Correlation of fungal penetration, CWDE activities and defense-related genes with resistance of durum wheat cultivars to Zymoseptoria tritici

Accepted Manuscript Correlation of fungal penetration, CWDE activities and defense-related genes with resistance of durum wheat cultivars to Zymoseptoria tritici L. Somai-Jemmali, A. Siah, K. Harbaoui, S. Fargaoui, B. Randoux, M. Magnin-Robert, P. Halama, P. Reignault, W. Hamada PII:

S0885-5765(17)30004-8

DOI:

10.1016/j.pmpp.2017.08.003

Reference:

YPMPP 1278

To appear in:

Physiological and Molecular Plant Pathology

Received Date: 3 January 2017 Revised Date:

10 July 2017

Accepted Date: 27 August 2017

Please cite this article as: Somai-Jemmali L, Siah A, Harbaoui K, Fargaoui S, Randoux B, MagninRobert M, Halama P, Reignault P, Hamada W, Correlation of fungal penetration, CWDE activities and defense-related genes with resistance of durum wheat cultivars to Zymoseptoria tritici, Physiological and Molecular Plant Pathology (2017), doi: 10.1016/j.pmpp.2017.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Correlation of fungal penetration, CWDE activities and defense-related genes with resistance

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of durum wheat cultivars to Zymoseptoria tritici

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L. Somai-Jemmalia*, A. Siahb, K. Harbaouic, S. Fargaouic, B. Randouxd, M. Magnin-Robert d,

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P. Halamab, P. Reignaultd and W. Hamadaa

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a

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Tunisie, 43 Avenue Charles Nicolle, 1082 Tunis, Tunisie.

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b

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F-59046 Lille cedex, France.

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c

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d

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Littoral Côte d’Opale, CS 80699, F-62228, Calais cedex, France.

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*E-mail: [email protected] (L. Somai-Jemmali).

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Laboratoire de Génétique et Amélioration des Plantes, Institut National Agronomique de

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Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, 48 Boulevard Vauban,

Regional Field Crops Research Center (CRRGC) Béja-Tunisia, BP.350 Béja 9000–Tunisia.

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Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du

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ABSTRACT

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We investigated here for the first time the infectious process and CWDE activities of Zymoseptoria

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tritici on durum wheat (DW), by using three Tunisian cultivars with different susceptibility levels to

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the pathogen: Karim (susceptible), Nasr (moderately resistant) and Salim (resistant). High DW

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resistance level was associated with decreased direct leaf penetration at early stage of infection and

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with reduced sporulation and production of CWDE (endo-β-1,4-xylanase and endo-β-1,3-

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glucanase) and protease activities during the necrotrophic phase. Furthermore, the expression

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pattern of six wheat defense-related genes, including GLUC, Chi 4, POX, Msr and Bsi1, exhibited

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significant differences between resistant and susceptible DW cultivars. Altogether, these results

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provide new insight into the nonspecific resistance mechanisms in durum wheat against Z. tritici.

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Key words: Triticum durum, Zymoseptoria tritici, defense, genes, expression.

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1. Introduction Septoria tritici blotch (STB), caused by Zymoseptoria tritici (Desm.) [1], previously known as

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Septoria tritici (teleomorph Mycosphaerella graminicola (Fuckel) J. Schröt.), is an important wheat

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disease in the Mediterranean region. Z. tritici is a hemibiotrophic pathogen with a long symptomless

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phase considered as biotrophic, followed by a necrotrophic phase. This later phase conducts to a

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tissue collapse which further results in chlorosis and necrosis development on the leaf and

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pycnidium formation [2,3]. The infection process of Z. tritici is characterized by a latent period of

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approximately 10-15 days during which the fungal germ tube grows on the leaf surface, penetrates

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either through the stomata or directly via the epidermal cell wall, and colonizes the intercellular

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space between mesophyll cells. The fungus then rapidly switches to necrotrophic growth, associated

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with an increase of fungal biomass, chlorotic and/or necrotic lesions on the leaf surface, and

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pycnidium formation in colonized substomatal cavities [4,5,6,7]. Although no haustoria are

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differentiated, Z. tritici obtains nutrients from the apoplastic space during the biotrophic period,

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whereas during the necrotrophic phase, the fungus releases cell wall degrading enzymes (CWDEs)

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leading to an increase of nutrient content in the apoplastic space [8]. It has been demonstrated that

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Z. tritici is able to produce a battery of CWDEs capable of degrading cell-wall polymers such as

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endo-β-1,4-xylanase, endo-β-1,3-glucanase and exo-β-1,4-xylosidase [9]. Douaiher et al. [10] and

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Siah et al. [7] revealed that the production of CWDEs, such as endo-β-1,4-xylanase, by Z. tritici, is

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correlated with symptom and sporulation levels. In their study, Tian et al. [11] showed that endo-β-

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1,4-xylanase and protease activities are highly associated with susceptibility level of the wheat

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cultivar.

