Search Results (17)

Witches’ broom disease (WBD), caused by the fungus Moniliophthora perniciosa, poses a major threat to cocoa production and little is yet known about how the fungus adapts at the molecular level, particularly in the apoplastic environment during early infection. Here, we investigated how apoplastic washing fluid (AWF) from two cocoa genotypes with contrasting resistance to WBD modulates the mycelial protein profile of two M. perniciosa isolates: (i) Mp553—low infection level; and (ii) Mp565—high infection level. A total of 1272 proteins were identified. Mp565, showed increased accumulation of proteins associated with oxidative stress response, energy metabolism, and virulence when exposed to AWF from the resistant variety TSH1188. Key proteins such as phosphoglycerate kinase, enolase, and heat shock were significantly modulated. Interestingly, AWF from the resistant variety promoted the suppression of metabolic proteins, suggesting an effective defense response in the resistant genotype. Furthermore, interaction network analysis revealed the central role of the MPER_11800 protein, a potential regulator of fungal adaptation. The findings underscore the importance of the T. cacao apoplast in both plant defense and fungal adaptation. The study also reveals key molecular targets, such as MPER_11800, for potential strategies to control WBD. These insights enhance our understanding of M. perniciosa pathogenicity and offer valuable directions for developing novel interventions to mitigate the impact of this devastating disease. Full article

Safflower (Carthamus tinctorius L.) is an economically important crop, yet its production is severely threatened by fungal diseases including Botrytis cinerea. The molecular mechanism underlying disease resistance in safflower remains largely unclear. Extracellular vesicles (EVs), as vital carriers for cross-kingdom communication and transport, play crucial roles in plant antifungal defense. Lipid transfer proteins (LTPs), members of the pathogenesis-related protein 14 family, have also been shown to be key players in plant disease resistance. The promising resistance-related candidate gene CtLTP8 was previously identified via genome-wide association study (GWAS). In this study, a genome-wide analysis of the LTP gene family in safflower was performed. EVs were isolated from the apoplastic washing fluid of B. cinerea-infected safflower leaves, and proteomic analysis was performed. Numerous proteins associated with disease resistance, including CtLTP8, were detected by proteomic profiling. CtLTP8 was found to be present in EVs through molecular biological experiments. Moreover, stable overexpression of CtLTP8 in safflower significantly increased resistance to B. cinerea. In summary, this study characterized the disease resistance-related proteome of safflower EVs, and verified the presence of CtLTP8 in EVs and its antifungal function, providing valuable gene resources and theoretical support for safflower disease-resistance breeding and research on EV-mediated plant immune mechanisms. Full article

Hemipteran plant sap feeders acquire food by inserting their piercing–sucking mouthparts into the apoplast (xylem sap feeders) or symplast (phloem sap feeders). When feeding, these insects seal their stylets inside the plant with a salivary sheath, minimising embolisms. The main impact of the insect on the host or food plant consists of sucking plant sap, transmitting phytopathogenic agents, or triggering galls as hypertrophic and/or hyperplastic neoformations. This paper proposes a rapid method for locating and counting the salivary sheaths of Philaenus spumarius (the Meadow Spittlebug), the primary Italian vector of Xylella fastidiosa, at the feeding point on its host or food plants. Hand-sliced stems of herbs hosting juveniles and olive twigs or leaves feeding the adult spittlebug, preserved while mounted in 75% ethanol (v/v), show the salivary sheaths. Alternative dyeing with acid fuchsin, chlorazol black, or phloroglucinol, and diaphanisation with Essig’s fluid alone or in combination with benzyl alcohol, may help with observations. The 75% EtOH solution provides a better compromise, yielding rapid evidence. The other methods offer similar outcomes but require more time and effort and expose the operator to harmful dyes. Assessing vectors’ access to plants may enable us to quantify the potential number of pathogen transmission events or to spot rejecting clones, thereby advancing the development of robust, effective control strategies that incorporate resistance. Full article

