Marie-Eva E Ors

Sameh Selim

A Siah

G Couleaud

C Maumené

K Sahmer

P Halama

Philippe Reignault
email: philippe.reignault@univ-littoral.fr

Béatrice Randoux

Cultivar-dependent partial resistance and associated defense mechanisms in wheat against Zymoseptoria tritici

Keywords: Triticum aestivum L., Zymoseptoria tritici, defense mechanisms, RT-qPCR, cell wall degrading enzymes

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Introduction.

Septoria tritici blotch (STB) is one of the most devastating foliar diseases of wheat (Triticum aestivum L.) and is caused by the hemibiotrophic fungus Zymoseptoria tritici (telemorph:

Mycosphaerella graminicola), previously known as Septoria tritici. The Z. tritici infection process is characterized by a latent period of approximately 10-15 days during which fungal germ tube grows on the leaf surface, penetrates either through the stomatal opening or directly via the epidermal cell wall, and colonizes the intercellular space between mesophyll cells [START_REF] Cohen | The histology of processes associated with the infection of resistant and susceptible wheat cultivars with Septoria tritici[END_REF][START_REF] Duncan | Cytological analysis of wheat infection by the leaf blotch pathogen Mycosphaerella graminicola[END_REF]Kema et al., 1996;[START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]. The fungus then rapidly switches to necrotrophic growth, associated to an increase of the fungal biomass, chlorotic and/or necrotic lesions on the leaf surface, and the formation of pycnidia in colonized substomatal cavities. Although no haustoria are differentiated, Z. tritici obtains nutrients from the plant apoplastic space during the biotrophic period, whereas during the necrotrophic and symptomatic phase, the fungus releases cell wall degrading enzymes (CWDEs) leading to an increase of nutrient content in this apoplastic space (Keon et al., 2005;[START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]. The production of cell-wall degrading enzyme (CWDEs), such as β-1,4-endoxylanase, by Z. tritici has been correlated with symptom and sporulation levels [START_REF] Douaiher | Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonase[END_REF][START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]. In their study, [START_REF] Tian | Correlation between cell wall-degrading enzymes in wheat leaves infected by Septoria tritici and disease severity[END_REF] showed that β-1,4endoxylanase and protease activities are highly associated with the susceptibility level of the infected wheat cultivar. Although carbohydrate degrading enzymes (CAZymes)-encoding genes are more frequently observed in the genomes of necrotrophic fungi, the Z. tritici genome possesses more genes coding for proteases than for CAZymes (Goodwin et al., 2011).

The control of STB in the field mainly relies on the use of conventional fungicides. However, their use is increasingly controversial because of their impact on both the environment and human health. In addition, their efficacy is compromised in the field because of the

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development and widespread of fungicide-resistant Z. tritici strains. An alternative to chemical control is breeding for new and more resistant wheat cultivars. However, such a strategy requires better knowledge of wheat defense mechanisms against STB.

Wheat resistance against Z. tritici can be controlled either by specific or non-specific partialresistance. The first one confers a complete resistance, observed during incompatible interactions, and is controlled by gene-for-gene relationships [START_REF] Brading | A gene-forgene relationship between wheat and Mycosphaerella graminicola, the septoria tritici blotch pathogen[END_REF]. To date, 18 specific stb R genes have been identified in wheat against Z. tritici, which should be capitalized in order to obtain a durable resistance [START_REF] Brading | A gene-forgene relationship between wheat and Mycosphaerella graminicola, the septoria tritici blotch pathogen[END_REF][START_REF] Simón | Population structure of Mycosphaerella graminicola and location of genes for resistance to the pathogen: recent advances in Argentina[END_REF].

On the other hand, partial resistance or quantitative disease resistance (QDR) is observed during compatible interactions. It is not isolate-specific and is controlled by several genes or by quantitative traits loci (QTL) (Chartrain et al., 2004). In this case, severity of symptoms caused by different strains of Z. tritici varies between wheat cultivars, leading to a wide range of susceptibility/resistance levels. It has been reported that Z. tritici populations can overcome specific resistances, which are less durable than QDR in the field. Breeding for several selected genes or loci involved in QDR can also lead to an increased durability of wheat resistance against STB in the field (Chartrain et al., 2004;Orton et al., 2011).

Wheat defense mechanisms against Z. tritici have been recently investigated by molecular and biochemical studies in order to identify host responses, but previous works focused on incompatibles interactions, with compatibles ones studied only as a reference. It has been suggested that pathogenesis-related (PR) proteins, reactive oxygen species (ROS) metabolism, cell wall reinforcement with callose, and signalling via the MAPK pathway are involved in wheat resistance against STB [START_REF] Adhikari | Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression[END_REF][START_REF] Diani | Induction of early oxidative events in soft wheat leaves inoculated with Septoria tritici and their relationship to resistance of Moroccan cultivars[END_REF][START_REF] Fazeli | Development of DNA Microarray Technique to Evaluation of transcriptional responses of wheat to Mycosphaerella graminicolla (septoria tritici)[END_REF]Rudd et al., 2008;Shetty et al., 2003[START_REF] Shetty | Role of hydrogen peroxide during the interaction between the hemibiotrophic fungal pathogen Septoria tritici and wheat[END_REF][START_REF] Shetty | Effects of β-1,3-glucan from Septoria tritici on structural defence responses in wheat[END_REF]Yang et al., 2013). Indeed,

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This article is protected by copyright. All rights reserved. most of studied defense-related genes were differentially expressed between compatible and incompatible interactions. Early expression, before penetration, of host genes encoding for PR1, chitinases, glucanases and peroxidases was suggested to contribute to wheat resistance during incompatible interactions [START_REF] Adhikari | Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression[END_REF]Yang et al., 2013). In STB resistant cultivars, no evidence of hypersensitive response (HR) was observed during the first steps of the infectious process (Kema et al., 1996), while HR occurs early on plants during incompatible interactions with other biotrophic pathogens, and refers to effector-triggered immunity (ETI) (Jones and Dangl, 2006). However, during the fungal switch from the biotrophic to the necrotrophic phase, necrosis consisting of a programmed cell death (PCD) showing similarities with HR was observed in susceptible wheat cultivars (Keon et al., 2007).

It has been suggested that in susceptible and only partially resistant cultivars, Z. tritici undergoes adaptation to wheat defence metabolites and manipulates defense mechanisms during its extracellular infection process (Keon et al., 2007;Hammond-Kosack and Rudd, 2008;Rudd et al., 2008). However, very little is known so far about the features of wheat defences expressed during compatible interactions and QDR leading to partial resistance.

In the present study, we therefore investigated wheat QDR expressed during compatible interactions with Z. tritici. We compared the expression of defence markers associated with this partial resistance in three distinct wheat cultivars characterized in the field by various resistance levels to STB. First, infection process and fungal pathogenicity were examined by cytological and biochemical approaches during both biotrophic and necrotrophic phases of the infection. Furthermore, wheat defenses were studied by molecular and biochemical tools during the early period following leaf inoculation. We measured by quantitative real-time PCR (RT-qPCR) the expression level of a set of genes involved in wheat defense activated either during specific resistance, after pathogen infection or resistance inducer treatment. The

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This article is protected by copyright. All rights reserved. expression of chitinase 2-and glucanase-encoding genes (AB029935; DQ090946) was induced in cultivars resistant to Z. tritici. [START_REF] Adhikari | Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression[END_REF][START_REF] Shetty | Effects of β-1,3-glucan from Septoria tritici on structural defence responses in wheat[END_REF], while the expression of pathogenesis-related protein 1 (PR1) encoding gene (HQ 848391) was induced earlier and with a higher level during an incompatible interaction between wheat and Puccinia triticina than in compatible interactions [START_REF] Gao | Expression and functional analysis of a Pathogenesis-Related Protein 1 Gene, TcLr19PR1, involved in wheat resistance against leaf rust fungus[END_REF]. The expression of a lipoxygenase-encoding gene (U32428) was also studied according to the induction observed after Benzothiadiazole (BTH;Gorlach et al, 1996) and trehalose (Tayeh et al., 2014) preventive treatments, which protected wheat against Blumeria graminis f. sp. tritici. The peroxidase (POX2) encoding gene (X85228) and the phenylalanine ammonia lyase (PAL) encoding gene (AY005474) were expressed in wheat leaves infected by Blumeria graminis f. sp. tritici [START_REF] Båga | Molecular cloning and expression analysis of peroxidase genes from wheat[END_REF]Tayeh et al., 2015). Expression of the chalcone synthase (CHS) gene (AY286097), encoding for a key enzyme involved in phenylpropanoid pathway, was partially reduced after inoculation by Z.tritici in both resistant and susceptible cultivars [START_REF] Shetty | Effects of β-1,3-glucan from Septoria tritici on structural defence responses in wheat[END_REF]. Likewise, the expression of the oxalate oxidase (OXO) encoding gene (AJ556991) was not induced by treatment by glucan extract from Z.tritici [START_REF] Shetty | Effects of β-1,3-glucan from Septoria tritici on structural defence responses in wheat[END_REF]).

The allene oxide synthase (AOS) gene (AY196004.1) encoding the first enzyme after LOX in the octadecanoid pathway, was isolated from wheat (Wu et al., 2004). The involvement of these defense mechanisms in the expression of the non-specific wheat resistances studied here against Z. tritici is discussed.

Experimental procedures

Plant and fungal growths and pathogenicity tests

Cultivars exhibiting distinct levels of partial resistance or susceptibility, i.e. Alixan, Premio and Altigo, were selected, with resistance notes of 4, 5.5 and 7, respectively, on a scale from 0 (fully susceptible) to 9 (fully resistant) at the field level (Arvalis info, 2009). Resistance notes were obtained after two years of field trials performed by GEVES (Groupe d'Etudes des

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Variétés et Semences), the French organism officially involved in cultivar characterization before they are released on the market. Symptom intensities were assessed at several developpemental stages and statistically validated for both individual and pooled trials. Postregistration resistance traits of cultivars were then monitored for two additional years for their stability at the field level by Arvalis-Institut du Végétal, the French Technical Institute dedicated to cereals.