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In Tunisia, durum wheat (Triticum turgidum L. subsp. durum [Desf.] Husn.) is the primary

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cultivated crop and occupies more than half of the area dedicated to cereal crops [12]. STB has

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become a major disease of durum wheat particularly during suitable growing seasons where it

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causes significant yield losses which can reach 50 % [13,14]. In Tunisia, fungicide application is

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ACCEPTED MANUSCRIPT commonly used as a control strategy; however, this strategy is costly, not reliable from year to year,

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and has become less effective over the years [15]. On the other hand, the prevalence of sexual

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recombination of the pathogen could also play a major role in the recurrent epidemics in durum

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wheat in Tunisia [16,17]. Host resistance is a major component of sustainable integrated disease

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management strategies; hence, breeding for resistance to STB became a major component of the

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national breeding program in Tunisia. However, the availability of effective sources of resistance is

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a key component of the breeding program, but limited sources of resistance are known and often

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confer only partial resistance [18,19]. In the last decade, a durum wheat variety, characterized by a

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better resistance to STB, has been registered under the name of Nasr [20]. Recently, a new durum

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wheat cultivar named Salim, with high level of resistance to STB, was released. This cultivar can

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also be used as parental material to eventually diversify resistance sources in breeding programs to

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STB [21].

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Understanding the defense-related pathways involved in disease resistance will help seek for

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new approaches to wheat breeding for disease resistance. Improving resistance would be a great

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advantage for farming areas where this disease causes significant financial losses. A Real-time PCR

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is based on the labeling of primers, probes, or amplicon with fluorogenic molecules and allows

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detection of the target fragment to be monitored while the amplification is in progress [22, 23]. The

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development of reliable methods of quantitative RT-PCR has opened up many further possibilities

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and promoted the expression patterns of defense-related genes in order to reveal their involvement

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in bread wheat resistance against STB [24, 25]. The early and strong accumulation of many

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defense-related genes-encoding transcripts was detected in incompatible interactions between wheat

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and Z. tritici leading to full resistance, such as Pathogenesis-related (PR)-protein including PR1

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(unknown function), PR2 (endo-β-1,3-glucanase), Chi 4 (chitinase IV precursor), PR4 (chitinase

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II), PR5 (thaumatin-like proteins) and POX (peroxidase) genes [24,25,26,27,28]. Indeed, other

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defense-related genes were differentially expressed between compatible and incompatible

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(vacuolar ATP synthase) and Bsi1 (Putative protease inhibitor) genes suggesting their contribution

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to resistance against STB in bread wheat [24, 25]. The purpose of this study was to compare the

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effect of cultivar resistance level on Z. tritici infection process and both CWDE and protease

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production on durum wheat plants. The secondary goal was to analyze the expression patterns of six

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defense-related genes, including GLUC, Chi 4, POX, Msr, ATPase and Bsi1 genes in order to

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determine whether they are associated with durum wheat resistance against Z. tritici. Differences

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and similarities with bread wheat findings reported in previous works will be discussed.

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2. Materials and methods

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2.1. Plant and fungal growths and pathogenicity tests

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We used three durum wheat cultivars selected for their different resistance levels to Z. tritici,

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including the susceptible (S) cv. Karim, the moderately resistant (MR) cv. Nasr and the resistant (R)

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cv. Salim. Wheat grains of each cultivar were germinated in Petri dishes on moist filter paper in

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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

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then placed into pots filled with 3 L loam in the greenhouse at 18 °C (±2°C) with a 16 ⁄ 8 h

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photoperiod with supplementary illumination. After 3 weeks (when the third leaf from the base of

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plant was fully expanded), plants of each pot were inoculated using hand sprayer with 30 mL of

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spore suspension in distillate water (106 spores mL-1) amended with 0.05% of Tween 20 (Sigma

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Aldrich, USA). Control plants were treated with sterile distillate water containing Tween 20.

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Immediately after inoculation, each pot was covered with a clear polyethylene bag for 3 days in order

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to ensure a water-saturated atmosphere compatible with a good fungal germination. For this

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experiment, the Z. tritici strain St-08-46 was used. St-08-46 strain was isolated in 2008 in Northern

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Tunisia from infected durum wheat plants and was chosen for its high pathogenicity. DNA sequences

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obtained from this strain were deposited in GenBank under the accession number KT336200. The

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ACCEPTED MANUSCRIPT disease level was scored at 22 days post inoculation (dpi) by noting the percentage of the third leaf

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area with symptoms (chlorosis and necrosis) and by noting pycnidium density (index of pycnidium

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coverage) on a scale from 0 to 5 (0, 1, 2, 3, 4, 5 correspond to 0% (absence), less than 20%, 20-40 %,

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40-60 %, 60-80 %, and 80-100 % of leaf areas with symptoms contain pycnidia, respectively).

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2.2. Histopathological analysis

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Monitoring of leaf penetration by Z. tritici was performed using Fluorescence Brightener 28

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(Calcofluor, Sigma Aldrich) according to Siah et al. [7]. Segments (2 cm) were harvested at 5 dpi

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from third inoculated leaves and then immersed for 5 min in a solution of 0.1 % (w ⁄ v) Calcofluor

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in 0.1 M Tris–HCl buffer pH 8.5. Leaf segments were washed for 2 min with sterile distilled water.

