# Understanding the mode of action of arsenic(III) on the fungus Neofusicoccum parvum: Target protein identification

Andréa Engel, Florence Ferrari, Maude Meyer, Jean-Marc Strub, Martin

Spichty, Christophe Bertsch, Christine Schaeffer-Reiss, Céline Tarnus,

Sébastien Albrecht, Mary-Lorène Goddard

► To cite this version:

Andréa Engel, Florence Ferrari, Maude Meyer, Jean-Marc Strub, Martin Spichty, et al.. Understanding the mode of action of arsenic(III) on the fungus Neofusicoccum parvum: Target protein identification. Analytical Biochemistry, 2025, 708, pp.115988. 10.1016/j.ab.2025.115988. hal-05313005

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# Understanding the mode of action of arsenic(III) on the fungus *Neofusicoccum parvum*: Target protein identification

Andréa Engel<sup>a,b,1,2</sup>, Florence Ferrari<sup>a,b,1,3</sup>, Maude Meyer<sup>a,b,1,4</sup>, Jean-Marc Strub<sup>c,d</sup>, Martin Spichty<sup>a</sup>, Christophe Bertsch<sup>b</sup>, Christine Schaeffer-Reiss<sup>c,d</sup>, Céline Tarnus<sup>a,5</sup>, Sébastien Albrecht<sup>a</sup>, Mary-Lorène Goddard<sup>a,b,*</sup>

## ARTICLE INFO

### Keywords:

Arsenic-based fluorescent probe

Arsenic-based affinity chromatography

Arsenic-binding proteins

Cysteine-rich proteins

## ABSTRACT

Before it was banned, sodium arsenite was the unique fungicide able to control fungi associated with grapevine trunk diseases. However, its mode of action has not been fully elucidated yet. This study focuses on the identification of arsenic(III)-binding proteins using an arsenic-based fluorescent probe and an arsenic-based affinity chromatography. To the best of our knowledge, this is the first comparative study of these techniques to demonstrate their complementarity. Mainly cysteine-rich proteins were identified, the majority of which are involved in the infection process, in particular in plant cell wall degradation, host-pathogen interaction, adhesion and pathogenicity. These proteins could therefore be relevant targets for the development of new ways of grapevine trunk disease control.

## 1. Introduction

Fungal diseases affecting grapevine wood, notably those caused by the Botryosphaeriaceae family, present a significant threat to global viticulture. Among these, *Neofusicoccum parvum* (*N. parvum*) is a key pathogen involved in grapevine trunk diseases (GTDs), causing severe wood decay and vine decline [1]. Historically, sodium arsenite was widely used to combat these cryptogamic diseases, demonstrating remarkable efficacy in controlling pathogens like *N. parvum* [2]. However, due to its high toxicity and environmental risks, sodium arsenite was banned in Europe in 2003 (https://eur-lex.europa.eu/eli/reg/2002/2076/oj).

Although this ban was essential for environmental and public health,

it unfortunately coincided with a resurgence of GTD symptoms, particularly those associated with *N. parvum*, in vineyards across France and worldwide (https://www.oiv.int/public/medias/188/grapevine-trunk-diseases-oiv-2015.pdf). Without an effective alternative treatment, winegrowers face significant economic losses and agricultural challenges. Despite research efforts, no solution has yet matched the effectiveness of sodium arsenite in managing these fungal pathogens [3–5].

Given these challenges, there is a renewed interest in understanding the mode of action of sodium arsenitetoxin develop new treatment strategies. While the mode of action of sodium arsenite on grapevines has not been clearly established, some studies report modifications in photosynthetic activity and the induction of plant defense responses [6,7]. More generally, As(III) is known to induce oxidative stress by generating

Abbreviations: (GTDs), Grapevine Trunk Diseases; (*N. parvum*), *Neofusicoccum parvum*; (V. vinifera), *Vitis vinifera*.

E-mail address: mary-lorene.goddard@uha.fr (M.-L. Goddard).

<footnote><sup>a</sup> Laboratoire d'Innovation Moléculaire et Applications, Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68093, Mulhouse cedex, France</footnote>

<footnote><sup>b</sup> Laboratoire Vigne, Biotechnologies et Environnement (LVBE, EA3991), Université de Haute-Alsace, 33, rue de Herrlisheim, 68000, Colmar, France</footnote>

<footnote><sup>c</sup> Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS, UMR7178, 25 Rue Becquerel, 67087, Strasbourg, France</footnote>

<footnote><sup>d</sup> Infrastructure Nationale de Protéomique ProFI - UAR2048, Strasbourg, France</footnote>

<footnote>* Corresponding author. Laboratoire d'Innovation Moléculaire et Applications, Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68093 Mulhouse cedex, France.</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² Present address: Eurofins Analyses pour l'Environnement France, 5, rue d'Otterswiller, 67700 Saverne, France.</footnote>

<footnote n="3">³ Present address: Collège Mont-Miroir, 1 rue de l'Europe, 25120 Maîche, France.</footnote>

<footnote n="4">⁴ Present address: Octapharma SAS, 72 rue du Maréchal Foch, 67380 Lingolsheim, France.</footnote>

<footnote n="5">⁵ Present address: Laboratoire Vigne, Biotechnologies et Environnement (LVBE, EA3991), Université de Haute-Alsace, 33, rue de Herrlisheim, 68000 Colmar, France.</footnote>
reactive oxygen species (ROS), leading to damage to lipids, DNA, and other cellular components. This oxidative stress can overwhelm the antioxidant defenses of fungal cells, contributing to cellular dysfunction and death [8]. Additionally, As(III) can disrupt mitochondrial function, particularly by inhibiting the electron transport chain, thereby impairing ATP production and inducing apoptosis [9,10]. Furthermore, As(III) can interfere with DNA repair mechanisms and disrupt the genomic stability of fungal cells. The arsenic-induced DNA damage may play a role in the cytotoxicity observed in pathogens like *N. parvum* [9]. Lastly, the antifungal activity of sodium arsenite is primarily attributed to the high affinity of arsenic in its trivalent form As(III) for the thiol groups of cysteine residues in proteins. This binding of As(III) to thiol groups leads to protein misfolding, inhibition of enzymatic activity, and disruption of various biological processes, resulting in cell dysfunction and death [11].

Our research has focused on identifying the specific protein targets of As(III) in *N. parvum*. By characterizing these interactions, we aim to uncover key insights into how sodium arsenite exerted its antifungal effects. This could potentially guide the development of non-toxic and effective alternatives to manage Botryosphaeriaceae-associated grape-vine trunk diseases. Several methods are employed for this purpose, each with its own advantages and disadvantages (For review, see Ref. [11,12]). Arsenic affinity chromatography is one common approach, in which arsenic is immobilized on a matrix to capture interacting proteins. This method reduces proteome complexity but may encounter the loss of low-affinity proteins, as well as steric hindrance and non-specific binding. Furthermore, it is imperative to safeguard protein thiols from the deleterious effects of oxidation. Another common method is the use of fluorescent arsenical probes, which provide a rapid response and can be used for *in vivo* visualization. The effectiveness of this technique is contingent on several factors, including the presence of light sources, pH level, and temperature conditions. Non-targeted chromatography is also a valuable tool for the separation of arsenic-bound from unbound proteins. Different types of column, such asgel filtration, anion exchange or size exclusion columns, can be used to achieve this. However, this technique is hindered by the potential instability of As-protein complexes and their co-elution, complicating identification. Finally, a more recent strategy involves biotin-streptavidin pull-down methods to overcome the issues of protein instability during chromatographic separation. It is important to note that this technique may also present problems related to the steric effects of the biotinylated moiety and non-specific binding.

Together, these techniques offer a comprehensive toolkit for unrav-
eling the proteins that interact with arsenic and understanding their
roles in cellular toxicity. To identify As(III)-binding proteins in the
extracellular proteome of *N. parvum*, we chose to develop and compare
two strategies, namely arsenic-based affinity chromatography and the
use of an arsenic-based fluorescent probe.

## 2. Materials and Methods

2.1. *Materials & reagents*

β-Mercaptoethanol (βME, 99 %), Bovine Serum Albumine (BSA, 66 kDa, 96 %), Dithiothreitol (DTT, 99 %), Epoxy Activated Sepharose™ 6B resin (Cytiva 17-0480-01), Ethanedithiol (99 %), Glutathione reduced (GSH, 98 %), Polyvinylpolypyrrolidone (PVPP) and Tris(2-carboxyethyl)phosphate (TCEP, 98 %) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Anhydrous Dimethylsulfoxide (DMSO, 99.8 %), (4-Aminophenyl)arsonic acid (p-Arsanilic acid or APAA, 98 %), 2,3-Dimercapto-1-propanol (BAL, 98 %), 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, 99 %) and Fluorescein isothiocyanate (FITC, 95 %) were purchased from Alfa Aesar and LC/MS grade Methanol (MeOH) was bought from Fisher Scientific (Thermo Fisher Scientific, Illkirch, France). Carbonic anhydrase (CA, 29 kDa, 95 %), Ovalbumin (OVA, 43 kDa, 94 %) and Conalbumin (CON, 75 kDa, 89 %) were bought

from Cytiva (Cytiva Europe, Saint-Germain-en-Laye, France).

¹H and ¹³C NMR spectra were recorded on a Bruker Avance I 400 spectrometer (400 MHz) (Bruker, Wissembourg, France) at 297 K. Chemical shifts were referenced to TMS. High-resolution electrospray ionization mass spectrometry (HRESI MS) was performed on an Agilent 3100 QToF using the ESI source in negative mode.