Wheat grains were germinated in Petri dishes on moist filter paper in darkness at 20 °C for 24 h, at 4 °C for 48 h and then at 20 °C for 24 h, and 12 germinated grains were then placed into pots filled with 3 L loam in the greenhouse at 18 °C (±2°C) with a 16 ⁄ 8 h photoperiod with supplementary illumination. After 3 weeks (when the third leaf from the base of the plant was fully expanded), plants of each pot were inoculated using hand sprayer with 30 mL of spore suspension in distillated water (10 6 spores.mL -1 ) amended with 0.05% polyoxyethylenesorbitan monolaurate (Tween 20, Sigma). Control plants were treated with sterile distillated water containing Tween20. Immediately after inoculation, each pot was covered with a clear polyethylene bag for 3 days in order to ensure a water-saturated atmosphere compatible with a good fungal germination. The disease level was scored at 21 days after inoculation (dai) by noting the percentage of the third leaf area with symptoms (chlorosis and necrosis) and by noting their pycnidial density on a scale from 0 (absence of pycnidia) to 5 [START_REF] Gao | Expression and functional analysis of a Pathogenesis-Related Protein 1 Gene, TcLr19PR1, involved in wheat resistance against leaf rust fungus[END_REF]2,3,4,5 correspond to less than 20%, 20-40 %, 40-60 %, 60-80 %, and 80-100 % of leaf areas bearing pycnidia, respectively)).

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Histopathological analysis

Monitoring of spore germination and leaf penetration by Z. tritici was performed using Fluorescence Brightener 28 (Calcofluor, Sigma) according to [START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]. Segments (2 cm) were harvested from the third fully expanded leaf at 1 and 5 dai from inoculated plants and then immersed for 5 min in a solution of 0.1 % (w ⁄ v) Calcofluor in 0.1 M Tris-HCl buffer pH 8.5. Leaf segments were washed for 2 min with sterile distillated water. After drying, they were placed on a glass slide, covered with a cover slip and observed microscopically (Nikon, Eclipse 80i) under UV illumination. Mesophyll colonization was investigated using Trypan blue (Shetty et al. 2003;[START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]) with few modifications.

Leaf segments (2 cm) were harvested from both control and inoculated leaves and cleared in a mixture of absolute ethanol: glacial acetic acid (3: 1, v ⁄ v) overnight. Cleared leaves were rehydrated for 4 h in distilled water and then fixed for 20 min in lactoglycerol (lactic acid:

glycerol: water, 1: 1: 1, v ⁄ v ⁄ v).
Staining of the fungus was carried out at 70 °C by immersing the leaf segments in 0.1% Trypan blue dissolved in lactophenol-ethanol (1: 2, v ⁄ v) for 6 h. After washing, leaf segments were stored in lactoglycerol, placed on a glass slide, covered with a cover slip and then observed microscopically under white light illumination.

Spores were recorded for each cultivar on three leaf segments and subsequently 150 randomly chosen spores were studied. Two independent experiments were performed. The percentage of germinating spores (GS) was recorded at 1dai. Total germinated spores with penetrated germ tube (PS) and within PS, penetrations through stomata (PSS) were accounted at 5 dai. At 21 dai, total colonized substomatal cavities (CS) among 150 randomly chosen cavities were recorded and, within CS, percentages of colonized substomatal cavities bearing pycnidia (CSP) were estimated according to the microscopic notations reported by [START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF].

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Protein extraction and enzymatic assays

CWDE and protease assays.

Total protein extractions were carried out at 1, 5, 11, 17 and 21 dai as previously described by Magro et al. (1984) and [START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF] with few modifications. Briefly, 5 g of inoculated and control third leaves were removed and immediately frozen in liquid nitrogen prior to grinding in a mortar. The resulting powder was then dispersed in 5 mL 0.05 M Tris-HCl buffer pH 7.8 containing 0.1 M KCl and 0.5 % cysteine. After cooling for 30 min at 4 °C, the mixture was squeezed through three layers of cheesecloth and centrifuged at 13 000 g for 20 min at 4 °C. From the supernatant, 100 µL were transferred to an Eppendorf tube and stored at 4°C for total protein quantification. Total protein extracts were then dialyzed against 0.5 L of distilled water for 24 h at 4 °C overnight (SpectrumLab Inc.). The dialyzed solution was then subjected immediately to β-1,4-endoglucanase and β-1,4-endoxylanase activity quantification using a modified 3,5-dinitrosalicylic acid (DNS) method [START_REF] Douaiher | Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonase[END_REF][START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF]. Absorbance was measured at 540 nm. Protease activity was measured according to the protocol described by Hellweg (2003) with slight modifications.

Six aliquots of 100 μL of total protein were heated to 80 °C for 10 min to inactivate enzyme activity. After cooling, 50 μL of azocasein solution (20 mg/mL Azocasein, SIGMA) and 50 μL of incubation buffer (200 mM Tris, 20 mM CaCl 2 pH 7.8) were added. After incubation at 40 °C under gentle agitation for 30 to 60 min, 500 μL of 5 % aqueous trichloroacetic acid were added to precipitate non-digested protein and the mixture was incubated for 10 min at room temperature. The precipitate was pelleted by centrifuging for 5 min at 13000 g and 500 μL of test buffer were added at 400 µL of the supernatant. Absorbance was measured at 400 nm. Total protein concentrations were determined at 595 nm using bovine serum albumin as a standard (Bradford, 1976).

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RNA extraction and gene expression relative quantification by real-time RT-qPCR

Wheat leaves were sampled at 3, 6, 12, 24, 72 and 96 hours after inoculation (hai) with Z.

tritici. The same sampling procedure was also conducted in non-inoculated conditions. All samples were immediately frozen and stored at -80 °C until use. Total RNA was extracted from 80 mg of plant tissue using RNeasy Plant Mini Kit (Qiagen, The Netherlands). Reverse transcription of total RNA was carried out using QuantiTect ® Reverse Transcription Kit (Qiagen, USA) according to the manufacturer's protocol. Real-time qPCR was performed using ABI Prism 7300 detection system (Applied Biosystems, USA). Table 1 describes primer pairs designed using the Primer Express ® program and tested for secondary structure using the AmplifX ® program. A set of 9 genes was tested: pr1 (HQ 848391), chit2 (AB029935), gluc (DQ090946), lox (U32428), aos (AY196004.1), pal (AY005474), chs (AY286097), pox2 (X85228) and oxo (AJ556991) encoding PR1, chitinase, glucanase, lipoxygenase, allene oxide synthase, phenylalanine ammonia lyase, chalcone synthase, peroxidase and oxalate oxidase respectively. The tub6 (accession number U76897) and GAPDH (accession number AF251217) genes were used as housekeeping genes (Scholtz and Visser, 2013). Cycling conditions consisted of a denaturation cycle (95 °C for 3 min) and an amplification and quantification cycle repeated 40 times (95 °C for 3 s, 60 °C for 30 s).

Expression ratios for each cDNA were calculated for each time point, relative to control at the same time using the 2 -ΔΔCt described by Livak and Schmittgen (2001), where

ΔΔCt = [Ct Target (Sample) -Ct Reference (Sample)] -[Ct Target (Control) -Ct Reference (Control)]
and Ct Reference = geometrical mean (Ct GAPDH : Ct Tub6). Similar amplification efficacies ranging between 90 and 110% were checked for all the tested primers and expressions ratio values of 2 were considered as a minimum to be significantly different from the (nI) control. The expression level of target gene in control sample sprayed with water

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Statistical analysis

Data from studies of disease scoring, infection process and enzyme activities were analyzed using ANOVA (Tukey test at P ≤0.05) assuming a normal distribution. When necessary, variances were stabilized with logarithmic transformation of data. Values correspond to the average of results obtained in two distinct experiments, on 20 and 3 leaves for disease level and infection process, respectively, and 3 measures for enzyme activity assays. Possible interaction between the two biological experiments and data was verified and no experiment in this study presented significant interaction. For gene expression studies, values correspond to the average of data from three technical repetitions from 2 biological samples. Statistical analyses were carried out with the statistical program Xlstat (Addinsoft, Paris, France).

Significant differences were evaluated using the Tukey test at P ≤0.05.

Results

Disease levels in Alixan, Premio and Altigo

In order to evaluate their resistance levels, the three cultivars Alixan, Premio and Altigo were inoculated with the T01193 Z. tritici strain and disease severity was assessed at 21 dai on the third leaf (Figure 1). Percentages of leaf area with symptoms recorded in Alixan (41.7 %) and in Premio (42.5 %) were significantly higher than in Altigo (27.7 %). In Alixan, the pycnidial density (2.9) was significantly higher than in Premio (1.1) and Altigo (1.1).

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Infectious process

Five cytological parameters related to the development of Z. tritici were quantified in Alixan, Premio and Altigo at various time points. Table 2 shows percentages of germinated spores (GS) at 1 dai, percentages of total germinated spores with penetrating germ tube (PS) and penetrations through stomata (PSS) at 5 dai, and at 21 dai, the percentage of total colonized substomatal cavities (CS) and within CS, the percentage of CS bearing pycnidia (CSP).

Alixan, Premio and Altigo cultivars presented close and non-significantly different percentages of GS at 1dai. At 5 dai, percentages of PS were significantly higher in Alixan and Premio (52.9 % and 55.2 %) than in Altigo (29.3 %). However, no significant differences were recorded for the percentages of PSS and no significant differences were obtained for the percentage of direct penetration attempts through the epidermis (data not shown). The percentage of CS recorded at 21 dai presented no significant difference between Alixan, Premio and Altigo, while the percentages of CSP recorded in Alixan (19.4 %) and Premio (23.9 %) were significantly higher than in Altigo (5.2 %).

