# Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and in silico studies

Camille Botcazon, Francisco Ramos-Martín, Nely Rodríguez-Moraga, Thomas Bergia, Sebastien Acket, Catherine Sarazin, Sonia Rippa

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## To cite this version:

Camille Botcazon, Francisco Ramos-Martín, Nely Rodríguez-Moraga, Thomas Bergia, Sebastien Acket, et al.. Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and in silico studies. Biophysical Chemistry, 2024, pp.107305. 10.1016/j.bpc.2024.107305. hal-04669883

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# Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of *Botrytis cinerea* and *Sclerotinia sclerotiorum*: Lipidomics profiles and biophysical studies

Camille Botcazon<sup>a,1</sup>, Francisco Ramos-Martín<sup>b,1</sup>, Nely Rodríguez-Moraga<sup>b</sup>, Thomas Bergia<sup>a</sup>, Sébastien Acket<sup>a</sup>, Catherine Sarazin<sup>b, *</sup>, Sonia Rippa<sup>a, *</sup>

## ARTICLE INFO

### Keywords:

Rhamnolipids

Fengycins

Botrytis cinerea

Sclerotinia sclerotiorum

Lipids

Membrane molecular dynamics

Solid-state NMR

## ABSTRACT

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

## 1. Introduction

Botrytis cinerea and Sclerotinia sclerotiorum are closely related phytopathogen ascomycetes of the Sclerotiniaceae family. They are respectively responsible for the gray mold and the white mold in more than 400 plant species, including crops such as soybean, rapeseed, and sunflower [1,2]. Synthetic pesticides are widely used to fight these pests and ensure yields. They act on essential biological functions, such as respiration, biosynthesis of ergosterol, or cell division, by targeting enzymes, which led to the emergence of pathogen resistances by gene mutations and have potential detrimental effects on human health and the environment [3].

Rhamnolipids (RLs) and Fengycins (FGs) are very promising natural biodegradable compounds suggested to reduce or replace synthetic pesticides to protect crops. They are mainly produced by Pseudomonas aeruginosa [4]. They are known to stimulate plant defense mechanisms and to have direct antifungal properties [5]. They are proposed to act through a direct interaction with the plasma membrane of the target cell by perturbing the lipid organization, a mode of action less prone to the induction of pathogen resistances [6].

RLs are amphipathic glycolipids composed of one or two 3-hydroxy fatty acid chains (from 8 to 16 carbons) and one (mono-RL) or two (di-RL) rhamnoses linked by a β-glycosidic bond [7]. At physiologic pH, RLs are negatively charged. The biocidal properties of RLs have been

<footnote><sup>a</sup> Unité Génie Enzymatique et Cellulaire, CNRS, UMR 7025, Alliance Sorbonne Université, Université de technologie de Compiègne, Compiègne, France</footnote>

<footnote><sup>b</sup> Unité Génie Enzymatique et Cellulaire, CNRS, UMR 7025, Université de Picardie Jules Verne, Amiens, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: catherine.sarazin@u-picardie.fr (C. Sarazin), sonia.rippa@utc.fr (S. Rippa).</footnote>

<footnote n="1">¹ These authors contributed equally to the work.</footnote>
demonstrated on pseudo-fungi and zoospores [8,9] and on filamentous fungi [10,9,11]. Their antimycelial effects have been evidenced on *B. cinerea* and *S. sclerotiorum* [12,13,14].

FGs are amphipathic cyclic lipopeptides mainly produced by *Bacillus subtilis* [15]. They are composed of 10 amino acids forming a lactone ring linked to a saturated or unsaturated fatty acid chain β-hydroxylated from 14 to 18 carbons, with a net charge of -1, resulting from 2 negative charges (glutamic acid residues) and one positive charge (ornithine residue). The FGs have a strong fungitoxic effect on different filamentous fungi but are inefficient on yeasts or bacteria [16,17]. Their antimicrobial activity against *B. cinerea* and *S. sclerotiorum* has also been assessed [18,12,19].

Interestingly, radial growth assays with different strains of *B. cinerea* and *S. sclerotiorum* have recently shown that RLs and FGs have opposite effects on both fungi. *B. cinerea* is globally more sensitive to FGs, whereas *S. sclerotiorum* is globally more sensitive to RLs [12]. The difference in sensitivity of both Sclerotiniaceae to RLs and FGs has been proposed to be due to a clear difference in the ergosterol content. Indeed, it was determined in this study that the strains of *S. sclerotiorum* and *B. cinerea* contain 1952.3 and 767.8 ng of ergosterol per mg of dry mycelia, respectively. The permeability induced by FGs on both fungi is increased in the presence of tebuconazole, which is an inhibitor of the ergosterol biosynthesis pathway. On the contrary, it is decreased in *B. cinerea* and not changed in *S. sclerotiorum* treated with RLs. These results suggest a different role of ergosterol in fungal sensitivity to both membranotropic compounds [12]. In the same way, ergosterol amount analysis has shown that *Alternaria solani*, which is less sensitive to FGs, contains more ergosterol than *Fusarium sambucinum* and *B. cinerea*, which are more sensitive to this antifungal compound [20]. Ergosterol is proposed to buffer the FG effects [21]. The interaction of mono- or di-RLs with POPC:POPG (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine: 1-palmitoyl-2-oleoyl-glycero-3-phosphoglycerol) plasma membrane models studied by solid-state Nuclear Magnetic Resonance (NMR) has shown a clear increase in fluidification with a higher ergosterol content [22]. This confirms a different effect of ergosterol on RL and FG activity on membrane model.

The interactions of RLs and FGs have been studied with phospholipids, the main class of plasma membrane lipids. The di-RLs intercalate in the bilayers of PC and induce structural perturbations [23]. Molecular dynamic simulations have shown that mono- and di-RLs insert in a phospholipid bilayer composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (POPC:PLPC) 50:50 model, near the phosphate group and close to the glycerol backbone of the phospholipids [22]. This location has recently been confirmed and explained by ionic interactions with polar head groups of PE and phosphatidylserine (PS) and van der Waals interactions with ergosterol, in more complex biomimetic membranes mimicking *Fusarium oxysporum* or *Saccharomyces cerevisiae* [24]. FGs disturb the structural and morphological characteristics of dipalmitoylphosphatidylcholine (DPPC) monolayer [25]. FGs would interact more easily with PE than with phosphatidylglycerol (PG) and PC [26] showing a role of the phospholipid composition of the membrane on the FG effects.

Some studies have shown that fatty acids containing 16 or 18 carbons (C16 and C18) with a variable number of unsaturations are predominant in *S. sclerotiorum* and *B. cinerea* [27,28,20]. The phospholipid composition of *B. cinerea* was assessed in two studies, showing numerous disparities. Griffith et al. quantified 54% of PC, 24% of PE, a pool of 18% of PA and PG, and a pool of 4% of phosphatidylinositol (PI) and PS (undistinguished). Later, Wise et al. [20] obtained mainly PG and PE (respectively 39 and 36%), 8% of PC, 15% of PI (quantified together with PS), and 2% of PA. Sumner and Colotelo [28] used a growth medium composed of dextrose, malt, yeast extract, and agar and grew the mycelium at 25 °C for 14 days. Griffith et al. [27] used cultures grown in malt agar 2% at 20 °C between 16 and 24 h. Wise et al. [20] used a tryptic soy broth at 28.5 °C for 96 h. Thus, the different studies on the lipids of *B. cinerea* and *S. sclerotiorum* were conducted in different culture

conditions, which are known to influence the lipid compositions [29]. Even though both methods were based on chloroform:methanol:water extractions and thin layer chromatography (TLC) analyses, differences in the protocols could also explain discrepancies in the phospholipid content. For instance, an inactivation step of lipid degradative enzymes was used in Griffith et al. [27] but not in Wise et al. [20]. Furthermore, the phospholipid composition of *S. sclerotiorum* is still missing. A comparison of phospholipid and fatty acid compositions is therefore necessary to understand the influence of the membrane lipid composition on RL and FG activities in these fungi.

