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.

Colletotrichum siamense is a predominant causal agent of anthracnose in rubber tree and numerous economically important crops, causing severe yield losses worldwide. Conidial germination represents a critical early step for successful infection, while the high-osmolarity glycerol (HOG) MAPK pathway and ergosterol biosynthesis individually govern fungal development, stress adaptation and fungicide responses. However, the molecular crosstalk between these two modules remains largely elusive in phytopathogenic fungi. Here, we identified CsErg5B, a sterol C-22 desaturase homolog, as a direct target of the HOG- regulated transcription factor CsAtf1 in C. siamense. CsErg5B was indispensable for ergosterol biosynthesis, conidial germination, appressorium formation, and full virulence. The {Delta}CsErg5B mutant showed increased conidiation but severely impaired germination, and exhibited elevated resistance to fludioxonil while hypersensitivity to azole fungicides. Epistasis analysis using the {Delta}CsErg5B/{Delta}CsCyp51G1 double mutant - where CsCyp51G1 serves as another downstream target of CsAtf1 - revealed that CsErg5B functions as the predominant downstream effector of CsAtf1 in modulating conidial development and fludioxonil sensitivity. Furthermore, overexpression of CsErg5B significantly rescued the defects in conidial germination and fludioxonil sensitivity in both {Delta}CsAtf1 and {Delta}CsPbs2 mutants. Taken together, our findings uncover a HOG MAPK - CsAtf1 - CsErg5B regulatory axis that connects HOG MAPK signaling to ergosterol homeostasis, thereby governing conidial germination and fungicide sensitivity in C. siamense. This study provides novel insights into the regulatory network underlying fungal development and fungicide response, and offers promising molecular targets for the integrated management of plant anthracnose.

Ubiquitin is a posttranslational modifier that is conserved among eukaryotes. Ubiquitination alters stability and folding of cellular proteins. Deubiquitinases (DUBs) reverse ubiquitination and often function as part of protein complexes. There are 32 predicted DUB-encoding genes present in the soil-borne phytopathogenic fungus Verticillium dahliae. Nuclear ubiquitin-specific protease 3 (Usp3) is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, whereas Usp1 is predicted to associate with the COP9 signalosome (CSN), which controls specificities of cellular E3 ubiquitin ligase activities. A proteomics approach using biotin capture and identification (BioID) supports that Usp3 regulates gene expression beyond the transcription level. Western experiments showed a dysregulation in ubiquitinated cellular proteins in corresponding deletion strains. Usp3 and Usp1 are both required for fungal development. They regulate microsclerotia formation based on different environmental cues and provide redundant functions in controlling conidiation. Absence of both corresponding genes resulted in significant impairment of conidiospore formation, which is required for fungal propagation within the plant vascular system. This paralysed spreading ability reduced virulence on tomato plants (Solanum lycopersicum). In summary, V. dahliae responds to environmental cues by Usp3- and Usp1-mediated adjustment of gene expression and protein stability. This is important for key developmental processes of the V. dahliae disease cycle and its virulence towards the host plant. Author summaryUbiquitination and deubiquitination of proteins enable cells to rapidly react to environmental cues and adjust protein stabilities and subsequently transcriptomic profiles. Usp3 is a nuclear deubiquitinase subunit of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex. Usp1 is predicted to be associated with the COP9 signalosome that regulates substrate specificities of the ubiquitin-proteasome system. BioID experiments suggest that other SAGA complex subunits, histone proteins, spliceosomal proteins, ribosomal proteins, a protein that tackles transcriptionally stalled RNAPII, and a protein that degrades mRNA with premature stop codons locate proximal to Usp3 within the cell. Deletion of USP3 led to the dysregulation of protein ubiquitination. A single deletion of USP1 did not significantly change ubiquitination profiles, however, a double deletion of USP1/3 significantly affected the ubiquitin-proteasome system. The altered ubiquitination profile correlated with a dysregulation of key developmental processes. Microsclerotia formation was decoupled from environmental cues in the {Delta}USP3 strain, whereas an additional deletion of USP1 reconnected it in a media-dependent manner. USP3 and USP1 contribute to a common governing process in conidiation, and the defect in spreading of the {Delta}USP1/3 strain is reflected by a significant reduction in plant pathogenicity.

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.

