# Characterization of Neofusicoccum parvum small secreted proteins belonging to Alt a1 and cerato-platanin families affecting defenses in Nicotiana benthamiana and Vitis vinifera

Alexia Laura Grau, Romain J G Pierron, Charlotte Doerr, Hélène Laloue, Mary-Lorène Goddard, Julie Chong

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Alexia Laura Grau, Romain J G Pierron, Charlotte Doerr, Hélène Laloue, Mary-Lorène Goddard, et al.. Characterization of Neofusicoccum parvum small secreted proteins belonging to Alt a1 and cerato-platanin families affecting defenses in Nicotiana benthamiana and Vitis vinifera. Physiological and Molecular Plant Pathology, 2025, 138, pp.102659. 10.1016/j.pmpp.2025.102659. hal-04996465

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# Characterization of *Neofusicoccum parvum* small secreted proteins belonging to Alt a1 and cerato-platanin families affecting defenses in *Nicotiana benthamiana* and *Vitis vinifera*

Alexia Laura Grau<sup>a</sup>, Romain J.G. Pierron<sup>a</sup>, Charlotte Doerr<sup>a</sup>, Hélène Laloue<sup>a</sup>, Mary-Lorène Goddard<sup>a,b</sup>, Julie Chong<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

* Grapevine trunk diseases
* Botryosphaeriaceae
* Cerato-platanin
* Major allergen Alt a1
* Defense responses

## ABSTRACT

Botryosphaeria dieback, which is associated with xylem-inhabiting *Botryosphaeriaceae* fungi, is one of the major Grapevine Trunk Diseases (GTDs) responsible for significant reduction of longevity and productivity of grapevine. Since the ban of sodium arsenite, no efficient treatment is available to fight against GTDs. *Neofusicoccum parvum* is considered as one of the most aggressive *Botryosphaeriaceae* causing wood decay. However, little is known about the molecular players of host-pathogen interaction. In this study, we characterize three small secreted proteins (NpCP1, NpAA1.1, and NpAA1.2) from *N. parvum*, previously isolated by their affinity for arsenite. Sequence and structure analysis revealed that these secreted proteins belong to cerato-platanin and major allergen Alt a1 families, and have strong structure similarity with known effectors from other pathogenic fungi. NpCP1, NpAA1.1, and NpAA1.2 expression was early induced after infection of grapevine wood with *N. parvum*, suggesting a role in the colonization process. Further investigation of the role of these secreted proteins was realized after transient overexpression in *Nicotiana benthamiana* and *Vitis vinifera*. Whereas their expression did not trigger extensive cell death in both systems, our work shows that these small secreted proteins could differentially manipulate the expression of defense genes after agroinfiltration, and could thus be considered as effectors. More specifically, downregulation of enzymes involved in ROS production and ethylene biosynthesis suggests a role during wood colonization.

## 1. Introduction

Grapevine trunk diseases (GTDs), caused by a complex of xylem-inhabiting fungi, are responsible for considerable economic losses for viticulture worldwide. GTDs threaten the future of vineyards since no efficient and environmentally friendly treatment is available to manage them [1]. Among these diseases, Botryosphaeria dieback (BD) is one of the most damaging and significantly reduces longevity and productivity of grapevine [2,3]. BD, which is associated with *Botryosphaeriaceae* fungi, is characterized by several symptoms, i. e. wood necrosis and cankers, foliar discolorations, as well as bud necrosis and fruit rots, finally leading to the death of the plant [4]. It is assumed that trunk disease fungi penetrate the plant through pruning wounds to colonize the host tissue [5]. However, *Botryosphaeria* spp. have also been isolated

from nurseries' grapevine plants, and could cause young grapevine decline observed in different vineyards [6,7]. In grapevine, BD has been associated with 26 *Botryosphaeriaceae* species in the genera *Botryosphaeria*, *Diplodia*, *Dothiorella*, *Lasiodiplodia*, *Neofusicoccum*, *Sphaeropsis*, and *Spencermartinsia* [3]. More generally, fungi from the *Botryosphaeriaceae* family are associated with wood decay in a large number of woody plants, especially fruit trees [8]. One of the most aggressive is *Neofusicoccum parvum*, which can rapidly colonize grapevine wood by provoking necrosis [4,9]. *N. parvum* has also been described as a vascular aggressive pathogen leading to dieback symptoms in a wide range of woody host plants [8]. As other *Botryosphaeriaceae* fungi, *N. parvum* is present as an endophyte with a quiescent passive life phase in the host tissue and switches to an active pathogenic phase [10]. The molecular mechanisms behind this switch in the lifestyle are still elusive.

This article is part of a special issue entitled: Botryosphaeria dieback published in Physiological and Molecular Plant Pathology.

* Corresponding author.

E-mail address: julie.chong@uha.fr (J. Chong).

<footnote><sup>a</sup> Laboratoire Vigne, Biotechnologies et Environnement, UR3991, Université de Haute Alsace, LVBE, 33 Rue de Herrlisheim, F-68000, Colmar, France</footnote>

<footnote><sup>b</sup> Laboratoire d'Innovation Moléculaire et Applications, Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, 68093, Mulhouse cedex, France</footnote>
However, Khattab et al. (2023) reported that it could result from
modified chemical signaling between the host and the pathogen favored
by abiotic stress [11].

In the past, sodium arsenite was an efficient treatment to control
GTDs but was banned in the 2000s due to its toxicity to humans and the
environment [12]. Sodium arsenite is known to have a direct fungicide
effect on GTD fungi. Recently, Bruez et al. (2021) showed that sodium
arsenite treatment has a major effect on wood fungal communities [13],
especially regarding *Fomitiporia mediterranea*, a GTD fungus responsible
for white rot [14]. In addition, Songy et al. (2019) reported that sodium
arsenite could stimulate some grapevine defense responses [15].

Despite the virulence of *N. parvum* on a wide host range, little is
known about the molecular players of pathogen recognition and host-
pathogen interaction. Concerning fungal determinants of pathoge-
nicity, Massonnet et al. (2018) determined the genome-wide expression
dynamics of putative virulence factors in *N. parvum*, and showed that
gene clusters involved in secondary metabolism and cell wall degrada-
tion were co-regulated during plant infection [16]. Indeed, several
studies reported that *N. parvum* can synthesize several phytotoxins
belonging to naphtalenone polyketides, dihydrotoluquinone, epox-
ylactone, dihydroisocoumarin, and hydroxybenzoic acid chemical fam-
ilies [17,18]. In addition to phytotoxins, it is known that
pathogen-secreted proteins, deployed at the host-pathogen interface,
are key players in plant-pathogen interaction [19,20]. *Botryosphaer-
iaceae* fungi have been shown to produce a high number of predicted
secreted proteins [21], but few studies reported their activity. Esteves
et al. (2014) highlighted that *Botryosphaeriaceae* fungi, including
*N. parvum*, produce several secreted extracellular enzymes, particularly
those involved in cell wall and lignin degradation [22]. In addition,
Stempien et al. (2017) reported that secreted proteins from *N. parvum*
and *Diplodia seriata* have polysaccharide and lignin-degrading enzy-
matic activities [23]. Interestingly, these secreted proteins are also able
to metabolize grapevine stilbenes and could be important aggressiveness
factors [23,24].