## 2.2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Protein samples (12 µL) were mixed with 10 µL of Laemmli buffer (Bio-Rad) containing 2.1 % SDS, 26.3 % (w/v) glycerol, 65.8 mM Tris-HCl, 0.01 % bromophenol blue, and 2 µL of βME as a reducing agent. The samples were heated at 95 °C for 5 min to ensure complete denaturation. Proteins were then separated on a SDS-PAGE gel (4–20 % Mini-PROTEAN® TGX Precast Gel (Bio-Rad, Marnes-la-Coquette, France)) at 200 V for approximately 90 min in a Tris-Glycine-SDS running buffer thanks to Mini-PROTEAN Tetra Cells with PowerPac Universal Power Supply (Bio-Rad. Molecular weight markers were run alongside the samples for size estimation (Precision Plus Protein Dual Color Standards, Bio-Rad).

For fluorescence detection, gels were scanned with the Gel-Doc XR System (Bio-Rad) for 10 s in *trans*-UV mode. Then, they were rinsed twice for 5 min in ultrapure water with orbital shaking, and stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) during 1 h and subsequently destained with ultrapure water before being scanned using the Molecular Imager GS-800 calibrated densitometer (Bio-Rad).

2.3. Fungal culture

Neofusicoccum parvum strain Bourgogne isolate S-116 was obtained from the collection of the IFV (Institut Français de la Vigne et du Vin, Rodilhan, France). It was isolated in 2009 from Chardonnay plants showing decline in nurseries. The genome of *N. parvum* strain UCR-NP2 has been sequenced (NCBI GenBank accession number AORE00000000) [13]. According to Labois [14], this strain was grown in Petri dishes on PDA (Potato Dextrose Agar) at 27 °C for 10 days in the absence of light. Then, liquid cultures were carried out by introducing the mycelium (half of a Petri dish) in a 500 mL Erlenmeyer flask containing 250 mL of liquid culture medium. Two different media were used: 20 g/L malt medium and 20 g/L malt medium supplemented with 2.5 g of *Vitis vinifera* (*V. vinifera*) cv. Gewurztraminer sawdust previously sterilized. Liquid cultures were grown at 220 rpm in the dark at 27 °C and after 21 days of incubation, the medium was successively filtered under vacuum on 0.8 µm glass fiber prefilter (Millipore, Merck, Darmstadt, Germany), 0.45, and 0.2 µm regenerated cellulose membrane filters (Whatman, Cytiva, Buckinghamshire, UK) to remove the mycelium.

2.4. *Extracellular protein isolation*

A treatment with Polyvinylpolypyrrolidone (PVPP) was carried out to complex polyphenols by adding 1 g per 100 mL of filtrate and stirring during 30 min et 4 °C. The medium was filtered under vacuum using a Whatman grade 3 qualitative paper filter (Cytiva, Buckinghamshire, UK). Treatment was repeated three times. The supernatant was filtered a final time on a 0.2 µm regenerated cellulose membrane filters (Whatman, Cytiva, Buckinghamshire, UK). Following this, proteins were precipitated by progressive addition of ammonium sulfate (60 % w/v, Carl ROTH) under strong stirring at 4 °C during 1 h. After centrifugation (11000 rpm, 4 °C, 30 min), the proteins were recovered in 20 mM Tris buffer, pH 7.5, and the entire mixture was dialyzed with a 3.5 kDa cutoff (regenerated cellulose dialysis membrane Spectra/Por, SpectrumLabs) overnight against Tris buffer to eliminate residual salts. The approximate protein concentration was then analyzed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 280 nm and expressed in mg/mL.
## 2.5. Arsenic-based affinity chromatography

### 2.5.1. Preparation

Firstly, the resin Epoxy Activated Sepharose™ 6B was washed with distilled water. Then, according to the procedure described by Carbajal et al. [15], 4 g of resin were placed in a round-bottom flask and 8 mL of a DMSO/water mixture (1:1) and 800 mg of APAA were added. The pH was set at 8 using a 67 % ammonia solution. Reaction was carried out with stirring at 40 °C overnight. After rinsing with distilled water, the reduction of As(V) to As(III) was performed by incubating the resin in 12 mL of 1 mol/L aqueous β-ME solution for 3 h at 60 °C. A portion of the resin was placed in a 20 cm × 20 mm column (28 mL bed volume) and preserved in the presence of a thiol compound to prevent hydrolysis and reoxidation.

The amount of As(III) immobilized on resin was determined thanks to Ellman's assay [16]. Firstly, a portion of the resin was placed in a column and washed with ultrapure water to remove β-ME excess and hydrolyzed As-S links. Then, the resin was incubated for 2 h under stirring at room temperature in excess BAL (2 mL, 20 mM MeOH solution). BAL reacted with As(III) and free BAL on supernatant was then reduced by DNTB and quantified by absorbance measurements of 2-nitro-5-thiobenzoate ion at 412 nm (ε = 14,150 mol⁻¹ cm⁻¹) thanks to a UV-Visible spectrophotometer (Agilent 8453 spectrophotometer, Agilent Technologies, Santa Clara, California, USA). The quantification was performed twice.

### 2.5.2. As(III) target protein identification

Arsenic-based affinity column was connected on ÅKTA Purifier system, equipped with a fraction collector and Unicorn software (Cytiva Europe, Saint-Germain-en-Laye, France). Three different buffers were prepared: Buffer A: 20 mM Tris pH 7.5 for regeneration, equilibration and wash steps, buffer B: 20 mM Tris 10 mM βME pH 7.5 and Buffer C: 20 mM Tris 10 mM DTT pH 7.5 as elution buffer.

Firstly, the resin was regenerated by running buffer A for 30 min at a flow rate of 5 mL/min. Then, protein mixture (1.0 mL) was deposited on the column and the assembly was incubated with TCEP (1.0 mL, 1 mM) (or 1 mL Tris 20 mM pH 7.5 in non-reductive conditions), during 30 min at room temperature.

To evaluate the specificity of arsenic-based affinity chromatography, an aqueous mixture of CA (500 µL, 1.29 mg/mL, 29 kDa) as negative control (no cysteine in its structure), and BSA (500 µL, 1.73 mg/mL, 66 kDa) as positive control (35 cysteine residues) was used.

After, the resin was successively eluted with 5 CV of Buffer A, and then successively with 5 CV of Buffer B and 5 CV of Buffer C. Separation was monitored by UV absorbance at 280 nm and conductimetry. Fractions of interest were concentrated at 4 °C for 30 min by ultrafiltration with a cut-off at 3 kDa thanks to Heraeus Multifuge 3SR centrifuge (Thermo Fisher Scientific France, Illkirch-Graffenstaden, France) and centrifugal filter devices (Amicon® Ultra-15 at 5000 g for volumes up to 15 mL or Amicon® Ultra-0.5 at 14,000 g for volumes up to 500 µL, Merck KGaA, Darmstadt, Allemagne). Each concentrated fraction was analyzed by SDS-PAGE, with or without the presence of fluorescent probe.

## 2.6. Arsenic-based fluorescent probe

### 2.6.1. Arsenic-based fluorescent probe synthesis

To a solution of p-arsanilic acid (APAA, 1.00 g, 3.9 mmol) in LC-MS grade MeOH (50 mL) was added Ethanedithiol (1.8 mL). After 3 h under stirring at room temperature, the mixture was concentrated under vacuum and recrystallized in absolute ethanol at 4 °C to lead in one step to 297 mg of 4-(1,3,2-Dithiarsolan-2-yl)benzamine (29 % yield). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 7.26 (q, J = 8.3 Hz, 2H), 6.55 (m, J = 8.6 Hz, 2H), 5.41 (s, 2H), 3.27–3.37 (m, 2H), 3.17–3.27 (m, 2H), in accordance to the literature [17].

In a round-bottom flask under an inert atmosphere, to 700 µL

solution of FITC (28.1 µmol) in anhydrous DMSO (25 µL/µmol), were added 700 µL of the crude 4-(1,3,2-Dithiarsolan-2-yl)benzamine (28.6 µmol) in anhydrous DMSO (25 µL/µmol). The reaction mixture was stirred at room temperature under an inert atmosphere for 2 h to obtain the Arsenic-based fluorescent probe or As probe (Fig. 1A). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 10.24 (s, 1H, NH), 10.17 (s, 1H, NH), 10.13 (s, 2H, OH), 8.19 (d, J = 2.0 Hz, 1H, H-12), 7.82 (dd, J = 8.3 and 2.0 Hz, 1H, H-10), 7.64 (dt, J = 8.6 and 1.8 Hz, 2H, H-17 or H-18), 7.53 (dt, J = 8.6, 1.8 Hz, 2H, H-17 or H-18), 7.21 (d, J = 8.1 Hz, 1H, H-9), 6.67 (d, J = 2.0 Hz, 2H, H-2), 6.60 (d, J = 8.8 Hz, 2H, H-5), 6.55 (m, 2H, H-4), 3.38 (m, 2H, H-20 or H-21), 3.20 (m, 2H, H-20 or H-21). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = 180.0 (C-15), 168.9 (C-14), 159.9 (C-3), 152.3 (C-1), 148.3 (C-8), 141.7 (C-16), 140.6 (C-19), 139.6 (C-11), 131.6 (C-17 or C-18), 131.0 (C-10), 129.5 (C-4), 127.0 (C-13), 124.5 (C-9), 123.6 (C-17 or C-18), 118.1 (C-12), 113.1 (C-5), 110.1 (C-6), 102.7 (C-2), 83.5 (C-7), 41.9 (C-20). HRESI MS (−) calculated for C₂₉H₂₀AsN₂O₅S₃ [M − H]⁻: 646.97403; found: 646.97558.