O_LIHost specialization is a major driver of genetic structure in fungal plant pathogens, but it remains unclear whether specialization on different cereal hosts prevents sexual recombination when mating-type compatibility is retained. We addressed this question in stripe rust, caused by Puccinia striiformis, by crossing wheat-adapted P. striiformis f. sp. tritici and barley-adapted P. striiformis f. sp. hordei, two divergent host-adapted forms that share common barberry (Berberis vulgaris) as a sexual host. C_LIO_LIControlled reciprocal crosses on barberry produced 18 aeciospore-derived progeny, demonstrating that wheat- and barley-adapted Puccinia striiformis can undergo sexual recombination despite strong host specialization during asexual infection. Chromosome-scale parental assemblies placed the homeodomain (HD) mating-type locus, containing bW-HD1 and bE-HD2, on chromosome 2 and the pheromone receptor (PR) mating-type locus, containing STE3 and mfa genes, on chromosome 6. HD restriction genotyping showed biparental inheritance in all progeny, with each progeny carrying one HD haplotype from each parent. Together with conservation of PR-associated coding sequences and amplification of STE3-associated markers in progeny, these results are consistent with retention of tetrapolar mating across the two host-adapted lineages. C_LIO_LIHost interaction phenotypes were assessed across wheat and barley differentials, near-isogenic lines and wild relatives. The parental isolates retained contrasting wheat- and barley-restricted profiles, whereas progeny did not reproduce either parental virulence profile, but instead showed recombinant infection patterns, including compatibility with both wheat and barley genotypes. C_LIO_LIThese findings indicate that host specialization in Puccinia striiformis does not necessarily prevent sexual compatibility on a shared alternate host. Together with retention of tetrapolar mating, alternate-host sexual reproduction may provide a route for genetic exchange between host-specialized pathogen populations, enabling recombination to generate new combinations of host-interaction traits when divergent pathogen lineages mate on a shared alternate host. C_LI

