Improving transient expression in N. benthamiana by suppression of the Nb-SABP2 and Nb-COI1 plant defence response related genes.

Currently transient expression is one of the preferred plant-based technologies for recombinant protein manufacturing, particularly in respect to pharmaceutically relevant products. Modern hybrid transient expression systems combine the features of Agrobacterium tumefaciens and viral vectors. However, host plant reaction to Agrobacterium-mediated delivery of gene of interest can negatively affect foreign protein accumulation. In this study, we investigated whether the modulation of plant immune response through knockdown of the Nb-SABP2 and Nb-COI1 N. benthamiana genes could improve recombinant protein yield. In plants, the SABP2 and COI1 proteins are involved in the salicylic acid and jasmonic acid metabolism, respectively. We exemplified the utility of this approach with the green fluorescence (GFP) and ß nerve growth factor (ßNGF) proteins: compared to the tobacco mosaic virus (TMV)-based vector the Nb-SABP2 and Nb-COI1-suppressed plants provided an increased recombinant protein accumulation. We also show that this strategy is extendable to the expression systems utilizing potato virus X (PVX) as the vector backbone: the enhanced amounts of ßNGF were detected in the Nb-SABP2 and Nb-COI1-depleted leaves co-infiltrated with the PVX-ßNGF. These findings suggest that modulating host plant reaction to agrodelivery of expression vectors could be useful for improving transient foreign protein production in N. benthamiana.

Mirids secrete a TOPLESS targeting protein to enhance JA-mediated defense and gossypol accumulation for antagonizing cotton bollworms on cotton plants.

Most coexisting insect species exhibit stunted growth compared to the single species on plants. This phenomenon reflects an interspecific antagonism that draws extensive attention while the underlying mechanisms remain largely unknown. Mirids (Apolygus lucorum) and cotton bollworms (Helicoverpa armigera) are two common pests in cotton fields. We identified a secretory protein, ASP1, from the oral secretion of mirids, which was found in the nucleus of mirid-infested cotton leaves. ASP1 specifically targets the transcriptional corepressor TOPLESS (TPL) and inhibits NINJA-mediated recruitment of TPL, thereby promoting plant defense response and gossypol accumulation in cotton glands. ASP1-enhanced defense inhibits the growth of cotton bollworms on cotton plants, while having little impact on mirids. The mesophyll-feeding characteristic allows mirids to avoid most cotton glands, thereby invalidating cotton defense. Our investigation reveals the molecular mechanism by which mirids employ cotton defense to selectively inhibit the feeding of cotton bollworms.

Impaired Brown midrib12 function orchestrates sorghum resistance to aphids via an auxin conjugate indole-3-acetic acid-aspartic acid.

Lignin, a complex heterogenous polymer present in virtually all plant cell walls, plays a critical role in protecting plants from various stresses. However, little is known about how lignin modifications in sorghum will impact plant defense against sugarcane aphids (SCA), a key pest of sorghum. We utilized the sorghum brown midrib (bmr) mutants, which are impaired in monolignol synthesis, to understand sorghum defense mechanisms against SCA. We found that loss of Bmr12 function and overexpression (OE) of Bmr12 provided enhanced resistance and susceptibility to SCA, respectively, as compared with wild-type (WT; RTx430) plants. Monitoring of the aphid feeding behavior indicated that SCA spent more time in reaching the first sieve element phase on bmr12 plants compared with RTx430 and Bmr12-OE plants. A combination of transcriptomic and metabolomic analyses revealed that bmr12 plants displayed altered auxin metabolism upon SCA infestation and specifically, elevated levels of auxin conjugate indole-3-acetic acid-aspartic acid (IAA-Asp) were observed in bmr12 plants compared with RTx430 and Bmr12-OE plants. Furthermore, exogenous application of IAA-Asp restored resistance in Bmr12-OE plants, and artificial diet aphid feeding trial bioassays revealed that IAA-Asp is associated with enhanced resistance to SCA. Our findings highlight the molecular underpinnings that contribute to sorghum bmr12-mediated resistance to SCA.

Exogenous salicylic acid treatment enhances the disease resistance of Panax vietnamensis by regulating secondary metabolite production.