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After drying, they were placed on a glass slide, covered with a cover slip and observed

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microscopically (Nikon, Eclipse 80i) under UV illumination. Mesophyll colonization was

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investigated using Trypan blue [2,7] with few modifications. Leaf segments (2 cm) were harvested

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from both control and inoculated leaves and cleared in a mixture of absolute ethanol: glacial acetic

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acid (3: 1, v⁄v) overnight. Cleared leaves were rehydrated for 4 h in distilled water and then fixed

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for 20 min in lactoglycerol (lactic acid: glycerol: water, 1: 1: 1, v ⁄v ⁄v). Staining of the fungus was

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carried out at 70 °C by immersing the leaf segments in 0.1% Trypan blue dissolved in lactophenol-

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ethanol (1:2, v⁄v) for 6 h. After washing, leaf segments were fixed in lactoglycerol, placed on a

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glass slide, covered with a cover slip and then observed microscopically under white light

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illumination. Spores were recorded for each cultivar on three leaf segments and subsequently 150

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randomly chosen spores were studied. Data represent means of two independent experiments. Three

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independent replicates were carried out for each experiment.

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2.3. Protein extraction and enzymatic assays

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Two CWDE activities (endo-1,4-xylanase and endo-β-1,3-glucanase) and protease activity

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were assessed. Total protein extractions were carried out at 4, 10, 16 and 22 dpi as previously

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described by Siah et al. [7] with few modifications. Briefly, 2.5 g of inoculated and control third

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ACCEPTED MANUSCRIPT leaves were removed and immediately frozen in liquid nitrogen prior to grinding in a mortar. The

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resulting powder was then dispersed in 5 mL 0.05 M Tris–HCl buffer pH 7.8 containing 0.1 M KCl

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and 0.5 % cysteine. After cooling for 30 min at 4 °C, the mixture was squeezed through three layers

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of cheesecloth and centrifuged at 13000 g for 20 min at 4 °C. From the supernatant, 100 µL were

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transferred to an Eppendorf tube and stored at 4°C for total protein quantification. Total protein

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extracts were then dialyzed against 0.5 L of distilled water for 24 h at 4 °C overnight (Spectrum Lab

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Inc.). The dialyzed solution was then subjected immediately to endo-β-1,3-glucanase and endo-β-

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1,4-xylanase activity quantification using a modified 3,5-dinitrosalicylic acid (DNS) method [7,

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10]. Endo-β-1,4-xylanase activity was measured at pH 4.8 and 45 °C using xylan (1%, w/v) (Sigma

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Aldrich, USA) as substrate and xylose (10 mM) (Sigma Aldrich, USA) as reducing control sugar

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[7]. Endo-β-1,3-glucanase activity was measured at pH 4.8 and 50 °C using Laminarin (Sigma

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Aldrich, USA) at pH 5 (0.5 %, w/v) as substrate and glucose (10 mM) as reducing control sugar

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[10,29]. One unit of endo-1,4-xylanase and endo-β-1,3-glucanase activity is defined as the amount

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of enzyme necessary to release 1 µmol of reducing sugar/p-nitrophenol min−1 mL−1 of enzyme

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solution. Absorbance was measured at 540 nm. Protease activity was assessed according to Hellweg

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[30]. Six aliquots of 100 µL of total protein were heated to 80 °C for 10 min to inactivate enzyme

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activity. After cooling, 50 µL of azocasein solution (20 mg/mL Azocasein, Sigma Aldrich) and 50

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µL of incubation buffer (200 mM Tris, 20 mM CaCl2 pH 7.8) were added. After incubation at 40 °C

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under gentle agitation for 30 to 60 min, 500 µL of 5 % aqueous trichloroacetic acid were added to

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precipitate non-digested protein and the mixture was incubated for 10 min at room temperature. The

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precipitate was peleted by centrifuging for 5 min at 13000 g and 500 µL of test buffer were added at

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400 µL of the supernatant. Absorbance was measured at 400 nm. Measures were performed in

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triplicates with denaturized samples as comparative samples using an azo-dye labelled substrate.

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One unit of protease activity is defined as a change in enzyme activity of 1 per hour. Total protein

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concentrations were determined at 595 nm using bovine serum albumin as a standard [31].

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2.4. RNA extraction and analysis of gene expression by semi-quantitative RT-PCR and real-time RT-PCR For gene expression study, three third leaves were used as independent replicates. Samples

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were collected at 1, 2 and 3 dpi from treated and control (H2O-treated) plants. Total RNA was

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extracted from 100 mg of plant tissue using RNeasy Plant Mini Kit (Quiagen, The Netherlands).

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Genomic DNA contaminating the samples was removed by treatment with DNase using RNase-

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Free DNase Set (Quiagen, The Netherlands). Reverse transcription of total RNA was carried out

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using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the

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manufacturer’s protocol.

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PCR reactions were conducted on subsequent obtained cDNA to amplify six wheat defense genes

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for GLUC (β-1,3-glucanase), Chi 4 (chitinase class IV precursor), POX (peroxidase), Msr

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(methionine sulfoxide reductase), ATPase (vacuolar ATP synthase) and Bsi1 (Putative protease

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inhibitor). TUB (wheat beta-tubulin) encoding gene is used as housekeeping gene. Primers pairs

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used for amplifying GLUC (U76895), Chi 4 (AF112966), POX (X56011) and TUB (U76895) were

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designed using the Primer Express® program and were tested for secondary structure using Net

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Primer® program. Msr, ATPase and Bsi1 primers were published by Adhikari et al. [24]. All gene-

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specific primers used in this study are presented in Table 1. The expression of Msr, ATPase and

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Bsi1 genes was investigated by semi-quantitative RT-PCR. PCR was carried out in a 20 µL reaction