While transposable elements (TEs) are recognized as major drivers of fungal genome structure, evidence of their direct involvement in the interaction with host plants and the environment is only beginning to emerge. Retrotransposons can generate small RNA (sRNA) that act through cross-kingdom RNA interference, while giant DNA TEs called Starships carry dozens of cargo genes that enrich the accessory gene compartment of fungal genomes. In the polyphagous pathogen Botrytis cinerea, the Vv3 strain and other strains specialized on grapevine display a specific repertoire of TEs, including the retrotransposons BcCopia4, BcGypsy6, and BcGypsy7. This study first explored the putative role of sRNA generated from these retrotransposons in the interaction between the Vv3 strain and its host of origin, grapevine. Putative targets were identified among the host mRNAs, but predicted cleavage sites could not be experimentally validated. Moreover, Dicer mutants unable to produce retrotransposons-derived sRNA remained fully pathogenic on grapevine, indicating that these sRNAs do not act as virulence factors on this host. In parallel, this study provides an updated RNA-seq-based annotation of the accessory genes of the Vv3 strain, which revealed a new 93 kb-Starship harboring 43 cargo genes, some of which are related to arsenic resistance. A formal genetic approach confirmed that this locus confers resistance to this metalloid. This giant TE, named Ariane, was also detected in additional grapevine-specialized strains resistant to arsenic but not in strains isolated from other hosts such as tomato. In conclusion, this study highlights how a Starship giant transposon shaped the accessory genes compartment of the polyphagous fungus B. cinerea and may have contributed to its adaptation to vine cultivation by conferring resistance to arsenic, a compound widely used in vineyards during the last century. IMPACT STATEMENTFungal genomes contain many families of transposons whose functional role in adaptation to the environment and in biotic interactions remained hidden for a long time. In the grey mold fungus Botrytis cinerea, strains specialized on grapevine, such as Vv3, carry a specific repertoire of transposons which provides a valuable opportunity to investigate their role in niche adaptation. In this study, we first investigated retrotransposon-derived small RNA, previously described as effectors capable of manipulating the immunity of the model plant Arabidopsis thaliana. Although in silico analysis of the specific repertoire of small RNAs of the Vv3 strain suggested that some could target the expression of grapevine genes, a genetic approach demonstrated that they do not play a significant role in virulence on this host. In contrast, this study identified a new transposon, named Ariane, that carries 43 cargo genes and confers a selective advantage to the Vv3 strain. Ariane belongs to a family of giant transposons called Starships, recently discovered in fungi and considered to be responsible for horizontal genes transfers between unrelated species. Ariane was detected only in some B. cinerea strains isolated from grapevine, and a genetic cross showed that it provides these strains with the ability to grow in presence of arsenic. Arsenic was used in vineyards until the beginning of the 21st century to control fungal trunk diseases and insect pests. Therefore, Ariane appears to have played an important role in the adaptation of B. cinerea strains to cultivated grapevine. Overall, these results underline the importance of considering Starships when predicting emergence of resistance to antifungal compounds. DATA SUMMARYThe novel data described in this study, i.e., RNA-Seq data and the Starship element are accessible under NCBI GEO accession GSE327899 and at https://doi.org/10.57745/HYWRNM, respectively. All information related to Botrytis cinerea genomes used in this study are centralized and kept up to date at the Bioinfo Bioger genomic web portal: https://bioinfo.bioger.inrae.fr/portal/genome-portal/. Direct links to individual portals are respectively https://bioinfo.bioger.inrae.fr/portal/genome-portal/3/ for B. cinerea Vv3 genome, https://bioinfo.bioger.inrae.fr/portal/genome-portal/2/ for B. cinerea Sl3 genome, and https://bioinfo.bioger.inrae.fr/portal/genome-portal/4/ for B. cinerea populations isolated on tomato or grapevine. Each portal provides: (i) a centralized access to public genomic resources, including the genome, transposon, and RNA repositories; (ii) a data browser to download the genomic files; (iii) a genome browser that enables visualization of features within their genomic context, along with associated expression data. As a summary, the prior main public B. cinerea genomic accessions and resources used in this study are: GCA_039644125 for VV3 genome, GCA_022560135 for Sl3 genome, GCA_000143535 for B05.10 genome, PRJNA624742 for populations, https://doi.org/10.57745/HYWRNM for transposons, and GSE181592 for small RNAs. Furthermore, table S1 summarizes the list and characteristics of the 64 B. cinerea genomes publicly available to date. The genomic data for Vitis vinifera genome PN40024.v4 used in this study are available at: https://integrape.eu/resources/genes-genomes/genome-accessions/.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, one of the most economically important wheat diseases worldwide. One of the few cloned wheat resistance genes against Z. tritici, Stb6, recognizes the secreted fungal effector AvrStb6. Although AvrStb6 has been extensively studied as an avirulence determinant, its biological function during host colonization remains unknown. Based on the amphipathic nature of the predicted structure of AvrStb6, we hypothesized the effector to function as a membrane-active antimicrobial protein. However, in vitro growth inhibition assays demonstrated that AvrStb6 does not directly inhibit the growth of wheat-associated bacteria across multiple bacterial genera and experimental conditions. Instead, microbiome analyses of wheat apoplastic fluid revealed shifts in bacterial abundance associated with the presence or absence of AvrStb6 in a susceptible cultivar (without Stb6-induced resistance). This prompted us to further explore other putative microbiome-related functions of AvrStb6. In vitro confrontation assays further showed that deletion of AvrStb6 increased the sensitivity of Z. tritici to antagonistic wheat-associated bacteria, particularly Pseudomonas and Pantoea spp. This phenotype was conserved across independent fungal genetic backgrounds and across virulent and avirulent AvrStb6 variants. Fluorescence-based co-culture assays additionally showed reduced fungal growth and increased bacterial proliferation in the absence of AvrStb6 during interactions with Pseudomonas spp., but not with the control bacterium Escherichia coli. Finally, biochemical assays demonstrated that AvrStb6 associates with the Z. tritici cell wall in vitro, whereas other secreted fungal effectors do not. Collectively, our findings identify a previously uncharacterized role of AvrStb6 in protecting Z. tritici from antagonistic wheat-associated bacteria by associating with the fungal cell wall. More broadly, this work highlights that fungal effectors may contribute to microbial competition and ecological adaptation beyond their established roles in host immune recognition. Author summaryPlant pathogens secrete proteins that help them colonize their hosts. Some of these proteins are recognized by plant immune receptors and trigger disease resistance, but their original biological functions often remain unclear. We investigated the role of AvrStb6, a protein produced by the wheat pathogen that causes Septoria tritici blotch. AvrStb6 is best known because it is recognized by a wheat resistance gene, yet its contribution to fungal growth and survival has remained unknown. We initially tested whether AvrStb6 directly inhibits bacteria that live on wheat leaves, but found no evidence that it acts as an antimicrobial protein. Instead, we discovered that AvrStb6 influences interactions between the pathogen and wheat-associated bacteria. Fungal strains lacking AvrStb6 were more sensitive to several bacterial species that naturally occur in wheat, particularly members of the genera Pseudomonas and Pantoea. We also found that AvrStb6 can associate with the fungal cell wall, suggesting that it helps protect the pathogen during encounters with antagonistic bacteria. Our findings reveal an unexpected role for a fungal effector in microbial competition and show that pathogen proteins traditionally studied in the context of plant immunity can also influence interactions with other microbes. This work highlights the importance of considering the broader microbial community when studying plant-pathogen systems.

