Fig. 6. Heterologous overexpression of AtALA1 in maize enhances resistance to F. graminearum and decreases DON contamination in seeds.

a DON inhibition assay of Ubi1::AtALA1 transgenic lines and wild-type seeds. Seeds were soaked with DON (60 μM) or sterilized water and then germinated in moisture chambers for 2 d. WT, wild-type maize (cultivar Hi II); # 4, # 13 and # 15, transgenic Ubi1::AtALA1 seeds (T₃). Scale bar, 0.5 cm. b Radicle length of Ubi1::AtALA1 transgenic lines and wild-type seeds in the absence or presence of DON (60 μM). The results are shown with box-and-whisker plots of three replicates (5 roots each). c Distribution of DON^5-FAM in root cells of Ubi1::AtALA1 transgenic lines and wild-type. Three-day-old seedlings were treated with DON^5-FAM (30 μg/mL) and FM4-64 (20 μM) for 12 h. Scale bar, 20 μm. d Statistics of the proportion of DON^5-FAM-containing vacuoles in the root cells, as shown with box-and-whisker plots of three replicates (nine roots each). **p < 0.01, significantly different with respect to wild-type according to Student’s t test (two-tailed). e Stalk rot symptoms on coleoptiles of Ubi1::AtALA1 transgenic lines and wild type. F. graminearum spores suspension (8 μL, 7.5 × 10⁷ spores/mL) was dropped onto the coleoptiles of 6-day-old seedlings. To promote infection, the coleoptiles were wounded with a scalpel. Scale bar, 1 cm. f Statistics for lesion length on the F. graminearum-infected coleoptiles (6 dpi) of Ubi1::AtALA1 transgenic lines and wild-type maize. Six-day-old seedlings were treated with F. graminearum (7.5 × 10⁷ spores/mL). Data are presented as box-and-whisker plots of three replicates (eight plants each). g Rot symptoms of ears (40 dpi) of Ubi1::AtALA1 transgenic lines and wild-type maize. Maize ears (three days after pollination) of Ubi1::AtALA1 transgenic lines and wild-type were inoculated with F. graminearum (5 × 10⁵ spores/mL). Mock, maize ears (three days after pollination) of Ubi1::AtALA1 transgenic lines and wild-type plants were inoculated with deionized water as a control. Scale bar, 2 cm. h Statistics for the incidence rate of F. graminearum-infected maize ears. Data are presented as box-and-whisker plots of three replicates (five maize ears each). i Statistics for the diseased area ratio of F. graminearum-infected maize ears, shown as dot plots of three replicates (four ears each from individual plants). j DON concentration in the seeds from F. graminearum-infected corncobs of Ubi1::AtALA1 transgenic lines and wild-type maize. DON concentration was determined by LC-MS/MS. The results are shown as dot plots of three replicates (each replicate had 80 g seeds from four ears, which were harvested from individual plants). Box-and-whisker plots show the medians (horizontal lines), upper and lower quartiles (box edges), and 1.5× the interquartile range (whiskers). Different letters in b, f, and h–j represent significant differences at P < 0.05 by one-way ANOVA with Tukey multiple comparisons test. n.s., not significant.