Among pathogen-secreted proteins, small secreted proteins (SSPs)
from fungi are proteins of less than 300 amino acids, containing a signal
peptide that permits secretion. In fungal vascular wilt diseases, SSPs
have a dual role in both pathogen virulence and plant immunity [25].
Few studies have been conducted on these SSPs in the *Botryosphaeriaceae*
family [19]. However, recent studies reported several genes encoding
necrosis and ethylene-inducing proteins (NLPs) in *D. seriata* and
*N. parvum* [26,27]. Expression of these proteins led to cytotoxicity and
cell death in grapevine, tomato, and mammalian cells, showing that they
are likely involved in *Botryosphaeriaceae* virulence [26,27]. Fungal small
cysteine-rich proteins (SCPs) have also been identified in the
*N. parvum-Liquidambar styraciflua* interaction and could contribute to
pathogenicity [28].

In an attempt to decipher the sodium arsenite mode of action on
*N. parvum* metabolism, we previously identified several SSPs charac-
terized by their affinity to As(III) that could play a role in grapevine-
pathogen interaction [29]. In this study, we characterized in detail the
structure and expression during infection of three secreted SCPs from
*N. parvum*. The effect of these SCPs on cell death and defense responses
was further characterized after transient expression in *Nicotiana ben-
thamiana* and *Vitis vinifera*.

**2. Material and methods**

2.1. Candidate effector identification based on their affinity with As(III)

*N. parvum* isolate Bourgogne small secreted proteins with As affinity
were isolated as described in Engel et al. (unpublished results) [29]. In
brief, secreted proteins were isolated from *N. parvum* liquid culture
medium as described in Bénard-Gellon et al. (2015) [30]. Secreted
proteins with affinity for As were subsequently isolated either by affinity
chromatography on Sepharose beads grafted with an

aminophenylarsenite motif or by using an aminophenylarsenite motif
coupled to a fluorescent probe. Secreted proteins with affinity for As(III)
were analyzed by SDS-PAGE and gel bands were digested with trypsin
before nano LC-MS/MS analysis and protein identification as described
in Labois et al. (2021) [31]. In this work, we focused on 3 candidate
effectors because of sequence similarity with known effectors in other
plant pathogenic fungi: *Neofusicoccum parvum* Cerato-Platanin 1
(NpCP1, GenBank: EOD45360.1) and two *Neofusicoccum parvum* pro-
teins containing Alt a1-like domain (NpAA1.1, GenBank: EOD43269.1
and NpAA1.2, GenBank: EOD43152.1).

## 2.2. Bioinformatic analysis of the amino acids' sequences

Proteins' sequences of NpCP1, NpAA1.1, and NpAA1.2 were ob-
tained from NCBI GenBank Database, signal peptide was predicted using
SignalP 4.1 [32] (https://services.healthtech.dtu.dk/services/Sig-
nalP-4.1/), and prediction of secretion localization was performed
using EffectorP-fungi 3.0 [33] (https://effectorp.csiro.au/). A functional
analysis of the sequences was performed using InterPro (https://www.
ebi.ac.uk/interpro/search/sequence/) to identify specific domains and
the families to which belong the candidate proteins [34]. AlphaFold
Protein Structure Database (AFD8; https://www.alphafold.ebi.ac.uk/)
was used to predict 3D structures and to perform structural alignment
with homologous proteins in other fungi [35-37]. Obtained PDB files
were then edited on ChimeraX 1.8 software [38-40]. *Verticillium dahliae*
elicitor-like protein (VdPeVD1) [41], *Verticillium longisporum* homolog
of SnodProt1 (VI SnodProt1, first described on *Stagonospora nodorum*
[42]), and *Trichoderma atroviride* eliciting plant response-like protein
(TaEpl1) [43] sequences and PDB files were obtained with AFDB. Amino
acid sequence alignments were performed using NCBI BLAST (https://
blast.ncbi.nlm.nih.gov/Blast.cgi) and MEGA11 software [44] to
construct phylogenetic trees. Sequence comparisons and phylogenetic
trees were realized with similar proteins from other fungi selected
because of a high amino acid sequence similarity predicted by NCBI
and/or high structure homology predicted by AFDB. Amino acid align-
ments were then constructed using MEGA11 software [44] with MUS-
CLE algorithm.

## 2.3. Biological material

2.3.1. Plant material

*N. benthamiana* plants were grown in a greenhouse up to the five-leaf
stage in potting soil at min 18 °C- max 26 °C with a 16 h/8 h (day/night)
photoperiod.

V. vinifera cv. Syrah *in vitro* cuttings were cultured on McCown
Woody Plant medium including vitamins (Duchefa Biochemie, Nether-
land) for 10 weeks in a culture chamber with 25 °C temperature and 16/
8h (day/night) photoperiod.

One-year-old dormant lignified canes were collected from *V. vinifera*
Chardonnay 96 plants grown in greenhouses and conducted as liana
vines in January 2022, and from Riesling (rootstock: 16149, Grand Cru
Schlossberg, granitic soil) and Gewurztraminer vines (rootstock: SO4,
Grand Cru Marckrain, marl-limestone soil) in Kaysersberg city (France)
in February 2023. Canes were shortly stored at 4 °C until further
processing.

2.3.2. Fungal and bacterial material

*N. parvum* isolate Bt67, obtained from a single spore collection pro-
vided by the Instituto Superior de Agronomia (Universidade de Lisboa,
Portugal), was cultured at 27 °C in the dark on potato-dextrose agar
(PDA) and sub-cultured every 10 days.

*Rhizobium radiobacter* (*Agrobacterium tumefaciens*) C58-CI was
cultured on solid or liquid lysogeny broth (LB) medium in the dark at
28 °C (under 250 rpm agitation for liquid cultures) with appropriate
selection antibiotics.
## 2.4. Inoculation of grapevine detached canes

Wood assays were performed on detached dormant lignified grapevine canes of the year. Canes were cut to obtain wood sections between two nods and inoculated using the procedure described by Guan et al. (2016) [45]. Canes were wounded with a power drill (Black and Decker, USA) and inoculated with a 5 mm diameter plug of PDA alone or with a 10-day old culture of *N. parvum* Bt67 on PDA. Wounds were sealed with Parafilm®, and the infected internodes were stored in the dark at 28 °C under saturated humidity, until sampling. At each kinetics time point, sections of 1 cm long were cut on each side of the inoculation point, excluding the inoculation point, then frozen in liquid nitrogen and lyophilized for 24 h. Dry wood was then ground by pools of 3 canes to a fine, homogeneous powder with a ball mill (MM400 Retsch GmbH, Germany). For each time point, 3 biological replicates composed of 3 plant samples were obtained.

## 2.5. Fungal genomic DNA extraction and absolute quantification in plant material

Ten days after subculture on Petri dishes (PDA was covered with a cellophane membrane to facilitate mycelium retrieval), *N. parvum* mycelium was collected, frozen in liquid nitrogen, and ground with a mortar and pestle. Total DNA extraction was performed using the NucleoSpin® DNA Plant II kit (Macherey-Nagel, Germany) following the manufacturer's instructions (protocol 5.2 Genomic DNA from fungi, 04/2023, Rev. 14).