In this study, we analyzed the fatty acid and phospholipid profiles of *B. cinerea* and *S. sclerotiorum* hyphae grown in the same conditions to propose lipid membrane models representative of each fungus. The key lipid components were then used to better understand and discriminate the mode of interaction of RLs and FGs with both pathogens by means of molecular dynamic (MD) simulations and solid-state NMR experiments.

## 2. Materials and methods

### 2.1. Biological materials and culture conditions

The *S. sclerotiorum* strain Ssc51 was provided by the technical institute Terres Inovia (Grignon, France). The *B. cinerea* strain Bc1 was supplied by the French National Institute for Agriculture, Food, and Environment (INRAe, Montfavet, France). Twenty-five µL of a *B. cinerea* spore suspension (stored at -20 °C at 1.3 × 10⁶ mL⁻¹) were first germinated on potato dextrose agar 39 g L⁻¹ plates (PDA, VWR Chemicals, France). *S. sclerotiorum* sclerotia were cut in half and the internal face of one piece was placed on 39 g L⁻¹ PDA plates. The pre-cultures were incubated for 4 days at 19 °C in darkness. Mycelium agar plugs were then excised with a 6 mm diameter cork borer, transferred to fresh PDA plates and incubated for 2 days at 19 °C in darkness until the mycelium took up half of the plate surface. To quantify lipid content, for each condition, mycelium was scraped off the plates with an inoculating loop, inoculated in 10 cultures of 5 mL of 12 g L⁻¹ potato dextrose broth (PDB, Sigma-Aldrich, Missouri, United States), and incubated for 4 days at 25 °C in darkness with 60 rpm shaking.

### 2.2. Extraction of fungal total polar lipids

Three independent cultures of each fungus were used to extract and analyze the lipid classes. Considering the difficulty of performing a reliable analysis of the plasma membrane lipids, which requires important quantities of hyphae and a risk of contamination, we chose to consider global phospholipid and fatty acid contents, representative of the plasma membrane. After a 2-day lyophilization of the mycelium, polar lipids were extracted and analyzed following the method of Folch et al. [30]. Each sample contained 5 batches of mycelium cultures grown in 5 mL of PDB, extracted in a chloroform/methanol/water mixture (2:1:0.5, v:v:v) with the addition of butylhydroxytoluene (BHT) at 0.01%. Samples were ground for 3 h with a metal bead under 50 Hz oscillation. After centrifugation, the lower organic phase of the samples was recovered. The aqueous phase was extracted a second time, overnight at 4 °C. The two organic phases from the two extractions were combined in a 2 mL sample, dried under a nitrogen stream, and stored at -20 °C.

### 2.3. Fatty acids analysis

Polar lipids from 4-day-old mycelial cultures of *B. cinerea* and *S. sclerotiorum* grown in liquid PDB medium at 25 °C were extracted using the Folch et al. [30] method. Fatty acids were then derivatized into volatile fatty acid methyl esters (FAMEs) analyzed and quantified by GC-FID.

To obtain volatile fatty acid methyl esters (FAMEs), total lipid extracts were derivatized by transmethylation, as described by Beisson
et al. [31]. Each sample was derivatized by 1 mL of reaction mixture (methanol:sulfuric acid:BHT, 95:5:0.01) and 20 µg of a C13 internal standard were added. Each sample was heated to 85 °C for 90 min. Extraction was performed using 1.5 mL of 0.9% NaCl and 1 mL of heptane. The samples were centrifuged at 1500 g and the heptane fraction was evaporated under a nitrogen stream. Fifty microliters of FAMEs were then analyzed and quantified by gas chromatography with flame ionization detection (GC-FID 2010 Plus, Shimadzu®, California, USA) as described in Tao et al. [32] and Seité et al. [33]. Briefly, 1 µL was injected into a 250 °C inlet with a split ratio of 20:1 into the GC. Separation was carried out using a BPX70 column (30 m × 0.25 mm, 0.25 µm, Fisher Scientific). The oven temperature program ramped from 120 °C to 250 °C at a rate of 10 °C/min, with helium serving as the carrier gas. A detection of methyl ester derivatives was performed using a Flame Ionization Detector (FID) at 280 °C.

The fatty acids were identified by comparison of the retention times of a standard solution of 37 fatty acid methyl esters (Sigma; 47,885-U Supelco) in GC-FID and confirmed by the high accuracy mass of molecular ions and their fragments after injection into a GC-MS (Q-Exactive™, Thermo) as described by Merlier et al. [34]. In summary, the GC-Q-Exactive system was outfitted with a capillary column IL60 (30 m × 0.25 mm i.d., 0.20 µm film thickness). One µL was injected into the system. The initial oven temperature was set at 150 °C for 2 min, followed by a ramp of 15 °C/min to reach 300 °C, where it was held for 4 min. The MS source temperature remained constant at 280 °C, and electron impact ionization (EI) at 70 eV was utilized for ionization. Data acquisition was conducted in the mass range of 50–500m/z in 60,000-resolution mode using Xcalibur™ software (Version 4.0.27). Spectral processing made use of the 2014 NIST mass spectral library and an in-house HRMS FAMEs database.

In each sample, the peak obtained for the 20 µg of C13 standard was used to determine the quantity of each fatty acid in µg, according to the following calculation:

$$ \frac{\text{(peak area of the corresponding FAME x 20 µg of C13)}}{\text{peak area of FAME C13}} \qquad (1) $$

Fatty acid profiles are expressed in mol% and the total amount of lipids is expressed in micrograms of fatty acids per milligram of dry weight of fungi (µg/mg DW). The average chain length percentage was determined from the sum of the quantities of each fatty acid multiplied by its chain length. The average unsaturation number percentage was determined from the sum of the quantities of each fatty acid multiplied by its unsaturation number. The ratio of unsaturated to saturated fatty acids was determined by dividing the sum of unsaturated fatty acids by the sum of the saturated.