Introduction: Salicylic acid (SA) is a phenolic compound widely found in plants. It plays a key role in exerting plant disease resistance. Panax vietnamensis Ha & Grushv., a valuable medicinal plant, contains high levels of phenolic compounds, which contribute significantly to the resilience of the plant against stress. However, the precise role of SA in regulating the synthesis of secondary metabolites in P.vietnamensis remains elusive. Methods: Two-year-old P. vietnamensis seedlings were treated with exogenous SA. We systematically assessed the changes in the physiological parameters of SA-treated P. vietnamensis leaves, employing transcriptome and metabolome analyses to elucidate the underlying mechanisms. Results: Our results revealed a significant improvement of the plant's antioxidant capacity at 6 h post-treatment. Furthermore, exogenous SA treatment promoted the biosynthesis of lignin and flavonoids such as rutin, coumarin, and cyanidin. In addition, it increased the levels of endogenous SA and jasmonic acid (JA), promoting the disease resistance of the plants. Thus, SA pretreatment enhanced the defense of P. vietnamensis against pathogens. Conclusions: Our study provided novel insights into the potential molecular mechanisms underlying SA-mediated biosynthesis of secondary metabolites. Furthermore, our results provided a theoretical foundation for optimizing the cultivation practices of P.vietnamensis and the application of SA as a plant immunomodulator.

Plant microRNAs regulate the defense response against pathogens.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, typically 20-25 nucleotides in length, that play a crucial role in regulating gene expression post-transcriptionally. They are involved in various biological processes such as plant growth, development, stress response, and hormone signaling pathways. Plants interact with microbes through multiple mechanisms, including mutually beneficial symbiotic relationships and complex defense strategies against pathogen invasions. These defense strategies encompass physical barriers, biochemical defenses, signal recognition and transduction, as well as systemic acquired resistance. MiRNAs play a central role in regulating the plant's innate immune response, activating or suppressing the transcription of specific genes that are directly involved in the plant's defense mechanisms against pathogens. Notably, miRNAs respond to pathogen attacks by modulating the balance of plant hormones such as salicylic acid, jasmonic acid, and ethylene, which are key in activating plant defense mechanisms. Moreover, miRNAs can cross boundaries into fungal and bacterial cells, performing cross-kingdom RNA silencing that enhances the plant's disease resistance. Despite the complex and diverse roles of miRNAs in plant defense, further research into their function in plant-pathogen interactions is essential. This review summarizes the critical role of miRNAs in plant defense against pathogens, which is crucial for elucidating how miRNAs control plant defense mechanisms.

Withania somnifera osmotin (WsOsm) confers stress tolerance in tobacco and establishes novel interactions with the defensin protein (WsDF).

Pathogenesis-related proteins (PR), including osmotins, play a vital role in plant defense, being activated in response to diverse biotic and abiotic stresses. Despite their significance, the mechanistic insights into the role of osmotins in plant defense have not been extensively explored. The present study explores the cloning and characterization of the osmotin gene (WsOsm) from Withania somnifera, aiming to illuminate its role in plant defense mechanisms. Quantitative real-time PCR analysis revealed significant induction of WsOsm in response to various phytohormones e.g. abscisic acid, salicylic acid, methyl jasmonate, brassinosteroids, and ethrel, as well as biotic and abiotic stresses like heat, cold, salt, and drought. To further elucidate WsOsm's functional role, we overexpressed the gene in Nicotiana tabacum, resulting in heightened resistance against the Alternaria solani pathogen. Additionally, we observed enhancements in shoot length, root length, and root biomass in the transgenic tobacco plants compared to wild plants. Notably, the WsOsm- overexpressing seedlings demonstrated improved salt and drought stress tolerance, particularly at the seedling stage. Confocal histological analysis of H2O2 and biochemical studies of antioxidant enzyme activities revealed higher levels in the WsOsm overexpressing lines, indicating enhanced antioxidant defense. Furthermore, a pull-down assay and mass spectrometry analysis revealed a potential interaction between WsOsm and defensin, a known antifungal PR protein (WsDF). This suggests a novel role of WsOsm in mediating plant defense responses by interacting with other PR proteins. Overall, these findings pave the way for potential future applications of WsOsm in developing stress-tolerant crops and improving plant defense strategies against pathogens.

Citrus Fruits Produce Direct Defense Responses against Oviposition by Bactrocera minax (Diptera: Tephritidae).