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mixture containing 1x PCR buffer, 2 mM dNTPs, 10 µM of each primer, 0.5 unit of Taq

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polymerase and 20 ng of cDNA using the following protocol: 94°C for 5 min, followed by 30

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cycles each with 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The PCR reaction was

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terminated at 72°C for 7 min. PCR products were analyzed by electrophoresis on ethidium-bromide

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stained 1.5% agarose gels run in 0.5 x Tris-acetic acid-EDTA buffer at 100 V for 45 min. Gels were

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visualized under UV light. Experiment was repeated 3 times and only results which were repeated

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at least twice were considered. The investigation of GLUC, Chi 4 and POX gene expression was

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ACCEPTED MANUSCRIPT performed by real time-RT-PCR analysis. The amplification specificity of each qRT-PCR was

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checked by a single peak in melt curve analysis, and no primer dimmers were detected using

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agarose gel electrophoresis. Real-time RT-PCR was performed using ABI Prism 7300 detection

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system (Applied Biosystems, USA), using the following thermal profile : after incubation for 10

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min at 95°C, 15s at 95°C (denaturation), 1 min at 60°C (annealing/extension) for 40 cycles and 30s

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at 72°C (data acquisition). After qPCR, a melt curve was generated to verify the specificity of each

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amplification reaction. The results were normalized with the TUB gene and expressed relatively to

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the control, for each time point, corresponding to a fixed value of 1. The experiment was conducted

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in triplicate and only results which were repeated at least twice were considered.

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2.5. Statistical data analysis

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For disease severity parameters, enzymatic activities and microscopy, an analysis of variance

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(ANOVA) was performed using the Tukey test at P = 0.05 using the XLSTAT software (Addinsoft,

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France). For quantitative gene expression, the analyses were performed using the relative

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expression software tool REST® as described by Pfaffl et al. [32] and the significant differences

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were tested at P = 0.05).

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3. Results

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3.1. Comparative of Z. tritici infection in Karim (S), Nasr (MR) and Salim (R)

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3.1.1. Infectious process

The present study allowed the investigation of the cultivar resistance level on Z. tritici infection

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process. Therefore, several cytological parameters related to the development of the Tunisian Z.

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tritici strain St-08-46 were quantified in Karim, Nasr and Salim at different time points (Table 2) :

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(i) 1 dpi: germinated spores (GS), (ii) 4 dpi: non germinated spores (NGS), spores leading to

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stomatal penetration (SP), spores leading to a direct penetration (DP), and germinated spores that

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did not penetrate (GSNP), (iii) 22 dpi: non colonized stomata (NCS), colonized stomata but not yet

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transformed into pycnidia (CS) and colonized stomata transformed into pycnidia (P). Our results

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ACCEPTED MANUSCRIPT exhibited that tested cultivars presented closed and non-significantly different percentages of GS at

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1 dpi. At 4 dpi, percentages of SP were significantly higher in Salim (13.8%) than in Karim and

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Nasr (9.8% and 5.8%), while, DP were significantly privileged in Karim and Nasr (28.9% and

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27.5%) than in Salim (11.4%). However, no significant differences were recorded for the NGS and

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GSNP. At 22 dpi, the percentage of NCS and CS were not significantly different between Karim,

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Nasr and Salim. Whereas, Karim revealed the highest percentage of P (65.1%), followed by Nasr

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(41.4%) and Salim (21.3%).

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3.1.2. Disease level

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Disease severity was estimated by measuring the percentage of leaf necrosis (PLN) (Fig. 1A)

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and Index of pycnidium coverage (IPC) (Fig. 1B), at 4, 10, 16 and 22 dpi for three durum wheat

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cultivar Karim (S), Nasr (MR) and Salim (R) on the third leaf (Fig. 1A). Symptoms were detected

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from 16 dpi. At 16 and 22 dpi, the PLN and IPC were significantly different between the three

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tested cultivars. At 22 dpi, Karim revealed the highest percentage of PLN and IPC (63.5% and 4.5),

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followed by Nasr (30.8% and 3.8) and Salim (13.4% and 1.9).

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3.1.3. Fungal cell-wall degrading enzyme (CWDE) and protease activities

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Endo-β-1,4-xylanase, endo-β-1,3-glucanase and protease activities produced by the fungus in

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plant tissues were monitored during the infection process at 4, 10, 16 and 22 dpi (Fig. 2). At 4 and

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10 dpi, infected (i) leaves exhibited weak CWDE activities levels which were not significantly

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different from those obtained in non-inoculated (ni) control plants. At 16 dpi, only endo-β-1,4-

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xylanase activity recorded significant difference between infected and non-infected Karim and Nasr

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leaves. At 22 dpi, we detected a strong increase of all enzyme activities in the three tested cultivars,

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except protease activity in Salim. At 22 dpi, endo-β-1,4-xylanase activity was 33.1-, 18.6- and 2.8-

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fold higher in (i) Karim, Nasr and Salim leaves, respectively (than corresponding (ni) leaves) (Fig.

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2A). Similarly, endo-β-1,3-glucanase activity was 7.9-, 7.6- and 5.2-fold increased in infected

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Karim, Nasr and Salim leaves, respectively (Fig. 2B). Besides, protease activity level recorded

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respectively (Fig. 2C). Moreover, endo-β-1,4-xylanase and endo-β-1,3-glucanase activities were

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significantly different between three durum wheat cultivar and were higher in Karim, followed by

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Nasr and Salim plants. While, for protease activity, Karim and Nasr leaves were significantly higher

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than Salim plants.