## 2.4. Phospholipid class analysis by HPTLC

Quantification of phospholipids was performed with a high-performance thin-layer chromatography system (HPTLC, CAMAG®, Switzerland). Sample deposits were made by an automated system (CAMAG® Automatic TLC Sampler 4, ATS 4) on silica plates measuring 10 × 20 cm with 60 Å pores (1.05642, Supelco® Bellefonte, United States). Ten mL of chloroform: methanol: acetic acid: water (50:20:10:10:3, v:v:v:v:v) migration solvent was used for the analysis of phospholipid contents. After saturating the development chamber (CAMAG® Automatic Developing Chamber 2, ADC 2) for 1 hour, the plate was immersed in the migration solvent, migrating capillarily up to 7 cm in height. Migrations were performed in a chamber at a humidity rate of 40%. The revelation of bands corresponding to phospholipid classes was done by spraying, using a derivatizer (CAMAG® Derivatizer), with 3 mL of commercial ninhydrin revealer (Carl ROTH®) for PS or copper sulfate for PE, PC, PA, PI, and PG. The copper sulfate revealing agent was prepared by dissolving 20 g of copper sulfate II in 200 mL of methanol at -20 °C and adding 8 mL of sulfuric acid (98%) and 8 mL 85% ortho-phosphoric acid (85%) on ice. After revelation, the plates

were heated at 140 °C for 3 min for the ninhydrin or 30 min for the copper sulfate revealer. The data were obtained by scanning the plates at 370 nm with a densitometer (CAMAG®, Densitometer Scanner 4). The quantification of each class of phospholipid was performed by the Visioncats software (CAMAG®), using concentration ranges of standards phospholipids PA (18:0; ref. 830865), PC (16:0; ref. 850355), PE (16:0; ref. 850705), PG (17:0; ref. 830456), PI (Soy PI containing mostly 18:2, then 16:0, 18:3, 18:0, 18:1; reference 840044) and PS (17:0; reference 840028) (Avanti Polar Lipids, Alabama, USA). They were deposited simultaneously with the biological samples. Data were expressed in micrograms of phospholipids per microgram of dry weight of fungi (µg/µg DW) and in percent of total phospholipids in fungi.

## 2.5. Phospholipid profiling by HPLC-QTOF

Lipids extracted from fungi were analyzed by HPLC-QTOF as described by Tsugawa et al. [35] and Tao et al. [32]. Briefly, data were acquired on an ultra-high-performance liquid chromatography system Agilent 1290 Infinity LC system (Agilent Technologies®, Santa Clara, CA, USA) with a pump (G4220A), a column oven (G1316C) and an autosampler (G4226A). Lipids were separated on an Acquity UPLC CSH C18 column (100 mm length, 2.1 mm id., 1.7 µm) coupled to an Acquity CSH C18 VanGuard pre-column (5 mm length, 2.1 mm id., 1.7 µm) (Waters®, Milford, MA, USA). The column and pre-column were maintained at 65 °C. The separation was done at 0.6 mL/min by using the mobile phase 60:40 (vol/vol) acetonitrile/water (solvent A) and 90:10 (v:v) isopropanol/acetonitrile (solvent B) implemented with 10 mM of ammonium acetate, as described before [32]. The gradient was: 0 min 15% (B); 0–2 min 30% (B); 2–2.5 min 48% (B); 2.5–11 min 82% (B); 11–11.5 min 99% (B); 11.5–12 min 99% (B); 12–12.1 min 15% (B); 12.1–15 min 15% (B). Ten microliters of samples were injected. MS/MS data-dependent acquisition was done in negative mode, and MS2 scans were performed on the sixth most intense ions. The source temperature, fragmentor, and skimmer were set up at 350 °C, 150V and 65V, respectively. The acquisition was made in full-scan mode between 100m/z and 1500 m/z, with a scan of two spectra per second. Two internal references were used for in-run calibration of the mass spectrometer (112.9856, 1033.9881). The MassHunter B.07 software allowed us to control the parameters of the machine.

MsDial v 4.0 software [35] was used for data processing and annotation of lipids. The data collection was performed by fixing the MS1 and MS2 tolerances, at 0.01 Da and 0.05 Da, respectively. The peak detection was fixed at 1000 amplitude and a mass slice width of 0.1 Da. The deconvolution parameters correspond to a sigma window value of 0.5 and a MS/MS abundance cut off at amplitude 10. Isotopic ions were kept until 0.5 Da. The peaks list was annotated based on their unique MS/MS fragmentation patterns using the in-built LipidBlast mass spectral library in MsDial v 4.0 software. The accurate mass tolerances MS1 and MS2 were fixed at 0.01 Da and 0.05 Da, respectively. The identification score cutoff was fixed at 80%. Lipids were normalized by the intensity of the internal standard corresponding to the phospholipid class (EquiSPLASH, Avanti® Polar). Data were finally expressed in molar percentages.

## 2.6. All-atom molecular dynamics simulations

We previously determined that the strains of S. sclerotiorum and B. cinerea used in this study contain 1952.3 and 767.8 ng of ergosterol per mg of dry mycelia, respectively [12]. To determine the composition of the lipid bilayer used as the model membrane of each fungus, the quantities of phospholipids analyzed by HPTLC and ergosterol determined by GC-MS were normalized by the dry weights of the corresponding samples. The values were summed to constitute the total lipids studied (Σ lipids). The proportion of each lipid class to this total was then calculated using the following equation:
$$ \frac{\text{Quantity of one lipid } \left( \frac{\mu g}{mg} \text{ of dry mycelium} \right)}{\sum \text{lipids}} \times 100 \qquad (2) $$

To be as close as possible to the determined compositions and representative of fatty acid amounts, ratios of unsaturated to saturated fatty acids, and polar head group of phospholipid contents, the PE and PC used in the models were made up of C18:2-C18:2 chains. Therefore, 1,2-dilinoleoyl-sn-glycero-3-phosphatidylethanolamine (DLIPE according to the FF nomenclature), 1,2-dilinoleoyl-sn-glycero-3-phosphatidyl-choline (DUPC according to the FF nomenclature), and PA 16:0-18:1 (1-palmitoyl-2-linoleyl-sn-glycero-3-phosphate, PLPA in the FF nomenclature) were chosen.

Two ternary models based on the PE, PA, and ergosterol ratios were used as follows:

- B. cinerea ternary model: DLIPE 18:2_18:2/PLPA 16:0_18:2/Ergosterol 79/20/1%

- S. sclerotiorum ternary model: DLIPE 18:2_18:2/PLPA 16:0_18:2/Ergosterol 52/44/4%

To take into account the PC role in the interaction of the RLs and FGs with the biomimetic lipid membrane models, we also constructed quaternary models as follows:

- B. cinerea quaternary model: DLIPE 18:2_18:2/PLPA 16:0_18:2/Ergosterol/DUPC 18:2_18:2 72/18/1/9%

- S. sclerotiorum quaternary model: DLIPE 18:2_18:2/PLPA 16:0_18:2/Ergosterol/DUPC 18:2_18:2 51/44/4/1%.

All-atom molecular dynamics simulations were performed with the CHARMM-GUI (https://charmm-gui.org/) (CHARMM 36 m, C36) force field (FF), containing different topologies of phospholipids [36]. A temperature of 25 °C (298 K) was used. A water layer of 40 Å thick was added above and below the membrane, representing approximately 11,000 water molecules, as described by Jorgensen et al. [37]. This environment was balanced with K⁺ or Cl⁻ counter-ions. In accordance with previous biophysical experiments conducted on representative models of plants [22] and yeast [24], a ratio of 1 antifungal molecule to 25 lipids was used (i.e., 6 molecules for 150 total lipids). In the different simulations, antifungal molecules were placed at different distances from the lipid bilayer of each model. Simulations were conducted without antifungal molecules (controls), with molecules at a distance of 10 Å sufficient to avoid intermolecular contacts or in direct contact within the membrane. These three configurations were tested for each of the four models and for the two types of molecules (RLs or FGs), with three repetitions for each case, totaling 72 simulations. Each simulation in this study represents 500 ns length dynamics (except in cases where a longer duration is specified).