The biotrophic pathogen Puccinia triticina is the causative agent of the most vulnerable foliar disease, namely leaf rust disease of wheat. The pathogen-secreted effectors are essential in modulating fungal virulence and host immune responses. Despite their significance, potential effectors and their underlying mechanisms governing host susceptibility remain elusive. In the present study, we employed an in silico approach to identify and characterise effector proteins from the P. triticina proteome. Later, performed temporal expression profiling to prioritise effector candidates associated with rust disease. Here, a total of 273 high-confidence effector candidates were identified and analysed their physicochemical properties, domains, motifs, and functional annotations, to assess their conservation and dynamics. Although most of the effectors were uncharacterised, the conserved motif virulence-associated [YFW]xC was notably enriched in the effector repertoire. Comparative PHI-base annotation highlighted similarities with known fungal virulence factors involved in host susceptibility. Effectors harbouring CAZyme activity indicate involvement in host cell wall modification. Promoter analysis identified multiple stress- and defence-related transcription factor binding sites, suggesting regulated expression during infection. Transcriptome analysis revealed that 20 effector genes were significantly upregulated during P. triticina infection. qRT-PCR validated the expression of 4 highly induced effector transcripts following P. triticina infection in susceptible wheat variety. Specifically, two of these candidates demonstrated biphasic expression pattern that aligns contrasting PTI- and ETI-mediated defense mechanisms critical for sustained virulence. Overall, this study provides a comprehensive framework for identifying functionally relevant P. triticina effectors and offers insight for future effector-target studies and effector-based leaf rust management strategies.

Plant immunity is, amongst others, mediated by receptor like proteins (RLPs), which are localized on the plasma membrane. RLPs recognize extracellular immunogenic patterns (ExIPs) originating from pathogens or derived from the host itself, which leads to extracellularly triggered immunity (ExTI). Cf proteins, which are well-known RLPs of tomato (Solanum lycopersicum) confer resistance against the fungal pathogen Fulvia fulva. Cf-9, Cf-4, Cf-2 and Cf-5 are well-known examples of Cf proteins, mediating recognition of the matching ExIPs Avr9, Avr4, Avr2 and Avr5, respectively, which are secreted effectors of F. fulva and trigger hypersensitive response (HR)-related cell death in tomato plants carrying these Cf proteins. Although all these Cf proteins confer proper resistance to the fungus, Cf-9 and Cf-4 trigger a stronger and faster cell death than Cf-5 and Cf-2. It is unknown whether these phenotypical differences arise from variations in the molecular mechanism of the cellular immune response that is initiated by the Cf proteins, and whether this phenotypic difference correlates with varying degrees in the intensity and timing of the triggered immune responses and robustness of the resistance. To try to answer these questions, in this study the immune responses triggered by Cf-4 and Cf-5 were compared. Cf-4 and Cf-5 share the same core upstream signaling components to trigger HR-related cell death in Nicotiana benthamiana. In tomato, both receptors induce rapid MAPK activation, which is more sustained for the Cf-5/Avr5 combination. Both Avr4 and Avr5 induce an apoplastic burst of reactive oxygen species (ROS), independently of the presence of their matching receptors, while remaining dependent on RBOHB for this ROS burst. Full transcriptome analysis at 3 and 7 hours after immune activation revealed a large shared set of differentially expressed genes, alongside qualitative and quantitative differences, with the Cf-5/Avr5 combination inducing a broader transcriptional reprogramming. Despite these differences, Cf-4 and Cf-5 confer a comparable level of resistance to F. fulva. These results demonstrate that Cf-4 and Cf-5 share conserved immune initiation mechanisms, but diverge in downstream signaling dynamics, and that the intensity and timing of the HR-related cell death do not affect the robustness of the resistance.

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.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

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.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most destructive diseases of wheat worldwide. Although the Z. tritici genome encodes hundreds of predicted effector proteins, functional characterization through the use of genome-editing techniques has been limited due to low homologous recombination efficiency and extensive effector redundancy. In this study, we established and evaluated a CRISPR/Cas9-based genome editing procedure for targeted effector gene disruption in Z. tritici using in vitro-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes combined with short (60 bp) homologous donor DNA flanks. Using this approach, we successfully generated knockout mutants for a selected candidate effector gene, the Hce2 domain-containing effector Mycgr3107904. Virulence assays on the susceptible wheat cultivar Taichung 29 revealed that two independent{Delta} Mycgr3107904 mutants exhibited a pronounced delay in symptom development compared to the wild-type strain IPO323, with disease onset and progression delayed by approximately 4-5 days. While mutant strains ultimately followed a similar disease trajectory, wild-type-infected leaves displayed extensive necrosis and pycnidia formation at earlier time points, indicating a significant reduction in virulence upon loss of Mycgr3107904. Together, our results demonstrate the feasibility of CRISPR/Cas9-mediated effector gene knockout in Z. tritici and provide functional evidence that Mycgr3107904 contributes to timely disease progression. This work advances genome editing tools for Z. tritici and facilitates systematic dissection of effector functions underlying fungal virulence.

