Rust fungi are significant threats to global food security, causing substantial damage to crops through their ability to evolve new strains that overcome plant resistance. These obligate biotrophs infect host plants by secreting effector proteins that manipulate host physiology to promote infection and colonisation. We used AlphaFold2 to investigate structural conservation among effector proteins for the secretomes of Melampsora lini and four Puccinia species. AlphaFold2 yielded high-confidence predictions for 45.7% of the 27,090 secreted proteins, while 19% were poor quality. Comparative analysis revealed extensive structural diversity across the rust secretomes, with all thirteen known rust Avr proteins belonging to different clusters apart from AvrSr13 and AvrSr33. Nevertheless, there were still numerous large clusters of structurally-related proteins, including 59 clusters with over 50 members each, three of which contained known Avr proteins. Of the major structural families defined in other fungi, the rust species studied here only contained FOLD and ToxA-like families. Structural analysis of cysteine-rich proteins revealed over a thousand effector candidates featuring predicted zinc-binding sites, with approximately 75% predicted to be cytoplasmic effectors. In contrast, cysteine-rich apoplastic effector candidates were characterized by a high frequency of disulfide bonds. One family of predicted metal-binding proteins was greatly expanded in P. graminis f. sp. tritici and includes AvrSr13 and AvrSr33. We confirmed that purified AvrSr13 and AvrSr22 proteins bind to zinc in vitro using biochemical assays. Taken together, structural modeling provides new avenues to study sequence-unrelated effectors and highlights the high degree of diversity in the effector repertoires of rust species.

Lasiodiplodia theobromae (Botryosphaeriaceae) is a cosmopolitan ascomycete that endophytically colonizes over 500 plant species and switches to necrotrophy under host stress. In mango (Mangifera indica), it causes stem-end rot, dieback, gummosis, and collar rot, driving substantial pre- and postharvest losses worldwide. This review integrates five thematic areas: (i) multilocus phylogenetics (ITS, tef1-α, tub2, and rpb2) resolving cryptic species within the L. theobromae species complex; (ii) disease epidemiology centered on latent colonization of inflorescences and stress-triggered activation; (iii) molecular pathogenicity mechanisms, including secreted effectors, cell wall-degrading enzymes, fungal phytohormones, and regulatory small RNAs; (iv) multi-omics advances revealing expanded CAZyme and secondary metabolite biosynthetic gene clusters, plus emerging multi-class fungicide resistance (MBC, QoI, and DMI) driven by β-tubulin mutations, alternative oxidase upregulation, and CYP51 overexpression; and (v) sustainable biological control using Streptomyces, Bacillus, and Trichoderma strains, alongside host priming with hexanal or methyl jasmonate. We propose a testable model wherein environmental triggers (temperature and drought) and host hormone balance (ethylene and jasmonic acid-salicylic acid crosstalk) converge temperature-regulated virulence gene expression to govern the endophyte-to-necrotroph transition. Critical gaps include the limited genomic resources for mango-derived isolates, infection-stage dual RNA-seq, population genetics, and climate-informed epidemiological models. Future directions encompass CRISPR-enabled susceptibility gene editing, RNAi biocontrol, and integrated pest management tailored to this latent pathogen. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ralstonia solanacearum, the causal agent of bacterial wilt, is a major threat to solanaceous crops in tropical and subtropical regions worldwide. While most strains are pathogenic at high temperatures (>28°C), a subset can cause disease at low temperatures (16-18°C), posing a risk to temperate solanaceous crops. Previous comparative genomics of cool virulent with regular tropical strains identified candidate transcriptional regulators associated with cool virulence. In this work, we tested function in virulence to tomato at high and low temperatures of four of the candidate transcription regulators identified previously and demonstrated that a LysR-type transcriptional regulator (Rsp673_21385) in the cool-virulent strain P673 significantly contributes to virulence at 18°C but not at 30°C. This regulator is located upstream of a conserved gene cluster resembling a 2-aminoethylphosphonate (2-AEP) degradation operon. This cluster codes for an alternative pathway recently characterized. P673 was unable to utilize phosphonate as a sole carbon source, however it used 2-AEP as a source of phosphorus in a temperature dependent manner. Relative gene expression analysis of representative genes in the cluster showed significant upregulation at 18°C in the wild-type strain but not in the regulator mutant, indicating that the LysR regulator activates expression of the putative 2-AEP degradation cluster under cool temperatures. These findings reveal a novel temperature-dependent virulence factor and highlight the need to explore the regulon controlled by Rsp673_21385, providing new insights into the adaptation of R. solanacearum to temperate environments.

