The three wheat cultivars (cv) used in this experiment were selected from previous field observations for their contrasting susceptibilities to FHB, including, in ascending order of susceptibility, cv. Renan, cv. Cadenza, and cv. Recital. Recital and Renan are known to be among the most contrasting cultivars of the French wheat collections for their responses to FHB, while Cadenza is considered as intermediate (Gervais et al., 2003; Zwart et al., 2008; Fabre et al., 2019a). For each wheat cultivar, the seeds were sown in buckets and kept at 20°C to allow germination. Plant vernalization was carried out at 4°C for 8 weeks, then they were transplanted in 4-L pots and transferred to a growth cabinet with optimal conditions to allow tillering and synchronized flowering. For each wheat cultivar, 12 plants were prepared and divided into three randomized complete blocks in the growth cabinet. Each block was surrounded by additional plants to control any edge effects. Automatic watering was installed, and the daily photoperiod was set at 16-h daylight for a temperature of 20°C and 8-h darkness at 18°C. Relative humidity was maintained at 80% during day and night. After 47 days, flowering of the main culm was observed in all plants. Seven additional days were awaited for F. graminearum infection in order to inoculate spikes showing the same ontogeny during the same day (mid-anthesis).

Three F. graminearum French strains, named MDC_Fg1, MDC_Fg13, and MDC_FgU1, were selected for their contrasting aggressiveness based on a previous study (Fabre et al., 2019a). In this previous work, fungal aggressiveness was characterized through the monitoring of symptom severity induced by each F. graminearum strain individually inoculated in the three wheat cultivars. This profiling allowed for the establishment of an unambiguous aggressiveness ranking, where the MDC_Fg1 strain produced systematically the strongest symptoms, MDC_Fg13 strain induced intermediate ones, and MDC_FgU01 strain produced the weakest ones (Fabre et al., 2019a). This ranking has further proven the strong relationships with fungal protein abundance differences and especially with the accumulation of effector proteins. For each F. graminearum strain, the inocula were prepared at a concentration of 105 spores/ml of water. The three strains were individually inoculated in three plants of each wheat cultivar, i.e., three plants × three F. graminearum strains for a total of nine plants per cultivar. For each cultivar, inoculation was performed at the mid-anthesis stage by depositing 10 μl of inoculum in the floral cavity of six contiguous spikelets located in the middle zone of three synchronized spikes per plant, as described in Fabre et al. (2019b). Three other plants per cultivar were inoculated with water following the same methodology and were used as controls. For each cultivar × strain combination, the point-inoculated spikelets of the three spikes of three independent plants were specifically collected 72 hpi. The 72-hpi time point was chosen on the basis of our previous analyses that highlighted synchronized regulation in both fungal and wheat proteomes, demonstrating massive changes in protein abundance as compared to the 48-hpi stage (Fabre et al., 2019b). For each cultivar × strain combination and control sample, three biological replicates corresponding to three individual plants characterized by the pool of all inoculated spikelets from three spikes were collected and stored at −80°C for proteomics.

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