mutants. Outcomes from this study establish a foundation for identifying, functionally characterising and validating the corresponding host susceptibility gene Snn8.

Rhizosphere microbial communities contribute to the growth and health of their host but may be altered by the incidence of soil-borne pathogens. In avocado, the oomycete Phytophthora cinnamomi, causal agent of Phytophthora root rot (PRR), has been shown to alter rhizosphere bacterial communities, although its effect on fungal communities has seldom been explored. Our objective was thus to determine whether P. cinnamomi induced shifts in diversity, composition and co-occurrence networks of fungal communities in the rhizosphere of avocado trees, and to identify potential antagonists of P. cinnamomi that could be further considered for disease management. Fungal communities associated with the rhizosphere of asymptomatic and PRR-symptomatic avocado trees were studied through ITS metabarcoding. Although -diversity metrics were not significantly different between asymptomatic and PRR-symptomatic trees, differences in {beta}-diversity of rhizosphere fungal communities were detected. Moreover, PRR led to the enrichment of saprotrophic taxa and opportunistic pathogens such as Fusarium, Cladosporium or Plectosphaerella in the avocado rhizosphere, which were possibly attracted by the release of resources from necrosed roots. Co-occurrence network analysis revealed that fungal networks in the rhizosphere of PRR-symptomatic trees were more complex and connected than those from asymptomatic trees, suggesting a response of fungal communities to the disturbance caused by the pathogen. Some connector taxa from the PRR-symptomatic networks (Gibellulopsis, Cladorrhinum or Mycenella) were also identified as members of the P. cinnamomi pathobiome. Their negative correlations with the pathogen indicate they may act as potential antagonists, which calls for further isolation efforts to confirm their biocontrol activity of PRR.

The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. We set here to develop light inducible molecular tools to enable dynamic studies on signaling networks and fungi-host communication, and for metabolic engineering approaches. In particular, light-controlled, optogenetic switches provide quantitative, spatio-temporal control capabilities, are minimal invasive and reversible. We engineered two blue light-inducible LOV-domain-based gene expression switches, to up- (Blue-ON) and down-regulate (Blue-OFF) gene expression, and performed a functional characterization in sporidia and hyphae of U. maydis. Profiting from the dynamic control ranges and rapid kinetics, we implemented the optoswitches to control cell morphology by initiating the transition from a haploid sporidial cellular morphotype to filaments upon regulation of the levels of the polarity factor Rac1 and its constitutive active mutant Q61L. In addition to showing how expression level of effectors can be precisely regulated as an approach to understand fungi-plants interaction, we show in two proof-of-principle applications targeted control over U. maydis filamentous fungal invasion of plant tissue and the mechanisms of tumor formation. For this we placed under Blue-ON and Blue-OFF control two U. maydis effectors, See1 (Seedling efficient effector 1) and TIN2 (Tumor inducing 2), and tumor formation was assayed on maize leaves. Taken together, this study established blue-light switches as effective tools to control morphogenesis and pathogenesis in U. maydis.

Seed quality is a complex trait shaped by morphological, biochemical and microbiological properties that are rarely characterised simultaneously, limiting our ability to identify robust predictive indicators of germination speed and seedling emergence across varieties. Here, we performed a multi-factor characterisation of eight common bean (Phaseolus vulgaris L.) varieties, combining seed morphometrics, untargeted GC-MS metabolomics on three seed organs, and amplicon sequencing of bacterial and fungal communities, to identify indicators of germination speed and emergence percentage. The eight varieties showed substantial variation in both traits, used as physiological seed quality proxies. Seed weight and size variation between varieties were correlated with germination speed. The intravariety variance of seed weight was independently correlated with emergence performance. Metabolome composition differed strongly across seed organs, with variety as the dominant driver. Individual-seed metabolomic profiles in the plumule and cotyledon were associated with germination speed but not emergence, yielding 16 plumule and three cotyledon candidate metabolite markers. Fungal community composition was associated with both germination speed and emergence, while bacterial communities were associated with emergence only. Nine fungal and four bacterial taxa were identified as candidate indicators. Inter-kingdom co-occurrence network analysis revealed that fungi with similar germination speed associations tend to cluster in the same modules, suggesting that community-level modules rather than individual taxa may constitute more robust microbial indicators. These results demonstrate that germination speed and emergence capacity are governed by distinct seed properties, and provide morphological, metabolic and microbial candidate indicators for integration into targeted seed quality assessment frameworks for common bean.

Abstract/SummaryInteractions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear. Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites. Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition. Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors.

O_LILegume nodule formation is induced by rhizobia secreted Nod factors (NFs). It has been shown that NF receptors also accumulate in the apex of Medicago truncatula nodules. However, the NF signaling induced transcriptional changes in there have never been studied. C_LIO_LIHere, we studied this by using NF signaling mutant TE7, a weak allele of IPD3, blocked in rhizobial release. Nodule apices were isolated with laser microdissection and used for transcriptional analysis. C_LIO_LIWe identified 1655 NF signaling controlled genes in nodule apex. By comparing this with the transcriptome data from VAMP721d&e RNAi nodule apices, we identified a subset of 445 genes whose expression depends on NF signaling and rhizobial release. Further, we compared the set of genes controlled by NF signaling in nodule apices with that controlled in root epidermis, and these showed only a small overlap. NIN is induced by NF signaling both in the root epidermis and in the nodule. By overexpression of NIN in TE7 and knock down of NIN in wildtype nodules we showed that NF signaling controlled rhizobial release depends on NIN. C_LIO_LINF signaling controls a distinct set of genes in nodules, the function of which depends at least in part on NIN. C_LI

In fungi, hyphal fusion is beneficial within an individual, but fusion between individuals comes with the risks of infection or exploitation. To manage this risk, fungi have developed mechanisms to restrict sustained fusion to be within a genetic individual, called allorecognition. In Ascomycete fungi, this recognition is based on allelic identity at several polymorphic allorecognition genes, often triggering cell death. However, the genetic basis of allorecognition is unknown in basidiomycetes, the clade that includes mushroom-forming fungi. Here, we map the first locus for this trait, which we call somA, in the mushroom-forming fungus Coprinopsis cinerea. We combined F1 offspring phenotypes with independent backcross lines to identify a region on chromosome 5 linked with the production of a barrage zone, a classic allorecognition phenotype. Fine-mapping of this region resulted in a region with a set of kinases and NACHT domain proteins, flanked by a leucine-rich repeat (LRR) protein. While the NACHT and kinase proteins are diverse between the parents, the LRR-encoding protein shows signs of purifying selection. Additional C. cinerea genomes show that this region contains several highly divergent alleles, consistent with long-term balancing selection. These polymorphic alleles all contain a single monomorphic LRR, which may indicate a novel mechanism for fungal nonself recognition. Based on a phylogenetic survey of related Basidiomycetes, this specific locus architecture appears to be restricted to closely related species. This finding of a multiallelic locus may explain the general trend of few nonself recognition loci in basidiomycetes. These results provide a first understanding of how individuality is maintained in basidiomycetes.