Plants perceive and orchestrate defense responses when herbivorous insects are ovipositing. Fruits, as a crucial reproductive organ in plants, have rarely been researched on the responses to insect eggs. Here, we found that oviposition by the specialist insect Bactrocera minax in navel oranges activated the lignin synthesis pathway and cell division, causing mechanical pressure that crushed the eggs. Transcriptome and metabolome analyses revealed an enrichment of oviposition-induced genes and metabolites within the lignin synthesis pathway, which was confirmed by histochemical staining. Furthermore, hydrogen peroxide (H2O2) accumulation was observed at the oviposition sites. Plant defense-related hormones jasmonic acid (JA) and salicylic acid (SA) exhibited rapid induction after oviposition, while indole-3-acetic acid (IAA) activation occurred in the later stages of oviposition. Additionally, secondary metabolites induced by prior egg deposition were found to influence larval performance. Our studies provide molecular evidence that host fruits have evolved defense mechanisms against insect eggs and pave the way for future development of insect-resistant citrus varieties.

A review of plant phenolics and endozoochory.

Phenolic compounds (phenolics) are secondary metabolites ubiquitous across plants. The earliest phenolics are linked to plants' successful transition from an aquatic to a terrestrial environment, serving as protection against damaging ultraviolet (UV) radiation, and as antioxidants to reduce oxidative stress in an atmosphere with an increasingly high O2:CO2 ratio. In modern plants, phenolics are best known for the defense against fungal and bacterial pathogens and as antifeedants that deter herbivory. Phenolics also play a role in seed dormancy, delaying germination, and lengthening viability in the seed bank. Many plants' seeds are endozoochorous - dispersed by animals, like birds, who eat and later excrete the seeds. Plants send visual signals to attract birds with UV-sensitive (UVS) vision for pollination and seed dispersal. As fruits ripen, antioxidant activity and phenolic content decrease. The waxy cuticle of fruits increases in UV reflection as phenolic rings, which absorb UV light, degrade. The UV contrast that birds detect may act as an honest signal, indicating nutritional changes in the fruit. However, there is little evidence to support the evolution of UV coloration during ripening being driven by frugivore selection. Antioxidant properties of fruit phenolics may be dually adaptive in plants and avian frugivores.

Priming of Exogenous Salicylic Acid under Field Conditions Enhances Crop Yield through Resistance to Magnaporthe oryzae by Modulating Phytohormones and Antioxidant Enzymes.

This study explores the impact of exogenous salicylic acid (SA) alongside conventional treatment by farmers providing positive (Mancozeb 80 % WP) and negative (water) controls on rice plants (Oryza sativa L.), focusing on antioxidant enzyme activities, phytohormone levels, disease resistance, and yield components under greenhouse and field conditions. In greenhouse assays, SA application significantly enhanced the activities of peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) within 12-24 h post-inoculation (hpi) with Magnaporthe oryzae. Additionally, SA-treated plants showed higher levels of endogenous SA and indole-3-acetic acid (IAA) within 24 hpi compared to the controls. In terms of disease resistance, SA-treated plants exhibited a reduced severity of rice blast under greenhouse conditions, with a significant decrease in disease symptoms compared to negative control treatment. The field study was extended over three consecutive crop seasons during 2021-2023, further examining the efficacy of SA in regular agricultural practice settings. The SA treatment consistently led to a reduction in rice blast disease severity across all three seasons. Yield-related parameters such as plant height, the number of tillers and panicles per hill, grains per panicle, and 1000-grain weight all showed improvements under SA treatment compared to both positive and negative control treatments. Specifically, SA-treated plants yielded higher grain outputs in all three crop seasons, underscoring the potential of SA as a growth enhancer and as a protective agent against rice blast disease under both controlled and field conditions. These findings state the broad-spectrum benefits of SA application in rice cultivation, highlighting its role not only in bolstering plant defense mechanisms and growth under greenhouse conditions but also in enhancing yield and disease resistance in field settings across multiple crop cycles. This research presents valuable insights into the practical applications of SA in improving rice plant resilience and productivity, offering a promising approach for sustainable agriculture practices.

Defense Responses Induced by Viral Movement Protein and Its Nuclear Localization Modulate Virus Cell-to-Cell Transport.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed 'binary movement block' (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport.