Genomic DNA from grapevine wood infected by *N. parvum* was extracted with NucleoSpin® Plant II kit (Macherey-Nagel, Germany) following the manufacturer's instructions (protocol 5.1 Genomic DNA from plant, 04/2023, Rev. 14) on 100 mg of dry lyophilized wood powder. DNA purity and quantity were checked by OD control with NanoDrop One® (Thermo Fisher Scientific, USA). Total DNA was diluted at 10 ng/µL concentration. PCR reactions were carried out in CFX96 Real-Time PCR detection System (BioRad, USA) with CFX Maestro software. Reactions were carried out in triplicates in a reaction buffer containing 1X iTaq Universal SYBR® Green Supermix (2X) (Bio-Rad, USA), 0.2 mM of forward and reverse primers (*Neofusicoccum Beta-tubulin* described by Luo et al. (2017) [46]; Supplemental Table 1), and 20 ng of total DNA in a final volume of 10 µl. Thermal cycling conditions were: 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C (denaturation) and 35 s at 65 °C (annealing and elongation). A plate read was performed before the next amplification cycle. The specificity of the individual PCR products was checked using a heat dissociation curve from 60 to 95 °C following the final cycle of the PCR. A standard range from $10^2$ to $10^8$ copies was performed with *N. parvum* genomic DNA samples known concentrations following the same procedure.

## 2.6. Cloning of *N. parvum* NpCP1, NpAA1.1 and NpAA1.2

NpCP1, NpAA1.1, and NpAA1.2 coding sequences were modified to remove the fungal signal peptide or replace it with the complete *V. vinifera* PR1 (pathogenesis-related protein 1) signal peptide to ensure that small *N. parvum* proteins are secreted to the apoplast after expression in planta [47]. The new constructs were synthesized and cloned by ProteoGenix (France) into a pBIN61 plasmid by restriction enzymes. pBIN61 is a pBIN19-derived vector containing the CaMV 35S expression cassette and resistance genes for kanamycin and tetracycline antibiotics [48]. The identity of the clones was confirmed by sequencing. Electro-competent *R. radiobacter* were electro-transformed with the different constructions in an Electroporation Cuvette, (0.1 cm, BioRad (USA)) using a Gene Pulser® II Electroporation System (BioRad, USA) set to the following parameters: capacitance = 50 µF, resistance = 200 Ω, and potential difference 1.8 KV. Transformed bacteria were then recovered in 1 mL liquid LB without antibiotics for 50 min at 28 °C before plating on LB Petri dishes containing antibiotics (kanamycin 50 µg/mL and

tetracycline 2.5 µg/mL) and incubating for 48 h at 28 °C.

## 2.7. Transient expression in *N. benthamiana* and *V. vinifera*

Because of some limitations of the grapevine model for transient expression through agroinfiltration, *N. benthamiana* was also used as an alternative non-host plant allowing strong expression of candidate SCPs.

Agroinfiltrations were performed directly on potted plants for *N. benthamiana* and on detached leaves kept in a humid atmosphere in the culture chamber (25 °C, photoperiod 16/8 h) for *V. vinifera*.

Grapevine and tobacco transient expressions were performed as previously described by Santos-Rosa et al. (2008) [49]. Briefly, an isolated colony was transferred to liquid LB with antibiotics for pre-culture, and the success of the transformation was checked by PCR using primers targeting pBIN61 (Supplemental Table 1) on colonies diluted in 50 µL of sterile ultrapure water. After 48 h, 1 mL of pre-cultured *R. radiobacter* was added to 5 mL of LB containing antibiotics, MES (10 mM), aceto-syringone (150 µM) and incubated at 28 °C for 24 h. Bacteria were subsequently collected by centrifugation (5 min at 6000 rpm) and incubated at 28 °C for 3–4 h in 5 mL of water-based induction buffer (MgCl₂ 10 mM, MES 10 mM (pH 5.6), aceto-syringone 150 µM, and sucrose 2 % (w/v)) until OD$_{600}$ = 0.8 for *N. benthamiana* infiltration or OD$_{600}$ = 0.4 for *V. vinifera* infiltration.

*N. benthamiana* potted greenhouse plants were infiltrated on leaves 2 and 3 starting from the apex (one construct on each side of the midrib) with a 1 mL syringe, using *R. radiobacter* transformed with pBIN61 expressing GUS construct as negative control. The L6$^{MHV}$-YFP construct previously described by Bernoux et al. (2016) [50] was used as a positive control. L6 gene codes for a *Linum usitatissimum* immune receptor modified to replace an aspartate with a valine and fused with yellow fluorescent protein (YFP). This construct was introduced by Bernoux et al. (2008) [51] in a pAM-PAT-35S plasmid (resistance to ampicillin, rifampicin, and gentamicin) and is able to induce constitutively programmed cell death when expressed in WT tobacco [51,52]. Agro-infiltrations with NpSCPs were first conducted with constructs with or without plant signal peptide. Constructs without signal peptide did not trigger any symptoms compared to the GUS construct. Only constructs with plant signal peptide were thus kept for following experiments and tested in *V. vinifera*, because of limited plant material availability. Plants were kept in the greenhouse for 5 days before harvesting leaves.

*V. vinifera* cv. Syrah leaves from *in vitro* cuttings were infiltrated by vacuum dipping (1 min at – 500 bar repeated 3 times) directly into agrobacterium solutions supplemented with 0.015 % Silwet L-77® (De Sangosse, France), following the method previously described by Santos-Rosa et al. (2008) [49]. GUS construct was used as a negative control. RXLR Pv 33Δsp from *Plasmopara viticola*, previously described as an HR inducer in *V. vinifera* [53] was used as a positive control. Leaves were incubated in sealed Petri dishes on dampened filter paper in a culture chamber at 25 °C, with a 16/8 h (day-night) photoperiod.

*R. radiobacter* transformed with pBIN61 containing GUS, RXLR Pv 33Δsp or L6$^{MHV}$-YFP constructs were kindly provided by Dr Pere MES-TRE (INRAE Colmar, France).

