Molecular Plant-Microbe Interactions®

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.

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.

A highly contiguous and complete reference genome of Cercospora cf. flagellaris, the causal agent of foliar disease on many plant hosts including Cercospora leaf blight of soybean, was assembled using a combination of PacBio and Illumina sequencing reads. The genome assembly is 33.72 Mb in length and consists of 14 nuclear scaffolds and one mitochondrial contig. Four scaffolds have telomeric repeats on both ends and represent fully assembled chromosomes, while nine scaffolds represent partially assembled chromosomes with telomeric repeats on one end. The assembly has an N50 of 2.90 Mb and an L50 of 5 scaffolds. Genome annotation identified 11,268 genes, of which 947 and 360 were predicted to encode secreted proteins and effectors, respectively. Additionally, 512 genes were predicted to encode carbohydrate-active enzymes and 60 biosynthetic gene clusters were annotated. Taken together, this annotated genome assembly will be a valuable resource for genomics, host-pathogen interactions, and population biology research in this economically important pathosystem.

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.

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.

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.

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.

To successfully infect their myriad hosts, generalist plant pathogens must tolerate a vast arsenal of plant specialized defense metabolites. To understand how host-specific metabolites influence plant-generalist pathogen interactions, we conducted a co-proteomic analysis of both Arabidopsis thaliana and Botrytis cinerea proteomes from the same samples during early infection. The Arabidopsis proteomic responses to Botrytis center around induction and suppression of defense metabolite pathways, particularly camalexin and glucosinolates. Several Botrytis proteins involved in key virulence pathways were induced within 32-48 hours, including potential defense metabolite detoxification proteins. Co-proteomic analysis using a panel of Arabidopsis genotypes with differing glucosinolate profiles revealed that disruptions to the glucosinolate pathway had broad changes on the Arabidopsis proteome, and that Botrytis induces specific proteins in response to presence/absence of Arabidopsis defense metabolites. Among the proteins that were induced quickly on infection and linked to the presence of glucosinolates, we validated a novel isothiocyanate hydrolase in Botrytis, BcSaxA, that catabolizes isothiocyanates in vitro. Gene expression data further indicated BcSaxA is expressed only in dicot hosts containing isothiocyanates. Our study describes a highly dynamic host proteome during infection with Botrytis and elucidates metabolite-specific infection strategies for a generalist pathogen.

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.

A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two "earliest" field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

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.

Bacterial leaf streak disease (BLS), caused by Xanthomonas translucens, is a re-emerging disease of cereals with few effective control measures. Although the disease was identified over 100 years ago, the fundamental molecular biology and mechanisms governing host-pathogen interactions, colonization, host responses, and disease development are poorly understood. To address these knowledge gaps, we studied the early stages of host-microbe interactions between cereal hosts (wheat and barley) and X. translucens pv. undulosa (Xtu). We found that, while the Type III Secretion System (T3SS) is essential for disease and is associated with a 12- or 150-fold increase in bacterial populations in barley and wheat, respectively, the T3SS is not sufficient for disease development. Xanthan, an Xtu-derived exopolysaccharide, strongly contributes to symptom development by suppressing the host immune response. However, in the absence of xanthan, bacterial populations in wheat are unaffected, and in barley xanthan only accounts for a 4-fold difference compared to the wildtype Xtu. Pathogen-associated molecular pattern (PAMP) inhibition bioassays that prime the plant immune response for subsequent infections revealed that xanthan suppresses defense priming. We also found that, in the absence of xanthan, the host immune system recognizes a potentially novel, unidentified microbe-associated molecular pattern (MAMP) of proteinaceous nature present in both X. translucens pathovars, undulosa (Xtu) and translucens (Xtt). Together, our work reveals both conserved and distinct X. translucens plant immune responses and pathogen elicitors, providing key insights into the host-pathogen interaction.

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.

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

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.

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.

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).

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/.

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.

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.