The phloem-colonizing bacterial pathogen Candidatus Liberibacter asiaticus (CLas) causes the devastating citrus Huanglongbing (HLB). Despite the important roles of roots, how CLas affects roots remains largely unknown. Here we dissected host responses in leaf and root at different time points after CLas inoculation. Our data showed that CLas triggers less reactive oxygen species (ROS) accumulation and phloem callose deposition, which are known to contribute to HLB symptom development in leaves, in roots than leaves. Increased chloroplastic ROS accumulation was detected in CLas-infected leaf compared to uninfected controls, which may explain the increased ROS levels in leaves than roots infected by CLas. qRT-PCR and RNA-seq analyses showed global induction of immune related pathways in both roots and leaves, whereas photosynthesis related pathways were only downregulated in leaves, but not in roots, by CLas infection. The differential expression pattern related to immunity and photosynthesis in the leaf and root tissues suggests that chloroplasts might be involved in the differences in ROS accumulation and callose deposition. Root cell death was observed in HLB symptomatic trees regardless of CLas infection status of roots. Significantly more starch accumulation was first observed in CLas positive leaves than healthy controls followed by starch depletion in CLas positive roots later, suggesting a causative relationship between them. Taken together, root cell death caused by CLas infection maybe caused by carbon starvation rather than via a similar mechanism as in leaves. Our data suggests chloroplasts play a critical role in the different pathogenicity mechanisms of CLas in leaf and root.

Hemibiotrophic fungi of the genus Colletotrichum provide valuable systems for studying plant immunity and pathogen lifestyle transitions; however, compatible and incompatible interactions have rarely been compared within the same host species. Here, we show that Arabidopsis thaliana exhibits contrasting responses to two phylogenetically distinct hemibiotrophic fungi, Colletotrichum coccodes KACC40803 and Colletotrichum scovillei KC05. Multilocus phylogenetic analyses using ITS, ACT, GAPDH, and TUB2 sequences confirmed their taxonomic identities. The two fungi also differed morphologically, particularly in colony characteristics and appressorial structures. Inoculation of Arabidopsis leaves with Colletotrichum coccodes caused progressive chlorosis, necrotic lesions, extensive fungal colonization, acervulus formation, and increasing fungal biomass, indicating a compatible susceptible interaction. Host defense-related genes associated with salicylic acid- and jasmonic acid-mediated pathways were suppressed during infection. By contrast, Colletotrichum scovillei induced restricted lesion development, rapid accumulation of callose and reactive oxygen species, localized plasma membrane disruption, and strong induction of defense-related genes in Arabidopsis leaves, consistent with an incompatible hypersensitive response (HR)-like cell death. Fungal biomass remained low throughout infection, demonstrating effective restriction of colonization. Together, these findings establish a dual Arabidopsis-Colletotrichum pathosystem that enables comparative analysis of susceptibility and HR-like resistance within a single host, providing a tractable model for dissecting host specificity, immune signaling, and regulated cell death during hemibiotrophic infection.

Plant-parasitic nematodes are a diverse group collectively capable of infecting most major crops. Amongst the most damaging are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii. We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins - small, plant-derived molecules which upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. While there were no observable morphological differences between early and late hatching nematodes on their day of hatching, the late hatchers displayed signs of faster utilisation of internal energy reserves after 7 days at 4°C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers - they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, non-genetic differences likely drive late hatchers to more rapidly utilise their internal energy reserves, to be differentially responsive to host-derived signals, and to be ultimately more infective than their early hatching counterparts.

