The necrotrophic fungal pathogen Sclerotinia sclerotiorum is distinguished by its exceptionally broad host range, infecting numerous plant species across diverse genera. Although historically characterized by a seemingly unspecialized infection strategy, increasing evidence indicates that S. sclerotiorum employs a diverse suite of virulence mechanisms, including effectors for host and environment manipulation. Leveraging recent advances in structural genomics and the growing availability of genomic and transcriptomic resources, we systematically re-examined the effector repertoire of the reference strain 1980 using updated computational approaches. We identified 215 effector-like proteins, which were classified into structural families based on predicted tertiary structures. Transcriptome meta-analysis across hundreds of publicly available samples revealed diverse expression patterns among effectors, highlighting both conserved and condition-specific expression dynamics. Several effector families were found to be expanded in S. sclerotiorum, including groups with predicted antimicrobial functions and unique domain architectures. Within families, structural and surface property variation suggests potential functional diversification. Furthermore, coexpression network analysis uncovered putative gene clusters that may operate synergistically with selected effectors. This work refines our understanding of effector diversity and organization in S. sclerotiorum and provides a framework for prioritizing candidates for functional studies, ultimately contributing to the broader understanding of effector evolution in generalist fungal pathogens. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Quantitative disease resistance (QDR) is an immune response limiting pathogen damage in plants. It involves transcriptomic reprogramming of numerous genes, each having a small contribution to plant immunity. Despite the broad-spectrum nature of QDR, the evolution of its underlying transcriptome reprogramming remains largely uncharacterized. Here, we analyzed global gene expression in response to the necrotrophic fungus Sclerotinia sclerotiorum in 23 Arabidopsis (Arabidopsis thaliana) accessions of diverse origin and contrasting QDR phenotypes. Over half of the species pan-transcriptome displayed local responses to S. sclerotiorum, with global reprogramming patterns incongruent with accession phylogeny. Due to frequent small-amplitude variations, only ∼11% of responsive genes were common across all accessions, defining a core transcriptome enriched in highly-responsive genes. Co-expression and correlation analyses showed that QDR phenotypes result from the integration of the expression of numerous genes. Promoter sequence comparisons revealed that variation in DNA-binding sites within cis-regulatory regions contributes to gene expression rewiring. Finally, transcriptome–phenotype maps revealed abundant neutral networks connecting diverse QDR transcriptomes with no loss of resistance, hallmarks of robust and evolvable traits. This navigability associated with regulatory variation in core genes highlights their role in QDR evolvability. This work provides insights into the evolution of complex immune responses, informing models for plant disease dynamics.

Acclimation enables plants to adjust to immediate environmental fluctuations and is therefore key to the resilience of plant disease resistance in a time of climate change. Here, we report on the acclimation of Arabidopsis thaliana quantitative immune responses against the fungal pathogen Sclerotinia sclerotiorum to daily environmental fluctuations. We analyzed disease resistance phenotypes and global gene expression in plants grown in three acclimation regimes, revealing the rewiring of regulatory networks during this process. We identified pathogen-induced genes weakly sensitive to acclimation as promising bases for acclimation-proof immunity. Fluctuations in Mediterranean-like acclimation resulted in an increased disease susceptibility and the misregulation of many pathogen-responsive genes. We identified A. thaliana mutants in novel immune components contributing positively to quantitative disease resistance following temperate but not Mediterranean acclimation. Quantitative disease resistance was maintained under Mediterranean acclimation in NAC42-like mutants and associated with a switch in the repertoire of pathogen-responsive targets of this transcription factor. Our work reveals the role of immune gene networks' plasticity in acclimation and suggests new strategies to maintain plant immune function in a warming climate.