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3.2. Induction of defense-related genes by Z. tritici infection in Salim (R) and Karim(S)

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We established transcript profiles of a set of six distinct defense-related genes including

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GLUC (endo-β-1,3-glucanase), Chi 4 (chitinase class IV precursor), POX (peroxidase), Msr

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(methionine sulfoxide reductase), ATPase (vacuolar ATP synthase) and Bsi1 (Putative protease

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inhibitor) in susceptible wheat cultivar (Karim) compared to resistant wheat cultivar (Salim) (Fig. 3

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and 4). Gene expression profiles in inoculated (i) plants of each cultivar were followed at 24, 48 and

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72 hours post inoculation (hpi) and were compared with their respective non-inoculated (ni)

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controls.

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Preliminary analyses of GLUC, Chi 4 and POX-encoding genes using semi-quantitative RT-

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PCR showed no stable and no exploitable results. Therefore, the expression level of these genes was

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investigated using real-time RT-PCR, which revealed their induction in (i) Salim (R), compared to

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Karim (S). Indeed, we found that GLUC gene was up-regulated at 48-72 hpi in both wheat cultivars,

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while this induction was higher in Salim (9.0- and 44.9-fold), than Karim (3.4- and 13.2-fold) (Fig.

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3A). However, Chi 4 and POX genes were significantly down-regulated at 24-48-72 hpi (3.5-, 4.7-

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and 3.9-fold) (Fig. 3B) and 72 hpi (3.4-fold) (Fig. 3C), respectively, in Karim leaves. Although, in

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Salim (R) leaves, Chi 4 and POX genes were highly up-regulated at 24, 48 and 72 hpi and marked

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maximal relative expression at 48 hpi (292.1-fold) (Fig. 3B) and 72 hpi (91.5-fold) (Fig. 3C),

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respectively.

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The semi-quantitative RT-PCR analysis exhibited that the expression of Msr, ATPase and

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Bsi1 genes were not induced in Karim leaves compared to (ni) control. Whereas, in infected Salim

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leaves, Msr gene was significantly enhanced at 72 hpi, Bsi1 gene was highly up-regulated at 24 and

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48 hpi, whereas ATPase was slightly enhanced at 72 hpi (Fig. 4).

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4. Discussion Three wheat cultivars characterized by different resistance levels to Z. tritici, including Karim

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(S), Nasr (MR) and Salim (R), inoculated by a high pathogenic Tunisian Z. tritici strain (St-08-46),

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were compared. Our study is the first investigating the effect of durum wheat resistance on fungal

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infection process and CWDE and protease production by Z. tritici. We also explored the

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involvement of six defense-related genes in durum wheat resistance against STB disease.

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4.1. Durum wheat resistance affect mode of leaf penetration, colonization and fungal sporulation

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No significant difference of spore’s germination capacity between the susceptible and

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resistant durum wheat cultivars was found, agreeing the findings of Shetty et al. [2] and Siah et al.

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[7] in bread wheat. However, in the susceptible (S) Karim and the moderately resistant (MR) Nasr

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cultivars, we observed high rates of direct penetration as reported by Siah et al. [7], whereas Shetty

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et al. [2] recorded lower rates of direct penetration attempts. These differences could be explained

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by the type of strain-cultivar interactions used and/or by the environmental conditions such as

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temperature, light intensity and relative humidity that can affect stomatal opening, thus the mode of

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fungal penetration. On the other hand, stomatal penetration was more frequent than direct

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penetration in the resistant cultivar (R) Salim than in Karim (S) and Nasr (MR) cultivars. This

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difference could be explained by the cultivar effect since wheat cultivars may differ on the traits

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related to stomatal density (abundance) and size [33]. As expected, the apparition and extension of

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necrotic symptoms and the importance of the plant invasion by the parasite was faster in Karim (S)

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followed by Nasr (MR) and Salim (R). Regarding tissue colonization, no significant difference of

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colonized and none colonized stomata were found between the three wheat cultivars at 22 dpi, while

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the frequency of colonized stomata transformed into pycnidia (Table 2) were significantly different

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ACCEPTED MANUSCRIPT in tested cultivars and were correlated to the index of pycnidium coverage at the macroscopic level.

277

Indeed, visually, Salim (R) was characterized by rare and small necrotic lesions bearing few

278

pycnidia, while Nasr (MR) showed large necrosis bearing more pycnidia, but not generalized on the

279

totality of leaf surface. By contrast, Karim (S) exhibited larger necrotic spots (with higher

280

pycnidium density) which occupied larger part of leaf area. This result suggests that in Salim (R),

281

the activation of defense mechanisms is more efficient to reduce necrosis and pycnidium coverage,

282

followed by Nasr (MR) and Karim (S). This observation could be explained, as reported by

283

Adhikari et al. [24] and Ray et al. [26], by the fact that wheat plants could activate their defense

284

mechanisms before penetration by the pathogen.

285

4.2. Durum wheat resistance is associated with reductions of CWDE and protease production by St-

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08-46 strain

Defense mechanisms take likely place during the early stages of infection, whose effective

288

expression can lead to an inhibition of intercellular hyphal growth, fungal biomass and fungal

289

pathogenicity compounds such as CWDEs, affecting disease symptoms and fungal sporulation. It

290

has been previously shown that the production of CWDEs is associated with Z. tritici pathogenicity

291

and impacts the disease level of wheat [7,10,11].