Plant-derived nanovesicles (PDNVs) are increasingly recognized as mediators of intercellular communication in plants, where they play roles in defense, signaling, and cell wall remodeling. In addition, PDNVs are gaining increasing attention for their biomedical potential, both as natural delivery systems and as bioactive entities, with promising applications in inflammatory disorders and cancer. In this study, we isolated carob nanovesicles (CbNVs) from the apoplastic fluid of carob pods (Ceratonia siliqua L.) using vacuum infiltration centrifugation followed by tangential flow filtration and size-exclusion chromatography. Morphological and biophysical analyses revealed spherical vesicles, while proteomic profiling identified 197 proteins, including suggested PDNV markers such as annexin, HSP70, GAPDH, elongation factors, malate dehydrogenase, and TET-8. These proteins were enriched in metabolic processes, stress responses, and cell wall modification pathways. Protein–protein interaction analysis further linked HSP70 to pectinesterases, reinforcing their role in cell wall remodeling and stress adaptation. Functionally, CbNVs were non-cytotoxic to human macrophages, keratinocytes, and intestinal cells. Notably, CbNVs significantly reduced LPS-induced NF-κB expression in macrophages and promoted wound closure in keratinocytes, with superior efficacy compared to the carob bioactive compound D-pinitol. These findings suggest that CbNVs harbor a synergistic cargo of bioactive molecules with anti-inflammatory and wound-healing properties, highlighting their potential as natural nanotherapeutics. Full article

Background/Objectives: Plants release extracellularly lipid bilayer-enclosed vesicles of nanometric size that can be retrieved in their fluids. Plant-derived extracellular vesicles (PDEVs) have mostly been involved in modulating host–pathogen interaction, making them a tool for cross-kingdom communication with a key role in plant immunity. In addition, PDEVs have demonstrated promising therapeutic features, not only in terms of intrinsic nutraceutical properties but also of active molecules’ delivery. Transgenic plants have been developed for a variety of purposes, i.e., to improve their functional properties like crops, but also to produce therapeutic molecules. However, it is unclear whether transgenes can end up in PDEVs, thus making them a vehicle for their cross-kingdom diffusion into the environment. Methods: Here, we investigated the association of transgenic DNA and RNA with PDEVs secreted by tobacco (Nicotiana tabacum) engineered to express the neomycine phosphotransferase II (Npt-II) gene. PDEVs were isolated from leaf apoplastic fluid by ultracentrifugation and characterized for their morphology and size. The association of DNA and RNA was assessed by qRT-PCR and their immunomodulatory properties by assaying PDEVs-induced IL1β and IL10 on THP1 monocytes. Results: Npt-II RNA, but not DNA, could be amplified from PDEVs, whereas no differences were observed between wt and transgenic tobacco PDEVs in terms of immunomodulatory properties. Conclusions: Although a different behaviour by other types of RNAs or DNAs could still be possible, our findings indicate that in this model, PDEVs are not associated with transgenic DNA, but they can protect RNA, including transgenic RNA, from degradation, contributing to their cross-kingdom spreading. Full article

Vacuum infiltration–centrifugation (VIC) is the most reproducible technique for the isolation of apoplast washing fluid (AWF) from leaves, but its effectiveness depends on the infiltration–centrifugation conditions and the anatomical and physiological peculiarities of leaves. This study aimed to elaborate an optimal procedure for AWF isolation from the leaves of Tartary buckwheat grown in in vivo and in vitro conditions and reveal the leaf anatomical and physiological traits that could contribute to the effectiveness of AWF isolation. Here, it was demonstrated that leaves of buckwheat plants grown in vitro could be easier infiltrated, were less sensitive to higher forces of centrifugation (900× g and 1500× g), and produced more AWF yield and apoplastic protein content than in vivo leaves at the same forces of centrifugation (600× g and 900× g). The extensive study of the morphological, anatomical, and ultrastructural characteristics of buckwheat leaves grown in different conditions revealed that in vitro leaves exhibited significant plasticity in a number of interconnected morphological, anatomical, and physiological features, generally driven by high RH and low lighting; some of them, such as the reduced thickness and increased permeability of the cuticle of the epidermal cells, large intercellular spaces, increase in the size of stomata and in the area of stomatal pores, higher stomata index, drop in density, and area of calcium oxalate druses, are beneficial to the effectiveness of VIC. The size of stomata pores, which were almost twice as large in in vitro leaves as those in in vivo ones, was the main factor contributing to the isolation of AWF free of chlorophyll contamination. The opening of stomata pores by artificially created humid conditions reduced damage to the in vivo leaves and improved the VIC of them. For Fagopyrum species, this is the first study to develop a VIC technique for AWF isolation from leaves. Full article