Phytopathogenic fungi must contend with host immune responses to successfully colonize plants. These responses include the production of heterogenous antimicrobial compounds (phytoalexins), which plants deploy to intoxicate infecting microbes. Among these host defense compounds are the coumarins, a family of aromatic lactones widely present in dicotyledonous plants (dicots). The fungal necrotroph Botrytis cinerea (gray mold) infects a plethora of dicots, causing large losses in the produce, viticulture, and ornamental flower industries. B. cinerea detoxifies numerous host defense compounds to support its virulence, including the coumarin scopoletin. However, B. cinerea coumarin detoxification factors have remained uncharacterized. Here, we employed transcriptomics in scopoletin-treated B. cinerea followed by heterologous expression of scopoletin-induced genes in budding yeast (Saccharomyces cerevisiae) to identify a reductase, BCIN14g01070 (Bcsaf1), which B. cinerea deploys to degrade scopoletin. Using liquid chromatography-tandem mass spectrometry, we demonstrate that Bcsaf1 reduces a carbon-carbon double bond within the coumarin backbone, converting coumarins to their nontoxic melilotic acid derivatives. We further show that Bcsaf1 orthologs are present in numerous fungi of the family Sclerotiniaceae and that an ortholog from another notorious phytopathogen, Sclerotinia sclerotiorum (white mold), possesses similar coumarin detoxification capacity. We constructed ΔBcsaf1 mutants that displayed compromised scopoletin degradation and increased scopoletin susceptibility. The ΔBcsaf1 mutants further displayed reduced virulence in Nicotiana tabacum (tobacco), which exerts a scopoletin defense response, whereas their virulence was only modestly compromised in Helianthus annuus (sunflower) seedlings, which produce much less scopoletin. Thus, Bcsaf1 is an important virulence factor that counters host coumarins to support B. cinerea infection. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Cercospora zeina causes gray leaf spot disease on maize in Africa. As a foliar fungal pathogen, C. zeina relies on effectors to facilitate successful infection and colonization of its host. Predicting effector function is therefore essential for understanding the molecular mechanisms underlying this plant-pathogen interaction. Although effector functions are typically predicted using sequence-based analyses, many effectors are subject to strong selection pressure and exhibit high sequence diversity, reducing the accuracy of such predictions. In this study, a structural phylogenetics approach was employed to identify effector structural families in C. zeina and to assign putative functions to C. zeina candidate secreted effector proteins based on their structural clustering with functionally characterized effectors from related phytopathogens. Twenty-two effector structural families were identified, several of which contained effectors shown to induce cell death in Nicotiana benthamiana. It was therefore hypothesized that C. zeina effectors belonging to these structural families would exhibit similar functions. Three families containing cell death-inducing effectors ZtCDI1, MgNLP, VdPEVD1with high structural similarity and low sequence identity to C. zeina effectors were selected for biological validation. Transient expression of C. zeina effectors from these families induced cell death in N. benthamiana. These C. zeina structural analogs were expressed in either the latent (CDI1-like) or necrotrophic (NLP-like, PevD1-like) phase of GLS disease in maize. This work shows that structural phylogenetics can be employed to predict effector function and identify structurally related, cell death-inducing effectors with divergent amino acid sequences in C. zeina.

