Molecular Plant Pathology

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.

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.

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.

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.

- 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.

Peach shoot blight (PSB), caused by Diaporthe amygdali, is one of the most devastating fungal diseases affecting peach production worldwide. Glutaredoxins (Grxs) are ubiquitous redox-regulatory proteins involved in antioxidant defense and iron metabolism, yet their functions in phytopathogenic fungi remain largely unknown. Here, we identified four Grxs (DaGrxA-DaGrxD) in D. amygdali. Among them, DaGrxB, a monothiol glutaredoxin, is the sole member essential for vegetative growth, asexual reproduction and virulence. DaGrxB interacts with iron-regulatory transcription factors DaHapX and DaSreA and modulates their activities via iron-dependent nucleocytoplasmic shuttling, coordinating iron acquisition and storage gene expression. During infection, D. amygdali encounters a biphasic iron environment: an inherently iron-limited host environment at early stages (1-2 dpi) followed by host-mediated iron excess (3-4 dpi), and DaGrxB is essential for navigating this transition. Consistently, deletion of either DaHAPX or DaSREA significantly reduced virulence, confirming that iron homeostasis is a critical determinant of D. amygdali virulence. Notably, deletion of DaGRXB disrupts Fe-S cluster biosynthesis, causing mitochondrial Fe2+ accumulation, impaired aconitase activity, and reduced ATP production, which triggers constitutive activation of the GSH-dependent antioxidant system, with elevated GSH/GSSG ratios, increased GR and GST activities, and reduced H2O2 levels. Exogenous GSH and H2O2 depletion both reduced virulence, suggesting that disturbed redox balance directly contributes to attenuated virulence. Additionally, the Trx domain assists the Grx domain in nucleocytoplasmic translocation through intramolecular interactions. Finally, LDH-delivered dsRNA targeting DaGRXB effectively controls PSB. Collectively, these findings reveal how a monothiol glutaredoxin integrates iron homeostasis, redox balance and virulence in a plant pathogen, providing a foundation for RNAi-based disease management.

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.

Pseudomonas species include important plant pathogens responsible for substantial agricultural losses. Although CRISPR-INTEGRATE has been applied for genome engineering in medically relevant Pseudomonas, its utility in plant-pathogenic Pseudomonas has not been established. Here, we demonstrate CRISPR-INTEGRATE, an RNA-guided Tn7-like transposition system that enables targeted chromosomal integration without double-strand breaks or host-dependent homologous recombination, across three plant-pathogenic Pseudomonas backgrounds. Targeted disruption of hrpJ, hrcC, and gacA was confirmed by PCR and Sanger sequencing and resulted in distinct phenotypes, including impaired type III secretion-associated virulence and hypersensitive response, altered colony morphology and biofilm formation, and reduced motility. We further coupled CRISPR-INTEGRATE with Cre-lox recombination to excise a ~40-kb prophage-associated region from the P. syringae pv. tomato DC3000 chromosome. Genomic characterization identified a 33.45-kb predicted prophage within this region, putative attL/attR sites sharing an identical 14-bp core sequence, an orthologous empty locus containing a candidate attB site, and conserved prophage-associated gene organization across P. syringae genomes. Deletion of the region altered bacterial growth and increased susceptibility to phage infection, with the strongest effect observed for phage Plaza, while having no detectable effect on the tested plant virulence phenotypes. Together, these findings establish CRISPR-INTEGRATE as a portable genome-engineering platform for plant-pathogenic Pseudomonas and demonstrate its utility for functional interrogation of individual genes and large accessory genomic elements.

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.

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.

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.