CHARMM-GUI contains topologies for polyunsaturated fatty acids, polar heads PC, PE, PG, PI, PA, and PS, and ergosterol for the C36 FF [38,39,40,41]. The topologies of mono- and di-RLs in C36 are available [42]. FGs molecules were modeled by CHARMM-GUI "Ligand Reader & Modeler" [43] and parametrized with Antechamber [44,45]. The order parameter S<sub>CD</sub> was calculated using the GROMACS gmx order tool according to the following equation [46]:

$$ S_{CD} = \frac{1}{2} < 3 \cos^2 \theta_z - 1 > \qquad (3) $$

For the last carbon, one of the hydrogen atoms of the last methyl was considered as C<sub>n+1</sub>. Area per lipid and thickness measurements were calculated by Fatslim software [47].

## 2.7. Sample preparation, solid-state NMR experiments and analysis

Multilamellar vesicles (MLVs) containing deuterated palmitoyl

chains were prepared as done in previous works [48] using the proportions of phospholipids and ergosterol determined as above. Due to constraints on deuterated lipid availability, we have needed to replace some of the lipids with their closest available counterparts. All phospholipids were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Ergosterol was from Cayman Chemical Company (Ann Arbor, Michigan, USA).

Two ternary models and two quaternary models were used as follows:

- B. cinerea ternary model: POPE:d31 (ref. 850757)/SoyPA (ref. 840074)/Ergosterol (ref. 19850), 79/20/1%

- S. sclerotiorum ternary model: POPE:d31/SoyPA/Ergosterol, 52/44/1%

- B. cinerea quaternary model: POPE:d31/SoyPA/Ergosterol/PLPC (ref. 850458), 72/18/1/9%

- S. sclerotiorum quaternary model: POPE:d31/SoyPA/Ergosterol/PLPC (51/44/4/1%).

Lipids were solubilized in chloroform, and solutions were mixed in order to obtain the right proportions in a total lipid amount of 60 mM. A concentration of 2.4 mM of antifungal molecules (corresponding to 1:25 lipids) were tested for the interaction studies. The resulting solutions were evaporated under nitrogen gas flow. The sample were hydrated with ultrapure water, well-vortexed to promote total hydration, and lyophilized overnight to remove the traces of solvents. The resulting powders containing lipids were hydrated by 80 µL of 50 mM HEPES (pH 6.6) and 100 mM NaCl, vortexed, and homogenized using four free-thaw cycles involving one step of freezing (−30 °C, 20 min) followed by thawing (40 °C, 15 min) and shaking. Finally, the MLV samples were placed in a 7-mm ss-NMR rotor to perform the experiments. Ss-NMR experiments were recorded at 298 K on a Bruker Avance Biospin 300 WB (7.05 T) equipped with a CP-MAS 7-mm probe. Static <sup>2</sup>H NMR was carried out by applying a phase cycled quadrupolar echo pulse sequence (90°x-τ-90°y-τ-acq) [49]. The parameters used are: spectral width of 500 kHz, π/2 pulse of 5.4 µs, an echo delay of 40 µs, a recycle delay of 0.5 s, and a number of acquisitions ranging from 8 k to 14 k depending on samples. Static <sup>31</sup>P NMR was carried out using a Hahn-echo sequence [50] with <sup>1</sup>H decoupling, a π/2 pulse of 6.5 µs, a recycle delay of 3 s, and a number of acquisitions ranging from 8 k to 14 k depending on samples. For all spectra, an exponential line broadening of 100 Hz was applied before Fourier-transform from the top of the echo signal.

## 2.8. Statistical analyses

Statistical analyses were performed using the R software [51]. Statistical differences were analyzed by paired t-tests using the pairwise.t. test function, with p-value adjustment according to the Bonferroni method.

# 3. Results

## 3.1. The fatty acid compositions of S. sclerotiorum and B. cinerea are slightly different

The fatty acid profiles of total lipids from B. cinerea and S. sclerotiorum grown in liquid growth medium under the same conditions were analyzed by GC-FID. For both pathogens, C18:2 (linoleic acid) is the largest fatty acid accounting for almost half of all fatty acids (48.37 ± 7.15 mol% in B. cinerea and 47.24 ± 13.44 mol% in S. sclerotiorum) (Table 1).

The distribution of the palmitic (C16:0), oleic (C18:1) and stearic (C18:0) acids differs between the two species. There is a predominance of C18:1 over C16:0 in B. cinerea (25.86 mol% ± 7.48 and 14.37 ± 0.94 respectively), while the amount of C16:0 is higher than C18:1 in S. sclerotiorum (21.14 ± 13.31 mol% and 6.39 ± 1.68 respectively).
<caption>**Table 1**</caption>

Fatty acid compositions of *B. cinerea* and *S. sclerotiorum*. The results represent mean and standard deviation values obtained from 3 independent experiments. The results are expressed in molar percentages relative to the total fatty acids per sample quantified by GC-FID. **, p < 0.01; *, p < 0.05; different letters correspond to statistically different groups.

<table><thead><tr><th>Fatty acid (mol%)</th><th><i>B. cinerea</i></th><th><i>S. sclerotiorum</i></th><th>Significance</th></tr></thead><tbody><tr><td>C14:0</td><td>Not detected</td><td>1.45 ± 0.47</td><td>**</td></tr><tr><td>C15:0</td><td>0.17 ± 0.13</td><td>1.94 ± 1.11</td><td>*</td></tr><tr><td>C16:0</td><td>14.37 ± 0.94</td><td>21.14 ± 13.31</td><td></td></tr><tr><td>C16:1</td><td>0.64 ± 0.10</td><td>0.26 ± 0.20</td><td></td></tr><tr><td>C17:0</td><td>Not detected</td><td>1.30 ± 0.71</td><td>**</td></tr><tr><td>C18:0</td><td>3.30 ± 0.68</td><td>11.54 ± 2.17</td><td>**</td></tr><tr><td>C18:1</td><td>25.86 ± 7.48</td><td>6.39 ± 1.68</td><td>**</td></tr><tr><td>C18:2</td><td>48.37 ± 7.15</td><td>47.24 ± 13.44</td><td></td></tr><tr><td>C18:3</td><td>6.40 ± 0.95</td><td>5.67 ± 3.28</td><td></td></tr><tr><td>C20:0</td><td>0.13 ± 0.18</td><td>Not detected</td><td></td></tr><tr><td>C20:1</td><td>0.06 ± 0.09</td><td>0.86 ± 0.41</td><td></td></tr><tr><td>C20:2</td><td>0.69 ± 0.08</td><td>0.62 ± 0.15</td><td></td></tr><tr><td>C22:1</td><td>Not detected</td><td>0.65 ± 0.24</td><td></td></tr><tr><td>C24:0</td><td>Not detected</td><td>0.94 ± 0.97</td><td></td></tr><tr><td>Average chain length</td><td>17.71 ± 0.02</td><td>17.55 ± 0.31 a</td><td></td></tr><tr><td>Average unsaturation number</td><td>1.44 ± 0.04 a</td><td>1.21 ± 0.22 a</td><td></td></tr><tr><td>Ratio of unsaturated/ saturated</td><td>4.59 ± 0.41 a</td><td>2.06 ± 1.34 b</td><td></td></tr></tbody></table>

There is a significant difference about the stearic acid (C18:0) between *S. sclerotiorum* (11.54 ± 2.17 mol%) and *B. cinerea* (3.30 ± 0.68 mol%). Linolenic acid (C18:3) is present at a lower amount in both fungi (6.40 ± 0.95 mol% and 5.67 ± 3.28 mol% respectively).