### 2.6.2. Control fluorescent probe synthesis

A negative control fluorescent probe was also synthesized using the same procedure as the arsenic-based fluorescent probe, replacing arsenic derivative with aniline. To 310 µL solution of FITC (12.4 µmol) in anhydrous DMSO (25 µL/µmol), were added 310 µL of distilled aniline (12.4 µmol) in anhydrous DMSO (25 µL/µmol). The reaction mixture was stirred at room temperature under an inert atmosphere for 2 h to obtain the control fluorescent probe (Fig. 1B). ¹H-MNR (400 MHz, DMSO-d₆): δ (ppm) = 10.16 (s, 1H, NH), 10.12 (s, 2H, OH), 10.08 (s, 1H, NH), 8.16 (d, J = 1.8 Hz, 1H, H-12), 7.80 (dd, J = 8.3 and 2.0Hz, 1H, H-10), 7.46 (d, J = 7.6 Hz, 2H, H-17), 7.35 (t, J = 7.6 Hz, 2H, H-18), 7.18 (d, J = 8.3 Hz, 1H, H-9), 7.14 (d, J = 7.3 Hz, 1H, H-19), 6.66 (d, J = 1.8 Hz, 2H, H-2), 6.58 (m, 4H, H-4 and H-5). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = 180.2 (C-15), 169.0 (C-14), 160.0 (C-3), 152.3 (C-1), 148.2 (C-8), 141.7 (C-16), 139.6 (C-11), 131.0 (C-10), 129.5 (C-4), 129.1 (C-18), 126.9 (C-13), 125.3 (C-19), 124.4 (C-9), 124.3 (C-17), 118.1 (C-12), 113.0 (C-5), 110.1 (C-6), 102.7 (C-2), 83.5 (C-3). HRESI MS (−) calculated for C₂₇H₁₇N₂O₅S₃ [M − H]⁻: 481.08704; found: 481.08637.

### 2.6.3. Fluorescent probe characterization

Solutions of each probe were prepared in 20 mM Tris, pH 7.5 at different concentrations (0.1, 0.5, 1, 2.5, 4, 5 and 8 µM) to determine the fluorescence linearity range of the two probes. A pH study was carried out on 1 µM As-probe and control-probe solutions prepared according to a range of different pH values from 3 to 11. The 50 mM buffer solutions at pH = 3, 4 and 5 were obtained from sodium acetate and hydrochloric acid, the pH = 6, 7 and 8 solutions from sodium dihydrogen phosphate and sodium hydroxide and the pH = 9, 10 and 11 solutions from sodium bicarbonate and sodium hydroxide.

For a constant final volume of 50 mL, either a 0.1 mM BAL or GSH solution was added at different volumes to obtain a 1 µM As-probe solution in 20 mM Tris pH 7.5 containing 1 mM TCEP with different molar ratios of sulfur reagents to As-probe (0.2, 0.5, 1, 2 and 2.5 ratios).

A Jasco FP-750 spectrofluorometer (Jasco France SAS, Lisse, France) equipped with Spectra manager software was used to measure the fluorescence and thus determine the conditions for using fluorescent probes. For all fluorescence measurements, the samples were placed in a quartz cuvette with a path length of 1 cm. The excitation wavelength was set to 480 nm based on the absorption spectrum of the fluorescent probe. Emission spectra were collected from 490 to 650 nm, with a slit width of 5 nm for both excitation and emission. All measurements were conducted at room temperature and expressed in relative fluorescence units (RFU).

### 2.6.4. As(III) target protein identification

20 µL protein solution were incubated with 20 µL of 1 mM TCEP solution (or 20 µL Tris 20 mM pH 7.5 in non-reductive conditions) under
<figure><img src="image_3.png" /><figcaption>Fig. 1. Chemical structures of As probe (A) and control probe (B).</figcaption></figure>

agitation at 500 rpm at room temperature for 1 h in the dark. 4 µL of 0.5 mM probe solution were added. The mixture was stirred again at room temperature for a further 30 min before being analyzed by SDS-PAGE.

To evaluate the specificity of arsenic-fluorescent probe, two protein solutions of BSA and CA at 1.5 mg/mL were chosen and to determine its sensitivity, three protein solutions of BSA (35 cysteines), CON (30 cysteines) and OVA (6 cysteines) were used at different concentrations (45.0, 22.5, 11.3, 4.5 and 2.25 nM for BSA and CON, 180.0, 90.0, 45.0, 22.5 and 11.3 nM for OVA).

## 2.7. MS-based proteomic analysis

### 2.7.1. Sample preparation

Excised gel bands were reduced with dithiothreitol (10 mM, 60 °C, 1 h) and alkylated with iodoacetamide (55 mM, RT, 20 min). After two dehydrations with acetonitrile (ACN), the proteins were cleaved with a modified porcine trypsin (Promega, Madison, WI, USA) solution at a 1:50 (w/w) enzyme: protein ratio. Digestion was performed overnight at 37 °C. Tryptic peptides were extracted using 60 % ACN in 0.1 % formic acid (FA). The excess of ACN was vacuum dried, and the samples were resolubilized in 0.1 % FA in water before nanoLC-MS/MS analysis.

### 2.7.2. NanoLC-MS/MS analysis

Proteolytic digests were analyzed on two different nanoLC-MS/MS systems, namely a nanoACQUITY Ultra-Performance-LC (Waters Corporation, Milford, USA) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and a nanoLC system (nanoACQUITY Ultra-Performance-LC) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (TripleTOF 5600, ABSciex).

The same chromatographic conditions were used on both systems. Peptides were trapped on a Symmetry C18 pre-column (C18, 20 × 0.18 mm, 5 µm particle size, Waters), and separation was performed on an ACQUITY UPLC® BEH130C18 column (250 mm × 75 µm with 1.7 µm diameter particles, Waters Corp.). The solvent system consisted of 0.1 % formic acid in water (solvent A) and 0.1 % formic acid in acetonitrile (solvent B). Trapping was performed during 3 min at 5 µL/min with 97 % of solvent A and 3 % of solvent B. Elution was performed at a flow rate of 300 nL/min, using 3–40 % gradient (solvent B) over 79 min followed by 80 % (solvent B) over 10 min at 60 °C.

The Q-Exactive mass spectrometer was operating in positive mode, with the following settings: spray voltage 1800 V and capillary temperature 250 °C. The system was operated in Data Dependent Acquisition mode with automatic switching between MS (mass range 300–1800 m/z with R = 140,000, automatic gain control (AGC) fixed at 3 × 106 ions and a maximum injection time set at 50 ms) and MS/MS (mass range 200–2000 m/z with R = 17,500, AGC fixed at 1 × 105 and the maximal injection time set to 100 ms) modes. The ten most abundant

peptides were selected on each MS spectrum for further isolation and higher energy collision dissociation fragmentation, excluding unassigned and monocharged ions. The dynamic exclusion time was set to 60 s. The normalized collision energy (NCE) was fixed at 27 V. The complete system was fully controlled by Thermo Scientific™ Xcalibur™ software.

The TripleTOF 5600 mass spectrometer was operating in positive mode, with the following settings: ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 25 psi, interface heater temperature (IHT) 75 °C, ion source gas 1 (GS1) 2 psi, declustering potential (DP) 100 V. Information-dependent acquisition (IDA) mode was used with top 5 MS/MS scans. The MS scan had an accumulation time of 250 msec on m/z [300–1800] range and the MS/MS scans 100 ms m/z [150–2000] range in high sensitivity mode. Switching criteria were set to ions with charge state of 2–4 and an abundance threshold of more than 150 counts, exclusion time was set at 12 s. IDA rolling collision energy script was used for automatically adapting the CE. Mass calibration of the analyser was achieved using peptides from digested BSA. The complete system was fully controlled by AnalystTF 1.6 (AB Sciex).

### 2.7.3. Protein identification

Mass data collected during nanoLC-MS/MS analyses were processed, converted into mgf peak list format with MSConvert, and interpreted using Mascot algorithm (v. 2.6.2, Matrix Science, London, UK) running on a local server. Searches were performed with an in-house generated protein database composed of protein sequences of *N. parvum* and *V. vinifera* and known contaminant proteins such as human keratins and trypsin, downloaded from UniprotKB (December 12, 2024) concatenated with reversed copies of all sequences (total 351928 entries). The following parameters were used: (i) Trypsin/P was selected as enzyme, (ii) one missed cleavage was allowed, (iii) methionine oxidation was set as variable modifications and the carbamidomethylation of cysteine was set as a fixed modification, (iv) mass tolerance for precursor ions was set at 10 ppm and at 20 ppm for fragment ions. Mascot results were loaded into the Proline software (Proline Studio Release 2.0, http://proline.pro脂proteomics.fr [18]). For validating PSM (Peptide Spectrum Matches), the following parameters were applied: a rank of 1, a minimal score of 25, and a minimal length of 7 amino acids. In addition, a maximum false discovery rate of 1 % was applied to the protein set.

*N. parvum* proteins listed in UniprotKB were initially annotated using BLAST (Basic Local Alignment Search Tool) similarity searches against the complete GenBank nr database (BLASTp) [13]. Terms like “probable”, “putative”, and “uncharacterized” are used to describe proteins based on the level of confidence in their functional annotation. In all cases, there is no experimental confirmation. A probable protein indicates that there is a stronger indirect evidence suggesting its function, such as well-conserved domains, expression patterns, or strong homology to experimentally characterized proteins in closely related
organisms. A putative protein is a protein whose function is predicted based on sequence similarity to known proteins, but lacks direct experimental evidence in the organism in question. “Uncharacterized” means that specific functional annotation of the protein is unavailable.