Efficient plant immune response requires concerted reprogramming of cellular transcriptomes both globally and locally at the site of infection. Upon inoculation by the fungal pathogen Sclerotinia sclerotiorum, plants show quantitative disease resistance characterized by transcriptional reprogramming of numerous genes with small phenotypic effect. To study transcriptional heterogeneity across cells during quantitative disease resistance, we combine end-point single-nucleus RNA-sequencing and time-course RNA-seq of mock-treated and S. sclerotiorum-inoculated Arabidopsis thaliana leaves. We observe heterogeneity of plant immune responses across cell types and in sub-populations of mesophyll cells, and reconstruct the sequence of immune responses activation over time. The quantification of gene expression heterogeneity reveals a transient increase in intrinsic transcriptional noise followed by the activation of key defense genes and the rise of extrinsic transcriptional noise in infected cells. Using the R-GECO1 cytoplasmic calcium reporter, we find that the intensity of calcium variations upon S. sclerotiorum inoculation coincides with variations to transcriptional noise in space and time. These results provide evidence that stochastic cell–cell variability plays a key role in commitment to plant immunity and in the coordination of plant defense at the organ scale. Our study offers new insights into the mechanisms underlying plasticity and robustness of plant immune responses that can inform the design of strategies to reduce pathogen damage to crops in unstable environments.

In response to necrotrophic fungal pathogens, plants often display quantitative disease resistance (QDR), an immune response with complex genetic determinants. Due to their diversity and small phenotypic effect, the genetic bases of QDR are challenging to characterize. Here, we used genome-wide association mapping in Arabidopsis thaliana natural populations to identify novel determinants of QDR against the fungal pathogen Sclerotinia sclerotiorum. We found that presence-absence polymorphism of the AT4TE56270 Copia transposable element (TE) upstream of the cysteine-rich receptor-like kinase 8 (CRK8) gene is associated with QDR. The presence of the TE associates with higher CRK8 expression in healthy and inoculated plants and increased QDR. The constitutive knockdown of CRK8 reduced QDR, hydrogen peroxide production, and the expression of defense genes upon inoculation. Transcriptome analysis revealed altered defense pathways and salt responses in CRK8 mutants, including impaired glutathione and camalexin biosynthesis, likely contributing to disease susceptibility. Mutants in CRK8 showed altered seed germination on salt, and the absence of AT4TE56270 is associated with enhanced seed germination under salt stress in A. thaliana natural populations. These results reveal a trade-off between salt tolerance and defense against S. sclerotiorum associated with presence-absence polymorphism of a TE.

Colletotrichum nymphaeae, the causal agent of anthracnose fruit rot, is globally recognized as a major pathogen of strawberries due to its economic impact. Fungal pathogens utilize secreted proteins to facilitate infection by acquiring host nutrients and suppressing plant immunity. Understanding the transcriptomic responses of C. nymphaeae during infection can provide critical insights into its pathogenic mechanisms. In this study, RNA sequencing (RNA-seq) was performed to profile the transcriptome of C. nymphaeae strain 02-179 during infection of leaf and fruit tissues of the susceptible strawberry (Fragaria x ananassa) cultivar Florida Beauty. Differential gene expression analysis identified fungal genes upregulated during these interactions. Transcriptomic profiling revealed a set of genes encoding secreted effector proteins, including NUDIX hydrolase and LysM domain-containing proteins. Additionally, genes associated with Carbohydrate-Active enzymes (CAZymes), such as multicopper oxidase, pectinesterase, pectate lyase, glycosyl hydrolase family 7, and endochitinase, were significantly upregulated. Notably, two novel tannase genes were identified among the top upregulated genes in strawberry-infected leaves and fruits. Tannase enzymes are hypothesized to degrade tannins, a group of plant secondary metabolites abundant in strawberries, known for their defensive roles against pests and pathogens. The identification of tannase genes and the other genes associated with virulence underscores the complex molecular strategies employed by C. nymphaeae to infect and colonize strawberry tissues. Genes involved in degrading plant cell walls, suppressing host defenses, and potentially overcoming chemical barriers such as tannins play critical roles in the pathogenesis of anthracnose. Further functional characterization of these genes will enhance our understanding of the disease mechanisms and could inform the development of improved management strategies for C. nymphaeae infections in strawberries.

Plant pathogens secrete effectors to facilitate infection and manipulate host physiological and immune responses. Effector proteins are challenging to characterize because of their sequence and functional diversity, rapid evolution, and host-specific interactions. Recent advances in artificial intelligence (AI), particularly in protein biology, offer new opportunities for identifying and characterizing effector proteins and understanding their evolutionary processes. This review discusses recent progress in applying AI to effector biology, focusing on identification, functional characterization, and evolution. Key areas include subcellular localization prediction, protein structural modeling with tools like AlphaFold, and the use of pretrained protein language models. AI promises to complement existing experimental and computational approaches and further accelerate the investigation of effector protein functions and their evolutionary histories, even in the absence of clear sequence similarity or known functional domains.

