Pinus-derived membrane vesicles disrupt pathogenic metabolism in fungi
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Wiley
Abstract
• Much of what we know about the biological impacts of vesicles (MVs) is derived from Arabidopsis thaliana. Our study focused on vesicles from species in the non-model plant group, Pinus (pine) (P. elliottii, P. radiata, and P. patula × Pinus tec (hybrid)). These plants have tougher tissues and strong, acicular-shaped leaves (needles).
• Herein, we first developed a protocol to guide effective collection of juice fluid from needles and roots in a clean and efficient manner. The effects of these vesicles were characterized in terms of the global nutrient profile of the pine pitch canker fungus, Fusarium circinatum, generated from growing fungal spores on ~400 substrates embedded across BioLog phenotypic microarray (PM) plates (PM1, PM2A: carbon sources; PM3B: nitrogen sources; PM9: osmolytes/pH; PM24C: chemicals).
• Our findings revealed that MVs, specifically needle-derived MVs (ndMVs) from P. elliottii, disrupt metabolite assimilation in several important pathways, including carbon and nitrogen metabolism. The PM data were also strongly correlated with observed phenotypic effects, including reduced viability and germination of spores in liquid media, as well as impaired filamentous growth on solid media. Importantly, these MV-induced phenotypic effects were reproducible in other filamentous pathogens (e.g., Botrytis cinerea, Chrysoporthe cubensis and F. graminearum) and during a glasshouse trial conducted with F. circinatum-infected P. elliottii seedlings, demonstrating the stable biological effects of ndMVs.
• Cumulatively, our results suggest that plant-derived vesicles can disrupt metabolism in pathogenic fungi and, therefore, serve as a cost-effective and sustainable source of novel plant protection molecules.
Description
SUPPLEMENTARY MATERIAL
DATA S1. Supporting Information.
FIGURE S1. Overview of membrane vesicle (MVs) isolation process from pine needle and root juice. (A, B) Pine leaves and roots cleaned and dried (C). Dried plant material mixed with vesicle isolation buffer (D). Juice extracted, filtered (E), and loaded into centrifuge tubes (F). Centrifugation at 700 × g to remove plant fibres (G), followed by filtration through a 0.22-μm membrane (H). A second centrifugation at 8000 × g concentrates fluid (I), removing excess buffer (J), then placed into Eppendorf tubes (K). (L) concentrated supernatant mixed with lipophilic dye FM4-64 and incubated (M). The sample is loaded onto a Sepharose CL2B column equilibrated with phosphate-buffered saline (PBS) (N). Fractions (200 μL) collected in 96-well plates (O), and fluorescence measured using a spectra max M2 plate reader (P). Fractions >3.3 RFU pooled, indicating presence of vesicles.
FIGURE S2. Correlation matrices of metabolite interactions across different PM plates (PM1, PM3B and PM24C), comparing treatments with (+MVs) and without vesicles (-MVs). Each subplot displays correlation coefficients (−1 to 1) among metabolites in different conditions, with positive correlations in red shades and negative correlations in blue shades. Strong positive or negative correlations in darker shades. Matrices highlight shifts in metabolite interactions potentially induced by vesicle treatment, illustrating how vesicles influence metabolic pathways over 1, 3, 7, 14, and 21 days. Data from 3 independent biological replicates.
FIGURE S3. Disease persistence on needle and stem samples after 14 days post-inoculation. Needles (a) and stems (b) plated on ¼ potato dextrose agar (PDA) plates after 14 days to check for disease growth and effect on microbial communities. Data from 3 independent biological replicates.
FIGURE S4. Radial growth of fungal colonies of other Fusarium strains (CMWF2552, CMWF2654, CMWF5689) over 14 days on PDA plates following a 24 h exposure to 15 μg mL−1 needle-derived membrane vesicles (ndMVs). Fungal spores were incubated with ndMVs and controls without ndMVs. Growth measured in 3 independent biological replicates at 3, 7, and 14 days to determine the impact of ndMVs on fungal proliferation (a,b). Light microscopy of vesicle-exposed and non-exposed cells (controls) on PDA plates at 3, 7 and 14 days (c). Plates examined at 20x magnification. P.r; Pinus radiata, P.h; Pinus hybrid, P.e; Pinus elliottii. scale bar = 20 μm.
FIGURE S5. Radial growth of fungal colonies of Botrytis cinerea (a,b), Chrysoporthe cubensis (c,d), Fusarium graminearum (e,f), Fusarium circinutum CMWF 2652 (g,h), Fusarium verticillioides (I,j) over 14 days on ¼ potato dextrose agar plates following a 24 h exposure to 15 μg mL−1 and 30 μg mL−1 P. elliottii needle-derived membrane vesicles, respectively. Schematic representation of radial growth (mm) with ±SD (b, d, f, h, j). Data from 3 independent biological replicates.
Keywords
Biocontrol agents, Filamentous pathogens, Needle-derived vesicles (ndMVs), Pine pitch canker disease, Pinus species, Vesicles (Vs)
Sustainable Development Goals
SDG-15: Life on land
Citation
Kunene, S., Mmushi, T.J., Steenkamp, E. & Motaung, T. 2026, 'Pinus-derived membrane vesicles disrupt pathogenic metabolism in fungi', Plant Biology, doi : 10.1111/plb.70069.