## 2.8. RNA extraction from grapevine detached canes and *N. parvum* absolute quantification of small protein expression by quantitative RT-PCR

RNA extractions were performed using a hybrid protocol based on RNA extraction protocol previously described by Gambino et al. (2008) [54] with the following modifications. Approximately 150–200 mg of ground freeze-dried samples were incubated for 10 min at 65 °C in 1.5 mL sterile extraction buffer (CTAB 2 %, PVPP 2 %, Tris-HCl 300 mM, EDTA 25 mM, and NaCl 2 M, pH 8.0) containing 2 % of β-mercaptoethanol added just before use. The supernatant was recovered after a long centrifugation at 4 °C, mixed with 1 volume of phenol-/chloroform/isoamyl alcohol (25:24:1), and centrifugated for 10 min at 4 °C. The aqueous phase was recovered, mixed with 1 volume of
chloroform/isoamyl alcohol (24:1), and centrifugated for 10 min at 4 °C. This last step was repeated a second time. The supernatant was then mixed with 750 µL of PFB buffer from NucleoSpin® RNA Plant and Fungi kit (Macherey-Nagel, Germany). Samples were then incubated at 4 °C for 2 h, and eventual precipitate was resuspended by vortexing. Extraction kit protocol was subsequently performed following the manufacturer's instructions (protocol 5.1 RNA isolation from plant and fungal material, starting from step 4. Bind RNA, 12/2021, Rev. 04). The amount of RNA extracted was measured by an optical density control (NanoDrop One®, Thermo Fisher Scientific, USA), and its purity was checked on a 1 % agarose gel. A DNase (DNase I, RNase Free kit, Thermo Fisher Scientific, USA) treatment was performed on 2 µg of RNA to remove all potential genomic DNA residues. 1 µg of DNA-free RNA was then converted in cDNA with a reverse transcriptase treatment (iScript™ Reverse Transcription Supermix for RT-qPCR kit, BioRad, USA) following the manufacturer's instructions.

In parallel, total RNA was extracted from 50 mg of *N. parvum* Bt67 frozen mycelium using the NucleoSpin® RNA Isolation kit (Macherey Nagel, Germany, protocol 5.1, 02/2013, Rev. 15). Quantity and purity controls, DNase treatment, and reverse transcription were carried out using the procedure described above. This sample was then diluted to create standard ranges for *NpCP1*, *NpAA1.1*, and *NpAA1.2* from 10 to 10⁵ cDNA copies.

Quantitative real-time PCR was performed on 20 ng of standard or test cDNAs in 1X iTaq Universal SYBR® Green Supermix kit (2X) (Bio-Rad, USA) with 0.2 mM of forward and reverse primers in a final volume of 25 µL. Amplification and quantification were carried out on a CFX Duet Real-Time PCR detections System (BioRad, USA) with the following cycling instructions: 3 min at 95 °C followed by 40 cycles of denaturation for 15 s at 95 °C, annealing for 20 s at 60 °C, elongation for 20 s at 72 °C, and 20 s at 76 °C. A plate read was performed before the start of the next amplification cycle. The specificity of the individual PCR products was checked using a heat dissociation curve from 60 to 95 °C following the final cycle of the qPCR. The absence of genomic DNA in the samples was confirmed using noRT mix provided in the BioRad iScript™ Reverse Transcription Supermix kit. RT-qPCRs were performed in 3 technical replicates.

## 2.9. RNA extraction from agroinfiltrated leaves and relative quantification of defense gene expression by quantitative real-time RT-PCR

*N. benthamiana* infiltrated half-leaves were collected at 5 dpi, photographed, and snap-frozen in liquid nitrogen before grounding to a fine, homogeneous powder in a ball mill (MM400 Retsch GmbH, Germany). Total RNA was extracted using NucleoSpin® RNA Plant and Fungi kit (Macherey-Nagel, Germany) following the manufacturer's instructions. *V. vinifera* detached leaves were photographed at 5 dpi, snap-frozen in liquid nitrogen by pools of 3 leaves, and grounded to a fine, homogeneous powder in a ball mill (MM400 Retsch GmbH, Germany). RNA extractions were performed using the RNeasy® Plant Mini Kit (Qiagen, Germany) following the "Isolation of total RNA from plant tissues using the RNeasy® Plant Mini Kit; alternative procedure" (available on https://www.qiagen.com/us/resources/). The concentrations and purity of the samples were controlled by an optical density control (NanoDrop One®, Thermo Fisher Scientific, USA) and migration on 1 % agarose gel. DNase treatment, reverse transcription, and RT-qPCR were performed as described in part 2.8. Relative expression of *PR-1a*, *PR-4*, *PR-5*, and *RBOHB* in *N. benthamiana* and *PR-1*, *PR-3*, *PR-4*, *CHIT4a*, *PR-5*, *PR-10*, *PR-14*, *RBOHC2*, *ACO1B*, *GST1*, *ACC*, *ACS3*, *AOS1*, *NRPB2*, *PAL*, *STS*, and *CHS* in *V. vinifera* were investigated using $log_2(2^{-\Delta\Delta Ct})$ method [55]. All normalizations were performed by comparison of test samples to the mean of non-infiltrated samples. *EF1α* was used as reference gene for both experiments. Detailed names of defense genes and primer information are listed in Supplemental Table 1.

## 2.10. Statistical analysis

All statistical analyses were performed using RStudio v.2024.04.2 and R ×64 4.2.1 software [56]. A descriptive analysis of the data was first carried out to remove experimental outliers. A Shapiro test verified the normal distribution and the variance distribution was checked by a Levene test. Relative expression data for plant defense genes were transformed to log base 2. A parametric one-way ANOVA was then performed, followed by a Tukey HSD post-hoc test. Comparison of absolute *Neofusicoccum Beta-tubulin* copies quantification was also tested by a one-way ANOVA and Tukey HSD post-hoc test, while comparison of surface necrosis was performed by a non-parametric Kruskal-Wallis test and Dunn's post-hoc test (Holm correction). In all null-hypothesis significance testing, the p-value was set at p.value < 0.05.

## 3. Results

### 3.1. *NpCP1 and NpAA1.1, NpAA1.2 belong to cerato-platanin and major allergen A1 families respectively*

NpCP1 is a 139 amino-acid sequence containing a signal peptide (1–19 aa) predicted by SignalP 4.1, suggesting secretion. The predicted molecular mass is 12.233 kDa and pI = 4.70. Four cysteine residues were found (C40, C79, C82, C137) in the sequence suggesting two disulfide bonds (Fig. 1A). Being shorter than 400 aa and containing 4 cysteine residues, NpCP1 presents all the characteristics of a SCP [57]. Further bioinformatic analysis revealed a double-psi beta-barrel domain (DPBB_CEPL-like), characteristic of the cerato-platanin family, and only existing in fungi [58]. An analysis performed using AFDB predicted a high confidence level 3D structure that presented high similarity with Eliciting Plant Response-Like 1 (Epl1) from *T. atroviride* (TM-score = 0.77914) and SnodProt1 from *V. longisporum* (TM-score = 0.93032) [59, 60] (Fig. 1B and C). The merging of NpCp1 and VLSnodProt1 3D structures shows slight differences in the N-terminal region, the first sheet of *Verticillium* protein being a bit longer. In addition, the NpCP1 sheet in positions 124 to 133 aa is divided in two, while it is only one part in VLSnodProt1. The superposition between NpCP1 and TaEpl1 is almost perfect. Only slight changes in sheet rotation were detected at positions 65–68 and 128–132 aa. The phylogenetic analysis highlighted putative homologs in several other fungi. Among the sequences we chose to perform a phylogenetic tree because of a high sequence or structure similarity, eight homologs were previously characterized as Epl1 protein (including the one found in *T. atroviride*), seven as SnodProt1 (including the one found in *V. longisporum*), and four were only described as cerato-platanin family member. We found homologs in several fungal species associated with wood diseases belonging to *Lasiodiplodia*, *Diploidia*, *Botryosphaeria*, *Macrophomina*, and *Aplosporella* genera, or vascular wilt (*Verticillium* and *Fusarium*) (Fig. 1D). NpCP1 belongs to a clade comprising several Epl1 proteins described in fungi associated with wood diseases.