Facilitating plant defense through priming holds significant promise for advancing sustainable crop health practices. The primed state of plants is correlated with a more rapid and efficient response, achievable by subjecting plants to recurrent stressors. Consequently, priming constitutes a dynamic transitional process between two distinct biological states, necessitating the establishment and maintenance of transcriptional memory mechanisms. Research on transcriptional memory has revealed molecular mechanisms including protein and transcription factor accumulation, as well as epigenetic modulations. Nonetheless, there remains a dearth of understanding regarding the interplay among the dynamics of transcriptional stress memory, its preservation, and the resultant emergent properties conferred upon the organism once primed. Here, we found that the robustness of transcriptional memory associated with repeated mechanical stimuli played an antagonistic role in the priming of plant defense. Incorporating experiments and computational modeling, we have analyzed the transcriptional stress memory of Arabidopsis thaliana subjected to daily acoustic stimulations. The observed enhanced resistance to the pathogen Sclerotinia sclerotiorum following three rounds of repeated acoustic stimulations is attributed to three primary mechanisms: the activation of a first line of defense in non-inoculated plants, an increase defense-associated gene diversification, and gene priming. These mechanisms are sustained by a transcriptional stress memory involving thousands of genes, likely regulated mostly by transcription factor cascades. Upon comparing the predictions of the transcriptional stress memory model with experimental outcomes, we propose that priming does not arise from the sequential activation of different pathways but rather from the simultaneous modulation of a broad spectrum of pathways. Pathway redundancy in the transcriptional memory topology imparts robustness to plant defense priming. Consequently, primed plants exhibit independence from limited genetic variations, preventing critical loss of resistance observed in naive plants. Pathway redundancies also imply strong limitations in increasing plant defense. Thus, plants remain unaffected by an increase in the daily rate of RAS and exhibited a stress memorization time limited to 1.5 days.### Competing Interest StatementThe authors have declared no competing interest.

Acclimation enables plants to adapt to immediate environmental fluctuations, supporting biodiversity and ecosystem services. However, global changes are altering conditions for plant disease outbreaks, increasing the risk of infections by pathogenic fungi and oomycetes, and often undermining plant immune responses. Understanding the molecular basis of plant acclimation is crucial for predicting climate change impacts on ecosystems and improving crop resilience. Here, we investigated how Arabidopsis thaliana quantitative immune responses acclimates to daily temperature fluctuations. We analyzed responses to the fungal pathogen Sclerotinia sclerotiorum following three acclimation regimes that reflect the distribution areas of both species. Mediterranean acclimation, characterized by broad diurnal temperature amplitudes, resulted in a loss of disease resistance in three natural A. thaliana accessions. Global gene expression analyses revealed that acclimation altered nearly half of the pathogen-responsive genes, many of which were down-regulated by inoculation and associated with disease susceptibility. Phenotypic analysis of A. thaliana mutants identified novel components of quantitative disease resistance following temperate acclimation. Several of these mutants were however more resistant than wild type following Mediterranean acclimation. Notably, mutant lines in the NAC42-like transcription factor did not show a loss of resistance under Mediterranean acclimation. This resistance was linked to an acclimation-mediated switch in the repertoire of NAC42-like targets differentially regulated by inoculation. These findings reveal the rewiring of immune gene regulatory networks by acclimation and suggest new strategies to maintain plant immune function in a warming climate. ### Competing Interest Statement The authors have declared no competing interest.