CWDEs activities

β-1,4-endoxylanase, β-1,4-endoglucanase and protease activities were monitored in leaves during the infection process at 1, 5, 11, 17 and 21 dai (Figure 2). At 1, 5 and 11 dai, these three enzymes showed weak activity levels which were not significantly different from those obtained in non-inoculated control plants for each cultivar.

In Alixan, all enzymatic activities increased significantly in inoculated plants from 17 dai. At 21 dai, they reached higher values than those measured in non-inoculated plants (17 fold, 7 fold and 72 fold higher for β-1,4-endoxylanase, β-1,4-endoglucanase and protease,

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This article is protected by copyright. All rights reserved. respectively). In Premio, β-1,4-endoxylanase and β-1,4-endoglucanase significantly increased by 17 dai, while protease activity was induced at 21 dai only. In Altigo, all activities were not modified in inoculated plants at 17 dai, but were produced at 21 dai.

The comparison between inoculated cultivars at 21 dai showed that values obtained in Alixan were 4.5 and 7.2 fold higher for β-1,4-endoxylanase than those obtained in Premio and Altigo respectively, 3.4 and 4.1 fold higher for β-1,4-endoglucanase and 3.7 and 5.2 fold higher for protease. Activities measured in Premio and Altigo were not significantly different, excepted for β-1,4-endoxylanase which was 1.6 fold higher in Premio.

Induction of wheat defense mechanisms by Z. tritici

Basal expression levels of defense-related genes in the three cultivars.

We established basal transcript profiles of a set of nine distinct genes associated with four types of wheat defenses: ROS metabolism (pox2, oxo), octadecanoid (lox, aos), phenylpropanoid (pal, chs) pathways and PR proteins biosynthesis (pr1, chit2, gluc) (Table 3). For a meaningful biological approach, significantly differentially expressed transcripts were reported according to a strict selection criterion; namely, a ≥2.0-fold change threshold.

Gene expression profiles in non-inoculated (nI) plants of Premio and Altigo were compared with Alixan (nI) controls. The basal levels of pr1, gluc and aos genes in both Premio and Altigo were significantly higher than in Alixan (approximately 10, 10 and 4-fold respectively), whereas those of chit2, pal, pox2 and oxo were significantly higher in Premio than in both Alixan and Altigo (approximately 2, 3, 2 and 5-fold compared to Alixan, respectively). On the other hand, concerning chs, the basal transcription level recorded in both Premio and in Altigo was lower than in Alixan (approximately 0.6-fold).

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Induction of defense-related genes by Z. tritici in Alixan, Premio and Altigo.

Transcript profiles in inoculated (I) plants of each cultivar were followed at 3, 6, 12, 24, 72 and 96 hai and were compared to their respective (nI) controls, and between (I) Alixan, Premio and Altigo plants ( Figures 3, 4, 5 and6). For a meaningful biological approach, significantly differentially expressed transcripts were reported according to a strict selection criterion; namely, a ≥2.0-fold change threshold.

Concerning ROS metabolism, the pox2 gene was induced in the Alixan cultivar all over the time course, except at 12 hai (Figure 3). This induction ranged from 7.4-to 24.8-fold compared to the (nI) control. On the other hand, the pox2 expression was induced at 3, 12 and 24 hai in Premio (from 6.6-to 16.9-fold) and at 12 hai only in Altigo (11.3-fold). Moreover, pox2 expression was significantly higher in Alixan compared to both Premio and Altigo at 6, 24, 72 and 96 hai. Likewise, the oxo gene expression was induced in Alixan at 6, 24 and 72 hai (from 3.8-to 6.4-fold), whereas in Premio, it increased from 12 to 72 hai (from 3.1-to 29.9-fold) and in Altigo at 6 and 24 hai (3.3-and 4.5-fold). The highest expression level of oxo gene was measured in Premio 12 hai.

Regarding the octadecanoid pathway, Premio showed an early induction of lox expression at 3 and 6 hai, 2.4-and 7-fold compared to the (nI) control (Figure 4). This gene expression was induced later in Alixan (2.4-fold 72 hai), and no up-regulation was observed in Altigo. In addition, the lox expression exhibited down regulations in Alixan 12 hai, and 24 hai in the three wheat cultivars. The aos gene expression was only induced in Premio 72 hai (2.7-fold), while it was down regulated in Premio and Altigo 24 hai, and in Alixan and Premio 96 hai.

Concerning the phenylpropanoid pathway, an induction of the pal gene expression was observed in Premio at 3, 6 and 12 hai from 3-to 16.6-fold compared to the (nI) control (Figure 5). On the other hand, the pal expression was induced later at 6 and 72 hai in Alixan

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(5.6-fold) and at 3 hai only in Altigo (4.5-fold). Regarding the chs gene, Premio showed an early induction of expression 12 hai (10.4-fold) and this gene was induced later in Alixan, at 96 hai (4.5-fold), but not in Altigo. The chs expression exhibited a down-regulation in Alixan 12 hai, and in Alixan and Altigo 24 hai. Moreover, the highest expression levels of both pal and chs genes were measured in Premio (16.6-and 10.4-fold, respectively) at 12 hai.

Concerning the PR-proteins, the pr1 gene expression in Alixan showed an induction all over the time course except at 6 and 12 hai, which ranged from 7.9-to 21.8-fold compared to the (nI) control (Figure 6). In Premio, this gene expression was induced at 6 and 96 hai, from 5.3to 6.2-fold, and no significant induction was observed in Altigo. Moreover, the pr1 expression level was significantly higher in Alixan than in both Premio and Altigo at 3, 24, 72 and 96 hai. Similarly, the chit2 gene expression was significantly induced in Alixan all over the time course, except at 6 and 12 hai, and the most drastic inductions were measured at 3 and 24 hai (10.1-and 38.1-fold, respectively). In Premio, this gene expression was slightly induced at 12 and 72 hai (from 3.9-to 4.1-fold) and no significant induction was observed in Altigo. The expression level of the gluc gene increased in Premio at 6 hai, in Altigo at 12 hai and in Alixan at 24 hai (7.4, 2.6 and 2.4-fold, respectively).

Discussion.

Wheat non-specific resistance expressed during a compatible interaction with Z. tritici is more durable, but harder to select and much less studied than specific resistance [START_REF] Chartrain | Partial resistance to septoria tritici blotch ( Mycosphaerella graminicola ) in wheat cultivars Arina and Riband[END_REF]Orton et al., 2011). Three wheat cultivars (susceptible, moderately and partially resistant) have been compared in the present study in order to characterize mechanisms involved in such a resistance against Z. tritici.

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Defense mechanism expression during compatible interaction varies depending on the wheat cultivar

In literature, studies often refer to comparisons between compatible and incompatible interactions, in order to highlight mechanisms involved in wheat specific resistance against Z. tritici. Molecular studies described in resistant cultivars an early up-regulation of defenserelated genes, e.g. those encoding for phenylalanine ammonia-lyase, peroxidase, chitinase, and various other pathogenesis-related (PR) proteins such as PR-1, PR-2 (for β-1,3endoglucanase), and PR-5 (for thaumatin-like protein). On the other hand, little or no induction of these genes was observed in susceptible cultivars during the first days after infection (Shetty et al., 2003;Ray et al., 2003;[START_REF] Adhikari | Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression[END_REF]. Recently, a transcriptome analysis during a compatible interaction revealed that many wheat genes related to defense responses were down-regulated 1 day after leaf inoculation with Z. tritici (Rudd et al., 2015). In our study, although Altigo is more resistant than both other studied cultivars, most of targeted genes were not induced or were repressed in this cultivar during the first 24 hours after infection, except for gluc, oxo, pox2 and pal genes at rare time points, By contrast, high levels of induction were observed in Alixan during the same period of time and until 4 dai, especially for pox2, chit2, and PR1, while this cultivar was the most susceptible cultivar among the three studied one. Similarly, in the transcriptomic study of a compatible Z. tritici-wheat interaction, Yang et al. ( 2013) noticed an up-regulation at 4 dai of some defense genes such as PR2, PR3 (chitinase) and genes involved in ROS metabolism.

Interestingly, in Premio, PR1, chit2, oxo and pox2 were moderately induced compared to Alixan. In Premio, the necrosis development recalled that observed in Alixan, while sporulation is reduced like in Altigo. Moreover, the percentages of direct penetration attempts through the epidermal cell wall were higher in Alixan and Premio. In various studies, the penetration of Z. tritici via stomata was described as more frequent than direct penetration

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This article is protected by copyright. All rights reserved. through the epidermis (Kema et al., 1996;Shetty et al., 2003;Selim et al., 2013). In Alixan and Premio, the direct penetration attempts may be responsible for the higher defense gene expression observed during the first hours after infection.