Uncovering the Mechanisms: The Role of Biotrophic Fungi in Activating or Suppressing Plant Defense Responses.

This paper discusses the mechanisms by which fungi manipulate plant physiology and suppress plant defense responses by producing effectors that can target various host proteins. Effector-triggered immunity and effector-triggered susceptibility are pivotal elements in the complex molecular dialogue underlying plant-pathogen interactions. Pathogen-produced effector molecules possess the ability to mimic pathogen-associated molecular patterns or hinder the binding of pattern recognition receptors. Effectors can directly target nucleotide-binding domain, leucine-rich repeat receptors, or manipulate downstream signaling components to suppress plant defense. Interactions between these effectors and receptor-like kinases in host plants are critical in this process. Biotrophic fungi adeptly exploit the signaling networks of key plant hormones, including salicylic acid, jasmonic acid, abscisic acid, and ethylene, to establish a compatible interaction with their plant hosts. Overall, the paper highlights the importance of understanding the complex interplay between plant defense mechanisms and fungal effectors to develop effective strategies for plant disease management.

Brandt's vole (Lasiopodomys brandtii) affects the dominant position of three gramineous species by altering defense traits and interspecific competition.

Rodents can cause considerable changes in plant community composition. However, relationships between shifts in species dominance and plant functional traits caused by rodents have seldom been investigated, especially for belowground functional traits. In this study, a set of enclosures was constructed to analyze the effects of 10 years of Brandt's voles' activities on the defense strategies and dominant position changes of three gramineous plants (Leymus chinensis, Stipa krylovii, and Cleistogenes squarrosa) in Inner Mongolia. Here, we measured the dominance, biomass, and fourteen functional traits of three plants. The effects of Brandt's voles on dominance, biomass, and functional traits were analyzed, and then we explored the effect of functional traits on plant dominance by using the structural equation model. Results showed that long-term feeding by Brandt's voles resulted in a significant decrease in the dominance of L. chinensis and S. krylovii, whereas C. squarrosa was positively affected. The belowground biomass of L. chinensis and S. krylovii was higher in the vole treatment, which showed that they were increasing their escape characteristics. The leaf thickness of L. chinensis and the leaf C:N ratio of S. krylovii significantly increased, while the specific leaf area of C. squarrosa significantly decreased. All three gramineous showed increased resistance traits in response to Brandt's voles, which positively affected their dominance. Tolerance-related traits of S. krylovii significantly increased, with the increasing growth rate of root length contributing to enhancing its dominance. We highlight that selective feeding by rodents led to the selection of different defense strategies by three gramineous plants, and that changes in biomass allocation and functional traits in the different species affected plant dominance, driving changes in the plant communities.

Post-Translational Modifications Control The Signal At The Crossroads Of Plant-Pathogen Interaction.

The co-evolution of plants and pathogens has enabled them to 'outsmart' each other by promoting their own defense responses and suppressing that of the other. While plants are reliant on their sophisticated immune signalling pathways, pathogens make use of effector proteins to achieve the objective. This entails rapid regulation of the underlying molecular mechanisms for prompt induction of the associated signalling events in both plants as well as pathogens. The last decade has witnessed the emergence of post-translational modification (PTM) of proteins as key players in modulating cellular responses. Their ability to expand the functional diversity of the proteome and induce rapid changes at the appropriate time enables them to play crucial roles in the regulation of plant-pathogen interactions. Therefore, this review will delve into the intricate interplay of five major PTMs in plant defense and pathogen countermeasures. The review discusses how plants employ PTMs to fortify their immune networks, and how pathogen effectors utilize/target host modification systems to gain entry into the plant and cause disease. The review also underscores the need for identification of newer PTMs and proposes to use PTM machineries as potential targets for genome editing approaches.

The Dual Role of Antimicrobial Proteins and Peptides: Exploring Their Direct Impact and Plant Defense-Enhancing Abilities.