Quantitative disease resistance (QDR) is an immune response limiting the damage caused by pathogen infection in plants. It involves the transcriptomic reprogramming of numerous genes each having a weak contribution to the plant immunity phenotype. Despite QDR widespread and broad-spectrum nature, the evolution of the underlying transcriptome reprogramming remains largely uncharacterized. Here, we analyzed global gene expression in response to the necrotrophic fungus Sclerotinia sclerotiorum in 23 Arabidopsis thaliana accessions of diverse origin and contrasted QDR phenotype. Over a half of the species pan-transcriptome displayed local responses to S. sclerotiorum , with global reprogramming patterns incongruent with accessions phylogeny. Due to frequent small-amplitude variations, only ∼11% responsive genes were common to all accessions. They formed protein-protein interactions networks densely connecting both old and recent genes. Clustering of accessions based on global regulation patterns identified four subsets unrelated to QDR phenotypes, highlighting at least four distinct paths towards regulatory evolution of QDR at the species level. We identified WRKY and MYB-related DNA binding sites enriched in the 5’-regulatory regions of core and subset-specific responsive genes respectively. Our findings show that the evolution of QDR involves distinct gene reprogramming trajectories at the species level, associated with contrasted patterns of cis-regulatory variation.### Competing Interest StatementThe authors have declared no competing interest.

Besides the well-understood qualitative disease resistance, plants possess a more complex quantitative form of resistance: quantitative disease resistance (QDR). QDR is commonly defined as a partial but more durable form of resistance and, therefore, might display a valuable target for resistance breeding. The characterization of QDR phenotypes, especially of wild crop relatives, displays a bottleneck in deciphering QDR’s genomic and regulatory background. Moreover, the relationship between QDR parameters, such as infection frequency, lag-phase duration, and lesion growth rate, remains elusive. High hurdles for applying modern phenotyping technology, such as the low availability of phenotyping facilities or complex data analysis, further dampen progress in understanding QDR. Here, we applied a low-cost (<1.000 €) phenotyping system to measure lesion growth dynamics of wild tomato species (e.g., Solanum pennellii or Solanum pimpinellifolium). We provide insight into QDR diversity of wild populations and derive specific QDR mechanisms and their cross-talk. We show how temporally continuous observations are required to dissect end-point severity into functional resistance mechanisms. The results of our study show how QDR can be maintained by facilitating different defense mechanisms during host–parasite interaction and that the capacity of the QDR toolbox highly depends on the host’s genetic context. We anticipate that the present findings display a valuable resource for more targeted functional characterization of the processes involved in QDR. Moreover, we show how modest phenotyping technology can be leveraged to help answer highly relevant biological questions.

Pathogens secrete effector proteins to subvert host physiology and cause disease. Effectors are engaged in a molecular arms race with the host resulting in conflicting evolutionary constraints to manipulate host cells without triggering immune responses. The molecular mechanisms allowing effectors to be at the same time robust and evolvable remain largely enigmatic. Here, we show that 62 conserved structure-related families encompass the majority of fungal orphan effector candidates in the Pezizomycotina subphylum. These effectors diversified through changes in patterns of thermodynamic frustration at surface residues. The underlying mutations tended to increase the robustness of the overall effector protein structure while switching potential binding interfaces. This mechanism could explain how conserved effector families maintained biological activity over long evolutionary timespans in different host environments and provides a model for the emergence of sequence-unrelated effector families with conserved structures.

Additional file 5: Table S1. Parameters values used to model the effect of fungal-derived hydrolysis heterogeneities on the principal strain patterning and the dependency of the overstretched length on the lesion radius.

Additional file 14: File S5. Pictures used in this work (raw and analyzed).

Plants use programmed cell death as a potent defense response against biotrophic pathogens that require living host cells to thrive. However, cell death can promote infection by necrotrophic pathogens. This discrepancy creates specific coevolutionary dynamics in the interaction between plants and necrotrophs. Necrotrophic pathogens produce diverse cell death-inducing effectors that act redundantly on several plant targets and sometimes suppress plant immune responses as an additional function. Plants use surface receptors that recognize necrotrophic effectors to increase quantitative disease resistance, some of which evolved independently in several plant lineages. Co-evolution has shaped molecular mechanisms involved in plant-necrotroph interactions into robust systems, relying on degenerate and multifunctional modules, general-pur pose components, and compartmentalized functioning.

Plants are engaged in a coevolutionary arms race with their pathogens that drives rapid diversification and specialization of genes involved in resistance and virulence. However, some major innovations in plant-pathogen interactions, such as molecular decoys, trans-kingdom RNA interference, two-speed genomes, and receptor networks, evolved through the expansion of the functional landscape of genes. This is a typical outcome of genetic co-option, the evolutionary process by which available genes are recruited into new biological functions. Co-option into plant-pathogen interactions emerges generally from (i) cis-regulatory variation, (ii) horizontal gene transfer (HGT), (iii) mutations altering molecular promiscuity, and (iv) rewiring of gene networks and protein complexes. Understanding these molecular mechanisms is key for the functional and predictive biology of plant-pathogen interactions.