Wheat blast, caused by Magnaporthe oryzae Triticum (MoT), is a devastating fungal disease threatening global food security. Emergence of fungicide-resistant MoT populations necessitates safe, eco-friendly alternatives. This study evaluates the biorational potential of 3-methylpentanoic acid (3-MP), a volatile organic compound (VOC) produced by Bacillus safensis NI2B against MoT. In vitro assays demonstrated that 3-MP inhibits MoT mycelial growth, conidiogenesis, conidial germination, and appressorium formation in a dose-dependent manner, achieving complete suppression at 100-125 µM. Furthermore, detached leaf, seedling, and spike assays revealed robust preventive and curative efficacy, indicating strong translational potential for wheat blast management. Mechanistic analyses indicated reduced fungal cellular viability following 3-MP treatment, as confirmed by fluorescein diacetate staining. In silico molecular docking and dynamics simulations predicted that 3-MP putatively interacts with UDP-glucose 4-epimerase (UGE), a key enzyme in galactose metabolism and cell wall integrity. 3-MP binds stably within the NAD[+]-associated Rossmann fold of UGE, sterically hindering substrate access and perturbing NAD[+] orientation. RT-qPCR expression analysis was consistent with this model, showing a biphasic response characterized by early induction followed by significant repression of UGE transcripts, which aligns with disrupted UDP-glucose metabolism and impaired cell wall biosynthesis. Collectively, this study establishes 3-MP as a potent biocontrol agent and provides preliminary mechanistic insights into its antifungal activity associated with UGE perturbation. These findings offer a promising molecular framework for developing VOC-based integrated pest management strategies against wheat blast.

The obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew, secretes effector proteins to suppress plant immunity and establish infection. Here, we characterize BgtH12, a conserved small secreted protein that is highly expressed during early infection (peaking at 48 h postinoculation [hpi]). Functional analysis using host-induced gene silencing with two independent fragments achieved 54 to 82% knockdown of BgtH12 transcripts, resulting in significantly reduced fungal sporulation, hyphal length, and branching. Stable transgenic RNAi lines confirmed these findings, with fungal biomass reduced to 0.6- to 0.7-fold that of the wild-type control. Conversely, stable overexpression of BgtH12 in wheat enhanced susceptibility, increasing fungal biomass 4- to 7-fold and accelerating hyphal growth. Histological examination revealed that BgtH12 promotes infection by suppressing early host defense responses: overexpression lines exhibited reduced reactive oxygen species (ROS) accumulation at 24 and 48 hpi, whereas RNAi lines showed enhanced ROS production. In Nicotiana benthamiana, BgtH12 suppressed BAX-triggered cell death, and delivery into wheat cells via the type-III secretion system attenuated callose deposition and the ROS burst, indicating broad-spectrum suppression of pattern-triggered immunity. Subcellular localization assays in wheat protoplasts and N. benthamiana leaves showed that BgtH12 associates with the cell periphery. Together, these results establish BgtH12 as a critical virulence factor that suppresses basal immunity to promote Bgt colonization. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plasmodiophora brassicae, the causal agent of clubroot disease, actively manipulates host hormone pathways to promote infection. Previous studies identified PbGH3 as a putative GH3-like effector with in vitro auxin-conjugating activity, although its biological function in planta was investigated but remained unresolved. Here, we studied the role of PbGH3 during host colonization using PbGH3-overexpressing Arabidopsis thaliana and Brassica napus lines. PbGH3 overexpression induced conserved developmental phenotypes in both species, including epinastic leaves, reduced apical dominance, enhanced lateral branching, and increased root hair formation, consistent with altered hormone balance. However, hormone profiling and exogenous auxin assays did not support a role for PbGH3 as a canonical auxin-conjugating enzyme in planta. Instead, structural analyses revealed a strong similarity between PbGH3 and the Arabidopsis clade III GH3 proteins GH3.12/PBS3 and GH3.7, which are associated with salicylic acid (SA) metabolism. Consistent with this observation, loss of GH3.12/PBS3 increased susceptibility to clubroot, whereas expression of PbGH3 in the gh3.12 mutant background partially restored SA accumulation and reduced disease susceptibility. PbGH3 expression peaked during the early stages of infection, and overexpressing lines displayed enhanced root hair colonization by P. brassicae, suggesting a role during primary infection rather than later gall development. Together, our results support a model in which PbGH3 modulates SA-associated hormonal crosstalk and root morphology that could be harnessed by the clubroot pathogen to ensure host colonization. These findings provide new insight into how P. brassicae manipulates host hormone homeostasis during infection. