How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System

Abstract

Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 μM to 25 μM. Inhibition of phytopathogenic bacteria - Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum - is also observed with MIC = 25-100 μM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Keywords: MALDI-MSI; Violaceae; antifungal defense; antimicrobial peptide; cyclotides; immunohistochemistry; plant host defense.

FIGURE 1

Isolation of different V. odorata tissues. (A) Petiole fragment with the vascular bundle (marked with an arrowhead) uncovered by removing the surrounding tissues. (B) Isolated fragments of lower (abaxial) and (C) upper (adaxial) leaf epidermis. Bar = (A) 1 cm; (B,C) 100 μm.

FIGURE 2

Permeabilization of a fungal cell membrane model by cycloviolacin cyclotides from V. odorata: cyO2, cyO3, cyO13, cyO19 (EC₅₀ = 72 nM; 67 nM; 130 nM; 80 nM respectively). All the tested cyclotides exhibited similar effectiveness at disrupting fungal lipid membranes, and were approximately 4–7 times more active than the bee venom antimicrobial peptide melittin (EC₅₀ = 490 nM).

FIGURE 3

Demonstration of cyclotides’ antifungal mode of action at the ultrastructure level –TEM images of F. oxysporum spores incubated with cyO2 and stained with immunogold. (A) Negative control – spores subjected to the complete immunogold procedure but not treated with the cyclotide. No gold particles are visible, demonstrating the antibodies’ specificity. The spores had normal cell walls (cw), cell membranes (arrowheads), mitochondria (∼) and nuclei (n). (B–D) Spore cells damaged by treatment with cyO2 – fragmented cell membranes (arrows) and plasmolysis (^∗). The cyclotide was found in all the cell structures (indicated by the presence of gold particles): the cytoplasm, mitochondria and nucleus, although only very small amounts were observed in the vacuole (v). (E) A single undamaged cell that did not bind cyO2 among the treated spores. The TEM images are representative of at least 3 stained grids per treatment and hundreds of observed spores. Bar = (C,D) 100 nm; (A,E) 200 nm; (B) 500 nm.

FIGURE 4

Cyclotides’ resistance to degradation by the fungal plant pathogen F. oxysporum. The proportion of non-degraded cyO2 (measured by integrating the AUC for the peptide peak at 280 nm) relative to that at time 0 after 2 h (h) to 3 days (d) of incubation with the fungus. All results are mean values of three replicate experiments, bars indicate standard deviations.

FIGURE 5

MS profiles of cyclotide extracts from different V. odorata isolated tissues and parts of the seed: adaxial and abaxial leaf epidermis, petiole vascular bundles, endosperm and embryo. Cycloviolacin cyclotides (cyO2, cyO3, cyO14, and cyO19) dominate in the leaf epidermis and vascular bundles. Quantities of kalata B1 and S found in the vascular bundle were much smaller than those in the petiole with the vascular bundle removed. Both kalata and cycloviolacin cyclotides were present in the dissected seed parts. The endosperm exhibited the highest cyclotide diversity. The figure shows results for triply charged (3+) ions.

FIGURE 6

Immunolocalization of cycloviolacin cyclotides (cyO2, cyO3, cyO13, cyO19) in the ovary, ovule, anther, mature embryo and endosperm of V. odorata. DAPI and Daylight 549 channels are merged, and the locations of cyclotides and nuclei are revealed by red and blue fluorescence, respectively. (A) Cross section of the ovary and (B) longitudinal section of the ovule; large amounts of cyclotides were found in the ovary wall and integuments (marked with arrowheads). (C) Cross section of the anther with visible pollen grains (pg) and cyclotides in the connective (cn) and the pollen sac walls (w). (D) Cross section of the appendix shielding the anther in the flower, with large amounts of cyclotides, especially in the epidermis (marked with an arrowhead). (E) Longitudinal section of the mature embryo – cyclotides are present in all the tissues, with higher amounts in the protodermis (arrowheads) and procambium (E1). (F) Close-up on the embryo protoderm and (G) endosperm cells with small vacuoles/protein bodies filled with cyclotides. Bar = (A,C,D,E) 250 μm; (B,E1) 50 μm; (F,G) 20 μm.

FIGURE 7

Preservation of tissue structure in V. odorata petiole cross sections obtained using different pretreatments, embedment techniques, and freezing temperatures. (A) Embedment in gelatin and freezing on dry ice. (B) Infiltration with PBS buffer under vacuum, embedment in gelatin, and snap freezing in liquid nitrogen. Optical images were taken before matrix application. Bar = 500 μm.

FIGURE 8

Distribution of the cyclotides kB1, cyO2, cyO3, and cyO19 in V. odorata leaf, petiole and root cross sections, depicted by MALDI-MSI. Warmer colors indicate higher abundance. kB1 was detected in the leaf mesophyll (m), close to the adaxial (ue) and abaxial (le) leaf epidermis, the outer layers of the petiole parenchyma (p), and the epidermis (e), but not in the roots. Cycloviolacin cyclotides were localized to the leaf mesophyll and epidermis, in and close to the vascular bundle (v), and in various tissues of the root - the pericycle (pr), cortex (c) and rhizodermis (r). Optical images were acquired after matrix application. Bar = 500 μm.

FIGURE 9

Distribution and co-localization of different cyclotides in V. odorata leaf, petiole and root cross sections, determined by MALDI-MSI. The distinct spatial distributions of kB1, cyO2, cyO3, and cyO19 are clearly visible in the co-localization images – cycloviolacins occur in the adaxial (ue) and abaxial (le) leaf epidermis, close to and in the petiole vascular bundles (v) and root cortex (c), rhizodermis (r), and pericycle (pr). Optical images were acquired after matrix application. kB1 found in leaf mesophyll (m) and petiole parenchyma (p). Bar = 500 μm.

FIGURE 10

Distribution of different cyclotides in V. odorata leaf, petiole and root cross sections as determined by MALDI-MSI. The locations of cyclotides were determined based on the distribution of the corresponding high intensity peaks in the average MALDI mass spectra, and were as follows: varv A - in the outer layers of the petiole parenchyma (p); cyO14 - leaf mesophyll (m), petiole parenchyma (p), and vascular bundles (v); 3263 - petiole parenchyma (p) and leaf epidermis (ue, le). varv A was absent from the roots. Warmer colors indicate higher abundance. Optical images were acquired after matrix application. Bar = 500 μm.