The fact that *S. sclerotiorum* exhibits a higher diversity of fatty acids than *B. cinerea* is also noteworthy. The fatty acids C14:0, C17:0, C22:1 and C24:0 are detected only in *S. sclerotiorum*, while C20:0 is only identified in *B. cinerea*.

Average fatty acid chain lengths and average number of unsaturations were calculated for each fungus because these parameters can alter membrane fluidity in adaptive response to temperature changes or other stressors [52]. However, no significant differences were observed for both fungi grown in the same conditions. On the other hand, a significantly higher ratio of unsaturated to saturated fatty acids was found in *B. cinerea* as compared to *S. sclerotiorum*.

## 3.2. The phospholipid contents of *S. sclerotiorum* and *B. cinerea* show differences

### 3.2.1. Analysis of the phospholipid classes

A total phospholipid analysis was performed by HPTLC to separate and obtain absolute quantifications of each class of phospholipids. The data are presented as mol% of total phospholipids (Table 2).

In both fungi, PE, PA, and PC are predominantly found, while PI, PS,

<caption>**Table 2**</caption>

Phospholipid compositions of *B. cinerea* and *S. sclerotiorum*. The results represent average values obtained from 3 independent experiments. The results are presented as molar percentages relative to the total quantified phospholipids in a sample, analyzed by HPTLC. ***, p < 0.001; **, p < 0.01; *, p < 0.05; different letters correspond to statistically different groups.

<table><thead><tr><th>Class (%)</th><th><i>B. cinerea</i></th><th><i>S. sclerotiorum</i></th><th>Significance</th></tr></thead><tbody><tr><td>PE</td><td>66.90 ± 4.15</td><td>55.98 ± 2.65</td><td>*</td></tr><tr><td>PA</td><td>22.98 ± 2.24</td><td>41.33 ± 3.89</td><td>**</td></tr><tr><td>PC</td><td>8.68 ± 0.27</td><td>1.67 ± 0.22</td><td>***</td></tr><tr><td>PI</td><td>0.72 ± 0.05</td><td>0.25 ± 0.06</td><td>*</td></tr><tr><td>PS</td><td>0.05 ± 0.00</td><td>0.05 ± 0.00</td><td></td></tr><tr><td>PG</td><td>0.67 ± 0.06</td><td>0.72 ± 0.16</td><td></td></tr><tr><td>Anionic (A)</td><td>24.42</td><td>42.35</td><td></td></tr><tr><td>Zwitterionic (Z)</td><td>75.58</td><td>57.65</td><td></td></tr><tr><td>Ratio A/Z</td><td>0.32 a</td><td>0.73 b</td><td></td></tr></tbody></table>

and PG are present in lower proportions. PE are the predominant phospholipids in both fungi, with 1.2 times more PE in *B. cinerea* than in *S. sclerotiorum* (66.90 ± 4.15 mol% and 55.98 ± 2.65 mol% respectively). The second class of phospholipids, PA, differs significantly between the two fungi, with 22.98 ± 2.24 mol% in *B. cinerea* and almost twice in *S. sclerotiorum* (41.33 ± 3.89 mol%). The PC values obtained show significant difference between the two fungi, with *S. sclerotiorum* having 1.67 mol ± 0.22 mol% and *B. cinerea* having 8.68 ± 0.27 mol%. The phospholipids PG, PS, and PI are minoritarian in both fungi.

Thus, the order of predominance for the different phospholipid classes of *B. cinerea* is PE > PA > PC > PG ≥ PI>PS and PE > PA > PC > PG > PI>PS for *S. sclerotiorum*. There is a significant difference between the ratio of anionic phospholipids to zwitterionic phospholipids between *B. cinerea* and *S. sclerotiorum* with values of 0.32 and 0.73 respectively (Table 2), especially due to the difference in the amount of PA.

The PA content quantified in the present work was higher than previously described data for both fungi (a pool of 18% of PA quantified with PG in Griffiths et al. [27] or 2% of PA in Wise et al. [20]). This could result from the hydrolysis of phospholipids, preferentially PC, into PA by the phospholipase D [53,54]. To check this point, additional experiments were performed, comparing two extraction methods on *S. sclerotiorum* mycelium. On a first sample, phospholipids were extracted by the Folch et al. [30] method (Fig. S1 A). On a second sample, 1 M isopropanol at 75 °C was added before the extraction solvent and it was heated for 15 min at 75 °C to deactivate the phospholipase D [27,55] (Fig. S1 B). The lipids were separated and analyzed by HPTLC. In all samples, the PC remains low compared to the PA content, around 1.5% molar of phospholipids, discarding that the measured amount of PA comes from the hydrolysis of PC or other phospholipids.

### 3.2.2. Analysis of the main molecular species of each phospholipid class

HPLC-QTOF analysis enables the identification of the molecular species constituting each class of phospholipids by comparing it with the LipidBlast database implemented into MsDial v4.0 software [35]. HPLC-QTOF analysis confirmed the proportions of the different phospholipid classes determined by HPTLC in both fungi (Fig. S2). The results of the HPLC-QTOF analysis (Fig. 1) are consistent with those obtained in GC-FID (Table 1) and confirm a predominance of unsaturated C18 and saturated C16 fatty acids in the phospholipids of both fungi.

We identified 17 different PEs, 20 PCs, 10 PAs, 11 PIs, 7 PGs, and 9 PSs. In *B. cinerea* and *S. sclerotiorum*, the major species for PE is PE 36:5 (corresponding to PE 18:2_18:3), PA 36:4 (PA 18:2_18:2), and PC 36:4 (PC 18:2_18:2). They are followed by PI 34:1 (PI 16:0_18:1) and PS 34:1 (PS 16:0_18:1) in *B. cinerea* and PI 34:2 (PI 16:0_18:2) and PS 34:2 (PS 16:0_18:2) in *S. sclerotiorum*. Next comes PG 34:2 (PG16:0_18:2) in both fungi.

The lysophospholipids were also quantified with this method. The majority of them are composed of saturated C16 or unsaturated C18 fatty acids, in accordance with the fatty acid analyses in both fungi (Fig. S3).

## 3.3. The role of the lipid compositions on the action of antifungal molecules studied with molecular dynamic biomimetic membrane models of *B. cinerea* and *S. sclerotiorum*

Lipid biomimetic membranes representative of *S. sclerotiorum* and *B. cinerea* lipid compositions were constructed to perform MD simulations for the study of the interaction of RLs and FGs. The selected compositions were based on our present data on the main phospholipids (PE, PA, PC) and previous results on ergosterol contents [12].