Similar analyses were performed on the two other proteins. NpAA1.1 (131 aa) and NpAA1.2 (146 aa) were both predicted to have a signal peptide (1–19 and 1–18 aa respectively), and their molecular masses were estimated to 11.520 kDa with a pI = 4.54 for NpAA1.1 and to 13.638 kDa with a pI = 4.30 for NpAA1.2. Both sequences contain 4 cysteine residues each (for NpAA1.1: C51, C67, C107, and C119 and for NpAA1.2: C66, C80, C121, and C133), and are therefore qualified as SCPs (Fig. 2A). The presence of an *Alternaria alternata* allergen 1 domain (Alt a1) was detected in the two sequences, this domain is only described in two fungal classes: Dothideomycetes (including *N. parvum*) and Sordariomycetes [61]. 3D structure predictions revealed a structural similarity of both proteins with the secretory effector PevD1 from *V. dahliae* (Fig. 2B and C) (TM-score = 0.64963 for NpAA1.1 and TM-score = 0.78568 for NpAA1.2). Strong 3D structure similarity is shown after merging both NpAA1.1 and NpAA1.2 structures with PevD1. However, for both NpAA1s, several variations were visible on helix structures but
A >NpCP1

MQFTTILASLASMVAMTSA TSVSYDTGYDDAGRSLTAVSCSDGVNGLITKYGWQTQGAVSKFPYIGG
SDTIAGWGSANCGKCYSLTYNGKTINVLAIDHAGSGFNIAQAALNDLTNGQAAALGRIEATTAEVPL
SNC GL*

B

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

C

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

NpCP1 SnodProt1
merged

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

NpCP1 Epl1
merged

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

D

<figure><img src="image_9.png" /><figcaption>**Fig. 1.** Analysis of cerato-platanin NpCP1. (A) Highlights of particular patterns on NpCP1 amino acid sequence: signal peptide (in green) and DPBB_CEPL-like domain (in blue). All cysteine residues are shown in light yellow. (B) Structures of NpCP1 (blue), *V. longisporum* SnodProt1 (yellow) and, *T. atroviride* Epl1 (green) predicted by AlphaFold Protein Structure Database (ID: R1G1Q3, A0A2J8F2T3, A0PCX2). Confidence scores were predicted very high (pLDDT >90) for all three proteins. (C) Structures of the proteins aligned with ChimeraX 1.8. (D) The evolutionary relationship of NpCP1 with homologs in other fungi was determined using the maximum likelihood algorithm of MEGA11. NpCP1, SnodProt1, and Epl1 are represented with blue, yellow and green squares respectively. Purple circles highlight trunk disease pathogens. NCBI IDs of the proteins are indicated in brackets.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 2.** Analysis of *N. parvum* Alt a1 small proteins. (A) Highlights of particular patterns on NpAA1.1 and NpAA1.2 amino acids sequences: signal peptide (in green) and Alt a1 motif (in orange). All cysteine residues are shown in light yellow. (B) Structures of NpAA1.1 (green), NpAA1.2 (blue), and *V. dahliae* PevD1 (yellow) predicted by AlphaFold Protein Structure Database (ID: R1GBM4, R1E6W1, A0A2J8D129). Confidence scores were predicted very high (pLDDT >90) for all three proteins. (C) Structures of the proteins aligned with ChimeraX 1.8. (D) The evolutionary relationship of NpAA1.1 and NpAA1.2 with homologs in other fungi was determined using the maximum likelihood algorithm of MEGA11. NpAA1.1, NpAA1.2 and PevD1 are represented with green, blue, and yellow squares respectively. Purple circles highlight trunk disease pathogens. NCBI IDs of the proteins are indicated in brackets.</figcaption></figure>

the two major domains containing two series of parallel sheets were highly conserved compared to VdPevD1. A comparison of NpAA1.1 and VdPevD1 structures revealed several small differences (slight rotations or shortening) as well as one supplemental sheet in position 56 to 62 aa in NpAA1.1. NpAA1.2 structure is closer to VdPevD1 since only one sheet (94–106 aa position) has a differential rotation, and 3 are shorter (42–45, 119–123, 131–135 aa positions) compared to VdPevD1. *V. dahliae* PevD1 has been reported to have a dual role during plant infection since it is known to both induce plant immune responses and promote fungal infection [62]. Phylogenetic analysis showed homologs in several other fungal species. Five of the homologs have been described as PevD1 proteins, three as major allergens, two as

HR-inducers, and the eleven others only as putative effectors or uncharacterized proteins. We found homologs in several genera including several wood pathogens: *Lasiodiplodia*, *Diplodia*, *Botryosphaeria*, *Macrophomina*, *Aplosporella*, and *Phyllosticta* or vascular wilt: *Verticillium* (Fig. 2D). *N. parvum* NpAA1.2 is phylogenetically more closely related to PevD1 proteins described in *Verticillium* and *Colletotrichum*, whereas NpAA1.1 belongs to a different clade comprising uncharacterized proteins and an HR-inducing protein from *Lasiodiplodia theobromae* (Fig. 2D).
## 3.2. Similar wood colonization but different SCPs expression kinetics depending on the cultivar after *N. parvum* infection

The inoculation of detached canes revealed rapid colonization by *N. parvum* Bt67 in the three cultivars tested. No significant difference was observed in the rate of colonization analyzed by *N. parvum* quantification at 72 hpi, and the size of the necrotic lesions that appeared on the infected wood was similar between the 3 grape varieties (Fig. 3). Despite similar colonization, the expression of the three SCPs varied widely between cultivars after *N. parvum* wood infection (Fig. 4). Indeed, while *NpCP1* expression seems strong and early in Chardonnay; in Riesling, although still early, the expression seems more moderate, while in Gewurztraminer it is barely detectable (Fig. 4A–C). *NpAA1.1* is expressed most consistently between grape varieties. Its expression is early, as early as 24 hpi in the three cultivars studied, reaching its maximum at 48 hpi for Chardonnay and 24 hpi for Riesling. *NpAA1.1* expression is slightly lower in Gewurztraminer, peaking at 48 hpi, then decreasing at 72 hpi, and finally increasing at 5 dpi (Fig. 4D–F). Finally, *NpAA1.2* expression is low but early induced in Chardonnay, while it is barely detected in Gewurztraminer and Riesling (Fig. 4G–I). Early and strong expression of *NpCP1* in all cultivars, *NpAA1.1* in Chardonnay and Riesling, and to a lesser extent of *NpAA1.2* in Chardonnay suggests that these SCPs are involved in *N. parvum* wood colonization process.

## 3.3. NpAA1.1, NpAA1.2, and NpCP1 do not induce extensive cell death after transient expression in *N. benthamiana* and *V. vinifera*

After transient expression in *N. benthamiana*, *NpCP1* does not cause the appearance of any symptoms. Concerning the two *NpAA1s*, half of the leaf infiltrated with the protein containing the plant signal peptide inducing secretion to the apoplast shows some necrosis locally at the point of infiltration and light chlorosis in the entire infiltrated zone. However, compared to HR symptoms induced by L6<sup>MHV</sup>-YFP expression,

symptoms induced after *NpAA1s* transient expression were far more moderated and hardly observable (Fig. 5A).

