Molecular Plant-Microbe Interactions®

Phakopsora pachyrhizi, the causal agent of soybean rust, is among the most aggressive and economically impactful pathogens affecting soybean (Glycine max) production worldwide. As a dikaryotic rust fungus, P. pachyrhizi maintains its genetic information as two discrete haplotypes that are sequestered within separate nuclei for most of its life cycle as it reproduces asexually via the production of infectious urediniospores. Previous genomes of three P. pachyrhizi isolates collected in South America revealed a large genome with a high transposable element content, measuring up to 1.25 Gb and 93%, respectively. Although these efforts provided valuable foundational resources, the complexity of the genome resulted in highly fragmented assemblies. Here, we generated a highly contiguous, substantially improved, annotated genome resource from the P. pachyrhizi isolate LA04-1, collected during the initial outbreak of soybean rust in the continental United States in 2004. The assembly is haplotype-phased, with each haplotype assembled into 18 chromosomes totaling 572 Mb and 569 Mb in size. The isolate contains unique mating-type genes and does not share haplotype genomes with any available isolates with whole-genome sequencing datasets. This resource will be valuable to future investigations into the underlying mechanisms of virulence in this important plant pathogen.

Despite the recent cloning of RLK-encoding resistance genes and the identification of a common point of hyphal arrest at the stomatal level in incompatible interactions, the molecular mechanisms leading to the establishment of wheat resistance against Septoria tritici blotch (STB) remain poorly characterized. Here, we use long-read RNA-sequencing data to compare the early transcriptomic responses of two wheat cultivars carrying major, isolate-specific resistances to Zymoseptoria tritici via differential expression, differential transcript usage (DTU), functional- and pathway enrichment analyses. We show that both magnitude and composition of the responses depend strongly on the combination of host cultivar and pathogen isolate and that DTU appears to be uncommon at this infection stage. We do not detect a set of genes exclusively differentially expressed across incompatible interactions, but genes coding for proteins involved in several defense-related processes consistently show higher expression levels in incompatible than compatible interactions, including receptors and ROS homeostatic, antifungal hydrolytic, phenylpropanoid and diterpenoid biosynthetic enzymes. Our results suggest that whilst interaction-specific differences in magnitude and composition may also shape resistance, different major STB resistances may quantitatively enhance overlapping defense machinery, providing a basis for its functional investigation.

Pectobacterium, a member of the soft rot Pectobacteriaceae (SRP), can cause highly destructive soft rot and blackleg diseases on many important crop and other host plants, particularly potato (Solanum tuberosum). Pectobacterium and its sister genus Dickeya have common virulence strategies, yet a highly diverse "open" pangenome containing many unique and hypothetical proteins. Here, we used randomly-barcoded transposon insertion-site sequencing (RB-TnSeq) on six Pectobacterium species to identify genes involved in bacterial survival and growth in potato tubers, as well as in minimal media conditions containing different single carbon sources. The strains evaluated varied broadly in genomic content as well as host plant and year of isolation. We focused on identifying conditionally essential genes across all strains tested, as well as genes that were important for growth in one or few of the tested strains. These results highlight the importance of many common genes involved in metabolism and maintaining cellular homeostasis, as well as shared and rare virulence traits including polygalacturonate catabolism, genes involved in polysaccharide biosynthesis, immunity proteins, and (for P. brasiliense) self-tolerance of the antibiotic carbapenem. This large dataset of conditionally essential genes, generated across Pectobacterium species during soft rot infection of the agriculturally important plant host potato, can serve as a rich dataset for further hypothesis generation and identification of important and/or novel in planta fitness factors.

- The ascomycete fungal pathogen Blumeria hordei (Bh) is predicted to secrete over 530 effector proteins to infect barley and cause powdery mildew disease, yet their individual and collective functions are largely unexplored. We used yeast two-hybrid next-generation interaction screening (Y2H-NGIS) to probe the host targets of 48 Bh candidate secreted effectors (CSEPs), including multiple AVRA effectors that are recognized by MLA nucleotide-binding leucine-rich repeat (NLR) receptors. - Y2H-NGIS and subsequent binary retesting identified 356 high-confidence protein-protein interactions (PPI) among Bh CSEPs, MLA NLR fragments, and barley proteins. We uncovered unique and shared host targets, including numerous proteins involved in ubiquitin-associated processes, gene expression, immune and kinase signaling, metabolism, and vesicle trafficking. - We identified a host protein (HvCAP2) that interacts with MLA and at least five Bh effectors, as well as additional effector-host PPI that correlate with loss of recognition of AVRA13 and AVRA6 by MLA13 and MLA6, respectively. - Results were integrated with an interolog-based barley interactome (HvInt) to assemble a host-pathogen network of 1248 proteins and 1701 interactions. This PPI resource can serve as a platform for functional investigations into the molecular basis of powdery mildew pathogenesis and host recognition.