### 3.3.1. Interaction of RLs with biomimetic membrane models of *S. sclerotiorum* and *B. cinerea*

Simulations were made first by placing the RLs on the outside of the membranes, with the rhamnose groups oriented toward or away from the membrane. Six molecules of RLs were used, 4 molecules of mono-RLs
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Distribution of molecular species in phospholipid classes of *B. cinerea* and *S. sclerotiorum* analyzed by HPLC-QTOF. The height of a bar represents the average value from at least 3 biological replicates. Error bars depict standard deviations. The red boxes represent the major molecular species in both fungi, the blue and green boxes represent the major molecular species of *B. cinerea* and *S. sclerotiorum*, respectively. **, *p* < 0.01; *, *p* < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

and 2 molecules of di-RLs. This proportion is close to the composition of the natural mixture of RLs (with a ratio of mono-RLs/di-RLs of 60/40%), produced by *P. aeruginosa* as previously used in other works [13,22].

The way in which RLs fit into model membranes was studied. An example involving a di-RL molecule and the *B. cinerea* ternary model is illustrated in Fig. 2, but mono-RL look to enter in a similar fashion. The insertion of RLs happens in several steps. One of the two fatty acids penetrates the membrane and sits parallel to the fatty acids of the phospholipids (Fig. 2A); later, the second fatty acid of the RL inserts in

the same way (Fig. 2B) and, finally, the amphiphilic group of one or two rhamnose(s) enters the membrane (Fig. 2C) by locating at the polar headgroups membrane region. The molecule gets completely inserted into the lipid bilayer (Fig. 2D). Similar behaviors were obtained with the models mimicking *S. sclerotiorum* lipid membranes. The outward-facing RLs of the model membranes enter in the same way as the inward-facing RLs. The initial orientation of the RLs (water- or bilayer-oriented rhamnoses) does not have a noticeable effect on their final localization in the bilayer, the rhamnose groups are always located near the
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Example of the insertion of RLs in a lipid bilayer. The arrow points to the di-RL molecule whose insertion kinetics is being monitored. Insertion process involves multiple steps (A–D). Model composition: DLIPE/PLPA/Ergosterol 79/20/1% (*B. cinerea* ternary model without PC), mixture of mono-RLs:di-RLs 4:2.</figcaption></figure>

polar heads of the phospholipids and the fatty acid chains of the RLs are located along the fatty acid chains of the phospholipids.

Every RL placed outside the pre-formed lipid membrane models of *B. cinerea* and *S. sclerotiorum* at the beginning of the simulation gets completely internalized into all the studied membrane models, with no differences among different lipid compositions (Fig. 3A). No aggregation of RLs is observed during the 500 ns simulation time and no membrane exit is observed. Other simulations were performed by placing the RLs initially inside the membranes. This does not change the way RLs are placed in the bilayer at the end of the simulation.

We proceeded to analyze some structural membrane properties, studying how those could get altered because of the internalization of the RLs. The order parameter ($S_{CD}$) was calculated to check the effect of RLs insertion on global order of phospholipid acyl chains (Fig. 3B). In the absence of RLs, independently of the presence of PC in ternary or quaternary models, the order parameter in *S. sclerotiorum* models is slightly higher than in models of *B. cinerea* (Fig. 3B, black lines), implying a slightly higher rigidity of the *S. sclerotiorum* models compared to the *B. cinerea* ones. This could be explained by the higher amount of ergosterol, as it is known to modify the fluidification of the lipid bilayers [56]. For all four models, there was a decrease in the $S_{CD}$ values in the presence of RLs (Fig. 3B, red lines), compared to models without RLs (black lines). The internalization of the different RLs decreases the order parameter, which can be interpreted as a rise in the fluidity of the hydrophobic core of all these membrane models,

regardless of the lipid composition. No membrane remodelling was observed.

Other membrane properties get altered too (Table S1). Area per lipid values get remarkably increased, and membrane thickness gets reduced in all models, as expected from the incorporation of the RLs into the bilayer.

### 3.3.2. Interaction of FGs with biomimetic lipid membrane models of *S. sclerotiorum* and *B. cinerea*

Simulations with the different fungal membrane models were first performed in the presence of a single molecule of FG (Fig. 4). The fatty acid chain of FG penetrates into the hydrophobic core of each model, while the peptidic part of the molecule takes a longer time to insert. In all the internalization cases, the process involves different steps. After the attachment to the surface of the bilayer (Fig. 4A), the lipidic part of the lipopeptide penetrates into the membrane parallel to the phospholipid fatty acid chains (Fig. 4B). Eventually, the peptidic part is able to establish some more contacts with the surface (Fig. 4C), but they are usually not stable enough to play a major role in the internalization process. We have extended the duration of the simulations for these systems up to 1.5 µs and, at least at the studied time scale, the peptide ring of the molecule stays located out of the bilayer while the lipidic tail stays internalized (Fig. 4D).

This positioning of the FGs is observed whether one or six FG molecules are simulated (Fig. 5). The rise in concentration highlights a
A

Ternary models

B. cinerea

<figure><img src="image_6.png" /></figure>

S. sclerotiorum

<figure><img src="image_7.png" /></figure>

Quaternary models

B. cinerea

<figure><img src="image_8.png" /></figure>

S. sclerotiorum

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

PE (DLIPE)

<figure><img src="image_11.png" /></figure>

PA (PLPA)

<figure><img src="image_12.png" /></figure>

Ergosterol

<figure><img src="image_13.png" /></figure>

PC (DUPC)

<figure><img src="image_14.png" /></figure>

Mono-RL

<figure><img src="image_15.png" /></figure>

Di-RL

B

Ternary models

B. cinerea

<figure><img src="image_16.png" /></figure>

S. sclerotiorum

<figure><img src="image_17.png" /></figure>

Without RLs
RLs

Quaternary models

B. cinerea

<figure><img src="image_18.png" /></figure>

S. sclerotiorum

<figure><img src="image_19.png" /></figure>

**Fig. 3.** Placement of RLs (6 molecules: 4 mono-RLs and 2 di-RLs) once they are internalized in different lipid bilayer models. Phospholipid fatty chains are not shown to facilitate visualization of the RLs and ergosterol molecules (A). S<sub>CD</sub> order parameters regarding the carbon position in the sn-1 palmitoyl chain of PLPA. The coordinates of the points correspond to the means measured for each carbon during the last 250 ns of simulation. Black curves (2 replicates) correspond to control bilayers without RLs; red curves (2 replicates) correspond to bilayers containing RLs (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
<figure><img src="image_20.png" /><figcaption>**Fig. 4.** Example of multiple steps (A–D) needed for the insertion of one FG molecule in a lipid bilayer. Model composition: DLIPE/PLPA/Ergosterol 79/20/1% (*B. cinerea* ternary model without PC).</figcaption></figure>

noteworthy phenomenon: FGs have a strong tendency to aggregate, leading to an impairment of their ability to penetrate the membrane. In all models, aggregates of FGs molecules are observed with different multimerization degrees. It appears that for the lipidic tail to internalize, a single molecule must detach from the aggregate and then insert itself. Focusing on the different models, we observe that the interaction with *B. cinerea* membrane models is more stable. The FG molecules stay attached to the surface for longer periods of time, even in cases where aggregation occurs.