However, despite a stronger induction of genes usually involved in PTI, Alixan exhibited an important infection rate. In this cultivar, potential effectors released by the fungus could block or suppress the resulting induced defense, therefore facilitating fungal growth during the biotrophic phase and leading to subsequent important diseases lesions, as suggested by Yang et al. (2013) andRudd et al. (2015). Apoplastic fungi such as Z. tritici are known to overcome basal plant induced defense involved in PTI by defensive strategies, for instance by releasing chitin binding effectors (Stotz et al., 2014). Indeed, during its endophytic phase of colonization, Z. tritici produces the Mg1LysM and Mg3LysM effectors, which bind chitin and protect hyphae against plant chitinases (Marshall et al., 2011). Mg3LysM-encoding gene was shown to be up-regulated from 1 day after infection in a susceptible cultivar (Rudd et al, 2015). In addition, this effector blocks the elicitation of chitin-induced plant defenses mediated by both chitin elicitor binding protein (CEBiP) and chitin elicitor receptor kinase (CERK) in wheat (Lee et al., 2014;Marshall et al., 2011). In Alixan, a first induction of chit2 was observed at 3 hai, followed by a stronger one at 24 hai. In summary, Z. tritici probably overcomes defenses mediated by chitin release during the fungal cell wall degradation by wheat chitinases, mainly from 1 dai in such a susceptible cultivar. ROS accumulation is considered as one of the major primary responses during the Z. triticiwheat interaction. However, these compounds are known to accumulate during the necrotrophic phase in compatible interactions, while their synthesis occurs earlier during incompatible interactions. The accumulation of hydrogen peroxide (H 2 O 2 ) is thought to arrest wheat penetration by biotrophic fungi and to inhibit colonization by Z. tritici in resistant cultivars (Shetty et al., 2003[START_REF] Shetty | Role of hydrogen peroxide during the interaction between the hemibiotrophic fungal pathogen Septoria tritici and wheat[END_REF]. In the three studied susceptible cultivars and mainly in

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Alixan, both oxo and pox2 expressions were induced after infection. Oxalate oxidase produces H 2 O 2 by the conversion of oxalic acid and O 2 [START_REF] Bolwell | Mechanisms for the generation of reactive oxygen species in plant defence -a broad perspective[END_REF] and is considered as a marker of pathogen infection in powdery mildew-cereal interactions. Our results suggest that this enzyme may be involved in the Z. tritici-wheat interaction as well.

However, since peroxidases use H 2 O 2 as a substrate during defense responses (O'Brien et al., 2012;Passardi et al., 2005;Torres and Dangl, 2005), the up-regulation of pox2 suggests that H 2 O 2 does not accumulate. Interestingly, the stronger and long-lasting induction of pox2 in the most susceptible cultivar Alixan indicates that such up-regulations may be considered as a physiological reaction to infection rather than an efficient defense reaction. This gene induction was previously described in wheat in response to powdery mildew infection [START_REF] Båga | Molecular cloning and expression analysis of peroxidase genes from wheat[END_REF].

On the contrary, pal expression was induced from 3 hai untill 12 hai in Premio, and was followed by an induction of chs at 12 hai, suggesting a potential synthesis of flavonoids in this cultivar. Resistance-related metabolites belonging to the flavonoids chemical class were recently identified in barley in response to Fusarium graminearum [START_REF] Bollina | Identification of metabolites related to mechanisms of resistance in barley against Fusarium graminearum, based on mass spectrometry[END_REF]. In Alixan, a slight biphasic induction of pal occurred (at 6 and 72 hai), while a 4-fold induction of pal was observed in Altigo 3 hai. Comparing wheat cultivars, [START_REF] Adhikari | Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression[END_REF] showed that a pal gene was induced during the first hours after infection by Z. tritici in a fully resistant cultivar, while its induction was less significant in a distinct other one and undetected in susceptible cultivars. The phenylpropanoid pathway may therefore be involved in PTI in wheat against Z. tritici in a cultivar-dependant manner. By contrast, the octadecanoid pathway is either slightly stimulated or even repressed, during the first steps of the interactions studied here. Acting upstream of the octadecanoid pathway, the lipoxygenase activity catalyses the dioxygenation of polyunsaturated fatty acid and leads, when followed by alene oxide synthase encoded by aos gene, to the downstream biosynthesis of jasmonic

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This article is protected by copyright. All rights reserved. acid (JA) [START_REF] Creelman | Biosynthesis and action of jasmonates in plants[END_REF]. In Alixan and Altigo cultivars, lox gene has not been up-regulated in the first hours after infection, and its expression was repressed at 12 and 24 hai in both cultivars. In Premio, a similar repression was observed at 24 hai, after an induction at 3 and 6 hai, recalling results obtained in a fully resistant cultivar by Ray et al. (2003). Similarly, aos was repressed at 24 hai in Premio and Altigo. Besides, Rudd (2015) described a down regulation 1 dai of five genes noted as OPDA reductases, enzymes that are involved in JA synthesis in a susceptible cultivar.

Using three susceptible cultivars, we showed here that basal defense during the biotrophic phase of the infectious process varies between genotypes, and probably results from a given expression pattern of various genes rather than from important specific up-regulations.

Nevertheless, in non-infectious context, the basal expression level of PR1, gluc and aos was higher in both Premio and Altigo than in Alixan, with means of 10-fold for PR1 and gluc, and 4-fold for aos. Such a constitutive expression could participate to the natural basal resistance of Premio and Altigo expressed during the first steps of the biotrophic phase of fungal development.

Wheat basal resistance levels differently affect symptom development and fungal sporulation

The extent of symptom development and sporulation of Z. tritici have been shown to depend on the resistance level of the host [START_REF] Cohen | The histology of processes associated with the infection of resistant and susceptible wheat cultivars with Septoria tritici[END_REF]Kema et al., 1996;[START_REF] Duncan | Cytological analysis of wheat infection by the leaf blotch pathogen Mycosphaerella graminicola[END_REF]. The range of symptoms caused by the T01193 Z. tritici strain used in the present study on Alixan, Premio and Altigo wheat cultivars is in accordance with disease levels previously reported for these cultivars in field conditions. Indeed, resistance to STB was scored 4, 5.5 and 7 in Alixan, Premio and Altigo respectively, on a scale from 1

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(susceptible) to 9 (resistant), in 2009 field trials by the French technical institute Arvalis-Institut du végétal. Under semi-controlled conditions, Altigo exhibited at 21 dai the lowest level of diseased leaf area with both symptoms and pycnidia, thus confirming its highest resistance level against Z. tritici. Levels of symptoms and sporulation recorded on the most susceptible cultivar, Alixan, were both higher than in Altigo. Interestingly, the moderately resistant cultivar, Premio, showed a percentage of leaf area with symptoms close to that of the susceptible wheat cultivar Alixan, and a pycnidial density similar to that of the resistant cultivar Altigo. Our microscopic study highlighted the stages of fungal infectious process that were different between the three cultivars. The rate of stomatal penetration (PSS) was similar among the three cultivars, which exhibited the same frequency of colonized substomatal cavities (CS) at the histological level at 21 dai. At the macroscopic level, necrosis was reduced in Altigo in comparison to Alixan and Premio. This reduction of macroscopic symptoms suggests an alteration in the switch to the necrotrophic phase, which can not be related to the extent of host colonization. In addition, the percentage of penetrating spore (PS) is reduced in Altigo in comparison with Alixan and Premio. This suggests that defense mechanisms take place earlier in Altigo than in Premio and Alixan, delaying the switch to necrotrophic phase. Moreover, the proportion of CS bearing pycnidia (CSP) within the total CS was found to be lower in Altigo only, while at the macroscopic level, both Premio and Altigo displayed similar and low sporulation levels. At the macroscopic level, only mature pycnidia are visible and could be considered in the assessment of the sporulation, whereas at the microscopic level, all pycnidia -young and mature -are visible and accountable. It can be suggested that late defense mechanisms in Altigo inhibit or delay both necrosis and pycnidium formation, whereas in Premio, they only slow down pycnidium development in substomatal cavities. This point is also interesting since at the field level, the reduction of sporulation contributes to reduce spore dissemination. All together, these results suggest that

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This article is protected by copyright. All rights reserved. the distinct basal resistance levels of wheat cultivars against Z. tritici may also depend on defense mechanisms expressed lately, as described by Rudd et al. (2015), and involved either in symptom or in sporulation inhibitions.

It is important to underline that defense mechanisms involved in wheat resistance also take place during the transition to the necrotrophic phase and during symptoms expression. Rudd et al ( 2015) described a peak in wheat defense activation during the fungal necrotrophic colonization phase. In parallel, fungal genes were highly expressed, such as those encoding for secreted CWDEs, including first proteases, followed by carbohydrate degrading enzymes (CAZymes). It has been previously shown that the activity of CWDEs was associated with Z. tritici pathogenicity and impacted the disease level of wheat [START_REF] Douaiher | Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonase[END_REF][START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF][START_REF] Tian | Correlation between cell wall-degrading enzymes in wheat leaves infected by Septoria tritici and disease severity[END_REF]. In the present work, we highlighted significantly higher levels of β-1,4endoxylanase, β-1,4endoglucanase and protease activities in the three cultivars from 17 dai, date from which symptoms can be observed (data not shown). At 21 dai, they reached their highest activity levels in Alixan and were associated with the highest pycnidium density.

Therefore, β-1,4-endoxylanase, β-1,4endoglucanase and protease activities seem to correlate with the degree of fungal sporulation, suggesting their involvement in providing the fungus nutrients for asexual reproduction. These observations confirm results of [START_REF] Siah | Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[END_REF] and [START_REF] Tian | Correlation between cell wall-degrading enzymes in wheat leaves infected by Septoria tritici and disease severity[END_REF], where CWDEs produced by Z. tritici were associated with the sporulation level of the fungus, and negatively correlated with the resistance level of wheat.

In conclusion, the occurrence of susceptible wheat response to Z. tritici infection is clearly cultivar-dependent However, an important and early expression of defense-related genes is not associated with a reduction of necrosis development when comparing susceptible or partially resistant wheat cultivars, suggesting that the pathogen manipulates defense gene

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Figures legends Figure 1 .

 legends1 Figures legends

Figure 2 .

 2 Figure 2. Time course of fungal β-1,4-endoxylanase (a), β-1,4-endoglucanase (b) and

Figure 3 .

 3 Figure 3. Relative expression of pox2 (peroxidase) and oxo (oxalate oxidase) wheat genes in

Figure 4 .