A complementary BLAST analysis was performed on the uncharacterized protein sequences to identify potential homologs and infer its functional and structural properties. The analysis was conducted against the UniProtKB reference proteomes and Swiss-Prot databases (http s://www.uniprot.org).

The prediction of sub-cellular localization was carried out using the DeepLoc - 2.1 software (Ødum et al., 2024). Upset plots were drawn using the UpsetR package [19] (v. 1.4.0) in RStudio 2024.12.0 [20] and R 4.4.2 [21] softwares.

## 3. Results

### 3.1. Arsenic-based affinity chromatography

To prepare the arsenic-based affinity chromatography, we reacted APAO with an epoxy-activated Sepharose™ 6B resin, followed by the reduction of As(V) to As(III) using βME as the reducing agent, as described in Fig. 2. The Ellman's assay was used to determine the quantity of As(III) grafted onto the resin at 40.9 ± 2.1 µmol/g of resin.

The specificity of the As(III) affinity chromatography towards proteins possessing thiol groups was tested with a mixture of two proteins, BSA as a positive control (BSA has 35 cysteine residues), and CA as a negative control, which possesses no cysteine residues. This was conducted under both reducing (presence of TCEP) and non-reducing (absence of TCEP) conditions. Under non-reducing conditions, BSA eluted throughout the separation (with buffer A and B, data not shown). However, after incubation with TCEP, carbonic anhydrase eluted completely with buffer A, and BSA eluted with buffer B and more effectively with buffer C (Fig. 3). It was noted that a small proportion of BSA was also eluted by buffer A.

### 3.2. Arsenic-based fluorescent probe

The arsenic and control fluorescent probes were synthesized from FITC and 4-(1,3,2-Dithiarsolan-2-yl)benzamine or benzylamine, respectively, as described in Fig. 4.

The working concentration of the arsenic and control fluorescent probes was first determined by measuring the fluorescence intensity of probe solutions ranging from 0.1 to 5 µM. Good linearity was obtained for the arsenic and control probes up to a concentration of 1 and 2.5 µM, respectively (Supplementary Fig. S1). Therefore, the working concentration was set at 1 µM. A study of fluorescence as a function of pH was then carried out over a range extending from 3 to 11. At pH 7.5, the physiological pH at which the extracellular protein mixtures are conserved, the fluorescence was almost maximal for both probes (Supplementary Fig. S2). Finally, the impact of the interaction of arsenic with a sulfur compound on fluorescence intensity was also evaluated. It is noticeable that, regardless of the ratio of sulfur compound to probe or the type of sulfur compound type (GSH, a monosulfur compound, and BAL, a disulfur compound), the fluorescence intensity was slightly lowered but stable ($I_f$ = 81 ± 2 RFU vs. 123 ± 1 without sulfur derivative) (data not shown).

<figure><img src="image_4.png" /><figcaption>Fig. 3. SDS-PAGE (4–20 % gel) analysis of CA and BSA mixture step elution from arsenic-based affinity chromatography. Lane 1 marker, Lanes 2 and 10 CA and BSA controls, lanes 3, 4, 5 tampon A elution fraction, lanes 6 and 7 tampon B elution fraction, lanes 8 and 9 tampon C elution fraction. As commercially available proteins were not pure, the band corresponding to the correct molecular weight was highlighted by a black box.</figcaption></figure>

As with arsenical-based affinity chromatography, the specificity of the arsenic-based fluorescent probe was demonstrated thanks to BSA vs. CA proteins. As shown in Fig. 5, no fluorescence was detected in the CA protein band, indicating that the arsenic probe does not bind to this cysteine-free protein via an As–S bond or any other type of interaction. The band corresponding to the multi-cysteine BSA protein fluoresced in the presence of the arsenic probe, but not in the presence of the control probe, indicating a specific interaction of the protein with arsenic.

To test the sensitivity of the arsenic probe, the same amount of the probe was incubated with solutions of three proteins at different concentrations. Ovalbumin OVA was used because it has fewer cysteines (6) than bovine serum albumin BSA and conalbumin CON (35 and 30, respectively). As can be seen in Fig. 6, the higher the protein concentration, the more intense the fluorescence. Moreover, at the same protein concentration (11.3 nM, for instance), the fluorescence depends on the number of cysteines contained in the protein. In addition, the difference in fluorescence intensity between BSA and CON might be explained by the difference in conformational accessibility between the two proteins (CON displays more cysteines on the surface of the protein (Supplementary Fig. S3)).

### 3.3. As(III) target protein identification in N. parvum

Both arsenic-based fluorescent probe and arsenic-based affinity chromatography were used on *N. parvum* secretome, grown in the presence or absence of *V. vinifera* cv. Gewurztraminer sawdust.

To detect arsenic-binding proteins using fluorescence, the mixture of *N. parvum* extracellular proteins from different cultures was incubated in the presence of the arsenic and control probes. Fig. 7 shows images of

<figure><img src="image_5.png" /><figcaption>Fig. 2. Preparation of As(III) dimercaptoethanol-immobilized Sepharose.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 4. Synthesis of fluorescein-based arsenic and control probes.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. SDS-PAGE (4–20 % gel) analysis of CA (band at 29 kDa) and BSA (band at 66 kDa), alone or in mixture, after incubation with As or control probe. Lane 10 marker, lanes 1 to 3 CA protein, lanes 4 to 6 CA and BSA mixture, lanes 7 to 9 BSA protein. Proteins incubated with arsenic probe (As) and control probe (C) were respectively analyzed in lanes 2, 5, 8 and lanes 3, 6, 9. Proteins not incubated with any probe (s) were analyzed in lanes 1, 4, 7. A: Coomassie blue revelation, B: Fluorescence revelation, C: superimposition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

SDS-PAGE of the complete *N. parvum* secretome, revealed by Coomassie blue staining and fluorescence detection. First of all, it is noteworthy to note the significant differences in protein content between cultures supplemented and not supplemented with *V. vinifera* sawdust, particularly in terms of the relative quantities of several proteins. Indeed, a protein of around 67 kDa showed intrinsic fluorescence (green boxes), while proteins at 85 kDa for the malt culture and 24 kDa for the malt + sawdust culture showed non-specific fluorescence (fluorescence was detected with both the arsenic probe and the control probe, blue boxes). Only a 14 kDa protein produced in both culture media fluoresced specifically with As(III) (red boxes). The same results were obtained with the secretome incubated without TCEP, showing that the latter had no impact on the affinity of the arsenic probe with proteins (Supplementary Fig. S4). In order to facilitate the visualization of proteins demonstrating an affinity for the arsenic probe, an additional analysis on SDS-PAGE was carried out with a high excess of probe (Fig. 8). This enabled the identification of a 22 kDa band that exhibited autofluorescence in all

lanes. More importantly, a new fluorescent band at 38 kDa in lanes 2 and 3 only was observed, indicating the presence of a protein specifically linked to As(III).

Affinity chromatography was performed after incubating the extracellular protein mixtures on the resin, with or without TCEP. Elution was monitored by UV absorbance, and the different fractions were pooled according to the elution buffer. SDS-PAGE was achieved to verify the results obtained (Fig. 9). As can be seen in the SDS-PAGE image, there were no more eluted proteins in the last wash fraction using 20 mM Tris buffer (lane 4, Fig. 9). In the elution fractions (lanes 5 to 9), whether with β-ME or DTT, a single band appeared at 39 kDa. This protein seemed to exhibit strong resin binding since DTT must be used to elute it perfectly (no more bands were revealed in lane 10). Another band at 14 kDa was weakly revealed by Coomassie blue in lane 5 (10 mM β-ME).

The different fractions were pooled and concentrated, then incubated or not with the arsenic probe. The resulting gel was revealed using Coomassie blue and fluorescence. As demonstrated in Fig. 10, the 14
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** SDS-PAGE (4–20 % gel) of proteins incubated with arsenic-based fluorescent probe. The concentrations of each protein solution are given in nM. A: BSA (35 cysteines), B: CON (30 cysteines), C: OVA (6 cysteines).</figcaption></figure>

kDa band was barely discernible in lanes 7 and 8 using Coomassie blue staining, and was not observable in lanes 9 and 10. However, upon addition of the arsenic probe, the band became clearly discernible under fluorescence (lanes 8 and 10). In contrast, the 38 kDa band was only revealed by Coomassie Blue, suggesting a difference in sensitivity between the two techniques. The same results were found without TCEP incubation (Supplementary Figs. S5 and S6).

Arsenic-based affinity chromatography was also carried out with the secretome of *N. parvum* grown in a malt culture supplemented with sawdust from *V. vinifera* cv. Gewurztraminer. Whereas Coomassie blue detection revealed a band at 38 kDa for proteins extracted from a culture in malt medium alone, no protein was detected in this sample. Indeed, a comparison of the bands observed in Fig. 7 between the secretome from a malt culture (lanes 2 to 7) and that from a malt + sawdust culture (lanes 8 to 10) suggested that there was little or no protein at 38 kDa in the malt + sawdust culture. However, after incubation with the arsenic

probe, a 14 kDa band was revealed under fluorescence in the β-ME and DTT elution fractions (Supplementary Fig. S7) but not in the Tris wash fraction, thereby indicating the presence of a protein that specifically binds to As(III).

### 3.4. MS-based proteomic analysis

To identify arsenic-binding proteins detected by affinity chromatography and/or the arsenic probe, gel bands around 14 kDa and 38 kDa were excised under UV light or after revelation with Coomassie blue (Table 1). Proteins were in-gel digested, peptides extracted and analyzed by nanoLC-MS/MS. All the identified proteins for each gel band analyzed are listed in Supplemental file.

