Fig 1.
Primary amino acid sequence and properties of NCR13.
(A) Primary amino acid sequences for Chickpea (C. arientinum) NCR13 and its homologs NCR15 and NCR07. Conserved cysteine residues are highlighted in red. The number of amino acids is indicated at the end of each sequence. (B) Peptide characteristics of CaNCR13, CaNCR15 and CaNCR07. (C) High-performance liquid chromatography (HPLC) analyses of NCR13 produced recombinantly in Pichia pastoris, monitored at 260 nm, reveals the presence of two major products. mAU represents milli-Absorbing Units. (D) Chemically synthesized NCR13 (NCR13_CS) elutes as one HPLC peak.
Fig 2.
Fingerprint 1H-15N HSQC spectra for oxidized and reduced NCR13.
Assigned 1H-15N HSQC spectra for oxidized (disulfide) NCR13_PFV1 (A) and NCR13_PFV2 (B). (C) The 1H-15N HSQC spectrum of fully reduced (dithiol) NCR13_PFV1 and NCR13_PFV2 were identical (the spectrum for NCR13_PFV2 is illustrated). All spectra collected on ~ 1 mM samples at 20°C in 20 mM sodium acetate, 50 mM NaCl, pH 5.3 at a 1H resonance frequency of 600 MHz. Amide side chain resonance pairs are connected by a red dashed line. Not within the displayed spectral boundaries is the G26 amide resonance for NCR13_PFV1 and NCR13_PFV2 at 8.66/103.1 ppm and 7.99/101.7 ppm, respectively, and the I24 amide resonance for NCR13_PFV1 at 8.72/132.0 ppm.
Fig 3.
Three-Dimensional NMR solution structures of NCR13_PFV1 and NCR13_PFV2.
Cartoon representation of the backbone superposition of the ordered regions in the ensemble of 20 structures calculated for oxidized NCR13_PFV1 (8ULM) and NCR13_PFV2 (7TH8). β-strands are colored blue and labeled sequentially starting from the N-terminal and the lone α-helix is colored wheat. Next to each ensemble is a backbone stick representation of a single structure with the six oxidized cysteine side chains highlighted in yellow, red, or cyan. Above the structures is a schematic summary of the elements of secondary structure observed for the two NMR structures, color-coded similarly. Also shown in the schematic are the disulfide bond connections in both isomers. One disulfide bond, between C15 and C30 (red), is identical in both isomers with C10 and C28 swapping disulfide bond partners.
Fig 4.
NCR13_PFV1 exhibits potent antifungal activity compared to NCR13_PFV2.
(A) Minimal Inhibitory Concentration (MIC) values of NCR13_PFVs for different fungal pathogens. (B) Antifungal activity of NCR13_PFV1, NCR13_PFV2, and reduced NCR13_CS against B. cinerea. Fungal cell viability assay using resazurin dye. Change from blue to pink/colorless signals resazurin reduction and indicates metabolically active B. cinerea germlings after 60 h. NCR13_PFVs are used at concentrations of 0.09–6 μM, N = 3, N indicates biological replicates. (C) Representative microscopic images showing the inhibition of B. cinerea growth 24 h after treatment with 0.09 μM NCR13_PFVs (Right). B. cinerea without peptide added served as a negative control (Left). Scale bar = 100 μm.
Fig 5.
Removing disulfide bonding in NCR13_PFV1 reduces antifungal activity against B. cinerea in vitro.
(A) Four NCR13 constructs used to assay the importance of disulfide bond formation for antifungal activity by substituting pairs of Cys residues with Ser (highlighted in red). The numbers above the sequence indicate the position of the Cys substitution. (B) HPLC analyses of NCR13_C4S-C23S, NCR13_C10S-C28S, and NCR13_C15S-C30S recombinantly produced in P. pastoris monitored at 260 nm (mAU = milli-Absorbing Units). (C) Fungal cell viability assay of disulfide knockout variants using the resazurin assay. A color changes from blue to pink/clear indicates of metabolically active (live) B. cinerea germlings after 60 h. (D) Summary of the MIC values for NCR13_PFV1 and NCR13 disulfide knockout variants.
Fig 6.
Active motif of NCR13 required for antifungal activity.
(A) Antifungal activity of reduced NCR13_CS and reduced NCR13 alanine variants against B. cinerea. Alanine scanning mutagenesis was done by substituting alanines within the NCR13 core sequence, leading to the creation of the NCR13_AlaV1-V5. MIC values and net charge of all peptides were determined. The amino acid substitutions that completely inactivate the peptide are shown in red. Representative pictures are shown in the S6 Fig. (B) Active motif of NCR13 (highlighted with a bold red font) required for its antifungal activity against B. cinerea. The secondary structure elements are shown above the amino acid sequence. The brown wavy symbol and blue arrow indicate α-helix (α1) and β-strand (β1) respectively.
Fig 7.
NCR13 PFV1 permeabilizes the cell membrane of B. cinerea rapidly.