Genomic structural variants (SVs) are important sources of genetic diversity and contribute to the adaptation and evolution of plant-pathogenic fungi. Colletotrichum graminicola, the causal agent of maize (Zea mays L.) anthracnose, causes significant yield losses and serves as an important model for genetic studies. To investigate genomic plasticity in this pathogen, long-read genome assemblies were generated for four C. graminicola field strains collected in France, Croatia, Brazil, and Argentina and compared with the chromosome-level reference genome from the USA. Comparative analyses revealed extensive structural variation across core chromosomes, including large-scale translocations, inversions, duplications, and insertions and deletions, whereas minichromosomes showed markedly reduced collinearity among strains. Secreted proteins and predicted effectors were located significantly closer to regions affected by SVs. Approximately 174 kb of segmental duplications lacking genes were shared between core chromosome 04 and minichromosomes 11 and 13 in the reference genome but were absent from the other field strains, revealing a strain-specific structural link between core and minichromosomes. Protein clustering identified 733 candidate strain-specific genes across the reference genome. Approximately 72% of the genes annotated on the minichromosomes were strain-specific, indicating that most of their annotated genes lacked orthologous gene models in the other strains. Of the 733 candidate strain-specific genes, 492 retained homologous genomic sequences in at least one other strain and many also showed evidence of gene disruption. This pattern is consistent with lineage-specific pseudogenization and suggests that apparent gene presence-absence variation may arise through several evolutionary processes, including gene gain, gene loss and loss of gene function. Insertions and deletions in potentially regulatory flanking regions were associated with plant-pathogen interaction genes. In addition, interpretable machine learning analyses revealed a co-occurrence of duplications and LTR retrotransposons in genomic regions associated with secreted genes. This study shows that structural variation, transposable elements, and lineage-specific pseudogenization are important features of genome evolution in C. graminicola and identifies associations between genomic variation and genes involved in plant-pathogen interactions.

Background: Plant defensins are small cysteine-rich antimicrobial peptides. In Arabidopsis thaliana, the inducible Class I gene AtPDF1.2a is widely used as a transcriptional marker of jasmonate/ethylene immunity, whereas the constitutively expressed Class II members have received less functional attention. AtPDF2.2 was compared with AtPDF1.2a during infection of Arabidopsis by the vascular fungal pathogen Verticillium longisporum, the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas syringae pv. tomato DC3000. Results: Profiling of the AtPDF family showed that AtPDF2.2, together with the Class II genes AtPDF2.3 and AtPDF2.5, was significantly reduced during V. longisporum infection, whereas AtPDF1.2a was induced. A promoter-reporter line showed a corresponding decrease in AtPDF2.2 promoter activity during V. longisporum infection, strongest in infected roots. KD-pdf2.2 plants contained more V. longisporum DNA, whereas OE-PDF2.2 plants contained less. The same reciprocal disease pattern was observed during S. sclerotiorum and Pst DC3000 infection. Histochemical Prom::AtPDF2.2::GUS reporter signal decreased during S. sclerotiorum infection but increased following Pst DC3000 infection. Basal AtPDF2.2 expression was lower in JA and ET-signaling mutants and higher in the SA-deficient sid2 background, while the analyzed promoter lacked the GCC-box that mediates inducible AtPDF1.2a regulation. AtPDF1.2a overexpression reduced symptoms across the three pathosystems, but loss of AtPDF1.2a increased disease only during V. longisporum infection under the conditions tested and did not alter Pst DC3000 DNA abundance. AtPDF2.2 overexpression was not associated with reduced vegetative growth under the conditions tested, whereas AtPDF1.2a overexpression reduced rosette size and leaf number. Conclusions: AtPDF2.2 is a constitutively expressed Class II defensin whose expression level is associated with disease outcome across two fungal pathogens and a bacterium. Its promoter shows tissue-dependent regulation during V. longisporum infection, while histochemical reporter signal decreases during S. sclerotiorum infection and increases during Pst DC3000 infection. This regulatory behavior differs from that of the inducible reference gene AtPDF1.2a. The reciprocal disease phenotypes of the AtPDF2.2 transgenic backgrounds support a protective association that may involve direct antimicrobial activity, modulation of host responses or both. The absence of reduced vegetative growth in OE-PDF2.2 under the conditions tested supports further evaluation of PDF2-type genes in Brassica.