Over recent years, extracellular vesicles (EVs), commonly termed exosomes, have gained prominence for their potential as natural nanocarriers. It has now been recognized that plants also secrete EVs. Despite this discovery, knowledge about EV biogenesis in plant cell cultures remains limited. In our study, we have isolated and meticulously characterized EVs from the callus culture of the model plant, Arabidopsis thaliana. Our findings indicate that the abundance of EVs in calli was less than that in the plant’s apoplastic fluid. This difference was associated with the transcriptional downregulation of the endosomal sorting complex required for transport (ESCRT) genes in the calli cells. While salicylic acid increased the expression of ESCRT components, it did not enhance EV production. Notably, EVs from calli contained proteins essential for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those found in intact plants. This suggests that plant cell cultures could serve as a feasible source of EVs that reflect the characteristics of the parent plant species. However, further research is essential to determine the optimal conditions for efficient EV production in these cultured cells. Full article

Over the past decade, there has been a significant expansion in the development of plant-derived extracellular nanovesicles (EVs) as an effective drug delivery system for precision therapy. However, the lack of effective methods for the isolation and characterization of plant EVs hampers progress in the field. To solve a challenge related to systemic separation and characterization in the plant-derived EV field, herein, we report the development of a simple 3D inner filter-based method that allows the extraction of apoplastic fluid (AF) from blueberry, facilitating EV isolation as well as effective downstream applications. Class I chitinase (PR-3) was found in blueberry-derived EVs (BENVs). As Class I chitinase is expressed in a wide range of plants, it could serve as a universal marker for plant-derived EVs. Significantly, the BENVs exhibit not only higher drug loading capacity than that reported for other EVs but also possess the ability to modulate the release of the proinflammatory cytokine IL-8 and total glutathione in response to oxidative stress. Therefore, the BENV is a promising edible multifunctional nano-bio-platform for future immunomodulatory therapies. Full article

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30–1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP⁺ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (−10.32 kcal/mol and −6.13 kcal/mol, respectively) as compared to phyllanthin (−6.42 kcal/mol and −2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv. Full article

Xanthomonas citri pv. citri (Xcc) (X. citri subsp. citri) type A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp. and close relatives. Two narrow-host-range strains of Xcc, A^w and A*, from Florida and Southwest Asia, respectively, infect only Mexican lime (Citrus aurantifolia) and alemow (C. macrophylla). In the initial stage of infection, these xanthomonads enter via stomata to reach the apoplast. Herein, we investigated the differences in chemotactic responses for wide and narrow-host-range strains of Xcc A, X. euvesicatoria pv. citrumelonis (X. alfalfae subsp. citrumelonis), the causal agent of citrus bacterial spot, and X. campestris pv. campestris, the crucifer black rot pathogen. These strains of Xanthomonas were compared for carbon source use, the chemotactic responses toward carbon compounds, chemotaxis sensor content, and responses to apoplastic fluids from Citrus spp. and Chinese cabbage (Brassica pekinensis). Different chemotactic responses occurred for carbon sources and apoplastic fluids, depending on the Xanthomonas strain and the host plant from which the apoplastic fluid was derived. Differential chemotactic responses to carbon sources and citrus apoplasts suggest that these Xanthomonas strains sense host-specific signals that facilitate their location and entry of stomatal openings or wounds. Full article