(A) Confocal microscopy images of SYTOX Green (SG) uptake in B. cinerea germlings at different time points. Germlings were treated with 0.09 μM NCR13_PFVs and SG dye simultaneously for 20 min. No peptide is used as a control. Scale bars = 10 μm. The experiment was repeated thrice with similar results. (B) Quantitative measurements of average fluorescence intensity over time to assess cell membrane permeability in B. cinerea germlings treated with 0.09 μM NCR13 PFVs. (C) PIP strip showing binding of NCR13 PFVs to multiple phospholipids, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), phosphatidylinositol (3,4,5)-trisphosphate PI(3,4,5)P3 and phosphatidylinositol monophosphates PI(3)P, PI(4)P, and PI(5)P, phosphatidylinositol bisphosphate PI(3,4)P2. (D) Integrated density measurement of PIP strip probed with NCR13_PFVs. Datapoints are means ± SE of two independent biological replicates (N = 2). (E) Binding of NCR13_PFVs to PI(4)P and PI(4,5)P2 (PIP2) containing liposomes. NCR13_PFV’s were incubated without liposomes (None) and liposomes bearing no phosphoinositides (control), S- supernatant fraction and P- pellet fraction.
Fig 8.
Internalization and localization of NCR13 PFVs in B. cinerea germlings.
(A) Confocal microscopy images of B. cinerea germlings at different points treated with 0.09 μM DyLight550-labeled NCR13_PFV1 (left) and NCR_PFV2 (right) for 30 minutes. Scale bars = 10 μm. Data represents representative results of three independent experiments. (B) Quantification of peptide internalization was conducted by analyzing the average fluorescence intensities over time (in minutes) for NCR13_PFVs. The upper threshold for background was set at 23 for Fiji analysis. (C) Confocal microscopy images of B. cinerea germlings treated with 1.5 μM DyLight550-labeled NCR13_PFVs for 30 minutes. White arrowheads indicate NCR13_PFV2 on the cell surface. Scale bars = 10 μm. (D) Quantitative measurements of peptide internalization in B. cinerea germlings treated with 1.5 μM NCR13_PFV1 (left) and NCR13_PFV2 (right). (E) Internalized DyLight550-labeled NCR13_PFV1 (left) and NCR13_PFV2 (right) concentrate in specific foci (white arrowheads) within B. cinerea germlings. Scale bars = 10 μm. (F) NCR13_PFV1 and NCR13_PFV2 localize to the nucleolus. DyLight550-labeled NCR13_PFV1 (upper panel) and PFV2 (lower panel) colocalize with rRNA-specific Nucleolus Bright Green stain. Hoechst 33258 is used for staining nucleus. White arrowheads indicate nucleolar localization. Scale bars = 5 μm. (G) Semi-quantitative analysis of NCR13_PFVs within B. cinerea germlings. Germlings were treated with 1.5 μM NCR13_PFV1 and NCR13_PFV2 for 0, 2, and 4 hours. Total proteins were extracted from fungal cells and immunoblotted using anti-NCR13 antibody. Equal loading of proteins was verified using ponceau staining. The bar graph represents integrated density measurements. Statistical significance was tested using paired Student’s t-test. Data are shown as mean ± SD (N = 3, where N refers to biological replicates).
Fig 9.
NCR13_PFV1 exhibits rRNA binding and higher translation inhibition activity.
(A) rRNA binding by NCR13_PFVs tested using electrophoretic mobility shift assay (EMSA). B.cinerea rRNA was used to assess the binding activity of the peptides NCR13_PFV1 and NCR13_PFV2. The binding reactions were subjected to electrophoresis, and the mobility shifts were visualized on an agarose gel (1%). The concentration gradient of peptides is indicated above the lanes. The first lane on the left (M) contains molecular weight marker. (B) Relative in vitro translational inhibition (%) in the presence of NCR13_PFVs at different concentrations. Sterile water was used as negative control and cycloheximide (96 μM) was used as positive control. N = 3, ‘N’ indicates biological replicates. Statistical significance is determined by using One-way ANOVA with Tukey multiple comparisons test (**** indicates p value < 0.0001).
Fig 10.
NCR13 PFV1 exhibits enhanced curative antifungal activity against gray mold in pepper (Capsicum annuum) plants.
(A) Curative antifungal activity of NCR13_PFV1 and PFV2. Four-week-old pepper plants were first spray-inoculated using 1 mL of 5 × 104 B. cinerea spores. At 8 hpi (hours post inoculation), the plants were sprayed with 2 mL of peptide (NCR13_PFV1 or PFV2) at a concentration of 1.5 or 3 μM or control (B. cinerea alone). Mock-treated plants were only sprayed with SFM (no fungal spore suspension). (B) Calculated photosynthetic quantum yield (Fv/Fm) for individual pepper plants. (C) Disease severity (%) was calculated as described in materials and methods. (B-C) Each data point represents the average of 4 leaves per plant for 4 plants for each treatment. Horizontal lines represent the median and boxes indicate the 25th and 75th percentiles. The Kruskal-Wallis test with Dunn’s multiple comparisons test is used to determine statistically significant differences between control and peptide-treated inoculated plants. Two independent experiments were performed with similar results.
Fig 11.
Graphical abstract illustrates the biosynthesis of disulfide linkage variants of NCR13 (PFV1 and PFV2) in P. pastoris, their structures, antifungal activity, and modes of action in B. cinerea germlings.
The figure depicts the differences in cell penetration, localization, rRNA binding and translation inhibition, membrane permeabilization, phospholipid binding, and overall fungal cell killing potency. Created using BioRender.com.