BACKGROUND:The ongoing adaptation of plants to their environment is the basis for their survival. In this adaptation, mechanoperception of gravity and local curvature plays a role of prime importance in finely regulating growth and ensuring a dynamic balance preventing buckling. However, the abiotic environment is not the exclusive cause of mechanical stimuli. Biotic interactions between plants and microorganisms also involve physical forces and potentially mechanoperception. Whether pathogens trigger mechanoperception in plants and the impact of mechanotransduction on the regulation of plant defense remains however elusive.RESULTS:Here, we found that the perception of pathogen-derived mechanical cues by microtubules potentiates the spatio-temporal implementation of plant immunity to fungus. By combining biomechanics modeling and image analysis of the post-invasion stage, we reveal that fungal colonization releases plant cell wall-born tension locally, causing fluctuations of tensile stress in walls of healthy cells distant from the infection site. In healthy cells, the pathogen-derived mechanical cues guide the reorganization of mechanosensing cortical microtubules (CMT). The anisotropic patterning of CMTs is required for the regulation of immunity-related genes in distal cells. The CMT-mediated mechanotransduction of pathogen-derived cues increases Arabidopsis disease resistance by 40% when challenged with the fungus Sclerotinia sclerotiorum.CONCLUSIONS:CMT anisotropic patterning triggered by pathogen-derived mechanical cues activates the implementation of early plant defense in cells distant from the infection site. We propose that the mechano-signaling triggered immunity (MTI) complements the molecular signals involved in pattern and effector-triggered immunity.

Sclerotinia sclerotiorum is a notorious generalist plant pathogen that threatens more than 600 host plants, including wild and cultivated species. The molecular bases underlying the broad compatibility of S. sclerotiorum with its hosts is not fully elucidated. In contrast to higher plants and animals, alternative splicing (AS) is not well studied in plant-pathogenic fungi. AS is a common regulated cellular process that increases cell protein and RNA diversity. In this study, we annotated spliceosome genes in the genome of S. sclerotiorum and characterized their expression in vitro and during the colonization of six host species. Several spliceosome genes were differentially expressed in planta, suggesting that AS was altered during infection. Using stringent parameters, we identified 1,487 S. sclerotiorum genes differentially expressed in planta and exhibiting alternative transcripts. The most common AS events during the colonization of all plants were retained introns and the alternative 3 ' receiver site. We identified S. sclerotiorum genes expressed in planta for which (a) the relative accumulation of alternative transcripts varies according to the host being colonized and (b) alternative transcripts harbour distinct protein domains. This notably included 42 genes encoding predicted secreted proteins showing high-confidence AS events. This study indicates that AS events are taking place in the plant pathogenic fungus S. sclerotiorum during the colonization of host plants and could generate functional diversity in the repertoire of proteins secreted by S. sclerotiorum during infection.

The host range of parasites is an important factor in assessing the dynamics of disease epidemics. The evolution of pathogens to accommodate new hosts may lead to host range expansion, a process the molecular bases of which are largely enigmatic. The fungus Sclerotinia sclerotiorum has been reported to parasitize more than 400 plant species from diverse eudicot families while its close relative, S. trifoliorum, is restricted to plants from the Fabaceae family. We analyzed S. sclerotiorum global transcriptome reprogramming on hosts from six botanical families and reveal a flexible, host-specific transcriptional program. We generated a chromosome-level genome assembly for S. trifoliorum and found near-complete gene space conservation in two representative strains of broad and narrow host range Sclerotinia species. However, S. trifoliorum showed increased sensitivity to the Brassicaceae defense compound camalexin. Comparative analyses revealed a lack of transcriptional response to camalexin in the S. trifoliorum strain and suggest that regulatory variation in detoxification and effector genes at the population level may associate with the genetic accommodation of Brassicaceae in the Sclerotinia host range. Our work proposes transcriptional plasticity and the co-existence of signatures for generalist and polyspecialist adaptive strategies in the genome of a plant pathogen.