 4 Figure 4. Relative expression of lox (lipoxygenase) and aos (allen oxide synthase) wheat

Figure 5 .

 5 Figure 5. Relative expression of pal (phenylalanine ammonia lyase) and chs (chalcone

Figure 6 .

 6 Figure 6. Relative expression of pr1, chit2 and gluc wheat genes in leaves of Alixan, Premio

Table 1 .

 1 Selected genes, their accession numbers (GenBank) and their corresponding primer sets, used for the analysis of transcript profiles from wheat leaves.

	Name	GenBank	Primer Foward 5'-3'	Tm	Primer Reverse 5'-3'	Tm	Amplicon
		Accession		(°C)		(°C)	lengh (pb)
		n°
	tub6	U76897	CTGCCTCCAAGGTTTCCAAGTA 59 .2	GTGTCCATCCCGGAACCA	59.7	53
	gapdh	AF251217	AAGGGCATTTTGGGTTACGTT 58.4 CCTGTTGTCACCCTGGAAGTC 58.1	53
	pr1	HQ848391 CATGCACCTTCGTATGCCTAACT 58.9 TGGCTAATTACGGCATTCCTTT 59.4	52
	chit.2	AB029935	GGGTGGACCTGCTGAACAAT	58.4 AGAACCATATCGCCGTCTTGA 58.3	75
	gluc.	DQ090946 TCCTGGGTTCAGAACAATGTCC 59.8 TTGATGTTGACAGCCGGGTAGT 60.4	50
	lox	U32428	GGGCACCAAGGAGTACAAGGA 59.9	GCTCGTGATGGTGTGGATGA	59.1	66
	aos	AY196004	AGGCCGGAGAGAAGTTCCAC 59.3	CCGACTTGGTCAGCTCCATC	59.2	119
	pal	AY005474 GTCGATTGAGCGTGAGATCAAC 58.0	CACGGGAGACGTCGATGAG	59.0	59
	chs	AY286097	GCGCCTGCGTACTCTTCATC	60.0	CCTCGGCGGAGCGTTT	59.0	51
	pox2	X85228	TGCTTTGTCCAAGGCTGTGA	59.0	GACCCGCGTTTTGTTCCA	59.1	61
	oxo	AJ556991	GCCAGAACCCCGGTATCG	60.0 GGTGGGTTGGAGCTGAAGAG 58.0

Table 3 .

 3 Relative expression of pr1, chit2, gluc, lox, aos, pal, chs, pox2 and oxo genes in non-inoculated leaves of Premio and Altigo compared to Alixan. For each gene, means in bold are significantly different from Alixan using the Tukey test at P ≤ 0.05. The expression level of target gene in Alixan used for relative expression was considered to be 1.

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This article is protected by copyright. All rights reserved. expression in susceptible cultivars during the first steps of infectious process in order to promote disease development. A similar mesophyll colonization that is followed by a delayed necrosis development and/or delayed sporulation of Z. tritici suggests a distinct regulation of wheat defense genes in the three susceptible cultivars at the transition phase to necrotrophic lifestyle. Basal levels of expression of genes encoding for QDR-associated genes are likely to be more accurate markers for the expression and the selection of partial but more durable resistance to Z. tritici in wheat.

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This article is protected by copyright. All rights reserved. resistance on Mycosphaerella graminicola and its sterol 14α-demethylation-inhibitorresistant genotypes. Pest Manag. Sci. 70, 60-69. Shetty, N.P., Kristensen, B.K., Newman, M.-A., Møller, K., Gregersen, P.L., and Jørgensen, H.J.L. (2003). Association of hydrogen peroxide with restriction of Septoria tritici in resistant wheat. Physiol. Mol. Plant Pathol. 62,[333][334][335][336][337][338][339][340][341][342][343][344][345][346].

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This article is protected by copyright. All rights reserved. Wu, J.-S., Chong, K., Xu, Y.-Y., and Tan, K.-H. (2004). Cloning and characteristics of an allene oxide synthase gene (TaAOS) of winter wheat. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30, 413-420. Yang, F., Melo-Braga, M.N., Larsen, M.R., Joergensen, H.J.L., and Palmisano, G. (2013).

Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics. Mol. Cell. Proteomics mcp.M113.027532.

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This article is protected by copyright. All rights reserved. Goodwin, S.B., Ben M'Barek, S., Dhillon, B., Wittenberg, A.H.J., Crane, C.F., Hane, J.K., Foster, A.J., Van der Lee, T.A.J., Grimwood, J., Aerts, A., et al. (2011). Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis. PLoS Genet 7, e1002070.

Hammond-Kosack, K.E., and Rudd, J.J. (2008). Plant resistance signalling hijacked by a necrotrophic fungal pathogen. Plant Signal. Behav. 3, 993-995. Hellweg, M. (2003). Molecular biological and biochemical studies of proteolytic enzymes of the cereal pathogen Fusarium graminearum. PhD thesis, University of Münster, Deutschland, 240 pages. Kema, G.H.J., Yu, D., Rijkenberg, F.H.J., Shaw, M.W., and Baayen, R.P. (1996). Histology of the pathogenesis of Mycosphaerella graminicola in wheat. Phytopathology 86, 777-786. Keon, J., Rudd, J.J., Antoniw, J., Skinner, W., Hargreaves, J., and Hammond-Kosack, K.

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This article is protected by copyright. All rights reserved. Selim, S., Roisin-Fichter, C., Andry, J.-B., Bogdanow, B., and Sambou, R. (2014). Real-time PCR to study the effect of timing and persistence of fungicide application and wheat varietal

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Klara Kovarski
email: klara.kovarski@parisdescartes.fr

Joëlle Malvy

Raoul K Khanna

Sophie Arsène

Magali Batty

Marianne Latinus

Reduced visual evoked potential amplitude in autism spectrum disorder, a variability effect?

Atypical sensory behaviours represent a core symptom of autism spectrum disorder (ASD). Investigating early visual processing is crucial to deepen our understanding of higher-level processes. Visual evoked potentials (VEPs) to patternreversal checkerboards were recorded in ASD children and age-matched controls. Peak analysis of the P100 component and two types of single-trial analyses were carried out. P100 amplitude was reduced in the ASD group, consistent with previous reports. The analysis of the proportion of trials with a positive activity in the latency range of the P100, measuring inter-trial (in)consistency, allowed identifying two subgroups of ASD participants: the first group, as control children, showed a high inter-trial consistency, whereas the other group showed an inter-trial inconsistency. Analysis of median absolute deviation of single-trial P100 (st-P100) latencies revealed an increased latency variability in the ASD group. Both single-trial analyses revealed increased variability in a subset of children with ASD. To control for this variability, VEPs were reconstructed by including only positive trials or trials with homogeneous st-P100 latencies. These control analyses abolished group differences, confirming that the reduced P100 amplitude results from increased inter-trial variability in ASD. This increased variability in ASD supports the neural noise theory. The existence of subgroups in ASD suggests that the neural response variability is not a genuine characteristic of the entire autistic spectrum, but rather characterized subgroups of children. Exploring the relationship between sensory responsiveness and inter-trial variability could provide more precise bioclinical profiles in children with ASD, and complete the functional diagnostic crucial for the development of individualized therapeutical projects.

Introduction

Autism spectrum disorder (ASD) is characterized by (i) disturbances in the social and communication domain, and by (ii) repetitive and restricted behaviours. Within this second group of symptoms, sensory abnormalities, as acknowledged in the Diagnostic and Statistical Manual-5 (DSM-5) are now considered a diagnostic feature of ASD [START_REF]APA. Diagnostic and Statistical Manual of Mental Disorders (DSM-5)[END_REF] . These might be defined as hypo-or hyperresponsiveness in all sensory modalities, making atypical sensory behaviours highly heterogeneous both within and between autistic individuals. In the visual modality, children with ASD might be fascinated by lights, they might stimulate their eyes with their hands or might inspect objects in atypical manners [START_REF] Mottron | Lateral glances toward moving stimuli among young children with autism: early regulation of locally oriented perception?[END_REF] . In parallel, autistic individuals might also avoid challenging stimuli, such as others' gaze or scenes rich in information. In line with this, an increasing number of investigations using electroencephalography (EEG) have revealed that visual abnormalities are found when social but also non-social stimuli are presented: from atypical evoked-related potentials (ERPs) to atypical frequency-band responses, to stimuli such as faces, gabors and gratings [START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF][START_REF] Constable | Motion and pattern cortical potentials in adults with high-functioning autism spectrum disorder[END_REF][START_REF] Milne | Independent component analysis reveals atypical electroencephalographic activity during visual perception in individuals with autism[END_REF][START_REF] Weinger | Low-contrast response deficits and increased neural noise in children with autism spectrum disorder[END_REF][START_REF] Kovarski | Brief report: early VEPs to pattern-reversal in adolescents and adults with autism[END_REF][START_REF] Frey | Atypical cortical representation of peripheral visual space in children with an autism spectrum disorder[END_REF][START_REF] Siper | Rapid and objective assessment of neural function in autism spectrum disorder using transient visual evoked potentials[END_REF] . Atypical brain responses are additionally supported by structural abnormalities in visual regions [START_REF] Anagnostou | Review of neuroimaging in autism spectrum disorders: what have we learned and where we go from here[END_REF] , as revealed by different size and organization of micro-columns in those with ASD compared with controls [START_REF] Casanova | Minicolumnar abnormalities in autism[END_REF] . Taken together, these findings have been acknowledged by recent reviews, suggesting that investigating early sensory responses might provide crucial information to understand higherlevel disturbances [START_REF] Thye | The impact of atypical sensory processing on social impairments in autism spectrum disorder[END_REF][START_REF] Robertson | Sensory perception in autism[END_REF] .