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In the present work, we highlighted significantly higher levels of endo-β-1,4-xylanase, endo-

293

β-1,3-glucanase and protease activities produced by the fungus in Karim, the most susceptible

294

cultivar, from 16 dpi, at which the symptoms begin to be observed. These activities reached their

295

highest activity levels in Karim at 22 dpi and were associated with the highest index of pycnidium

296

coverage and percentage of leaf necrosis. Endo-β-1,4-xylanase activity also increased in the

297

partially resistant cultivar Nasr, from 16 dpi, while endo-β-1,3-glucanase and protease activities

298

were significantly increased only at 22 dpi, but at lower levels than in Karim. However, in Salim

299

(R), significant increase of endo-β-1,4-xylanase and endo-β-1,3-glucanase was detected only at 22

300

dpi, whereas no significant change of protease activity was observed. Therefore, β-1,4-

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endoxylanase, endo-β-1,3-glucanase and protease activities seem to be correlated with the degree of

302

fungal sporulation and symptoms. These observations confirm the studies of Siah et al. [7] and Tian

303

et al. [11], where CWDE production by Z. tritici was associated with sporulation level, and

304

negatively correlated with the resistance level. This finding supports the hypothesis that defense mechanisms affect disease development and

306

pycnidium formation. In response to fungal CWDE production, plants have evolved a diverse

307

battery of defense responses including protein inhibitors of these enzymes such as TAXI (Triticum

308

aestivum xylanase inhibitor) and XIP (xylanase inhibitor protein) [34,35]. Here, we could suggest

309

that the little and insignificant increase of protease activity in resistant cultivar Salim plants could

310

be due to an early up-regulation of Bsi1 gene which could enhance the production of protease

311

inhibitor proteins. The discovery of these plant inhibitors and the recent resolution of their three-

312

dimensional structures, free or in complex with their target enzymes, provide new lines of evidence

313

regarding their function and evolution in plant–pathogen interactions. On the other hand, the

314

suppression of protease activity in Salim (R) could be due to the repression, by the plant, of

315

protease gene expression within the pathogen.

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4.3. Effect of cultivar resistance on defense-related gene expression

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To our knowledge, all works investigated the expression patterns of defense-related genes in

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bread wheat resistance against Z. tritici, but no information is available regarding durum wheat.

319

Therefore, this study allowed us to highlight the expression of several defense-related genes in

320

durum wheat cultivars against Z. tritici and discuss the similarities and differences with expression

321

profiles previously found in bread wheat cultivars in response to STB. Thus, we compared the

322

expression profiles of six distinct genes, including GLUC (endo-β-1,3-glucanase), Chi 4 (chitinase

323

class IV precursor), POX (peroxidase), Msr (methionine sulfoxide reductase), ATPase (vacuolar

324

ATP synthase) and Bsi1 (Putative protease inhibitor) in Salim (R) and Karim (S).

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ACCEPTED MANUSCRIPT The endo-β-1,3-glucanase catalyzes hydrolytic cleavage of the 1,3-β-D-glucosidic linkages in

326

β-1,3 glucan, which is a component of fungal cell walls [36]. Here, the expression of PR-2 (GLUC)

327

gene was significantly induced in both susceptible and resistant cultivars. Higher level of expression

328

was noted in Salim (R) than in Karim (S) at 48-72 hpi and reached a maximum at 72 hpi, coincided

329

with spore generation on leaf surface, production of many branched hypha and leaf penetration by

330

hyphae. PR-2 gene, which encode for class II endo-β-1,3-glucanase [37], was significantly induced

331

in both susceptible and resistant cultivars. Our results indicated that this gene might have an

332

important role in the defense response and may directly inhibit the pathogen and protect the host

333

against fungal enzymes. Our findings corroborates with that of Shetty et al. [27], which showed that

334

endo-β-1,3-glucanase gene up-regulation correlates well with wheat resistance against Z. tritici.

335

Similar result was reported by Ray et al. [26], which found an accumulation of endo-β-1,3-

336

glucanase during the early stages of infection. Its level was significantly higher in resistant wheat

337

cultivar, suggesting its potential role in resistance against Z. tritici. Besides, Mohammadi et al. [28]

338

highlighted an earlier GLUC accumulation of transcripts in resistant genotype, suggesting the role

339

of glucanase enzyme in degradation of pathogen cell walls and in preventing its growth. Chitinases

340

hydrolyze β-1,4-glycoside bounds in chitin to produce N-acetylglucosamine and N, N+

341

diacetylchitibiose [38]. In our experiment, Chi 4 gene was significantly down regulated in the

342

susceptible cultivar at all studied time points, whereas it was strongly up-regulated in the resistant

343

cultivar following Z. tritici infection. Chi 4 gene encodes a precursor of an anapoplatic class IV

344

chitinase. Once fungal penetration has occurred, apoplastic chitinases release eliciting molecules

345

from the hyphae that penetrate into the intracellular space [39]. Our findings corroborate with those

346

of Shetty et al. [27], which showed that an induction of Chi 4 gene up-regulation in resistant wheat

347

cultivar at 24-72 hpi. They also concluded that chitinase appears to be less important than