The Arabidopsis PROSCOOP genes belong to a family predicted to encode secreted pro-peptides, which undergo maturation steps to produce peptides named SCOOP. Some of them are involved in defence signalling through their perception by a receptor complex including MIK2, BAK1 and BKK1. Here, we focused on the PROSCOOP10 gene, which is highly and constitutively expressed in aerial organs. The MS/MS analyses of leaf apoplastic fluids allowed the identification of two distinct peptides (named SCOOP10#1 and SCOOP10#2) covering two different regions of PROSCOOP10. They both possess the canonical S-X-S family motif and have hydroxylated prolines. This identification in apoplastic fluids confirms the biological reality of SCOOP peptides for the first time. NMR and molecular dynamics studies showed that the SCOOP10 peptides, although largely unstructured in solution, tend to assume a hairpin-like fold, exposing the two serine residues previously identified as essential for the peptide activity. Furthermore, PROSCOOP10 mutations led to an early-flowering phenotype and increased expression of the floral integrators SOC1 and LEAFY, consistent with the de-regulated transcription of PROSCOOP10 in several other mutants displaying early- or late-flowering phenotypes. These results suggest a role for PROSCOOP10 in flowering time, highlighting the functional diversity within the PROSCOOP family. Full article

Maize is an important cereal crop which is severely affected by Spodoptera frugiperda. The study aims to identify endophytic bacteria of maize root and leaf apoplastic fluid with bioprotective traits against S. frugiperda and plant growth promoting properties. Among 15 bacterial endophytic isolates, two strains—namely, RAF5 and LAF5—were selected and identified as Alcaligenes sp. MZ895490 and Bacillus amyloliquefaciens MZ895491, respectively. The bioprotective potential of B. amyloliquefaciens was evaluated through bioassays. In a no-choice bioassay, second instar larvae of S. frugiperda fed on B. amyloliquefaciens treated leaves (B+) recorded comparatively lesser growth (1.10 ± 0.19 mg mg⁻¹ day⁻¹) and consumptive (7.16 ± 3.48 mg mg⁻¹ day⁻¹) rates. In larval dip and choice bioassay, the same trend was observed. In detached leaf experiment, leaf feeding deterrence of S. frugiperda was found to be greater due to inoculation with B. amyloliquefaciens than Alcaligenes sp. The phenolics content of B. amyloliquefaciens inoculated plant was also found to be greater (3.06 ± 0.09 mg gallic acid g⁻¹). However, plant biomass production was more in Alcaligenes sp inoculated treatment. The study thus demonstrates the potential utility of Alcaligenes sp. and B. amyloliquefaciens for improving growth and biotic (S. frugiperda) stress tolerance in maize. Full article

Although animal-derived extracellular vesicles (EVs) are moving increasingly into scientific focus, EVs from other kingdoms remain underestimated and our knowledge of them is still expandable, probably due to the lack of an easy and broadly executable isolation, purification and visualization method. Using differential centrifugation with subsequent agarose gel electrophoresis, we were able to simplify the terms of EV isolation. EVs from Nicotiana tabacum L., Vinca minor L., and Viscum album L. were purified, even though they did not migrate into the gel matrix. If 3,3- Dihexyloxacarbocyanine iodide (DiOC ${}_6$ ) is added to the specimen in excess, membranous components can already be detected by eye, or with higher sensitivity, using a UV transilluminator. The sample preparation can be adjusted to the EV species of interest. Moreover, EVs are separated from small charged contaminants and dye excess, because these impurities can pass the gel matrix, while EVs themselves are retained in the pocket. Significantly, we isolated EVs from dried plant material, which is—to our knowledge—the first proof that EVs are stable enough to overcome the drying process of plant material. Full article

Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing. Full article

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc₂(Xy_l)Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. Full article