After transient expression in *V. vinifera* cv. Syrah, *NpCP1*, *NpAA1.1*, and *NpAA1.2* fused to plant signal peptide triggered the appearance of necrotic spots. Some necrotic spots were also observed in the negative GUS control but a higher number was observed for the three SCPs. These necrotic spots were also observed after RXLR Pv 33Δsp expression (Fig. 5B).

## 3.4. NpAA1.1, NpAA1.2, and NpCP1 transient expression have limited impact on defense responses in *N. benthamiana*

A control of the expression of the target genes encoding SCPs highlighted the success of transient expression. Indeed, high levels of *NpCP1*, *NpAA1.1*, and *NpAA1.2* transcripts were detected 5 days after agro-infiltration (Fig. 6A). We focused on the effect of SCPs on genes encoding PR proteins and enzymes regulating oxidative stress, since these candidates have been described as the targets of Epl1 or PevD1 in other systems [62–65]. Notwithstanding, no strong effect on the expression of defense genes could be observed. Expression of *NpCP1* led to a significant increase in *PR1a* expression, while expression of *RBOHB* was downregulated (Fig. 6B). In contrast, *NpAA1.1* overexpression triggered the downregulation of *PR1a*, but a strong increase in the expression of *PR4* and *RBOHB* (Fig. 6C). Finally, *NpAA1.2* triggered less significant changes in the expression of the defense gene studied than *NpAA1.1*: only repression of *PR1a* expression was evidenced in leaves infiltrated with the *NpAA1.2* construct (Fig. 6D).

## 3.5. Contrasted effect of NpAA1.1, NpAA1.2, and NpCP1 transient expression on defense responses in *V. vinifera*

In *V. vinifera* cv. Syrah leaves, agroinfiltration led to high levels of SCPs expression, confirming the success of transient transformation

<figure><img src="image_11.png" /><figcaption>**Fig. 3.** Wood infection by *N. parvum* Bt67 in 3 grapevine cultivars. (A) Necrotic symptoms 72 hpi. PDA-inoculated plants are used as a negative control. (B) Absolute quantification of *N. parvum* Bt67 beta-tubulin 72 hpi. Cultivars are shown in different colors: Chardonnay in light purple, Gewurztraminer in green, and Riesling in blue. Data represent the mean ± SE of 3 biological replicates. No significant differences were detected between cultivars (p.value < 0.05, one-way ANOVA and Tukey HSD post-hoc test, n = 3). Dashed lines represent residual beta-tubulin copies detected in PDA negative control for each cultivar. (C) Mean necrosis surface in cm² ± SE at 72hpi (for Chardonnay: n<sub>Bt67</sub> = 6 and n<sub>PDA</sub> = 3, and for Gewurztraminer and Riesling: n<sub>Bt67</sub> = 12 and n<sub>PDA</sub> = 6). No significant differences in necrosis surface in NpBt67-inoculated samples were detected between cultivars (p.value < 0.05, Kruskal-Wallis and Dunn post-hoc test).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 4.** Absolute quantification of *N. parvum* small secreted protein expression: *NpCp1* (A, B, C), *NpAA1.1* (D, E, F), and *NpAA1.2* (G, H, I). The number of transcript copies in the wood was determined by q-RT-PCR at different times after inoculation. Data were normalized to transcript copy numbers in mock-inoculated canes. Cultivars are shown in different colors: Chardonnay in light purple (left column), Gewurztraminer in green (center column), and Riesling in blue (right column). The horizontal black line indicates the lower limit of the standard quantification; all lower values are detected but cannot be quantified. Data represent the mean ± SE of 3 biological replicates.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Observation of agroinfiltrated leaves of *N. benthamiana* (A) and *V. vinifera* subsp. Syrah (B) 5 days post-infiltration. *N. benthamiana* leaves were infiltrated in two spots on each side of the midrib. (A) Agroinfiltrations with L6<sup>MHV</sup>-YFP construct and GUS construct were used as a positive control of HR induction and as a negative control (right side of the midrib). Leaves were infiltrated with NpCP1, NpAA1.1, or NpAA1.2 construct fused to plant signal peptide allowing secretion on the left side of the midrib or without signal peptide (right of the midrib). Pictures are representative of 5 independent leaves. (B) *V. vinifera* were infiltrated by soaking and placed on *Petri* dishes, top side against damp paper. Leaves agroinfiltrated with the GUS construct are used as negative control and leaves infiltrated with RXLR Pv 33Δsp are used as positive control of cell death induction. Leaves were infiltrated with NpCP1, NpAA1.1, or NpAA1.2 construct fused to plant signal peptide allowing secretion.</figcaption></figure>

(Figs. 7A and 8A and B). To get more detailed information on the functions and targets of these putative effectors, several classes of plant defense genes were investigated: genes involved in Ethylene and JA pathways, Oxidative stress, Secondary metabolism, and genes encoding PR proteins. Among all the tested defense genes, NpCP1 only triggered downregulation of *RBOH.C2*, *ACO1b* (involved in ethylene biosynthesis), and *PR-4* compared to GUS control (Fig. 7B, D and 7E). Interestingly, the *RBOHC2* regulation response is consistent to what was observed with its homolog in tobacco leaves.

Concerning the two NpAA1 proteins, a significantly higher number of defense genes were impacted after their transient expression. A general investigation of genes involved in secondary metabolism showed overexpression of *PAL* and *STS* after expression of both NpAA1 constructs compared to GUS control (Fig. 8C). On the other hand, NpAA1.1 overexpression led to significant repression of *ACS3* involved in ethylene biosynthesis and of *RBOH.C2* expression involved in ROS production

(Fig. 8D and E). The expression of several pathogenesis-related proteins was also impacted by the expression of both *NpAA1s* (Fig. 8F). *PR-1*, *CHIT4a*, and *PR-10* expression were significantly higher after expression of *NpAA1.1* only. However, *PR-3*, *PR-4*, and *PR-5* were overexpressed in the presence of both NpAA1.1 or NpAA1.2. Finally, NpAA1.1 and NpAA1.2 had contrasted effects on *PR14* expression: a high overexpression of *PR-14* was evidenced following overexpression of *NpAA1.2* while overexpression of *NpAA1.1* led to *PR14* repression (Fig. 8F).