Cucurbit yellow vine disease (CYVD) is a phloem disease caused by a group of Serratia ureilytica bacteria within the S. marcescens complex. The CYVD pathogen is distinct from other phloem pathogens in two fundamental ways: i) unlike other bacterial phloem pathogens, the CYVD pathogen readily grows on standard laboratory media; and ii) whereas most bacterial phloem diseases are transmitted solely by hemipteran insects, CYVD can be transmitted by both hemipteran (squash bug) and non-hemipteran (cucumber beetle) vectors. The phloem of cucurbits is also distinct, with cucurbits having a unique secondary phloem system, the extrafascicular phloem (EFP), for which a role in CYVD has not been investigated. The distinctive features of this pathosystem prompted a re-evaluation of pathogen localization in planta using a green fluorescent protein-tagged CYVD strain during infection of squash (Cucurbita pepo). In epifluorescence and confocal microscopy images of transverse and longitudinal sections of stem and petiole tissues, the fluorescence patterns of the CYVD pathogen were consistent with pathogen colonization of all four known EFP tissues, namely the peripheral, entocyclic, ectocyclic, and commissural sieve tubes. The CYVD pathogen may enter the EFP during herbivory by pathogen-infected vectors that cause mechanical damage, as the EFP exhibits much slower blockage following injury than the conventional fascicular (bundle) phloem. Moreover, although EFP has been proposed to have an antimicrobial function, EFP exudate collected from the stem and petioles of C. pepo supported rapid pathogen growth. These results provide evidence for a unique tissue tropism of the newly emerged plant pathogen S. ureilytica.

Plant pathogen genomes adapt across coding and non-coding regions in response to their hosts, environments, and disease management practices. Venturia inaequalis, the causal agent of apple scab, has evolved alongside its Malus host throughout apple domestication. We generated 17 high-quality genome assemblies, including the first chromosome-scale assembly of V. inaequalis, to define the species' complete gene content and investigate the role of structural variation in genome evolution. Together with a previously published scaffold-level reference, these assemblies were used to construct a gene-based pangenome containing 215,301 predicted protein-coding genes clustered into 13,240 orthogroups, 88.11% of which were core (73.86%) or soft-core (14.25%), 11.88% shell (present in 2-16 isolates), and 0.1% private, indicating a highly conserved gene repertoire. Virulence-associated effectors were significantly enriched in the soft-core and shell components, and accessory apoplastic effectors displayed elevated nucleotide diversity and nonsynonymous-to-synonymous substitution ratios (Ka/Ks) relative to core effectors, consistent with diversification. We further identified a putative accessory chromosome of variable size (23-132.6 kb) in European and U.S. isolates encoding genes with virulence function. A graph-based pangenome identified 38,799 non-redundant structural variants (SVs), largely driven by Gypsy LTR retrotransposons. We used the graph-based pangenome to genotype 136 globally distributed V. inaequalis isolates and identified variants differentiating populations associated with domesticated apple (Malus domestica) from those associated with its Central Asian wild progenitor, M. sieversii. Together, these resources establish a foundation for future studies of diversity, host adaptation, and genome evolution in V. inaequalis.

Microbial biological control agents (mBCAs) offer a promising alternative to conventional disease management, but identifying effective strains requires screening large microbial collections against many pathogens. Here, we developed a scalable screening approach to characterize the antagonistic activity of potato-associated bacteria against multiple pathogens and investigated how bacterial origin and phylogeny relate to antagonistic phenotypes. A collection of 600 bacterial strains isolated from two potato cultivars, three plant compartments using four cultivation media was screened against six major potato pathogens: the bacteria Dickeya solani and Pectobacterium carotovorum, the fungi Alternaria solani and Rhizoctonia solani, and the oomycetes Phytophthora infestans and Pythium ultimum. A scalable confrontation method was developed for non-filamentous bacteria, while complementary lower-throughput assays were used for filamentous strains. This approach enabled successful assessment of 92% of non-filamentous and 97% of filamentous strain-pathogen combinations. Isolation cultivar, plant compartment and cultivation medium had only limited effects on the proportion of antagonistic strains. In contrast, pathogen sensitivity varied markedly: P. infestans was the most susceptible pathogen, whereas D. solani and P. carotovorum were generally resistant to bacterial antagonism. Antagonistic activity clustered phylogenetically, with Bacillus and Streptomyces containing many broad-spectrum inhibitors. In addition, several taxa showed preferential activity against the oomycetes, including P. infestans-specific antagonists from the Pseudomonas genus and P. ultimum-specific antagonists from the Frigoribacterium, Curtobacterium, Pedobacter and Phyllobacterium genera. Overall, multi-pathogen screening revealed distinct generalist and specialist antagonistic profiles and identified candidate strains and taxa for further evaluation as mBCAs.