348

glucanase, but they suggested a synergistic effect between them. However, in this study, Chi 4 gene

349

showed the highest up-regulation, which could be explained by the importance of chitin in Z. tritici

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ACCEPTED MANUSCRIPT cell wall. However, a synergistic effect could be expected. Moreover, Adhikari et al. [24] found an

351

early and strong induction of chitinase II (PR4) gene expression following inoculation of two

352

resistant cultivars with Z. tritici. The peroxidase which use hydrogen peroxide (H2O2) as a substrate

353

in reactions involved in biosynthesis of lignin and other phenolic compounds that act as additional

354

antimicrobial barriers [40, 41]. In this experiment, we observed significant downregulation of POX

355

gene in Karim (S), while its expression was strongly enhanced in Salim (R) following the infection

356

at studied time points and reached a maximum at 72 hpi. The observed early induction of POX gene

357

in the resistant cultivar suggests that this gene might play a significant role in the generation of

358

H2O2 and lignin deposition during resistant response, which could decrease or stop fungal progress.

359

Adhikari et al. [24] also found early induction (3 hpi) of POX gene expression in resistant cultivars

360

to Z. tritici. Farsad et al. [25] reported a higher up-regulation of POX gene in resistant cultivar

361

suggesting its effectiveness in the conferral of resistance to STB in wheat. Similarly to our findings,

362

Shetty et al. [27] found significant down-regulation of Chi 4 gene following infection in susceptible

363

wheat cultivars. However, Adhikari et al. [24] and Farsad et al. [25] didn’t show down-regulation of

364

Chi 4 and POX genes. They exhibited slight induction of these genes in susceptible cultivars, but

365

much lower level compared to those of the resistant cultivars. On the other hand, we detected an

366

early and strong up-regulation of Bsi1 gene (at 24-48 hpi) coincided with the spore germination and

367

propagation on the leaf surface and the beginning of penetration into the wheat leaves. Therefore,

368

we could suggest that Bsi1 might have an antifungal activity and could inhibit growth of the

369

penetrating hyphae within the cell wall as previously proposed by Stevens et al. [42]. Our findings

370

confirm those of Adhikari et al. [24] and Farsad et al. [25], which also showed early induction of

371

Bsi1 gene up-regulation in resistant bread wheat cultivars, suggesting its association with the wheat

372

resistance against Z. tritici. Interestingly, Bsi1 gene expressed constantly in the inoculated Salim

373

cultivar, while it was downregulated in Karim cultivar after inoculation. This downregulation could

374

be explained by plant resistance overcoming or circumvent by the pathogen.

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ACCEPTED MANUSCRIPT The Msr enzyme catalyzes the reduction of methionine sulfoxides to methionine.

376

Consequently, it has been reported to act as a last-chance antioxidant that repairs proteins damaged

377

by oxidative stress [43]. Up regulation of the Msr gene in the resistant wheat cultivar suggests that

378

the product of this gene might repair proteins that have been damaged following Z. tritici

379

inoculation. Here, we found that Msr gene was constantly expressed in Salim (R) cultivar, while it

380

was constantly downregulated in Karim (S) cultivar (in both inoculated and non-inoculated plants),

381

suggesting its strong involvement in plant resistance. Our results agree with those of Adhikari et al.

382

[24] and Farsad et al. [25], which reported that Msr gene expression in bread wheat is correlated

383

with resistance to Z. tritici. ATP synthases are multi-subunit enzyme complexes that help to

384

maintain the acidic environment of cellular components by transferring protons across membranes

385

through ATP hydrolysis [44]. Wilkens et al. [45] reported that the vacuolar ATPase protein

386

complexes are several units that function as ATP hydrolysis pumps and transporters proton H+.

387

ATPase is involved in several vital processes including active transport proteins and metabolites. In

388

this experiment, we detected weak and insignificant expression of ATPase gene at 72 hpi. The study

389

of Adhikari et al. [24] supports our results and showed a very low up-regulation of vacuolar ATPase

390

gene at biotrophic phase, followed by a strong up-regulation during the necrotrophic phase, in

391

resistant genotypes. Therefore, we suggest that the vacuolar ATPase gene could not be involved in

392

the early protection when the fungus enters the stomatal cavities of wheat leaves but could have an

393

important role at necrotrphic phase in order to prevent the fungus from growing or producing

394

disease symptoms.

395

5. Conclusion

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In conclusion, the obtained molecular data explained the infection process and the inhibition

397

of the pathogen in resistant cultivar. During the first 24 h of inoculation, fungal spores germinate

398

and then penetrate and colonize host leaves. This initial contact stimulates the induction of PR

399

protein-encoding genes such as GLUC, Chi 4, POX and other defense-associated genes such as Msr

16

ACCEPTED MANUSCRIPT and Bsi1 genes in resistant cultivar. By contract, susceptible cultivar responds weakly or not at all to

401

the fungal infection, because either the pathogen was not recognized or it was able to shut off the

402

defense response. At this stage, we detected in the resistant cultivar that the fungus prefers the

403

penetration through the stomatal route rather than direct way, suggesting that enhanced cell wall

404

degrading proteins and cell wall reinforcement makes stomatal penetration easier than direct

405

penetration for the fungus. This early defense response explains the decrease of mesophyll invasion

406

and sporulation as observed at the microscopic and macroscopic scales in the resistant cultivar.