These differences are reflected in the analyzed membrane properties. In the ternary and quaternary models of *S. sclerotiorum*, the addition of FGs does not perturb the lipid bilayer order parameter, as the $S_{CD}$ values of the black (without FGs) and red (with FGs) curves are perfectly superimposed (Fig. 5B). On the other hand, in *B. cinerea* models, which contain more PE and less PA than the *S. sclerotiorum* models, the addition of FGs seems to decrease the order parameters. The same tendencies are observed when reviewing the area-per-lipid and membrane thickness properties. Area per lipid values get remarkably increased for *B. cinerea* models while remaining nearly unchanged for the *S. sclerotiorum* ones. The thickness of the membrane gets reduced, especially when a higher degree of FG penetration is observed, as is the case for *B. cinerea* models (Table S2).

### 3.4. The role of the different lipid compositions on the action of antifungal molecules studied with ss-NMR biomimetic membrane models of *B. cinerea* and *S. sclerotiorum*

Multilamellar vesicles (MLVs) provide more accurate models of lipid membranes suitable for ss-NMR, allowing to study systems that are similar to those studied by MD. Static $^2$H ss-NMR of liposomes provides an overall information on the internal dynamics of the bilayer when the acyl chains of phospholipids are deuterated. Tests were conducted on phospholipids that were solely deuterated in the palmitoyl chain. Inserting molecules into the bilayer is frequently accompanied by a change in the degrees of freedom of acyl chains and can be used as an indirect experiment to explore the depth of the interaction. We also performed static $^{31}$P studies to understand how antifungal compounds affect liposomes, possibly altering their lipidic phases [57].

#### 3.4.1. Interaction of RLs with biomimetic lipid membrane models of *S. sclerotiorum* and *B. cinerea*

We investigated how RLs affect MLVs that mimic the two types of fungal models. The static $^2$H ss-NMR experiments show a strong fluidification effect on the ternary models (Fig. 6A). In those cases, a strong resolution loss together with a narrowing of the spectra was observed (comparison between the sample with RLs in red and the reference in blue). When looking at the static $^{31}$P spectra, we observe a phase change from lamellar to hexagonal (Fig. 6B), as expected from the big changes
<figure><img src="image_21.png" /><figcaption>**Fig. 5.** Representative snapshots illustrating the interaction of multiple (6) FG molecules in different lipid bilayer models. Phospholipid fatty chains are not shown to facilitate visualization of the FGs and ergosterol molecules (A). S<sub>CD</sub> order parameters regarding the carbon position in the sn-1 palmitoyl chain of PLPA. The co-ordinates of the points correspond to the means measured for each carbon during the last 250 ns of simulation. Black curves (2 replicates) correspond to control bilayers without FGs; red curves (2 replicates) correspond to bilayers containing FGs (6 molecules) (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
A

Ternary models

<figure><img src="image_22.png" /><figcaption>**Fig. 6.** Static ²H NMR spectra (frequency, kHz) of various MLV liposomes in the absence (blue) and the presence (red) of RLs. Liposomes were deuterated at the level of palmitoyl chains (A). Static ³¹P NMR spectra (chemical shift, ppm) of various MLV liposomes in the absence (blue) and the presence (red) of RLs (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

B

in the ²H spectra.

On the other hand, the effect of RLs on quaternary models is much lower, even if the membranes are still damaged, as can be observed from the narrowing of the ²H spectra (Fig. 6A) and the change in ³¹P data (Fig. 6B), pointing to some phase transition still. It is clear from this that adding PCs to the models has a significant impact.

### 3.4.2. Interaction of FGs with biomimetic lipid membrane models of S. sclerotiorum and B. cinerea

Comparable experiments were performed using FGs. The addition of 2.4 mM of FGs significantly damaged the MLVs with a strong fluidification effect for all the studied models (Fig. 7A). To achieve whether FGs could modify membrane dynamics differently whether they are

A

Ternary models

<figure><img src="image_23.png" /><figcaption>**Fig. 7.** Static ²H NMR spectra (frequency, kHz) of various MLV liposomes in the absence (blue) and the presence (red) of FGs (A). Liposomes were deuterated at the level of palmitoyl chains. Static ³¹P NMR spectra (chemical shift, ppm) of various MLV liposomes in the absence (blue) and the presence (red) of Fengycin (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

B
targeting membrane models of *S. sclerotiorum* or *B. cinerea*, we tested FG effect at lower concentrations (0.5 and 1 mM). The effect is clearly larger against *B. cinerea* models (Fig. S4). The results of the ³¹P spectra are consistent with the ²H results, indicating a rise in the amount of hexagonal phase after the concentration of FGs increased (Fig. 7B, Fig. S4).

## 4. Discussion

RLs and FGs are antifungal compounds with an affinity for plasma membrane lipids [58,59]. We determined the lipid profiles of *B. cinerea* and *S. sclerotiorum* fungi, including the fatty acid and phospholipid compositions, in order to select contrasted lipid compositions for mimicking the lipid membranes of the two fungi by molecular dynamics simulations and solid-state NMR.

The main fatty acids in both fungi, grown in the same culture conditions, are composed of 18 saturated or unsaturated carbon chains and 16 unsaturated carbon chains. This confirms the results from previous studies ([28,27]). The C18:2 fatty acid is predominant in both fungi. After that, *B. cinerea* contains more C18:1, but *S. sclerotiorum* contains more C16:0. Haskins et al. [60] also described that the C18:1 amount is higher than C16:0 in *B. cinerea*. On the contrary, other studies found that the C16:0 amount is higher [27,61,20]. Taking all the data together, we can conclude that there are few differences in terms of main fatty acid content between both Sclerotiniaceae fungi. However, it appears that *S. sclerotiorum* contains a higher diversity of fatty acids from C14 to C24 chains. Our analysis also reveals the presence of odd-chain fatty acids (C15 and C17) in both fungi, as previously described in *B. cinerea* [60,20].

A high number of unsaturations in fatty acids is known to increase the fluidity of the membrane and could affect the activity of membraneotropic compounds [62]. On the other hand, it has also been reported that this parameter does not always influence the sensitivity of different fungi to FGs [20]. Sensitivities to RLs and FGs are unaffected by the average unsaturation number because the values for *S. sclerotiorum* and *B. cinerea* are comparable. Nevertheless, the ratio of unsaturated to saturated fatty acids is higher in *B. cinerea* than in *S. sclerotiorum*. It could be involved in a higher FG effect on *B. cinerea* but does not seem to be involved in the RL effect because there is no difference between the RL penetration between the four membrane models. The fatty acid chain length has also been described as correlated to the sensitivity of FGs. Fungi with shorter acyl chains, as in *B. cinerea* or *Pythium sulcatum*, would be more sensitive [20]. Given that there is no difference between *B. cinerea* and *S. sclerotiorum* fatty acid average chain length in our study, this parameter cannot explain on its own the difference in sensitivity.

The analyses and quantifications of the different phospholipid classes by HPTLC show that PE, PA and PC are predominant in both fungi and represent >98% of the phospholipids. A previous study in *B. cinerea* quantified the PG, PE, PC and PA as totalizing 85% of the phospholipids by thin layer chromatography (TLC) and colorimetric microphosphorus assays [20]. Another study quantified PC, PE, PA and PG as representing 96% of the phospholipids by TLC and lipid radiolabelling [27]. The differences between the studies can come from the fungal strains used, the growth culture conditions, the extraction process, or the method used for the quantification. It shows the importance of using the same conditions to compare fungal lipid compositions. Our experiments inhibiting the phospholipase D also show that the high PA content does not come from PC or PE degradation.