4. Discussion

It is well known that pathogenic fungi secrete proteins during plant colonization. SSPs, especially cysteine-rich proteins, often play an important role in interactions with other organisms and are of major importance in the pathogenicity of fungi involved in vascular diseases
<figure><img src="image_14.png" /><figcaption>**Fig. 6.** Relative expression of plant defense genes induced by transient expression of *N. parvum* small proteins in *N. benthamiana* leaves. (A) Control of *NpCP1*, *NpAA1.1*, and *NpAA1.2* transient overexpression. (B, C, D) Relative expression of *PR-1a*, *PR-4*, *PR-5*, and *RBOHB* following the overexpression of *NpCP1* (B), *NpAA1.1* (C), and *NpAA1.2* (D). Transcript levels of each defense gene were normalized to *N. benthamiana* *EF1α* transcript levels. Relative expression log₂(2⁻⁰·⁰⁵ΔΔCt) indicates normalized expression levels in agroinfiltrated leaves compared to normalized expression levels observed in control non-infiltrated leaves at the same time point. Plants infiltrated with GUS construct are used as negative controls. Different letters overhead the boxplot indicate statistically significant differences (p.value < 0.05, one-way ANOVA and Tukey HSD post-hoc test; n = 3). Results were obtained from the mean of three technical replicates on three biological replicates.</figcaption></figure>

[20,21]. Numerous SSPs have been detected in *Botryosphaeriaceae* but only a few have been fully characterized [21]. Recently, Vázquez-Avendaño et al. (2021) identified new cysteine-rich proteins during the interaction between *N. parvum* and *L. styraciflua*, including two fungal proteins with a CFEM domain that may be involved in pathogenesis [28]. However, the exact role of these SCPs remains to be characterized.

Among the different secreted proteins identified by their affinity with As (Engel et al., unpublished results) [29], we focused on 3 SCPs that are members of two protein families containing already known effectors in other plant pathogenic fungi, especially those involved in vascular wilts. These SCPs have not been described in *N. parvum*. In

detached cane infection experiments, we show that *NpSCPs* were differentially expressed depending on the grapevine cultivar, contrasting with necrotic symptoms as well as fungal growth which were similar in the 3 grapevine cultivars studied (Chardonnay, Riesling, and Gewurztraminer) 3 days after inoculation. These results show that *NpSCPs* expression does not reflect *N. parvum* growth in the wood. *NpSCPs* were very early overexpressed during wood colonization (as early as 24 h), indicating that they may be involved in *N. parvum* pathogenicity. Different expression of *NpSCPs* depending on the cultivar used also shows that cultivar-dependent factors may influence their expression.

The SCPs studied in *N. parvum* could be classified into already-known
<figure><img src="image_15.png" /><figcaption>**Fig. 7.** Relative expression of plant defense genes induced by transient expression of *N. parvum* NpCP1 protein in *V. vinifera* cv. Syrah *in vitro* leaves. (A) Control of NpCP1 transient overexpression. (B, C, D, E) Relative expression of genes implied in secondary metabolism (B), ethylene and jasmonic acid pathways (C), oxidative stress (D), and pathogenesis-related proteins (E). Transcript levels of each defense gene were normalized to *V. vinifera* Cabernet Sauvignon *EF1α* transcript levels. Relative expression (log₂(2⁻⁰·⁰⁵ΔΔCt)) indicates normalized expression levels in agroinfiltrated leaves compared to normalized expression levels observed in control non-infiltrated leaves at the same time point. Leaves infiltrated with the GUS construction are used as a negative control. Different letters overhead the boxplot indicate statistically significant differences (p.value < 0.05, one-way ANOVA and Tukey HSD post-hoc test; n = 4). Presented results were obtained from the mean of three technical replicates on four biological replicates.</figcaption></figure>

families of effectors. Structural analysis confirmed that NpCP1 is part of the cerato-platanin family, a family of SCP and non-catalytic proteins secreted by filamentous fungi which have very varied roles [66,67]. A comparison of the 3D structure shows that NpCP1 is very similar to both *T. atroviride* Epl1 and *V. longisporum* Snodprot1. According to sequence

comparison and phylogenetic analysis, cerato-platanins similar to Epl1 were identified in several fungi associated with wood diseases, although their exact role remains to be elucidated. In other fungi, most of the cerato-platanins were characterized as PAMPs since they act as elicitors inducing cell death and other defense responses in plants [66].
<figure><img src="image_16.png" /><figcaption>**Fig. 8.** Relative expression of plant defense genes induced by transient expression of *N. parvum* NpAA1.1 or NpAA1.2 proteins in *V. vinifera* cv. Syrah *in vitro* leaves. (A, B) Control of NpAA1.1 or NpAA1.2 transient overexpression. (C, D, E) Relative expression of genes implied in secondary metabolism (B), ethylene and jasmonic acid pathways (C), oxidative stress (D), and encoding pathogenesis-related proteins (E). Transcript levels of each defense gene were normalized to *V. vinifera* Cabernet Sauvignon *EF1α* transcript levels. Relative expression (log₂(2⁻⁰·⁰⁵ΔΔCT)) indicates normalized expression levels in agroinfiltrated leaves compared to normalized expression levels observed in control non-infiltrated leaves at the same time point. Leaves infiltrated with the GUS construct are used as a negative control. Different letters overhead the boxplot indicate statistically significant differences (p.value < 0.05, one-way ANOVA and Tukey HSD post-hoc test; n = 4). Presented results were obtained from the mean of three technical replicates on four biological replicates.</figcaption></figure>

Cerato-platanins could also exert a defense response priming effect in plants, as it was reported for the Epl1 protein identified in *T. atroviride* [43,68]. Other roles have been described for cerato-platanins: in *Fusarium graminearum*, it has been reported that SnodProt proteins protect fungal cell wall polysaccharides from enzymatic degradation during the

infection process [69]. In addition, some cerato-platanins could loosen plant cellulose and facilitate host colonization [58,70]. Several hypotheses can thus be put forward concerning the roles of NpCP1 in plant-pathogen interaction. Since cerato-platanins are known for their expansin-like activity, NpCP1 could be involved in cell-wall loosening
and facilitate growth of the trunk pathogen. In accordance with other studies, our work also emphasizes that NpCP1 could be involved in *N. parvum* pathogenicity by modulating basal defenses (see below).

The other two identified SCPs, NpAA1.1 and NpAA1.2, were identified as carrying the major allergen motif Alt a1 initially identified in the genus *Alternaria* [71]. It was then found in other pathogenic fungi such as *Magnaporthe oryzae*, *Stemphylium botryosum*, *Ulocladium atrum*, *Botrytis cinerea*, and in *V. dahliae*. Analysis of NpAA1.1 and NpAA1.2 structures revealed that they have a high level of similarity with PevD1 effector from *V. dahliae*, a pathogenic fungus causing cotton wilt. The highest structural similarity was identified for NpAA1.2. Several studies reported a contrasted role for this Alt a1-like secreted effector. PevD1 has been originally described as an elicitor inducing several defense responses and conferring systemic acquired resistance to tobacco mosaic virus in tobacco [41]. Bu et al. (2014) further demonstrated that recombinant PevD1 enhanced cotton resistance to *V. dahliae* and systemic defense responses such as ROS production, cell wall reinforcement, phenylpropanoid pathway, and PR proteins [72]. On another hand, Zhang et al. (2019) showed that *V. dahliae* PevD1 knockout mutants have attenuated pathogenicity on cotton compared to wild types [62]. More recently, it was also demonstrated that PevD1 is involved in leaf senescence triggered by *V. dahliae* after Arabidopsis infection: over-expression of PevD1 accelerated Arabidopsis leaf senescence, whereas disruption of PevD1 resulted in impaired leaf senescence [73].