Salivary secretions of herbivorous insects contain proteins that modulate plant defence and facilitate herbivory. Despite the broad host range of Spodoptera frugiperda, a major agricultural pest, its salivary molecular arsenal involved in interactions with different host plants remains poorly understood. In particular, the salivary proteins induced during feeding on different host plants and their potential interactions with plant immune components remain largely unexplored. To address this gap, we generated salivary gland transcriptomes from larvae reared on an artificial diet as well as larvae fed on tomato or chickpea plants. Comparative transcriptomic analysis identified salivary genes that were differentially expressed in response to plant feeding compared with the artificial diet. The encoded proteins were subsequently analysed using a secretory protein prediction pipeline to identify host-induced putatively secreted proteins with potential effector functions. Candidate salivary effectors were further investigated using large-scale in silico protein-protein interaction analyses with AlphaPulldown to predict their interactions with immune-pathway associated proteins of tomato and chickpea. This analysis identified multiple putative salivary effector candidates whose expression was induced by plants feeding, with distinct candidate repertoires associated with tomato and chickpea. Predicted interactions between these candidates and plant immune-associated proteins further identified potential host targets. Together, these findings suggest that S. frugiperda may adjust its salivary molecular repertoire in response to the host plant and provide a transcriptome-wide framework for validating effector-target interactions and elucidating the molecular mechanisms underlying S. frugiperda-plant interactions.

Fusarium graminearum is the causative agent of Fusarium head blight of wheat, yet genomic resources for Brazilian populations, where it is the dominant species, remain limited to a single reference genome. We report fifteen near telomere-to-telomere (T2T) assemblies of Brazilian F. graminearum isolates generated with Oxford Nanopore sequencing. Each genome was assembled into four nuclear chromosomes, with 118 of 120 telomeres resolved, BUSCO completeness of 99.199.3%, and 14,80214,894 predicted genes per isolate. The genomes are structurally conserved, showing no chromosome-scale rearrangements and uniform transposable element content. Orthology assignment placed 90.4% of genes in the core genome. This suggests that the large phenotypic variance in aggressiveness observed between isolates likely resides in regulatory differences and/or within the small and largely uncharacterised accessory fraction. Ultimately, these assemblies provide a high-resolution resource for the global FHB community and a foundation for comparative analysis of a previously underrepresented population.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

Fusarium oxysporum f. sp. lactucae (Fola), the cause of lettuce fusarium wilt, poses an increasing threat to global production due to the rapid emergence of resistance breaking isolates. However, the evolutionary origins and mechanisms underpinning this diversification remain poorly understood. We characterised 103 Fola isolates using molecular profiling, selecting ten key isolates for long-read whole-genome sequencing and performed in planta virulence assays for selected isolates. This included the first assemblies for Fola2 and Fola3, recent resistance-breaking variants and a highly divergent isolate from France designated as a putative novel race, FolaX. Phylogenomic analysis revealed that Fola is polyphyletic, with lettuce pathogenicity acquired independently across divergent F. oxysporum clades. Despite this, all lineages share a conserved "PanFola" effector set with near-identical sequences, indicating extensive horizontal gene transfer. Resistance-breaking Fola variants within established races were linked to copy number variations and targeted deletions, such as the loss of the SIX8/PSE1 locus in Fola4+. Conversely, FolaX possessed a divergent core genome and a chimeric accessory genome derived from multiple Fola lineages via segmental chromosome transfer and transposon-mediated reshuffling. Our findings highlight the remarkable evolutionary plasticity of Fola and the need for breeding strategies that address the dynamic nature of effector repertoires.