Double-stranded RNA (dsRNA) biopesticides that silence essential fungal genes by spray-induced gene silencing (SIGS) are advancing towards registration, underpinned by the claim that silencing is confined to sequence-matched target organisms, a claim that has never been tested systematically against beneficial fungi. We screened twelve dsRNA constructs (the whole transcripts of eleven canonical antifungal target genes of Fusarium graminearum: three CYP51 sterol 14-demethylases, six chitin synthases and two {beta}-tubulins, plus a concatenation of the three CYP51 transcripts modelling the flagship CYP3RNA design) for perfect 21-nucleotide (21-nt) identity, the primary match criterion of recent regulatory bioinformatics frameworks, against the transcriptomes of seven non-target fungi, including commercial biocontrol agents (Trichoderma harzianum, T. virens, Beauveria bassiana, Metarhizium anisopliae, M. brunneum), the arbuscular mycorrhizal fungus Rhizophagus irregularis and Saccharomyces cerevisiae. Eleven of the twelve constructs carried off-target hazard; only CYP51C was completely clean. TUB2b carried 580 perfect 21-nt matches and contained no hazard-free window of 250 consecutive 21-mer start positions, whereas 26.6-60.0% of screened windows were hazard-free in the designable genes. Hits landed overwhelmingly on orthologues of the targeted gene and concentrated in the Hypocreales; R. irregularis and S. cerevisiae were nearly clean. These are hazard findings, not risk findings: perfect siRNA-length matches to beneficial fungi are common in canonical antifungal dsRNA targets, and current regulatory screening panels contain no fungus that would detect them.

The Unfolded Protein Response (UPR) maintains endoplasmic reticulum (ER) homeostasis during proteotoxic stress while also contributing to plant immunity. However, the functional relationship between UPR signaling, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA) remains poorly understood. Here, we investigated this interplay using Arabidopsis mutants impaired in the two major UPR branches. Mutants defective in the INOSITOL-REQUIRING ENZYME 1 (IRE1)-bZIP60 branch displayed enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and to the type III secretion-deficient strain hrcC-, indicating potentiation of PTI. Consistently, the ire1a ire1b double mutant exhibited increased flg22-induced callose deposition, whereas reactive oxygen species production was unaffected. Despite enhanced basal resistance, all UPR mutants retained full responsiveness to benzothiadiazole, demonstrating that downstream SA signaling remains functional independently of UPR integrity. Conversely, acute ER stress induced by tunicamycin promoted SA accumulation and extensive leaf necrosis. Reverse genetics revealed that SA-deficient mutants were largely protected from ER stress-induced cell death, whereas mutants over-accumulating SA exhibited enhanced susceptibility, demonstrating that endogenous SA is required for the full execution of this response. Analysis of an independent transcriptomic dataset further showed that loss of IRE1 de-represses genes involved in SA biosynthesis, PTI and defense under ER stress. Together, our results identify IRE1 as a key regulator coordinating ER homeostasis with immune signaling by limiting SA-dependent defense activation during ER stress, while simultaneously preventing excessive cell death.

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.

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.

Canonical ribonucleases such as Argonaute (AGO) participate in RNA-based gene regulatory processes. They are important for small RNA (sRNA) association and the formation of RNA-induced RNA silencing complexes. Potato (Solanum tuberosum) has 14 AGO-encoding genes, including the Solanum-specific StAGO15, which is activated upon infection by filamentous pathogens. The resistant phenotype conferred by ectopic expression of StAGO15 was abolished in plants in which a 21-nt sequence in the PAZ-encoding region of StAGO15 was targeted using an artificial miRNA. We then analyzed the sRNAs associated with StAGO15 in Phytophthora infestans leaf infection. Small RNAs were coimmunoprecipitated, subjected to deep sequencing, and analyzed against the P. infestans and potato genomes, in which cleavage sites were predicted by applying the R package, smartPARE. The most notable difference between pathogen- and water-inoculated StAGO15 samples was observed in the transposon (TE) category. Here, two categories of nonautonomous TE dominated: the miniature inverted-repeat transposable elements (MITEs) and short interspersed nuclear elements. The StAGO15-associated sRNAs bind and target complementary endogenous potato RNAs. One example is siRNA122, which acts on a cleavage site in the resistance gene PGSC0003DMG400015681 (StRES1). siRNA122 is derived from a MITE; upon deletion of this source sequence, resistance is restored. Our findings provide new insights into the plant immune surveillance network and demonstrate that deleting a 21 nt RNA sequence from a MITE transposon released a defense response, against two important filamentous pathogens.

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.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.