Concerning the 14 kDa bands, a total of 342 different proteins were identified, and 217 of which had a probability of being extracellular of 0.8 (the prediction of sub-cellular localization was performed with the DeepLoc - 2.1 software as specified in Materials and Methods part). Among these proteins, 162 proteins were revealed in the course of experiments with the fluorescent probe (IDs E5, E6, E2, H1). More precisely, the upset plot in Fig. 11 A showed that 11 proteins were found in all four bands analyzed, with an additional 11 detected in three out of four bands. However, 51 proteins (31 % of the total analyzed proteins) were exclusively found in H1 band, which relates to the sawdust-supplemented culture. Examination of the proteins identified following arsenic-based affinity chromatography (bands P2 and Q2 for malt culture, bands U2 and V2 for malt supplemented with sawdust) revealed a total of 124 unique protein sequences by LC-MS/MS analysis. A focus subsequently shifted to the proteins found in both replicates P2 and Q2 as well as U2 and V2, highlighting 29 proteins (Table 2). More precisely, as shown in Table 2 and Fig. 11 B, 8 proteins were identified in all 4 bands (in red), 9 in the two U2 and V2 replicates but also in band P2 (in green), 3 in the two P2 and Q2 replicates but also in U2 or V2 bands (in blue). Finally, 9 proteins were specifically identified in the U2 and V2 bands from a culture supplemented with sawdust (orange). Moreover, only 6 out of 29 proteins were not detected in the fluorescent probe experiments (indicated by § or # symbols in Table 2). Furthermore, the prediction of sub-cellular localization confirmed that 27 proteins were extracellular with at least 85 % probability.

Three bands at around 38 kDa were also analyzed: the E3 band, which was obtained from a gel run on the complete mixture of extracellular proteins and incubated with the arsenic probe, and the J1 and J2 bands, which were excised after arsenic-based affinity chromatography.

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** SDS-PAGE (4–20 % gel) analysis of complete *N. parvum* secretome after incubation with TCEP and with or without incubation with the arsenic probe or the control probe. Lane 1 marker, lanes 2 to 4 secretome from malt culture A, lanes 5 to 7 secretome from malt culture B, lanes 8 to 10 secretome from malt + sawdust culture. Proteins incubated with arsenic probe and control probe were respectively analyzed in lanes 3, 6, 9 and lanes 4, 7, 10. Proteins not incubated with any probe were analyzed in lanes 2, 5, 8. A: Coomassie blue revelation, B: Fluorescence revelation. Boxes in red highlight a specific interaction with arsenic probe, boxes in blue indicate a non-specific interaction and boxes in green concern proteins with intrinsic fluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** SDS-PAGE (4–20 % gel) analysis of complete N. parvum secretome with fluorescence revelation. Lane 1 secretome alone, lane 2 secretome incubated with arsenic probe, lane 3 secretome incubated with arsenic probe and TCEP, lane 4 secretome incubated with control probe. Fluorescence revelation. Boxes in red highlight a specific interaction with arsenic probe, boxes in blue indicate a non-specific interaction and boxes in green concern proteins with intrinsic fluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** SDS-PAGE (4–20 % gel) analysis of N. parvum secretome after arsenic-based affinity chromatography with TCEP incubation. Lane 1 marker, lane 2 diluted complete secretome, lanes 3 to 4 Tris wash fractions, lanes 5 and 6 β-ME 10 mM elution fractions, lanes 7 and 8 β-ME 20 mM elution fractions, lanes 9 and 10 DTT elution fractions. Coomassie revelation.</figcaption></figure>

A total of 95 unique proteins were annotated (Fig. 11C). 22 of these 95 proteins were identified in both bands excised after arsenic-based affinity chromatography, and are listed in Table 3. Fig. 11C also showed that only 4 proteins were identified in the J2 band (experiment without TCEP), whereas 18 proteins were specifically found in the J1 band (experiment with TCEP as a reducing agent). Among the 22 proteins identified in both J1 and J2 bands, 10 were not detected in the E3 band (fluorescent probe experiment on complete extracellular protein extract). Moreover, 18 proteins have an extracellular localization with at least 87 % probability.

As can be seen in both lists in Tables 2 and 3, half of the proteins in both datasets were hydrolases (Fig. 12A and B), the 14 kDa bands were found to contain no enzymes other than hydrolases, but a quarter of the proteins were signaling proteins, which were also strongly represented in the 38 kDa bands. The other present proteins were structural and transport proteins. (Fig. 12 A). However, the 38 kDa bands contained other enzymes, such as oxidoreductases and transferases. There were no transport proteins, and only one structural protein was present (Fig. 12 B).

# 4. Discussion

## 4.1. Complementary tools for the detection of arsenic-binding proteins

The initial objective of the study was to enhance the preparation of the tools used, namely the fluorescent probe and the affinity chromatography). Thus, in order to synthesize the fluorescent probe with optimal purity, it was necessary to modify the protocol described in the literature. Indeed, Femia et al. (Femia et al., 2012) initially coupled APAA to FITC and then reduced As(V) to As(III) using β-ME. The first step proved to be incomplete, resulting in a product that could not be purified. Therefore, we initially synthesized 4-(1,3,2-Dithiarsolan-2-yl) benzamine through the reaction of APAA with ethanedithiol, as opposed to the conventional method involving β-ME, yield a more stable As(III) derivative. The subsequent step involved the coupling of this latter arsenical derivative with FITC. However, to process efficiently, it was necessary to prepare solutions of FITC and 4-(1,3,2-Dithiarsolan-2-yl) benzamine in DMSO prior to bringing the two solutions into contact.

Affinity chromatography with As(III) form was first described by Ref. [22]. It was prepared by covalently linking p-aminophenylarsine oxide (APAO) to CNBr-activated Sepharose 6B. The type of resin was later modified by various research groups: Sepharose 4B-CL [23], Glycidyl methacrylate [15] or Eupergit C [24]. In all cases, the resin was coupled to APAO, which was obtained by the reduction of APAA. However, this reaction performed in the presence of Triphenylphosphine/Iodine, Ascorbic acid/Iodine [25] or Phenylhydrazine [26] was not quantitative, and the arsine formed was not very stable and was readily re-oxidized. Consequently, adopting a method similar to that employed by Ref. [27], we prepared an As(III)-Sepharose using As(V)-Sepharose, which was synthesized from phenylarsonic acid (PAA) and NHS-activated Sepharose gel. We opted to initially couple an Epoxy Activated Sepharose™ 6B type resin with APAO, followed by a reduction of As(V) to As(III) in a second step using β-ME. By reducing the As(V) after grafting, a more efficient synthetic route was achieved. In addition, the resin was kept in an excess of thiol compound until use in order to preserve the reduced As(III) form.

The two techniques, arsenic-based fluorescent probe and arsenic-based affinity chromatography, were used to identify arsenic(III) affinity proteins in the extracellular protein extracts from the fungus *N. parvum*. To our knowledge, this was the first time that two different tools have been evaluated for the same study. Both methods revealed the same protein bands at 14 and 38 kDa. However, they did reveal differences in selectivity and sensitivity.

This study demonstrated a high level of selectivity in the affinity chromatography technique. Indeed, PAO affinity chromatography has been extensively proven for the identification of arsenic(III)-binding
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 10.** SDS-PAGE (4–20 % gel) analysis of affinity chromatography elution fractions (TCEP addition). Lane 1 marker, lanes 2 and 3 diluted complete secretome, lanes 4 and 5 Tris wash fractions, lanes 7 and 8 β-ME 10 mM elution fractions, lanes 9 and 10 DTT elution fractions. The mixtures in lanes 3, 5, 8 and 10 were first incubated with the arsenic probe. A: Coomassie blue revelation, B: Fluorescence revelation). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<caption>**Table 1**</caption>

List of gel bands excised and analyzed by nanoLC-MS/MS. * This gel is equivalent to the gel at Fig. 10 but has been specially prepared to excise the bands under fluorescence, the 14 kDa band not being revealed by Coomassie blue.

<table><thead><tr><th>ID</th><th>SDS-PAGE</th><th>Lane</th><th>Molecular weight (kDa)</th><th>Culture medium</th><th>TCEP</th><th>Affinity chromatography</th></tr></thead><tbody><tr><td>E2</td><td>Fig. 7</td><td>7</td><td>14</td><td>malt</td><td>yes</td><td>no</td></tr><tr><td>E5/E6</td><td>Fig. 8</td><td>2/3</td><td>14</td><td>malt</td><td>no/yes</td><td>no</td></tr><tr><td>P2/Q2</td><td>Figure S7*</td><td>8/10</td><td>14</td><td>malt</td><td>no</td><td>yes</td></tr><tr><td>H1</td><td>Fig. 7</td><td>10</td><td>14</td><td>malt + sawdust</td><td>yes</td><td>no</td></tr><tr><td>U2/V2</td><td>Fig. S8</td><td>8/9</td><td>14</td><td>malt + sawdust</td><td>no</td><td>yes</td></tr><tr><td>E3</td><td>Fig. 7</td><td>7</td><td>38</td><td>malt</td><td>yes</td><td>no</td></tr><tr><td>J1</td><td>Fig. 9</td><td>5</td><td>38</td><td>malt</td><td>yes</td><td>yes</td></tr><tr><td>J2</td><td>Fig. S5</td><td>5</td><td>38</td><td>malt</td><td>no</td><td>yes</td></tr></tbody></table>

proteins, in particular for proteins with vicinal thiols [28–30] but also with isolated cysteines, albeit with lower affinity [24]. This was also shown in studies involving a PAO linked to a biotin [31,32]. This selectivity could be improved through the use of a TCEP-based incubation protocol, as suggested by Ref. [33], prior to the implementation of the arsenic-based affinity chromatography. TCEP is a non-thiol reducing agent capable of reducing disulfide bridges, such as those formed by vicinal thiols. Indeed, we were able to obtain better binding of BSA to the As(III)-grafted resin by using TCEP. Furthermore, it was observed that of the 18 proteins found specifically in the J1 band (experiment with TCEP), 5 had vicinal thiols (data not shown). However, with regard to the selectivity of the arsenical-based probe, it appeared that the use of an arsenic-free control probe was imperative to limit false positives. As demonstrated in Fig. 8, bands at 24 and 85 kDa exhibited a fluorescence response with the control probe. Attention must also be paid to the intrinsic fluorescence of proteins, as evidenced by the bands observed at 22 and 67 kDa. It should also be noted that selectivity may be more severely decreased when the arsenical-based probe was used to analyze highly complex mixtures. This issue of non-specific binding and increased fluorescent background was addressed in the context of FlAsH probes, which were engineered to bind specifically to tetracycline motifs (CCXXCC) [34,35].