407

However, vacuolar ATPase gene seems to be not involved in early durum wheat defense against Z.

408

tritici. This study allowed the identification of markers that can be used to differentiate varieties

409

with difference response patterns to STB.

410

6. Acknowledgement

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This work was supported by Campus France, in the framework of a PhD internship performed

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by Lamia Somai-Jemmali in both Institut Supérieur d'Agriculture (Lille, France) and Université du

413

Littoral Côte d'Opale (Calais, France). Also, we thank Dr Mohamed Salah Gharbi from National

414

Institute of Agronomic Research of Tunisia for providing wheat seeds.

415

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Fig. 1. Evolution of disease severity of St-08-46 strain on Karim (S), Nasr (MR) and Salim (R)

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durum wheat cultivar. The percentage of leaf necrosis (A) and index of pycnidium coverage (B)

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were evaluated at 4, 10, 16, 22 dpi. Data represent means of two independent experiments (±s.d).

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Means tagged with the same letter are not significantly different using the Tukey test at P ≤ 0.05.

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Fig. 2. Fungal endo-β-1,4-xylanase (A), endo-β-1,3-glucanase (B) and protease (C) activities

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measured at 4, 10, 16 and 22 days post inoculation (DPI) of Karim (S), Nasr (MR) and Salim (R) by

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Z. tritici St-08-46 strain. Data represent means of two independent experiments (±s.d). Means

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tagged with the same letter are not significantly different using the Tukey test at P ≤ 0.05.

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Fig. 3. Quantitative expression of PR genes analyzed by real time RT-PCR at 0, 24, 48 and 72 hours

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post inoculation (hpi) of Karim and Salim by Z. tritici St-08-46 strain. Three genes were

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investigated such as GLUC (A), Chi 4 (B) and POX (C) genes. The experiment was conducted in

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triplicate. Similar results were obtained at least twice. Presented results were obtained in a second

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independent experiment. Results correspond to means (±s.d) of duplicate reactions. Means of

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infected samples designed by asterisks are statistically differentially regulated (up- or down-) in

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comparison to their corresponding H2O-treated controls using Tukey test at P ≤ 0.05.

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Fig. 4. Expression of Msr, ATPase and Bsi1 genes expression analyzed by semi-quantitative RT-

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PCR analysis at 0, 24, 48 and 72 hours post inoculation (hpi) of Karim and Salim by Z. tritici St-08-

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46 strain. At each time point, infected leaves (i) were compared to their reference non infected (ni)

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leaves. RT-PCR products were generated using primers specific for β-tubulin (TUB), Msr, ATPase

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and Bsi1 first-strand cDNAs obtained from total wheat RNA, and visualized on agarose gel (1.5%).

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TUB gene was used as a house-keeping gene. Experiment was repeated three times and only results

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which were similar from two independent experiments were considered.

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Tables

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Table 1

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List of primers encoding for wheat genes analyzed by quantitative and semi-quantitative RT-PCR.

GLUC (β-1,3-glucanase) Chi4 precursor (chitinase class IV precursor) POX (peroxidase) Msr (putative methionine sulfoxide reductase)

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ATPase (vacuolar ATP synthase)

Primer sequences (5’-3’) GGAGTACCCTGACCGAATGATG AACGACGGTGTCTGAGACCTTT CTCGACATCGGTAACGACCAG GCGGCGATGTACTTGATGTTC TTCTGGTTCTGGATGACCAAC ACTGCTTGCAGTACTCCGTG GAGATTCCACAGATGCAAACGAG GGAGGCCCTTGTTTCTGAATG CATGCAGATGTTTCGGACAAA ACCATCGCGTCCCATGTAAA GCTTTCGGATCTCTGGCAATT TGATGAGCACGGCCAGATT GGGCCCTGCAAGAAGTACTG ACACGCATAGGCACGATGAC

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Gene TUB (beta-tubulin, housekeeping gene)

Bsi1 (putative protease inhibitor)

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Table 2

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Frequencies of cytological parameters measured at 1, 4 and 22 dpi in Karim (S), Nasr (MR) and Salim (R).

GS

1

NGS SP DP GSNP

4

NCS CS P

22

Durum wheat cultivars Karim 72±11,5a

Nasr 67,7±0,6a

Salim 70,7±1,9a

20,4±3,5a 9,8±0,2b 28,9±5,9a 41±7,2a

24,2±11,3a 5,8±1,7c 27,5±7,3a 42,4±4,2a

26,4±0,8a 13,8±0,6a 11,4±2,2b 48,4±2,5a

17,4±2,1a 41,2±4,4a 41,4±5,3b

33,0±13,2a 45,6±17,2a 21,3±4,3c

13,8±0,2a 21,6±2,9a 65,1±3a

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Results with one similar letter did not have significant difference at P ≤ 0.05. GS: germinated spores; NGS: not germinated spores; DP: spores leading to a direct penetration; SP: stomatal penetration; GSNP: germinated spores that did not penetrate; NCS: non-colonized stomata; CS: colonized stomata but not yet transformed into pycnidia; P: colonized stomata transformed into pycnidia.

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DPI

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Parameters (%)

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Research highlights Non specific resistance of DW against Z. tritici was first investigated.

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Effect of resistant level of DW on fungal infection process was first examined.

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Genes involved in DW non specific resistance against Z. tritici were first determined.

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