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rhizoctonia solani AG1-IA is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. Efforts are being made to identify pathogenicity-associated genes in R. solani and modulate them to develop a disease control strategy. Here, we investigated the roles of some predicted pathogenicity-associated genes of R. solani that have previously been reported to be upregulated during infection in rice. The tobacco rattle virus-based host-induced gene silencing of the selected pathogenicity-associated genes revealed that silencing of Rs_MEP1, a zinc-containing Peptidase_M43 domain metalloprotease, severely compromises R. solani infection in tomato. Moreover, double-stranded RNA-mediated silencing of Rs_MEP1 prevented R. solani infection in rice. The signal sequence trap assay indicated the secretory nature of Rs_MEP1, and the reporter assay suggested its localization in the plant apoplast. Notably, Agrobacterium-mediated transient overexpression of Rs_MEP1 induces necrotic cell death responses in plants. We provide evidence that Rs_MEP1 interacts with the GH19 family of rice chitinases and potentially modulates their functions. Overall, our study emphasizes that Rs_MEP1 facilitates R. solani in promoting necrotic responses and targets rice GH19 chitinases to impart disease susceptibility in plants. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tomato spotted wilt virus (TSWV) is an economically devastating pathogen that rapidly overcomes genetic resistance in major crops. Reverse genetic systems are crucial for investigating plant-virus interactions and resistance-breaking mechanisms, and developing these tools for segmented ambisense RNA viruses remains a crucial challenge. Current TSWV clones rely on extensively modified Asian isolates requiring co-delivery of multiple replication helpers and viral silencing suppressors. Streamlining these systems for regionally significant strains with minimal genetic alterations is essential. Here, we developed the first infectious clone of a U.S. TSWV isolate (PA01). Three binary plasmids contain cDNAs for the antigenomic L and S segments, as well as the genomic M segment, with enhanced GFP replacing NSs on the S segment. Co-delivery of the cucumovirus 2b alone or in combination with tombusvirus P19 or begomovirus AL2 achieved a high proportion of systemically infected Nicotiana benthamiana and Capsicum annuum plants. In N. tabacum, co-delivering the Caenorhabditis elegans cell death suppressor CED-9 or using NahG transgenic plants produced 30 to 33% systemically infected plants. Co-delivery of 2b boosted infection levels in NahG plants to 62%. These data indicate that in addition to the antiviral RNA-silencing machinery, additional host defense pathways influence TSWV rescue and systemic infection from cDNA. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Leaf rust (LR), caused by Puccinia recondita f. sp. secalis (Prs), is considered one of the most dangerous rye (Secale cereale L.) diseases, causing yield losses exceeding 35%. This review summarizes all currently available data about this disease: pathogen characteristics (including its life cycle, natural variation, and disease symptoms), resistance resources, and the background of the plant immune response at the genome, transcriptome, and metabolome levels. The research conducted so far has allowed for the identification of dozens of genes that play a significant role in the rye immune response to Prs infection. Among them, genes encoding NBS-LRR proteins (including SECCE1Rv1G0014220, the most likely Pr3 candidate), glycosyltransferase, β-1,3-glucanase, 1-deoxy-D-xylulose 5-phosphate synthase, β-1,3-glucanase, UDP-glycosyltransferase, pathogenesis-related protein 1, ammonium transporter, and cytochrome P450 enzymes are candidates for seedling and all-stage resistance, whereas ScLr_ABC25 currently represents the most promising candidate associated with adult-plant resistance. Among the metabolites differentially accumulated in response to Prs, those related to phenylpropanoids, diterpenoids, and thiamine branches seem to play the most important role in the immune response. Finally, we suggest how the knowledge acquired so far about the rye-Prs interaction can be used in modern breeding programs aimed at obtaining cultivars with enhanced resistance to LR, such as through the use of functional gene markers and/or metabolic biomarker-assisted selection and, in the more distant future, by developing and applying new genomic techniques for precise editing of resistance and susceptibility genes, engineering synthetic immune receptors and decoys, and pan-genomic exploration for identification of rare or lineage-specific resistance alleles. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.