Regarding the sensitivity of the As(III) probe, this depended on the protein concentration, the number of cysteines and their accessibility. Fig. 6 illustrates this through the fluorescence evaluation of three proteins at varying concentrations and cysteine numbers. Furthermore, the band at 37 kDa was very weakly revealed by the As(III) probe (Fig. 8), while the proteins demonstrated strong binding to the resin (Fig. 9). In contrast, the 14 kDa band was strongly revealed through the use of the fluorescent probe, facilitating its detection following resin elution

(Fig. 10).

## 4.2. Identification of signaling proteins involved in N. parvum pathogenicity

A large number of studies have been carried out to identify arsenic-binding proteins using arsenic-biotin conjugates, arsenic-based probes or arsenic-based chromatography. These investigations have resulted in the identification of cysteine-rich proteins (CRPs) (for review, see Ref. [11,36,37]), but very few focused on fungi. Fungal proteins reported in the literature to interact with trivalent arsenic are mainly proteins involved in arsenic resistance. This includes ArsC, the ArsB and Acr transporters, the metal-binding protein TaGlo1 and metallothioneins, and even the stress-activated protein kinase Hog1 in yeast [11,38,39]. These proteins are either intracellular or membrane-bound, and among them, metallothioneins are considered to be CRPs.

The present study has focused on the proteins secreted by N. parvum, hypothesizing that they may be involved in plant interactions, such as recognition, adhesion, or penetration, all of which are necessary steps in the colonization and propagation of the fungus. The change in three-dimensional conformation resulting from the binding of arsenic to these proteins can lead to inhibition of enzymatic activity or disruption of signaling pathways [11,36,40].

The more selective arsenic-based chromatography allowed us to highlight potential arsenic-binding proteins, which are listed in Tables 2 and 3. After proteomic analysis, most of the proteins could be identified from the N. parvum genome sequence [13]. As shown in Fig. 12, most of the identified proteins are hydrolases, including several plant cell wall-degrading enzymes (PCWDEs), which are essential for fungi to overcome the barrier of the plant cell wall [41,42]. These include
<table><caption>**Table 2**

List of 29 proteins identified in 14 kDa bands (P2 and Q2 bands and U2 and V2 bands).</caption><thead><tr><th>Uniprot entry</th><th>Protein name</th><th>Protein type</th><th>Gene name</th><th>MW</th><th># cysteine</th><th>Cysteine motif</th><th># specific peptides</th><th>P2</th><th>Q2</th><th>U2</th><th>V2</th><th>Extracellular probability</th></tr></thead><tbody><tr><td>R1E8I6</td><td>Putative surface protein 1 protein</td><td>Signaling protein</td><td>UCRNP2_9221</td><td>15362</td><td>4</td><td>NA</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1G1Q3</td><td>Putative epl1 protein</td><td>Signaling protein</td><td>UCRNP2_7919</td><td>14190</td><td>4</td><td>C..C</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1EP88</td><td>Putative glycoside hydrolase family 55 protein</td><td>Hydrolase</td><td>UCRNP2_3629</td><td>84093</td><td>10</td><td>NA</td><td>20</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.88</td></tr><tr><td>R1EXU8</td><td>Putative gpi-anchored cell wall beta-endoglucanase protein</td><td>Hydrolase</td><td>UCRNP2_626</td><td>60373</td><td>7</td><td>NA</td><td>10</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1GM42</td><td>Putative aspartic endopeptidase pep1 protein</td><td>Hydrolase</td><td>UCRNP2_6229</td><td>41788</td><td>3</td><td>NA</td><td>7</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.92</td></tr><tr><td>R1H0S9</td><td>beta-glucosidase</td><td>Hydrolase</td><td>UCRNP2_1147</td><td>74639</td><td>7</td><td>NA</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.84</td></tr><tr><td>R1G5R2</td><td>Putative exo-beta-glucanase protein</td><td>Hydrolase</td><td>UCRNP2_9781</td><td>31462</td><td>9</td><td>C..C, CC</td><td>9</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.95</td></tr><tr><td>R1FV17<sup>#</sup></td><td>Cell wall protein</td><td>Structural protein</td><td>UCRNP2_10381</td><td>19147</td><td>5</td><td>NA</td><td>2</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.99</td></tr><tr><td>R1EPB0</td><td>Putative cell wall protein</td><td>Structural protein</td><td>UCRNP2_3869</td><td>18527</td><td>4</td><td>NA</td><td>4</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1E5V7</td><td>Putative lactonohydrolase protein</td><td>Hydrolase</td><td>UCRNP2_10238</td><td>47907</td><td>2</td><td>NA</td><td>6</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.85</td></tr><tr><td>R1GGZ6</td><td>Putative acid phosphatase protein</td><td>Hydrolase</td><td>UCRNP2_8019</td><td>40494</td><td>0</td><td>NA</td><td>7</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.91</td></tr><tr><td>R1G8H7</td><td>Putative cell surface spherulin 4-like protein</td><td>Signaling protein</td><td>UCRNP2_8856</td><td>27337</td><td>2</td><td>NA</td><td>2</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.93</td></tr><tr><td>R1GDJ0</td><td>chitinase</td><td>Hydrolase</td><td>UCRNP2_6952</td><td>37646</td><td>7</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1H3B2</td><td>beta-glucosidase</td><td>Hydrolase</td><td>UCRNP2_282</td><td>100513</td><td>9</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.33<sup>#</sup></td></tr><tr><td>R1E6Y2</td><td>Carboxylic ester hydrolase</td><td>Hydrolase</td><td>UCRNP2_10119</td><td>59873</td><td>4</td><td>NA</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.91</td></tr><tr><td>R1G4Q6</td><td>Putative-binding protein</td><td>Signaling protein</td><td>UCRNP2_10189</td><td>24218</td><td>6</td><td>NA</td><td>1</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.96</td></tr><tr><td>R1FZJ6<sup>#</sup></td><td>Uncharacterized protein<sup>*</sup></td><td>Signaling protein<sup>*</sup></td><td>UCRNP2_8740</td><td>19798</td><td>2</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1GE12</td><td>Probable Endo-1,3-beta-glucanase egIc</td><td>Hydrolase</td><td>UCRNP2_3376</td><td>31910</td><td>4</td><td>NA</td><td>6</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.96</td></tr><tr><td>R1H157<sup>#</sup></td><td>Cell wall protein</td><td>Structural protein</td><td>UCRNP2_994</td><td>22009</td><td>2</td><td>NA</td><td>1</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.96</td></tr><tr><td>R1EHU0<sup>#</sup></td><td>Vacuolar protein sorting-associated protein</td><td>Transport protein</td><td>UCRNP2_6183</td><td>357885</td><td>12</td><td>CC</td><td>1</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.12<sup>#</sup></td></tr><tr><td>R1GPA2</td><td>alpha-amylase</td><td>Hydrolase</td><td>UCRNP2_5425</td><td>56053</td><td>9</td><td>NA</td><td>14</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1FZC0</td><td>Putative npp1 domain protein</td><td>Signaling protein</td><td>UCRNP2_8830</td><td>26507</td><td>2</td><td>NA</td><td>4</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1EJE5</td><td>Putative alkaline phosphatase protein</td><td>Hydrolase</td><td>UCRNP2_5592</td><td>33879</td><td>0</td><td>NA</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1GFU6</td><td>Putative endonuclease exonuclease phosphatase family protein</td><td>Hydrolase</td><td>UCRNP2_6114</td><td>46629</td><td>6</td><td>NA</td><td>8</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.92</td></tr><tr><td>R1GHX8</td><td>alkaline phosphatase</td><td>Hydrolase</td><td>UCRNP2_7729</td><td>73974</td><td>5</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.93</td></tr><tr><td>R1GKI5</td><td>Phosphatidylglycerol/ phosphatidylinositol transfer protein</td><td>Transport protein</td><td>UCRNP2_4429</td><td>18449</td><td>4</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.89</td></tr><tr><td>R1GN84</td><td>Pectate lyase</td><td>Hydrolase</td><td>UCRNP2_3453</td><td>25245</td><td>8</td><td>C..C</td><td>3</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1GVH9<sup>#</sup></td><td>Uncharacterized protein<sup>*</sup></td><td>Structural protein<sup>*</sup></td><td>UCRNP2_845</td><td>20961</td><td>7</td><td>NA</td><td>1</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.95</td></tr><tr><td>R1E6W1<sup>#</sup></td><td>Putative major allergen alt</td><td>Signaling protein</td><td>UCRNP2_10157</td><td>15359</td><td>4</td><td>NA</td><td>1</td><td>×</td><td>×</td><td>×</td><td>×</td><td>0.98</td></tr></tbody></table>