Sudden Death Syndrome (SDS), caused by Fusarium virguliforme, can significantly impact soybean yields. To advance the understanding of SDS resistance in plant introductions, we conducted genome-wide association studies (GWAS) on a set of 351 accessions from the USDA Soybean Germplasm Collection. The analysis revealed seven single nucleotide polymorphisms (SNPs) significantly associated with foliar SDS resistance; three significant QTL (on chromosomes 3, 9 and 11) were detected with Fixed and Random Model Circulating Probability Unification (FarmCPU) and four (on chromosomes 5, 6 and two on chromosome 7) with Bayesian-information and Linkage disequilibrium Iterative Nested Keyway (BLINK). Localized linkage disequilibrium blocks were estimated for these regions. Notably, all of them overlapped with regions where QTL associated with SDS resistance had been previously reported and where within, or close to, sequences annotated as implicated in plant defense mechanisms, such as toxin transporters and transcription factors. The architecture of SDS resistance appears quantitative and polygenic, with most loci explaining modest phenotypic variance. Six plant introductions in this study--PI 175188, PI 307889A, PI 323559, PI 578307C, PI 445683, and PI 323558--harbor favorable alleles at all seven loci, providing high-value resources for resistance introgression. This study underscores the value of leveraging the USDA Soybean Germplasm Collection for the discovery of resistance loci and provides a foundation for marker-assisted breeding to enhance SDS resistance in elite soybean lines.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana. Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1/sobir1-like N. benthamiana. These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

The Fusarium oxysporum species complex (FOSC) comprises numerous plant pathogens infecting crops such as tomato, lettuce, melon, and banana. Despite its economic and ecological importance and decades of research, the species names linked to different phylogenetic lineages within this species complex remain largely controversial. Earlier taxonomic frameworks relied on a limited number of marker genes, variable taxon sampling and inconsistent species definitions, leading to a fragmented and unstable taxonomy. To address these challenges, we here analysed nearly 2,500 conserved marker genes from 660 genomes, including genome assemblies from 22 ex-type strains that were sequenced using long-read sequencing technology, representing nearly half of the 51 currently described species within FOSC. Maximum likelihood- and coalescence-based analyses of the full phylogenomic data matrix as well as subsets yielded a robust phylogeny of FOSC, which was furthermore corroborated by whole genome comparisons. Many previously described species are redundant, while two novel lineages remain undescribed. Direct comparisons to traditional marker gene-based approaches demonstrate their inadequacy for species delimitation in FOSC. We show that our phylogenomic framework can guide the detection of novel marker genes able to distinguish the major lineages within FOSC, which will be essential for taxon delineation and classification in future studies. Thus, our phylogenomic analyses provide a stable cornerstone for revising FOSC taxonomy and offers a critical resource for the broader research community.

Fungal diseases cause devastating crop infestations and millions of deaths annually. To improve disease treatment development, we must understand the biological processes these treatments target and the fungal cellular responses they elicit. Current methods to identify genetic responses of fungi to antifungal drugs and fungicides typically involve laborious molecular genetics experiments or transcriptomics. We utilized high-throughput screens in the basidiomycete yeast Rhodotorula (Rhodosporidium) toruloides to rapidly identify genes associated with the fungal response to a copper-zinc fungicide developed by VM Agritech and compared identified genes to those transcriptionally activated in response to the fungicide in R. toruloides and the filamentous ascomycete Neurospora crassa. These 'omics technologies combined with molecular genetics and biochemical analyses showed copper and zinc synergize to inhibit fungal growth due, at least in part, to an increase in cellular zinc concentrations when copper is present. Intriguingly, although we identified genes involved in the copper-zinc fungicide response using both high-throughput screens and transcriptomics, there was little overlap in genes identified through the two methods. Our findings demonstrate the power of combining functional genomics and transcriptomics approaches in identifying a more comprehensive map of the genetic mechanisms necessary for responding to fungicides.

Common tansy (Tanacetum vulgare), a significant agricultural invasive species, threatens forage productivity, livestock carrying capacity, and plant biodiversity across North American agroecosystems. Despite being a close relative to the ornamental Asteraceae Chrysanthemum, T. vulgare genomic studies are severely lacking, but remain critical to the development of novel weed management tools and the investigation of plant invasion evolution. In this study, we performed the assembly and annotation of the diploid genome (2n=2x=18) of an invasive North American T. vulgare genotype using long-read PacBio HiFi and Iso-Seq sequencing technology. The assembled haplotype genome comprised 9 pseudomolecules with a total size of ~4.8 Gb and a scaffold N50 of 546 Mb. In addition, both the plastid and mitochondrial genome sequences were assembled. BUSCO analysis revealed a high overall genome completeness with 69,343 predicted protein-coding genes (AED<0.6: 98%). A high-quality North American invasive reference T. vulgare genome, complementing a recent one assembled for a native European accession, is critical for the investigation of plant invasion biology, genome evolution, and the future development of novel genetic biocontrol weed management technologies.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.