Aphanomyces euteiches, the causative agent of Aphanomyces root rot (ARR), is of major concern for pea and other legume crops globally. This oomycete pathogen causes substantial decreases in crop yields, is unaffected by most fungicides, and persists in the soil for many years via its resilient oospores. Given the significance of pea crops in sustainable agriculture, namely the ability to fix nitrogen and act as a sustainable protein source, solutions to ARR are of high importance. We used RNA-seq in a novel strain of Pseudomonas donghuensis to identify two biosynthetic gene clusters under GacA/S control that are involved in producing bioactive molecules capable of inhibiting A. euteiches. Based on similarity to other reported clusters in Pseudomonas, the first is predicted to encode for a pseudoiodinine compound, while the second is predicted to produce the siderophore 7-hydroxytropolone. Individual knockouts of each cluster showed loss of inhibitory action of P. donghuensis NRC29 against A, euteiches in vivo. This is the first report highlighting the potential of P. donghuensis and the products of the two identified biosynthetic pathways as biocontrol agents for A. euteiches. Further investigations into the efficacy of P. donghuensis NRC29 and its metabolites in inhibiting A. euteiches in field trials will be of high value in developing sustainable strategies for ARR mitigation. ImportanceModern fungicidal treatments for control of root rot in pulse crops are ineffective for control of A. euteiches, leaving limited strategies for management of A. euteiches infected fields. We describe a novel P. donghuensis strain with potential for biocontrol against this persistent pathogen. Given the economic value of peas and other pulses globally, further work into harnessing the bioactive metabolites produced by this strain into a practical in-field treatment will be valuable.

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.

The fungal pathogen Pyricularia oryzae is notorious for causing blast disease in various important cereal crops, including wheat, rice, millet, and oat. Whole-genome-informed data on this pathogen are necessary to better understand the host adaptability of the fungus, including identifying key determinants of infection to enable more precise disease control. Here, we report highly contiguous genome sequences (using long-read PacBio technology) of two isolates from rice paddies in Portugal, M22.7 and T22.2, which exhibit distinctly aggressive symptoms in rice. Both mitochondrial and nuclear sequences were characterised in this study. The resulting nuclear genomes have assembly lengths of 46.4 Mb for M22.7 (198x coverage) and 46.3 Mb for T22.2 (163x coverage), with near-complete BUSCO completeness (98.8%) and a 0% contamination score (EukCC). Phenotypic analysis showed M22.7 to be more virulent than T22.2, which may be explained by the lower number of predicted effector genes and higher transposable element content in M22.7 relative to T22.2. This announcement represents the first genome resource for natural isolates of P. oryzae from Portugal in over 20 years, filling an important data gap from a major European rice-producing country that produces locally adapted rice varieties under specific agro-environmental conditions (near the Atlantic coast).

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.

Botrytis cinerea is a necrotrophic plant pathogen with an extremely wide host range. During invasion, the fungus induces rapid host cell death and proliferates in the necrotic tissue. The mechanisms of host killing are still incompletely understood, they involve secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs). We have previously shown that the sequential knockout of up to 12 CDIPs leads to a substantial reduction of virulence of B. cinerea mutants. In this study, we have identified additional CDIPs and generated an extended mutant series culminating in a 29x mutant which is deficient in most currently known CDIPs and two phytotoxic metabolites. These mutants are strongly reduced in infection, but still induced necrosis and grey mould symptoms, demonstrating that additional determinants of host killing remain unidentified. Overexpression of the highly phytotoxic Nep1 in a 22-fold CDIP mutant failed to increase its virulence. Reevaluation of several CDIPs previously described as virulence factors revealed for most tested CDIPs no major contribution to pathogenesis. Together with the observation that none of the CDIPs are specific to B. cinerea, our data question a particular role of CDIPs for necrotrophic pathogenesis. In contrast, generation of a mutant lacking all six predicted endo-polygalacturonases confirmed their major but not exclusive role for tissue degradation and infection.