MW: molecular weight, # cysteine: number of cysteins present in protein sequence, cysteine motif: indicates the presence of vicinal thiols, # specific peptides: indicates the number of unique peptide sequences used to identify a protein. “Probable” means that the functional annotation was based on strong sequence homology or conserved features, “putative” indicates that the function is predicted based on sequence similarity to known proteins, and “uncharacterized” means that specific functional annotation of the protein is unavailable. $^*$ proteins not observed in bands E5, E6 or E2. # proteins not observed in the band H1. $^*$ The subcellular prediction proposes a lysosome/vacuole localization at 0.80 and 0.64 for R1H3B2 and R1EHU0 Uniprot entries respectively. $^*$ The BLAST analysis revealed 40 % similarity between R1GVH9 and cell wall protein PhiA (Uniprot entry A0A5N5DMH5) of Lasiodiplodia theobromae, 44 % similarity between R1GVH9 and Gamma-glutamyltransferase (Uniprot entry A0A1J9S3U6) of Diplodia corticola, 36 % similarity between R1GVH9 and cell wall protein (Uniprot entry A0A8H4IP00) of Botryosphaeria dothidea and 87 % similarity between R1FZJ6 and small secreted protein (Uniprot entry A0A8H4J6Q3) of Botryosphaeria dothidea. Lasiodiplodia theobromae and Botryosphaeria dothidea are of family of Botryosphaeriaceae, like N. parvum. Cell wall proteins are structural proteins and small secreted proteins are signaling proteins.

β-glucosidases (R1H0S9, R1H3B2) and other cellulases (R1G8D0, R1EXU8, R1GPA2, R1GE12) involved in cellulose and hemicellulose degradation, some hydrolases responsible for pectin degradation (R1EP88, R1GN84), and feruloyl esterase R1EFK4, which hydrolyzes feruloyl-polysaccharide. The function of the putative exo-β-glucanase protein (R1G5R2) remains unknown. In the work of [43], R1GN84 and

R1G5R2 proteins are differentially secreted by *N. parvum* in the presence of the *Eucalyptus* stem. In our study, pectate lyase was found exclusively in experiments resulting from the culture of *N. parvum* in the presence of *V. vinifera* sawdust. In addition, both proteins possess vicinal thiols of the CxxC (Table 2).

Other highly represented protein classes, include structural proteins
<figure><img src="image_14.png" /></figure>

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

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

(caption on next page)
<caption>**Fig. 11.** Upset plots illustrating the intersection of proteins sets identified in (A) experiments with the fluorescent probe (E5, E6, E2 and H1 bands) (A) or (B) after As-based affinity chromatography (P2, Q2, U2 and V2 bands) of the 14 kDa bands, and (C) after the fluorescent probe (E3 band) and As-based affinity chromatography (J1 and J2 bands) of the 38 kDa bands. The horizontal bars on the left indicate the total number of proteins in each dataset, while the vertical bars represent the number of proteins shared among different combinations of datasets. The colored bars highlight proteins common to certain datasets. The colors within the intersection bars denote protein annotations: red for annotated proteins, orange for putative proteins, and purple for uncharacterized proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</caption>

<table><caption>**Table 3**
List of 22 proteins identified in 38 kDa J1 and J2 bands.</caption><thead><tr><th>Uniprot entry</th><th>Protein name</th><th>Protein type</th><th>Gene name</th><th>MW</th><th># cysteine</th><th>Cysteine motif</th><th># specific peptides</th><th>J1</th><th>J2</th><th>Extracellular probability</th></tr></thead><tbody><tr><td>R1GQT1</td><td>Putative fad binding domain-containing protein</td><td>Oxidoreductase</td><td>UCRNP2_4898</td><td>38470</td><td>0</td><td>NA</td><td>6</td><td>×</td><td>×</td><td>0.90</td></tr><tr><td>R1GE12</td><td>Probable glucan endo-1,3-beta-glucosidase egIC</td><td>Hydrolase</td><td>UCRNP2_3376</td><td>31910</td><td>4</td><td>NA</td><td>4</td><td>×</td><td>×</td><td>0.96</td></tr><tr><td>R1G8H7</td><td>Putative cell surface spherulin 4-like protein</td><td>Signaling protein</td><td>UCRNP2_8856</td><td>27337</td><td>2</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>0.93</td></tr><tr><td>R1G8D0</td><td>Cellulase</td><td>Hydrolase</td><td>UCRNP2_8897</td><td>25616</td><td>5</td><td>NA</td><td>7</td><td>×</td><td>×</td><td>0.19<sup>‡</sup></td></tr><tr><td>R1G1Q3</td><td>Putative epl1 protein</td><td>Signaling protein</td><td>UCRNP2_7919</td><td>14190</td><td>4</td><td>C...C</td><td>4</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1EXU8</td><td>Putative gpi-anchored cell wall beta-endoglucanase protein</td><td>Hydrolase</td><td>UCRNP2_626</td><td>60373</td><td>7</td><td>NA</td><td>7</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1ESA8</td><td>Amine oxidase</td><td>Oxidoreductase</td><td>UCRNP2_2536</td><td>51681</td><td>0</td><td>NA</td><td>12</td><td>×</td><td>×</td><td>0.95</td></tr><tr><td>R1EJE5</td><td>Putative alkaline phosphatase protein</td><td>Hydrolase</td><td>UCRNP2_5592</td><td>33879</td><td>0</td><td>NA</td><td>2</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1EFK4</td><td>feruloyl esterase</td><td>Hydrolase</td><td>UCRNP2_6745</td><td>32419</td><td>7</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>0.94</td></tr><tr><td>R1E8I6</td><td>Putative surface protein 1 protein</td><td>Signaling protein</td><td>UCRNP2_9221</td><td>15362</td><td>4</td><td>NA</td><td>4</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1E6Y2</td><td>Carboxylic ester hydrolase</td><td>Hydrolase</td><td>UCRNP2_10119</td><td>59873</td><td>4</td><td>NA</td><td>7</td><td>×</td><td>×</td><td>0.91</td></tr><tr><td>R1E5V7</td><td>Putative lactonohydrolase protein</td><td>Hydrolase</td><td>UCRNP2_10238</td><td>47907</td><td>2</td><td>NA</td><td>6</td><td>×</td><td>×</td><td>0.85</td></tr><tr><td>R1GMD5<sup>§</sup></td><td>Transaldolase</td><td>Transferase</td><td>UCRNP2_3761</td><td>35620</td><td>2</td><td>NA</td><td>10</td><td>×</td><td>×</td><td>0.04<sup>‡</sup></td></tr><tr><td>R1GJT0<sup>§</sup></td><td>Peroxidase</td><td>Oxidoreductase</td><td>UCRNP2_7003</td><td>32232</td><td>6</td><td>NA</td><td>4</td><td>×</td><td>×</td><td>0.98</td></tr><tr><td>R1GHB2<sup>§</sup></td><td>Carnosine N-methyltransferase</td><td>Transferase</td><td>UCRNP2_2217</td><td>65346</td><td>4</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>0.18<sup>‡</sup></td></tr><tr><td>R1GGZ6<sup>§</sup></td><td>Putative acid phosphatase protein</td><td>Hydrolase</td><td>UCRNP2_8019</td><td>40494</td><td>0</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>0.91</td></tr><tr><td>R1GAU0<sup>§</sup></td><td>1,3-beta-glucanosyltransferase</td><td>Transferase</td><td>UCRNP2_4537</td><td>48367</td><td>6</td><td>NA</td><td>9</td><td>×</td><td>×</td><td>0.92</td></tr><tr><td>R1G7D5<sup>§</sup></td><td>Putative gpi anchored cell wall protein</td><td>Structural protein</td><td>UCRNP2_5844</td><td>21239</td><td>7</td><td>C...C</td><td>1</td><td>×</td><td>×</td><td>0.96</td></tr><tr><td>R1G5R2<sup>§</sup></td><td>Putative exo-beta-glucanase protein</td><td>Hydrolase</td><td>UCRNP2_9781</td><td>31462</td><td>9</td><td>C...C, CC</td><td>4</td><td>×</td><td>×</td><td>0.95</td></tr><tr><td>R1FZJ6<sup>§</sup></td><td>Uncharacterized protein*</td><td>Signaling protein*</td><td>UCRNP2_8740</td><td>19798</td><td>2</td><td>NA</td><td>3</td><td>×</td><td>×</td><td>0.97</td></tr><tr><td>R1EBD7<sup>§</sup></td><td>Putative lipase b protein</td><td>Hydrolase</td><td>UCRNP2_8492</td><td>38070</td><td>4</td><td>NA</td><td>2</td><td>×</td><td>×</td><td>0.87</td></tr><tr><td>R1E8Z9<sup>§</sup></td><td>3-phytase</td><td>Hydrolase</td><td>UCRNP2_9401</td><td>123316</td><td>16</td><td>C...C</td><td>6</td><td>×</td><td>×</td><td>0.26<sup>‡</sup></td></tr></tbody></table>