HPLC-QTOF analyses confirmed the predominance of unsaturated C18 and saturated C16 fatty acids in phospholipids determined in GC-FID experiments and allowed us to highlight the presence of LPA, LPE and LPC in both fungi. Nevertheless, these last compounds do not seem to be involved in RL or FG sensitivity because there are few significant differences between their contents in both fungi.

Molecular dynamic simulations on the lipid membrane models of *B. cinerea* and *S. sclerotiorum*, based on our data lipid analyses, show that all RLs placed outside enter and fluidify all the model bilayers in similar

ways. The RLs are located just below the plane drawn by lipid phosphate head groups, promoting membrane fluidification of the hydrophobic core as already described ([63,10,24]). This localization has been shown previously to be promoted by the formation of ionic bonds between the carboxylate of RLs and the amino group of the PE headgroups [24]. All models used in the present study contain >55% of PE. That could explain the lack of differences observed between the fluidification of the different lipid bilayer models with RLs. It is possible that the high ratio of PE conceals the already observed effect of sterols increasing the interaction of RLs with membrane lipids [64]. With this high ratio of PE, the higher PA content in *S. sclerotiorum* than in the *B. cinerea* model, resulting in a higher anionic/zwitterionic phospholipid ratio, does not seem to influence the RLs insertion, which are negatively charged in our pH conditions of simulations. RLs are described to penetrate PE-containing membranes much more often and easily than PC-one [24], which cannot be seen here since the quantity of PC compared to PE in the quaternary models is very low accordingly to lipidomic results.

Molecular dynamic experiments performed with a single FG molecule have shown that the peptidic cycle inserts and stands out the lipid membrane periodically in all the models without affecting fluidification. On the other hand, the acyl chain is usually able to penetrate and be placed toward the interior of the lipid bilayer, while the peptidic ring stays near the surface, interacting with the lipid headgroups as already described in molecular simulations with DPPC, POPC or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG systems [65].

The order parameter is not modified when a single FG is inserted in a lipid bilayer model of *S. sclerotinia* and *B. cinerea*, but it is lowered when a higher concentration of FGs is used in the simulation performed with the *B. cinerea* models, which contain more PE and less PA than the *S. sclerotiorum* models. It has previously been shown by MD that FGs interact preferentially with PE than PC, probably due to the amine group of PE that could act as a hydrogen bond donor, unlike the choline group in PC, making favorable interactions with the peptide anionic side chains [26]. On the contrary, the PA could have an electrostatic repulsive effect on FGs, which contain 2 negative charges due to the glutamate residues charged negatively and one ornithine charged positively, resulting in a global charge of -1. This could explain the observed lower affinity of FGs for *S. sclerotiorum* models. This effect is slightly higher in the *B. cinerea* quaternary model containing PC. As PE are zwitterionic phospholipids this could explain a higher effect of FGs on *B. cinerea*.

No insertion of FGs has been observed in our simulations with *S. sclerotiorum* lipid membrane models. They contain more PA and more ergosterol than the *B. cinerea* models. As previously mentioned, the anionic charge of the PA head probably has a repulsive effect on the FG insertion, while the steric hindrance can also play a role in this interaction. It has also been shown on lipid bilayers that a higher ergosterol content buffers the FG action by reducing its effects [21]. Biological analyses and experiments comparing the sensitivity to FGs of different fungi confirmed this hypothesis [12,20].

Aggregation is observed in all simulations with 6 FG molecules. It seems that, at the interface, there is a competing effect between the FG aggregation and their insertion into lipid bilayers. This is probably because the peptidic part tends to stay outside the membrane, increasing the chance for a single molecule to interact with other FGs. This aggregation process depends on the competition between two favoring interactions: the hydrophobic interactions between the tyrosine and isoleucine of FGs to form aggregates and the electrostatic interactions between FG-charged residues and the lipid headgroups of phospholipids [26]. This aggregation behavior is thought to be part of the mode of action of FGs [58], as observed in our *B. cinerea* models.

ss-NMR experiments with MLV liposomes allowed us to study membrane mimetic systems in more realistic conditions. A notable alteration was observed in membrane dynamics upon RLs interaction with *B. cinerea* and *S. sclerotiorum* models. Interestingly, differences became more significant without PC in ternary models. Indeed, a higher fluidification effect was observed than with quaternary models. Even at
low percentages, PC appears to have a "stabilizing" role, even though this role was not seen in MD experiments. The results for ³¹P and ²H ss-NMR experiments are consistent, indicating that PC prevents transition from the lamellar to hexagonal phase in ternary models. Remarkably, this apparent "stabilization" of the MLVs by PC was not observed with FG.

As MD simulations, ss-NMR data performed with lower FG concentrations confirm that FGs have a stronger effect on *B. cinerea* models compared to *S. sclerotiorum*, in accordance with biological experiments [12].

## 5. Conclusion

Our results give new insights into the phospholipid compositions of two phytopathogens against which RLs and FGs have a great protective potential due to their membranotropic modes of action. Interaction studies with lipid membrane models have shown differences in RLs and FGs effects in accordance with fungal sensitivities. Our experiments were performed on models based on phospholipid and ergosterol contents and it has been shown that highly rigid sphingolipid domains seem to play a role in the action of antifungal agents [66]. Taking into account sphingolipids in models in future would allow us to get as close as possible to the lipid compositions of the fungi to be even closer to biological reality.

## Funding

This research was supported by the Hauts-de-France (HdF) Council and was carried out in the context of the COALA project. It was also partly funded in the frame of the SHIELD Project ANR Ecophyto-Maturation (ANR-21-ECOM-0006), co-funded by the OFB (Office français de la biodiversité) via the fee for diffuse pollution under the French plan Ecophyto. Camille Botcazon PhD thesis was co-funded by the HdF Council and the French Ministry of Higher Education, Research and Innovation (MESRI). This study was supported by equipments co-founded by the Regional Council of Picardy and European Union within the CPER 2007-2020.

## CRediT authorship contribution statement

**Camille Botcazon:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization.
**Francisco Ramos-Martín:** Writing – review & editing, Visualization, Methodology, Formal analysis, Conceptualization. **Nely Rodríguez-Moraga:** Writing – review & editing, Visualization, Methodology, Investigation.
**Thomas Bergia:** Writing – review & editing, Methodology.
**Sébastien Acket:** Writing – review & editing, Visualization, Methodology.
**Catherine Sarazin:** Writing – review & editing, Validation, Supervision, Project administration, Data curation, Conceptualization.
**Sonia Rippa:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Funding acquisition, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

## Acknowledgments

We sincerely thank Philippe Nicot (Unité de Pathologie Végétale,

INRAe Montfavet, France) and Annette Penaud (Terres Inovia, Grignon, France) for kindly providing us the *B. cinerea* and *S. sclerotiorum* strains, respectively. We would like to thank Thomas Dayez for his assistance in sample preparation for the NMR experiments.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bpc.2024.107305.

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