It should be noted that the 3 NpSCPs do not induce clear necrosis symptoms after transient expression in *N. benthamiana*. Whereas NpCP1 expression had no significant impact on cell death compared to GUS control, slight chlorosis observed after agroinfiltration of NpAA1.1 and NpAA1.2 in *N. benthamiana* may indicate induction of leaf senescence, in accordance with the senescence-promoting activity reported for PevD1 [73]. In *V. vinifera*, transient expression of NpSCPs resulted in a different response with the appearance of necrotic spots on some infiltrated leaves. These spots were rather sparse and non-visible on all infiltrated leaves. However, the transient expression of the RXLR Pv 33Δsp effector, which was reported as a cell death inducer in grapevine [53], also shows the same pattern. We hypothesize that this weak cell death response may be linked to the low efficiency of *Agrobacterium* transient transformation in grapevine. Indeed, the vacuum soaking method generally only allows partial infiltration of whole leaves, often concentrated near the stomata, the only entry point for agrobacteria [49,74].

Interestingly, the two types of SCPs also induced different types of responses concerning the expression of defense genes in tobacco and more particularly in grapevine. Apart from PR1 induction in *N. benthamiana*, NpCP1 only repressed the expression of some defense genes in grapevine, especially genes encoding enzymes involved in ROS and ethylene synthesis (RBOHC2 and ACO1b). Downregulation of RBOH by NpCP1 was also found in *N. benthamiana*. Repression of candidate defense genes is consistent with the absence of extensive cell death observed after NpCP1 expression, especially in *N. benthamiana*. These results contrast with other studies concerning Epl1 and SnodProt, showing that these proteins could trigger cell death and defense responses in several systems. Indeed, Epl1 was involved in the synthesis of salicylic acid-regulated proteins such as PR-P2 in tomato (*Solanum lycopersicum*), and PR-1 and PAL1 in *Arabidopsis thaliana* [64,75]. In addition, Epl1 may be implied in ROS accumulation [64] and in up-regulation of genes of the jasmonic acid pathway [76]. Induction of defense responses has also been reported for SnodProt1 from *Verticillium* and homologs [77].

Compared to grapevine, NpAA1.1 and NpAA1.2 expression did not highly impact the expression of defense genes in *N. benthamiana*. This could be linked to the fact that tobacco is not a host plant for *N. parvum* which normally contaminates woody plants. These results suggest that NpAA1s are recognized by specific grapevine factors. Concerning the impact of NpAA1s overexpression on expression of defense genes in grapevine, our results highlight a dual effect for *N. parvum* Alt a1-like proteins. First, it should be noted that both NpAA1s could have a

similar or different impact, depending on the defense genes studied. Both NpAA1.1 and NpAA1.2 stimulate the expression of PAL and STS involved in phenylpropanoid and stilbene synthesis respectively. Concerning PR proteins, both NpAA1s stimulated the expression of PR1, PR3, PR4, and PR5; however, NpAA1.2 highly induced the expression of PR14 whereas an opposite effect was observed with NpAA1.1, with significant repression of PR14. In addition, NpAA1.1 expression also triggered repression of RBOHB, involved in ROS production [78], and of ACS3, which could be involved in ethylene biosynthesis [79]. In contrast, no significant effect on these 2 genes was evidenced after NpAA1.2 expression. In addition, no change in the expression of gene encoding asparagine-rich protein NRPB2 expression was observed after NpAA1s overexpression. This result contrasts with those described in the literature concerning PevD1 from *V. dahliae*, which induces strong NRPs expression in *N. benthamiana* and *A. thaliana* [80,81].

Overall, we show that NpSCPs could manipulate the basal expression of grapevine defense genes after agroinfiltration and could thus be considered as effectors. However, it is difficult to conclude on their roles in triggering plant immunity or favoring wood colonization by *N. parvum*. Induction of secondary metabolism and PR proteins by both *N. parvum* Alt a1 proteins indicates that they may be considered as PAMPs and stimulate grapevine defenses. It is known that defense responses such as PR proteins production and stilbene biosynthesis are induced in the wood following colonization with trunk disease pathogens [2]. However, these defense responses may not be sufficiently efficient since vascular pathogens are still able to colonize the wood. In this way, we showed that *Botryosphaeriaceae* fungi are able to rapidly metabolize stilbenes such as resveratrol, δ-viniferin, and piceatannol [23,24]. In addition, it has been reported that *N. parvum* could inhibit STS activity by secreting 4-HPA [82].

Finally, induction of cell necrosis and repression of the expression of RBOHB (involved in ROS production) and ACS3 (involved in ethylene biosynthesis) by NpAA1.1 and NpCP1 in grapevine leaves suggests that it could favor wood colonization by the fungus. Indeed, ROS and ethylene biosynthesis may take part in signaling pathways regulating response to *N. parvum*. It is known that ROS production via RBOHB plays an important role in defense signaling during plant immunity [83]. In addition, a possible role for ethylene in the formation of tyloses in the stem of grapevines has been reported [84]. Since tyloses constitute a barrier for fungal spread in vascular tissues, one could hypothesize that the downregulation of ethylene biosynthesis could favor *N. parvum* development. Finally, PR14 is a lipid-transfer protein belonging to the antimicrobial peptide class [85] and its downregulation by NpAA1.1 could also favor *N. parvum* colonization. In contrast, PR14 is induced by NpAA1.2; however, the expression of NpAA1.2 in the wood following infection is much lower compared to NpAA1.1.

## 5. Conclusions

In accordance with the literature, *N. parvum* effectors belonging to cerato-platanin and Alt a1 families could have a dual role during grapevine wood colonization, either as inducers or inhibitors of plant defense pathways. Affinity of these SCPs for As(III) suggests that sodium arsenite, in addition to a direct antifungal role, could also inhibit effector activity involved in wood colonization. Future work will focus on these effectors' knockout in *N. parvum* and the resulting effect on wood colonization.

### CRediT authorship contribution statement

**Alexia Laura Grau:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Romain J.G. Pierron:** SuperVISION, Software, Methodology, Investigation, Formal analysis, Data curation. **Charlotte Doerr:** Methodology, Formal analysis, Data curation. **Hélène Laloue:** Methodology, Formal analysis, Data curation. **Mary-Lorène Goddard:** Resources, Methodology, Investigation, Data
curation, Conceptualization. **Julie Chong:** Writing – original draft, Supervision, Project administration, Investigation, Data curation, Conceptualization.

## Funding

This work was supported by a PhD grant from the French Ministry of higher education and research to Alexia GRAU and by the Université de 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.

## Acknowledgements

We are grateful to Dr. Pere MESTRE, Laurie MARSAN, and Marie-Christine PIRON (INRAE, UMR SVQV, COLMAR) for providing agrobacteria transformed with GUS, L6<sup>MHV</sup>-YFP, and RXLR Pv 33Δsp constructs and for help and advices in transient transformation experiments. We thank INRAE UMR SVQV (Colmar, France) for providing *N. benthamiana* plants. We thank Yann Leva (LVBE-UHA) for his technical support.

# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pmpp.2025.102659.

## Data availability

Data will be made available on request.

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