MW: molecular weight, # cysteine: number of cysteins present in protein sequence, cysteine motif: indicates the presence of vicinal thiols, # specific peptides: indicates the number of unique peptide sequences used to identify a protein. “Probable” means that the functional annotation was based on strong sequence homology or conserved features, “putative” indicates that the function is predicted based on sequence similarity to known proteins, and “uncharacterized” means that specific functional annotation of the protein is unavailable. § proteins not observed in bands E3. ‡ The subcellular prediction proposes cytoplasm localization at 0.66, 0.88 and 0.71 for R1G8D0, R1GMD5, R1GHB2 Uniprot entries respectively and cell membrane or lysosome/vacuole localization at 0.59 and 0.59 respectively for R1E8Z9 Uniprot entry. *The BLAST analysis revealed 87 % similarity between R1FZJ6 and small secreted protein (Uniprot entry A0A8H4J6Q3, signaling protein) of Botryosphaeria dothidea.

and proteins involved in cell signaling. The identified structural proteins (R1FV17, R1EPB0, R1H157, R1G7D5, and R1GVH9) are all cell wall proteins (CWPs). All of them, with the exception of R1H157, might be considered as secreted cysteine-rich proteins (CRPs). These CRPs are typically characterized by: (i) their relatively small size (a sequence length usually less than 200 amino acids), (ii) the presence of a secretion peptide signal, and (iii) and a large number of cysteine residues (4–16 cysteine residues usually in a C-terminal domain) (Supplementary Table S1) [44]. These effectors are essential for modulating host immune responses and promoting fungal infection [45–47]. A BLAST analysis of R1FV17, R1EPB0, and R1GVH9 showed that they might be related to the cell wall protein PhiA from other Botryosphaeriaceae, such as Lasiodiplodia theobromae (R1FV17 55.6 % similarity with Uniprot entry A0A5N5CY59; R1EPB0 78 % with A0A5N5CYX6; R1GVH9 40.1 % with A0A5N5DMH5), Macrophomina phaseolina (R1FV17 50.8 % similarity with Uniprot entry K2SBI4; R1EPB0 67.2 % with K2RQS3) or Botryosphaeria dothidea (R1EPB0 71.3 % with A0A8H4J3I2). The BLAST analysis of R1G7D5 indicated a high degree of similarity with the ubiquitin 3 binding protein But2 protein from several

Botryosphaeriaceae fungi (82.6 % with A0A8H4J1V3 Uniprot entry of Botryosphaeria dothidea, 76.2 % with A0A8H4J1V3 of Macrophomina phaseolina, 70.1 % with A0A5N5D999 of Lasiodiplodia theobromae, 68.7 % with A0A1S8BM88 of Diplodia seriata). The former PhiA is a cell wall protein involved in development of asexual structures such as phialide and conidium development and is thus required for spore formation [48]. It plays a role as a general stress protectant produced by the fungus in competition with antagonistic bacteria or in the presence of antifungals in Aspergillus spp. [49,50]. The latter protein, R1G7D5, binds to Uba3 and is involved in the NEDD8 signaling pathway, which is involved in the neddylation process (a biochemical process involving the covalent attachment of a small ubiquitin-like molecule, NEDD8, to designated target proteins) [51,52].

Among the proteins classified as signaling proteins, the uncharacterized protein R1FZJ6 (87 % similarity to a small secreted protein from Botryosphaeria dothidea) and the binding protein R1G4Q6 have no defined functions. The surface protein cell surface spherulin 4-like protein (R1G8H7) and surface protein 1 protein (R1E8I6) are probably involved in adhesion or recognition and are therefore potential
<figure><img src="image_17.png" /><figcaption>**Fig. 12.** Pie chart representing the distribution of protein types for (A) the proteins listed in Table 2 and (B) the proteins listed in Table 3. The chart shows the different categories of and the numbers indicate the total count of each type, with percentages reflecting their proportion within the dataset.</figcaption></figure>

virulence factors. The major allergen alt protein R1E6W1, observed only in experiments with grapevine sawdust, shows strong similarity (67–84 %) and identity (68–83 %) with effector protein PevD1 of *Lasioplodia theobromae* or *Verticillium dahliae*, or AA1-like domain containing protein of *Botryosphaeria dothidea* or *Verticillium alfalfae*. The effector protein PevD1 of *Verticillium dahlia* is known to elicits a hypersensitive response in cotton and tobacco plants [53–55]. The npp1 domain protein R1FZC0 was also identified only in experiments with grapevine sawdust. Proteins with the NPP1 (necrosis-inducing Phytophthora protein 1) domain induce hypersensitive cell death [56]. discovered, through structure-activity relationship studies that two cysteine residues conserved in all sequences studied are essential for elicitor activity. The BLAST analysis of epl1 protein R1G1Q3, presenting a vicinal thiol motif, showed a very high similarity and identity with protein Snod-Prot1 and cerato-platanins. Cerato-platanin proteins are small secreted proteins with four conserved cysteines and are known to induce plant defense responses [57,58]. Furthermore, these cysteine-rich proteins R1G1Q3 (C-X38-C-X2-C-X54-C) and R1E6W1 (C-X13-C-X40-C-X11-C) (Supplementary Table S1) exhibit a highly similar cysteine pattern to SnodProt1 (Uniprot entry O74238, C-X38-C-X2-C-X54-C) and PevD1 (Uniprot entry G0Y276, C-X13-C-X40-C-X9-C), respectively. As in both proteins, these four conserved cysteines are reported to form two disulfide bonds, it might also be the case for both R1G1Q3 and R1E6W1. Disulfide bonds represent a pivotal structural element that stabilizes the three-dimensional structure of mature proteins in diverse extracellular environments and protects these effectors against degradation by apoplastic plant proteases [59,60]. Consequently, this assists pathogenic fungi in surviving harsh environments and promoting infections in hosts [61,62]. On the other hand, binding to arsenic(III), in a reducing environment, prevents the formation of disulfide bridges, destabilizing the conformational structure of these effectors and their function during host infection. Studies in molecular biology, including transient over-expression assays in *Nicotiana benthamiana* and *Vitis vinifera*, as well as expression monitoring during *N. parvum* infection of grapevine wood, are currently underway to validate the biological role of candidate proteins such as R1G1Q3 and R1E6W1. Due to time constraints, the results of these experiments will be reported in a subsequent manuscript.

**Conclusion. Conclusion**

Our studies demonstrated that, although the two techniques converged to identify the same proteins, they exhibited different sensitivity thresholds. Although fluorescent probes are straightforward to use and allow rapid detection, affinity chromatography offers a more

selective method for detecting arsenic-binding proteins. Combining these two approaches could be advantageous: affinity chromatography could be used first for selective isolation, followed by detection using the fluorescent probe to complement traditional methods such as Coomassie blue staining.

To further refine the identification process, in-silico methods such as molecular dynamics and covalent molecular docking could help to predict and refine the binding modes of arsenic compounds to target proteins [63]. To experimentally confirm these predictions, biophysical techniques such as thermal shift assays or isothermal titration calorimetry (ITC) could be used to validate direct binding and provide quantitative data on binding affinity and specificity.

Several proteins from the *N. parvum* secretome have been identified, particularly those that act as virulence factors at different stages of the infection process including adhesion, cell wall degradation, and pathogenesis. Interfering with the function or secretion of these proteins could hinder the pathogen's ability to manipulate the host plant's defense mechanisms, preventing or reducing the severity of GTDs. Such interference could destabilize the pathogen's ability to establish a successful infection, offering a promising avenue for developing plant protection strategies. However, it is important to note that the strong affinity of arsenic for thiol groups underlies its specific mode of action, which may not be fully replicated by safer compounds. Future studies should therefore investigate whether alternative molecules or inhibitors can target the same pathways or proteins in a more environmentally sustainable way.

Further molecular biology studies are planned to validate the biological role of these key arsenic(III)-binding proteins. In addition to transient overexpression assays in *Nicotiana benthamiana* and *Vitis vinifera*, and expression monitoring during grapevine wood infection by *N. parvum*, future approaches may include RNA interference (RNAi), clustered regularly interspaced short palindromic repeats (CRISPR), or overexpression directly in *N. parvum* to modulate protein expression and assess their impact on fungal growth or virulence. Since the in vitro secretome approach using sawdust does not fully reproduce the physiological conditions of living grapevine tissue, future experiments will also involve inoculating woody cuttings with *N. parvum*, cultivated with or without arsenic(III), to confirm the expression and functional relevance of these candidate proteins under conditions close to the natural environment. Together, these complementary approaches will strengthen the identification of relevant targets and support the development of innovative and safer strategies to control GTDs.
### CRediT authorship contribution statement

**Andréa Engel:** Investigation. **Florence Ferrari:** Investigation. **Maude Meyer:** Investigation. **Jean-Marc Strub:** Writing – review & editing, Investigation, Formal analysis. **Martin Spichty:** Visualization, Software. **Christophe Bertsch:** Writing – review & editing, Funding acquisition. **Christine Schaeffer-Reiss:** Writing – review & editing, Formal analysis, Data curation. **Céline Tarnus:** Resources, Funding acquisition, Conceptualization. **Sébastien Albrecht:** Writing – review & editing, Funding acquisition, Conceptualization. **Mary-Lorène Goddard:** Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation.

### Ethics declaration

The authors confirm that all the research meets the ethical guidelines, including adherence to the legal requirements of the study country. They confirm that approval by a local ethics committee was not required.

### Funding

This work was supported by "Université de Haute-Alsace" (EcoVino project) and "Fondation Partenariale Haute-Alsace".

### 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.

### Acknowledgments

The authors acknowledge funding from the French proteomics infrastructure (ProFI FR2048, ANR-10-INBS-08-03). We are very grateful to Jacques Lalevée (IS2M, UHA, France) for giving us access to the Jasco spectrofluorometer. We would also like to thank Dr. Mélanie Gellon and Dr. Sibylle Farine for their help and advices for fungal culture and protein isolation.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ab.2025.115988.

### Data availability

Data will be made available on request.

### References

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