Among the different visual evoked potentials (VEPs) used for testing the integrity of the visual pathways, pattern-reversal checkerboards VEPs provide highly reliable waveforms. Accordingly, this visual response, recorded over Oz, represents a clinical tool to test the functional integrity of the visual system at the individual level [START_REF] Odom | ISCEV standard for clinical visual evoked potentials (2009 update)[END_REF] . In a previous study using pattern-reversal checkerboards VEPs, we showed that P100 amplitude was decreased in adolescents and young adults with ASD [START_REF] Kovarski | Brief report: early VEPs to pattern-reversal in adolescents and adults with autism[END_REF] . However, as ASD is a neurodevelopmental condition, investigating brain responses in young children is necessary to understand the developmental trajectory of this condition and to determine whether this reduced response is a genuine feature of ASD. More precisely, if a reduced P100 amplitude is also found in younger subjects, this leads to conclude that this atypicality is an early feature of autism. However, the reduced P100 in adults could result from a progressive modification of sensory processing in childhood as a consequence of the difficulty to deal with sensory information in everyday life; under this assumption, no reduction of amplitude would be observed in young children. This is particularly important as previous studies of VEPs with gratings in school-aged children and adolescent did not report differences in VEPs between typically developing (TD) and ASD children [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF] , which could suggest that VEPs alter with age.

Easy to record in young participants, these VEPs represent an ideal tool to evaluate early stages of visual processing in those with ASD, especially at the low-end of the spectrum, often neglected in research studies [START_REF] Brown | Vision research literature may not represent the full intellectual range of autism spectrum disorder[END_REF][START_REF] Chakrabarti | Commentary: critical considerations for studying lowfunctioning autism[END_REF][START_REF] Tager-Flusberg | Minimally verbal school-aged children with autism spectrum disorder: the neglected end of the spectrum[END_REF] . Although ERPs might be easily computed, they reflect an average response that masks inter-trial variability, known to be an important feature of ASD [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Dinstein | Unreliable evoked responses in autism[END_REF][START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF][START_REF] Milne | Atypical EEG in autism spectrum disorder: comparing a dimensional and a categorical approach[END_REF] and in other neurodevelopmental disorders such as Attention Deficit Hyperactivity Disorder (ADHD) [START_REF] Weinger | Low-contrast response deficits and increased neural noise in children with autism spectrum disorder[END_REF][START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Dinstein | Unreliable evoked responses in autism[END_REF][START_REF] Gonen-Yaacovi | Increased ongoing neural variability in ADHD[END_REF][START_REF] Dinstein | Normal movement selectivity in autism[END_REF] . In ASD, this increased variability has been explained by an increased neural noise, as described by the neural noise theory [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Rubenstein | Model of autism: increased ratio of excitation/inhibition in key neural systems[END_REF][START_REF] Simmons | Response to Davis and Plaisted-Grant: low or high endogenous neural noise in autism spectrum disorder?[END_REF][START_REF] Simmons | Vision in autism spectrum disorders[END_REF][START_REF] Dinstein | Neural variability: friend or foe?[END_REF] (but see ref. [START_REF] Butler | An examination of the neural unreliability thesis of autism[END_REF] ). This account interprets response variability reported in neuroimaging studies as a signature of abnormalities in the synchronization of neural activity, which might be crucial in ASD [START_REF] Dinstein | Neural variability: friend or foe?[END_REF] . Accordingly, single-trial analyses of EEG data represent a useful tool in order to assess neural response variability. However, only few previous studies have investigated neural responses variability in children with ADHD [START_REF] Gonen-Yaacovi | Increased ongoing neural variability in ADHD[END_REF] and in school-aged children and adolescents with ASD [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] .

Although response variability has been previously described in school-age children and adolescents with autism [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF] , here we deepen our understanding by linking differences in VEPs to inter-trial variability [START_REF] Kovarski | Brief report: early VEPs to pattern-reversal in adolescents and adults with autism[END_REF] . In addition, investigating this variability of visual responses in children with ASD might help provide further information on the developmental aspects of early visual impairments, but also on the compensatory mechanisms that might develop with age [START_REF] Livingston | Conceptualising compensation in neurodevelopmental disorders: reflections from autism spectrum disorder[END_REF] .

Materials and methods

Participants

Children were recruited from the Child Psychiatry Department of the University Hospital of Tours, France. The initial ASD group included 29 children diagnosed with ASD by an experienced clinical team according to DSM-5 criteria [START_REF]APA. Diagnostic and Statistical Manual of Mental Disorders (DSM-5)[END_REF][START_REF]APA. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR)[END_REF] , and Autism Diagnostic Interview-Revised and/ or the Autism Diagnostic Observation Schedule-generic assessments [START_REF] Lord | The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism[END_REF][START_REF] Lord | Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders[END_REF] . Epilepsy was the exclusion criterion. The ASD group received additional comprehensive ophthalmological and orthoptic examinations. These included tests for visual acuity refractive errors measurements under cycloplegia, as well as a slit-lamp and fundus examination, and an oculomotor exam. Seven children diagnosed with an ophthalmological disorder that could affect VEP responses were not included in the analysis (i.e., refractive errors, strabismus and amblyopia). Three other children were not included in the experiment, because they could not tolerate the electrodes cap, and the recording of one child was excluded because of an insufficient number of trials after artifacts rejection. The final group included 18 children aged 42 to 130 months (mean age ± SD: 89 ± 32 months; 1 girl and 17 boys; Childhood Autism Rating Scale scores from 24.5 to 38, mean: 30 ± 4.4) [START_REF] Schopler | Toward objective classification of childhood autism: Childhood Autism Rating Scale (CARS)[END_REF] . The ASD group was matched for chronological age to a group of eighteen TD children aged 37 to 143 months (mean age: 90 ± 31 months, t (34) = -0.14, P = 0.89; 3 girls and 15 boys). The local ethics committee approved the study (Clinical Trial: NCT02444117) and informed written consent was obtained from the parents of the children. The study was conducted according to the ethical principles of the Declaration of Helsinki.

Procedure

Participants were seated in a chair 110 cm away from the screen and were presented with black and white pattern-reversal stimulations (1 Hz frequency presentation) for a maximum of 2 min. The stimulus consisted in 11 × 11 checks that fitted a screen of 19.8°(wide) × 15.6°( high) visual angle. Each check measured 1.8°x 1.4°v isual angle.

EEG recording and processing

A 64-channel ActiveTwo system (Biosemi ® , The Netherlands) was used for EEG recording. Blinks and saccades were monitored using electrodes placed at the outer canthi of the eyes and below the left eye. For offline reference an electrode was applied to the tip of the nose. The signal was recorded at a sampling frequency of 512 Hz and filtered at 0-104 Hz. A 0.3 Hz digital high-pass filter was applied to the EEG signal. Ocular artifacts were corrected by applying independent component analysis (ICA) as implemented in EEGLab [START_REF] Delorme | EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis[END_REF] , Matlab, The Mathworks, Inc. Blink artifacts were captured into components that were automatically removed via the inverse ICA transformation. Other artifacts (i.e., muscle and movements) were rejected manually with the Elan software [START_REF] Aguera | ELAN: a software package for analysis and visualization of MEG, EEG, and LFP signals[END_REF] . Continuous EEG signal was time-locked to trial onset and trials were extracted between -100 ms pre-stimulus and 400 ms post-stimulus. Baseline correction (-100 to 0 ms) was applied and ERPs were digitally filtered with a low-pass frequency cut-off of 30 Hz.

Data were re-referenced offline to the potential of the tip of the nose. The number of trials averaged to compute ERPs was 110.4 ± 30.8 in ASD children and 99.5 ± 16.8 in TD children; the groups did not differ (t(34) = 1.32, P = 0.19; see ref. [START_REF] Odom | ISCEV standard for clinical visual evoked potentials (2009 update)[END_REF] ). Single trials were extracted with Matlab from the preprocessed data (all steps but baseline correction and low-pass filtering). Baseline correction was performed on the 100 ms preceding stimulus onset and a low-pass filter (cut-off frequency: 30 Hz) was applied on each trial using an EEGLab function (eegfilt with a Finite Impulse Response).

Statistical analysis

Three occipital electrodes were selected for the analyses: O1, Oz and O2. These electrodes were selected based on recommendations regarding analysis of pattern-reversal VEPs [START_REF] Odom | ISCEV standard for clinical visual evoked potentials (2009 update)[END_REF] and on previous studies reporting hemispherical differences between ASD and TD individuals [START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF] .

Two analyses were carried out: the first was based on the usual peak latency and amplitude (ERPs analysis), whereas the second aimed at considering intra-participant variability by looking at inter-trial variability (single-trial analyses).

ERPs analysis

P100 was defined as the greatest positive deflection in the 70-140 ms time window. Two analyses of variance (repeated-measure analysis of variance (ANOVA); one for amplitude and one for latency) were carried out on the P100 with Group (ASD and TD) as a between-subject factor and Electrodes (O1, Oz and O2) as a within-subject factor. Data met ANOVA assumptions: normality of residuals was assessed by visual inspection of a q-q plot in Statistica and a Shapiro-Wilk's test (W > 0.94; P > 0.09); variance was homogeneous across groups for the withinsubject factor (Levene's test: all F(1,34) > 3.5; all Ps > 0.075). For all ANOVAs performed, Bonferroni post-hoc corrections were applied where needed. F and corrected P-values are provided, as well as effect sizes (partial eta squared η 2 p ). Significance was considered for P < 0.05.

Single-trial analyses

First, for each participant, we measured the proportion of trials showing a positive activity at each time point for each of the three electrodes. Significance was assessed by means of bootstrapping with replacement (5000 bootstraps) of all trials in a single subject, and calculating the proportion of trials with a positive activity after each bootstrap. A 95% confidence interval was then estimated around the 'true' proportion of trials showing a positive activity. A subject was deemed as having a significant proportion of trials with a negative/positive activity if the confidence interval did not include 50% (e.g., the proportion obtained by chance) on 5 consecutive time points on the three electrodes of interest between 70 and 140 ms (for a similar analysis, see ref. [START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] ).