Plants face numerous environmental stresses that hinder their growth and productivity, including biotic agents, such as herbivores and parasitic microorganisms, as well as abiotic factors, such as cold, drought, salinity, and high temperature. To counter these challenges, plants have developed a range of defense strategies. Among these, plant antimicrobial proteins and peptides (APPs) have emerged as a promising solution. Due to their broad-spectrum activity, structural stability, and diverse mechanisms of action, APPs serve as powerful tools to complement and enhance conventional agricultural methods, significantly boosting plant defense and productivity. This review focuses on different studies on APPs, emphasizing their crucial role in combating plant pathogens and enhancing plant resilience against both biotic and abiotic stresses. Beginning with in vitro studies, we explore how APPs combat various plant pathogens. We then delve into the defense mechanisms triggered by APPs against biotic stress, showcasing their effectiveness against bacterial and fungal diseases. Additionally, we highlight the role of APPs in mitigating the abiotic challenges associated with climatic change. Finally, we discuss the current applications of APPs in agriculture, emphasizing their potential for sustainable agricultural practices and the need for future research in this area.

New Insights into Involvement of Low Molecular Weight Proteins in Complex Defense Mechanisms in Higher Plants.

Dynamic climate changes pose a significant challenge for plants to cope with numerous abiotic and biotic stressors of increasing intensity. Plants have evolved a variety of biochemical and molecular defense mechanisms involved in overcoming stressful conditions. Under environmental stress, plants generate elevated amounts of reactive oxygen species (ROS) and, subsequently, modulate the activity of the antioxidative enzymes. In addition, an increase in the biosynthesis of important plant compounds such as anthocyanins, lignin, isoflavonoids, as well as a wide range of low molecular weight stress-related proteins (e.g., dehydrins, cyclotides, heat shock proteins and pathogenesis-related proteins), was evidenced. The induced expression of these proteins improves the survival rate of plants under unfavorable environmental stimuli and enhances their adaptation to sequentially interacting stressors. Importantly, the plant defense proteins may also have potential for use in medical applications and agriculture (e.g., biopesticides). Therefore, it is important to gain a more thorough understanding of the complex biological functions of the plant defense proteins. It will help to devise new cultivation strategies, including the development of genotypes characterized by better adaptations to adverse environmental conditions. The review presents the latest research findings on selected plant defense proteins.

Trades-offs between pollinator attraction and florivore defense maximize reproductive success in the self-incompatible Rivea ornata (Convolvulaceae).

BACKGROUND: Rivea ornata, a rare species from the morning glory family, exhibits uncommon characteristics compared to other typical morning glories, including nocturnal flowers that fit the classic moth pollination syndrome. However, the accuracy of its predicted pollination syndrome and its mating system have never been assessed. Additionally, R. ornata flowers attract not only pollinators but also florivores, potentially reducing plant reproductive success. Therefore, this study examined two populations of R. ornata in Thailand and assessed traits related to pollinator attraction and reward, determined its mating system, identified floral visitors and effective pollinators, and investigated the effect of florivory on reproductive success. RESULTS: Rivea ornata is highly fertile but self-incompatible and an obligate outcrosser, rendering it highly dependent on pollinators. Lepidopterans, particularly nocturnal hawk moths, were found to account for a significant proportion of all visits and were the sole effective pollinators of this plant species, in correspondence with its predicted pollination syndrome. Surprisingly, florivory did not significantly reduce reproductive success. This phenomenon may be explained by the strategies employed by R. ornata, which align with the optimal defense hypothesis and functional trade-offs. Specifically, R. ornata appears to invest resources in defending key floral structures while, simultaneously, guard ants are conspicuously absent from flowers, resulting in some florivore damage to non-vital floral organs but ensuring that pollinators are not deterred by ants and thus maintaining high pollinator visitation rates. CONCLUSIONS: Our findings indicate that reproduction-related traits in R. ornata, including those involved in pollinator attraction and reward and florivore defense, are highly effective and work in concert to maximize plant reproductive success. Therefore, a main risk that R. ornata faces is the decline or disappearance of hawk moths and other lepidopterans given its extreme specialization and high dependence on pollinators, and conservation efforts should include habitat protection for both R. ornata and its pollinators.

Green Routes: Exploring Protein-Based Virus-like Nanoparticle Transport and Immune Activation in Nicotiana benthamiana for Biotechnological Applications.