It has been previously shown that trial-by-trial variability of P100 latency and amplitude was increased in those with ASD [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF] . Accordingly, in the current study additional analyses were performed on the single-trial P100 (st-P100) component (latency and amplitude) to investigate the influence of trial-by-trial variability on the ERPs [START_REF] Ouyang | Reconstructing ERP amplitude effects after compensating for trial-to-trial latency jitter: a solution based on a novel application of residue iteration decomposition[END_REF] . Single-trial peak analysis was performed with Matlab. st-P100 was considered as the maximum peak in the 70-140 ms time window, closest to the latency of the individual P100 component if several peaks were identified within this time window. If no peak was identified, the peak was measured as the maximal value in the predefined time window for the P100. Mean latencies and amplitudes (including all trials) of the components were measured for each participant and repeated-measure ANOVAs were performed for both components with Group (ASD and TD) as a between-subject factor and Electrodes (O1, Oz and O2) as a within-subject factor. To evaluate inter-trial variability in latency and amplitude, the median absolute deviation (MAD) was computed for each subject at each electrode (for a similar analysis see [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Gonen-Yaacovi | Increased ongoing neural variability in ADHD[END_REF] ).

Results

ERPs results

Grand average responses over the three electrodes of interest are shown in Fig. 1a, whereas individual VEPs for both groups are shown in Fig. 1b.

Regarding P100 latency, a significant effect of Electrode was found (F(1.62,55.2) = 5.14, P = 0.013, η 2 p = 0.131), due to latency being shorter over Oz than O2 (P = 0.037) and O1 (P = 0.037). There was neither an effect of Group, nor an Electrode by Group interaction on P100 latency (P > 0.20).

On P100 amplitude, main effects of Group (F(1,34) = 6.00, P = 0.019, η 2 p = 0.15) and Electrodes (F(1.67,56.8) = 23.79, P < 0.001, η 2 p = 0.412) were observed, further characterized by a Group by Electrodes interaction (F(1.67,56.8) = 12.1, P < 0.001, η 2 p = 0.263). Planned comparisons for the interaction (within-group across electrodes and betweengroups at each electrode; n = 9) revealed a smaller amplitude in the ASD group as compared with the TD group on O2 (P = 0.029) and Oz (P = 0.023) but not on O1 (P > 0.5), and that only the TD group presented greater amplitude on O2 and Oz compared with O1 (both P < 0.001).

Inter-subject variability, measured as the proportion of participants of each group showing a positive activity around the P100 (Fig. 1c), highlighted that all participants of both groups showed a positive deflection on their averaged ERPs between 90 and 100 ms, suggesting that the differences observed on the P100 are not driven by the absence of a positive response on the ERPs in some participants.

To provide further understanding of these differences we investigated intra-subject variability by performing single-trial analyses.

Single-trial results

Inter-trial consistency

Figure 2 illustrates this single-trial approach for one TD and one ASD child. The maximal proportion of positive trials observed on Oz in the TD group was 94% and 79% in children with ASD (Fig. 3). A proportion of trials with positive activity significantly greater than chance in the latency range of the P100 was observed in 78% of TD participants and 44% of children with ASD. This analysis allowed identifying two subgroups of ASD participants: in the first group (n = 8), there was a significant proportion of trials with positive amplitude reflecting a high intertrial consistency (P100 amplitude, on O2, was 11.7 µV ± 3.2), whereas the other subgroup (n = 10) was characterized by a nonsignificant proportion of positive trials traducing a low inter-trial consistency (P100 amplitude on O2 was 5.6 µV ± 2.9).

Peak variability

The ANOVA performed on the mean amplitude of the st-P100 revealed a Group by Electrodes interaction (F (2,68) = 9.85, P < 0.001, η 2 p = 0.225); yet, planned comparisons revealed no between group differences (all corrected Ps > 0.16). It is noteworthy that in the TD group, the effect of electrodes remained (all corrected Ps < 0.001). On O2, mean st-P100 amplitude was 15.3 ± 4.1 in ASD and 19.7 µV ± 5.9 in TD.

We then measured the MAD of st-P100 amplitudes and latencies in each child.

Although no Group effect was found on the MAD of st-P100 amplitudes, a main effect of Electrodes, further characterized by an interaction between Group and Electrodes, was observed (F(1.56,52.99) = 6.38, P = 0.006, η 2 p = 0.157); planned comparisons revealed no relevant between-groups differences, but MAD was greater on O2 and Oz than on O1 in the TD group (corrected P < 0.005).

The MAD of st-P100 latencies was significantly larger for the ASD group than the TD group (F(1,34) = 7.73, P = 0.0088, η 2 p = 0.18). Electrodes also affected the MAD of latencies (F(1.58,53.9) = 19.9, P < 0.001, η 2 p = 0.37), as it was the smallest on Oz and the largest on O1; all pairwise comparisons were significant (Bonferroni corrected). MAD of st-P100 latencies was within 1 SD from the mean for all three electrodes in 89% of the TD children and in 44% of the subjects with ASD. Based on the MAD of st-P100 latencies, two subgroups of ASD participants can also be identified: in the first group (n = 8) the MAD was within 1 SD (P100 amplitude, on O2, was 10.9 µV ± 3.9); in the second group the MAD (n = 10) was larger than 1 SD (P100 amplitude, on O2, was 6.3 µV ± 3.6).

By measuring st-P100 amplitudes, we showed that the group difference was no longer significant, suggesting that the single-trial approach leads to different results as compared to ERPs. The latency MAD analysis revealed that children with ASD presented a larger inter-trial latency variability than TD children. VEPs result from an averaging procedure strongly relying on the assumption of inter-trial consistency, i.e., an ERP is elicited at roughly the same latency for each trial. A larger trial-to-trial latency variability has the potential to blur the average VEP waveform. To go further in our understanding, we tested whether controlling inter-trial variability allows reducing group differences.

Controlling analyses Controlling amplitude variability at each time point

To control the effect of inter-trial inconsistency, we reconstructed ERPs with only trials with a positive response on five consecutive time points in the latency range of the P100 (i.e., between 70 and 140 ms) and on the three electrodes of interest. The reconstructed ERPs included on average 73 trials for the children with ASD (range: [33 121]) and 70 for the TD children (range: [36 104]). An ANOVA on amplitude of reconstructed P100 revealed a main effect of electrodes and a Group by Electrodes interaction (F(2,68) = 12.75, P < 0.001, η 2 p = 0.27) as TD children presented a larger P100 on O2 and Oz. It is noteworthy that the main effect of group and the group differences on O2 and Oz observed on the original ERPs were no longer significant.

Controlling for latency variability

To control for latency variability, we rebuilt the trimmed mean ERPs by selecting only trials that presented a homogeneous P100 latency. This analysis allows reducing the influence of the tails of a distribution by removing them [START_REF] Wilcox | Introduction to robust estimation and hypothesis testing[END_REF] . Practically, for each electrode, the 15% of trials that had the earliest latency and the 15% that had the longest latency were excluded from the ERPs generation (see Fig. 4). Amplitude and latency of the P100 component were then measured on the reconstructed ERPs (N trials [Range]: 55 [30 93] for ASD and 60 [36 87] for TD children). This analysis revealed a main effect of Electrodes further characterized by a Group by Electrodes interaction (F(2,68) = 10.53, P < 0.001, η 2 p = 0.24) due to TD children presenting a larger P100 on Oz and O2 than O1. Again, the main effect of Group was no longer significant. When keeping only trials with a more homogeneous latency range, 78% of children with ASD and 78% of TD children presented a significant proportion of trials with a positive activity.

Discussion

The present study aimed at investigating early visual responses in children with ASD. To this end, P100 amplitudes and latencies evoked by pattern-reversal checkerboards were measured in children with ASD and in TD peers.

Using an ERPs analysis, we report a smaller P100 amplitude to a pattern-reversal checkerboard stimulation in children with ASD, similar to that observed in adults [START_REF] Kovarski | Brief report: early VEPs to pattern-reversal in adolescents and adults with autism[END_REF] , in accordance with a vast literature showing atypical visual functioning in ASD [START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF][START_REF] Constable | Motion and pattern cortical potentials in adults with high-functioning autism spectrum disorder[END_REF][START_REF] Milne | Independent component analysis reveals atypical electroencephalographic activity during visual perception in individuals with autism[END_REF][START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Kornmeier | A different view on the checkerboard? Alterations in early and late visually evoked EEG potentials in Asperger observers[END_REF] . Importantly, by controlling for ophthalmological issues that were present in an unexpectedly high number of ASD participants [START_REF] Little | Vision in children with autism spectrum disorder: a critical review[END_REF] , we are sure that our observations are not biased by the inclusion of participants with ophthalmological pathologies. Therefore, the current study suggests that these atypical sensory responses are not linked to ophthalmological pathologies and represent a genuine feature of ASD, present at all ages, rather than a compensatory mechanism observed only in adulthood consecutive to atypical visual experience.