Viral, bacterial, fungal, and nematode infections cause significant agricultural losses, with limited treatment options, necessitating novel approaches to enhance plant defense systems and protection against pathogens. Virus-like nanoparticles (VLPs), extensively used in animal and human therapies (e.g., vaccines and immune enhancers), hold potential for novel agricultural solutions and advancing plant nanotechnology. This study employed various methodologies, including VLP production, confocal microscopy, and real-time qPCR. Our findings demonstrated the presence of 30 nm Qß-VLPs, fluorescently labeled, within the intercellular space of Nicotiana benthamiana leaves one hour post-infiltration. Furthermore, infiltration with Qß-VLPs led to an upregulation of key defense genes (NbPR1a, NbPR5, NbNPR, NbERF1, NbMYC2, and NbLRR2) in treated plants. Using RT-qPCR, a significant increase in the relative expression levels of defense genes was observed, with sustained high levels of NbERF1 and NbLRR2 even after 24 h. These findings suggest that Qß-VLPs effectively upregulate genes crucial for pathogen defense in N. benthamiana, initiating PAMP-triggered immunity and launching signaling cascades that enhance defense mechanisms. This innovative application of VLPs to activate plant defense programs advances plant nanobiotechnology, offering new agricultural solutions.

Plant-nematode battle: engagement of complex signaling network.

Plant-parasitic nematodes (PPNs) are widely distributed and highly adaptable. To evade the invasion and infection of PPNs, plants initiate a series of defense responses. In turn, PPNs secrete effectors into the host tissues to suppress plant defense. In this ongoing battle between PPNs and plants, complex signal transduction processes are typically involved. This article aims to review the plant signaling network involved in host perception by the nematode, nematode perception, and downstream activation of plant defense signaling and how nematodes attempt to interfere with this network. Our goal is to establish a foundation for elucidating the signaling and regulatory mechanisms of plant-nematode interactions, and to provide insights and tools for developing PPN-resistant crops and technologies.

Alleviating protein-condensation-associated damage at the endoplasmic reticulum enhances plant disease tolerance.

Disease tolerance is an essential defense strategy against pathogens, alleviating tissue damage regardless of pathogen multiplication. However, its genetic and molecular basis remains largely unknown. Here, we discovered that protein condensation at the endoplasmic reticulum (ER) regulates disease tolerance in Arabidopsis against Pseudomonas syringae. During infection, Hematopoietic protein-1 (HEM1) and Bax-inhibitor 1 (BI-1) coalesce into ER-associated condensates facilitated by their phase-separation behaviors. While BI-1 aids in clearing these condensates via autophagy, it also sequesters lipid-metabolic enzymes within condensates, likely disturbing lipid homeostasis. Consequently, mutations in hem1, which hinder condensate formation, or in bi-1, which prevent enzyme entrapment, enhance tissue-damage resilience, and preserve overall plant health during infection. These findings suggest that the ER is a crucial hub for maintaining cellular homeostasis and establishing disease tolerance. They also highlight the potential of engineering disease tolerance as a defense strategy to complement established resistance mechanisms in combating plant diseases.

Unraveling the interactions of Esteya vermicola, pinewood nematode, and pine hosts: Insights into population dynamics and molecular responses.

Esteya vermicola has shown promise as an efficient biological control agent against pine wilt disease, a devastating disease in pine forests caused by the pinewood nematode (PWN, Bursaphelenchus xylophilus). However, the in vivo interactions among E. vermicola, PWN, and pine hosts are less understood, both at the population and molecular levels. In this study, we performed a series of bioassays to investigate E. vermicola colonization patterns in pine xylem and its population responses to PWN invasion in healthy and PWN-induced wilting trees. Our results demonstrated that although E. vermicola exhibits slow growth, its conidia germinate and grew along the pine tracheid, even producing lunate conidia capable of initiating PWN infections within the xylem. Interestingly, while fungal hyphae became undetectable in pine sapling xylem after inoculation, the E. vermicola population increased immediately in response to PWN invasion. Furthermore, we observed a "leap-frog" dispersal pattern of fungal colonization in PWN-induced wilting pines, facilitated by the migration of fungal-infected nematodes. Moreover, we explored the molecular mechanisms underlying fungal tolerance to pine defense systems using transcriptomic analysis. Comparative transcriptomics revealed that carbohydrate metabolism and abiotic stress-induced oxidoreductive activities are involved in the fungal tolerance to the pine defense compound ß-pinene. This study enhances our understanding of how E. vermicola colonizes and persists within pine xylem, its molecular responses to plant defense compounds, and its population dynamics upon PWN invasion, validating its efficacy as a biocontrol agent against pine wilt disease.