ASD children differed from TD children regarding the lateralization of P100 component as an early hemispherical effect was found in TD children only: amplitude was larger in the right than in the left hemisphere. Previous studies have reported a lack of hemispherical difference in those with ASD [START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF][START_REF] Lazarev | EEG photic driving: righthemisphere reactivity deficit in childhood autism. A pilot study[END_REF] . Pei et al. [START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF] found in the ASD group a selective amplitude reduction over the right hemisphere to steady-state VEPs pattern reversal. Abnormal hemispheric responses or lack of lateralization have also been described in the auditory domain [START_REF] Orekhova | Auditory cortex responses to clicks and sensory modulation difficulties in children with autism spectrum disorders (ASD)[END_REF][START_REF] Orekhova | The right hemisphere fails to respond to temporal novelty in autism: evidence from an ERP study[END_REF][START_REF] Charpentier | Emotional prosodic change detection in autism spectrum disorder: an electrophysiological investigation in children and adults[END_REF] , suggesting that this effect is not visual-specific, but could be a core feature of ASD. A smaller activity to faces is often reported in the right hemisphere [START_REF] Puce | Differential sensitivity of human visual cortex to faces, letterstrings, and textures: a functional magnetic resonance imaging study[END_REF][START_REF] Kanwisher | The fusiform face area: a module in human extrastriate cortex specialized for face perception[END_REF] ; this hypo-activation is often discussed as an atypical social processing. As face perception is dominated by the right hemisphere [START_REF] Puce | Differential sensitivity of human visual cortex to faces, letterstrings, and textures: a functional magnetic resonance imaging study[END_REF][START_REF] Kanwisher | The fusiform face area: a module in human extrastriate cortex specialized for face perception[END_REF] , an atypical face processing could be the consequence to this atypical lateralization of sensory processing.

To go further in our understanding of the reduced P100 amplitude in ASD, we performed two types of analyses on single trials as previously suggested for ASD [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF][START_REF] Dinstein | Neural variability: friend or foe?[END_REF][START_REF] David | Variability of cortical oscillation patterns: A possible endophenotype in autism spectrum disorders?[END_REF] , but also for ADHD [START_REF] Gonen-Yaacovi | Increased ongoing neural variability in ADHD[END_REF] . We measured inter-trial consistency [START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] by computing the proportion of trials showing a positive activity in the latency range of the P100 component. This method allows quantifying the consistency in the neural response recorded across trials and is more robust to inter-trial amplitude variation by categorizing trials in a binary fashion. ERPs are obtained by averaging many epochs with the underlying assumption that the response evoked by a stimulus is similar from trial-to-trial [START_REF] Ouyang | Reconstructing ERP amplitude effects after compensating for trial-to-trial latency jitter: a solution based on a novel application of residue iteration decomposition[END_REF] . If this was the case, one would expect to observe 100% of positive trials in the latency range of the P100. It is noteworthy that no participant showed 100% of inter-trial consistency, although the proportion reached 90% in three TD children. In children with ASD, the maximum proportion observed was 79% highlighting an overall smaller inter-trial consistency in ASD. Inter-trial (in) consistency reflects variability that could be attributable to inter-trial desynchronization. Seventy-eight per cent of TD children presented a high inter-trial consistency in the P100 time window, but only 44% of ASD children presented a similar pattern. Therefore, 66% of children with ASD presented an inter-trial inconsistency possibly reflecting an inter-trial desynchronization. In the second analysis on single trials, we evaluated inter-trial latency variability of the st-P100, as measured by the MAD, and showed that the latency MAD was increased in children with ASD compared with controls [START_REF] Milne | Atypical EEG in autism spectrum disorder: comparing a dimensional and a categorical approach[END_REF] . Again, although 89% of TD children presented latency MAD values within 1 SD of the mean, only 44% of children with ASD presented a similar pattern.

Both approaches highlighted an heterogeneity in intertrial variability in the autistic group: although a subgroup of autistic children (n = 6) presented patterns similar to the typically developing children, eight children presented altered responses regardless of the measure of inter-trial variability used; finally, a third group of ASD children (n = 4) presented atypical response in either measures (atypical inter-trial consistency: n = 2; atypical MAD value: n = 2). As this increased inter-trial variability may explain the amplitude reduction observed in the P100 measured on the ERPs, additional analyses were performed on reconstructed ERPs following the exclusion of trials with negative activity and trials with outlier latencies. Both controlled analyses abolished group differences in P100 amplitude, suggesting that the reduction in amplitude is solely driven by inter-trial variability. This important result demonstrates that amplitude reduction of the P100 is not the major difference between typical and autistic children, but is rather the consequence of an increased inter-trial variability in subgroups of children on the autism spectrum, consistent with what has been described in the auditory domain [START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] . This increasing evidence of neural heterogeneity in ASD both at the individual and population level [START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF][START_REF] Milne | Atypical EEG in autism spectrum disorder: comparing a dimensional and a categorical approach[END_REF][START_REF] Hahamy | The idiosyncratic brain: distortion of spontaneous connectivity patterns in autism spectrum disorder[END_REF] could explain the failure in identifying biomarkers that are both discriminant with regard to the typical population but also sensitive to variation of clinical symptoms within the spectrum. These results also further support the necessity of developing models that combine multiple clinical scores and neural profiles [START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] to better understand the complexity of the spectrum [START_REF] Al-Jawahiri | Atypical neural variability in carriers of 16p11.2 copy number variants[END_REF] .

The present study relates the reduced P100 amplitude to increased latency variability, suggesting that combined analyses of ERPs and single trials should be carried out when investigating visual responses in ASD across age. This increased variability in ASD is consistent with previous reports [START_REF] Milne | Increased intra-participant variability in children with autistic spectrum disorders: evidence from single-trial analysis of evoked EEG[END_REF][START_REF] Latinus | Atypical sound perception in ASD explained by inter-trial (in) consistency in EEG[END_REF] and supports the neural noise theory [START_REF] Simmons | Vision in autism spectrum disorders[END_REF][START_REF] Dinstein | Neural variability: friend or foe?[END_REF] . Enhanced response variability might be explained in turn by changes in attention [START_REF] Heinze | Visual event-related potentials index focused attention within bilateral stimulus arrays. I. Evidence for early selection[END_REF][START_REF] Mangun | Allocation of visual attention to spatial locations: tradeoff functions for event-related brain potentials and detection performance[END_REF][START_REF] Fontanini | Behavioral states, network states, and sensory response variability[END_REF] , but also by atypical ocular functioning [START_REF] Zhang | Effects of fixation instability on multifocal VEP (mfVEP) responses in amblyopes[END_REF][START_REF] Dalton | Gaze fixation and the neural circuitry of face processing in autism[END_REF][START_REF] Perlman | Experimental manipulation of face-evoked activity in the fusiform gyrus of individuals with autism[END_REF] . As adults with ASD present abnormal and more heterogeneous fixation [START_REF] Frey | Atypical cortical representation of peripheral visual space in children with an autism spectrum disorder[END_REF][START_REF] Johnson | Ocular motor disturbances in autism spectrum disorders: systematic review and comprehensive meta-analysis[END_REF][START_REF] Shirama | Ocular fixation abnormality in patients with autism spectrum disorder[END_REF] , future studies should combine eye-tracking with EEG to explore the link between visual fixation and VEPs variability. The existence of subgroups in our sample of autistic children suggests that the neural response (in)consistency in terms of either amplitude or latency variability is not a characteristic of the entire autistic spectrum, but rather characterized specific children. The neural response inconsistency helps defining neural profiles which, when combined with other clinical markers provide a multidimensional functional assessment of each child. In turns, this would provide support for targeted interventions.

Fig. 1

 1 Fig. 1 VEPs over the three electrodes of interest in the ASD (black line) and the TD groups (grey line). a Grand average of both groups (dark: ASD; light: TD). Note that on O2, P100 amplitude was 8.3 µV ± 4.3 for children with ASD and 14.3 µV ± 6.3 for TD children. b Individual data. c Percentage of participants presenting positive VEPs at each time point.

Fig. 2

 2 Fig. 2 Individual examples for a TD participant (top) and an ASD participant (bottom) showing the percentage of trials showing a positive deflection at each time point on O2. Green colour indicates the percentage of trials with positive activity, whereas red colour indicates (minus) the percentage of trials with negative activity. Note that the length of each 'bicolour' bar is therefore 100%.

Fig. 3

 3 Fig. 3 Individual data of inter-trial consistency illustrated on O2. Left side: proportion of trials with a positive activity in all TD children. Right side: proportion of trials with a positive activity in all ASD children. Shaded area represent 95% confidence intervals around the proportion of positive trials.

Fig. 4

 4 Fig. 4 Illustration of single-trial P100 latencies in each individual for TD and ASD groups. Latencies are sorted from the smallest to the largest. A line represents single-trial latencies measured for one participant. The black stars mark the limit of the latencies used in the analysis of homogenous latencies. [Note the larger variability of singletrial latencies in ASD participants seen in less steep slopes.].

Acknowledgements

We thank Céline Courtin and Luce Corneau for their help during the recordings. This study was supported by the Fondation individualisée John Bost pour la Recherche (2016) to ML.

Author details 1 UMR 1253, iBrain, Université de Tours, Inserm, Tours, France. 2 CNRS (Integrative Neuroscience and Cognition Center, UMR 8002), Paris, France.

3 Université Paris Descartes, Sorbonne Paris Cité, Paris, France. [START_REF] Constable | Motion and pattern cortical potentials in adults with high-functioning autism spectrum disorder[END_REF] Fondation Ophtalmologique A. de Rothschild, Paris, France. [START_REF] Milne | Independent component analysis reveals atypical electroencephalographic activity during visual perception in individuals with autism[END_REF] CHRU de Tours, Centre Universitaire de Pédopsychiatrie, Tours, France. [START_REF] Weinger | Low-contrast response deficits and increased neural noise in children with autism spectrum disorder[END_REF] CHRU de Tours, Département d'Ophtalmologie, Tours, France. [START_REF] Kovarski | Brief report: early VEPs to pattern-reversal in adolescents and adults with autism[END_REF] Université de Toulouse, CERPPS, Toulouse, France

Conflict of interest

The authors declare that this study was conducted in the absence of any commercial or financial relationship that could represent a potential conflict of interest.

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