Wheat Varietal Response to Tilletia controversa J. G. Kühn Using qRT-PCR and Laser Confocal Microscopy

Abstract

Tilletia controversa J. G. Kühn is a causal organism of dwarf bunt in wheat. Understanding the interaction of wheat and T. controversa is of practical and scientific importance for disease control. In this study, the relative expression of TaLHY and TaPR-4 and TaPR-5 genes was higher in a resistant (Yinong 18) and moderately resistant (Pin 9928) cultivars rather than susceptible (Dongxuan 3) cultivar at 72 h post inoculation (hpi) with T. controversa. Similarly, the expression of defensin, TaPR-2 and TaPR-10 genes was observed higher in resistant and moderately resistant cultivars after exogenous application of phytohormones, including methyl jasmonate, salicylic acid, and abscisic acid. Laser confocal microscopy was used to track the fungal hyphae in the roots, leaves, and tapetum cells, which of susceptible cultivar were infected harshly by T. controversa than moderately resistant and resistant cultivars. There were no fungal hyphae in tapetum cells in susceptible cultivar after methyl jasmonate, salicylic acid and abscisic acid treatments. Moreover, after T. controversa infection, the pollen germination was of 80.06, 58.73, and 0.67% in resistant, moderately resistant and susceptible cultivars, respectively. The above results suggested that the use using of resistant cultivar is a good option against the dwarf bunt disease.

Keywords: TaPR genes; laser confocal microscopy; pollen grain; tapetum; wheat dwarf bunt.

Figure 1

Expression profiles of TaLHY, TaPR-4, and TaPR-5 in resistant (Yinong 18), moderately resistant (Pin 9928), and susceptible (Dongxuan 3) wheat cultivars at different time intervals after T. controversa infection. (a) relative expression of TaLHY; (b) relative expression of TaPR-4; (c) relative expression of TaPR-5. The T. controversa treatment at every time point is normalized at 0 hpi. The significant differences were statistically analyzed based on three biological replications and four technical replications (Tukey’s test: p < 0.05). Bars indicate the standard errors. Lettering above the bars showed level of significance.

Figure 2

Transcriptional patterns of defensin, TaPR-2 and TaPR-10 after treatment with hormones. The resistant (Yinong 18), moderately resistant (Pin 9928) and susceptible (Dongxuan 3) wheat cultivars were sprayed with Methyl jasmonate (MeJa), salicylic acid (SA), and abscisic acid (ABA). The plants treated with ddH₂O were used as control in the study. (a) relative expression of defensin; (b) relative expression of TaPR-2; (c) relative expression of TaPR-10. The significant differences were statistically analyzed based on three biological replications and four technical replications (Tukey’s test: ** p < 0.05). Bars indicate the standard errors.

Figure 3

Infestation of T. controversa in wheat roots and leaves as indicated by staining with WGA-AF 488 (for hyphae) and propidium iodide (for roots and leaves cell) (a) severity of fungal hyphae in roots of resistant (Yinong 18) cultivar; (b) severity of fungal hyphae in roots of moderately resistant (Pin 9928) cultivar; (c) severity of fungal hyphae in roots of susceptible (Dongxuan 3) cultivar; (d) severity of fungal hyphae in leaves of resistant (Yinong 18) cultivar; (e) severity of fungal hyphae in leaves of moderately resistant (Pin 9928) cultivar; (f) severity of fungal hyphae in leaves of susceptible (Dongxuan 3) cultivar. Scale bar = 25 µm.

Figure 4

Examination of fungal hyphae in tapetum cells of anther in resistant (Yinong 18), moderately resistant (Pin 9928) and susceptible (Dongxuan 3) wheat cultivars. (a) examination of fungal hyphae in tapetum cells of resistant cultivar; (b) examination of fungal hyphae in tapetum cells of moderately resistant cultivar; (c) examination of fungal hyphae in tapetum cells of susceptible cultivar. Scale bar = 25 µm.

Figure 5

Effect of exogenous hormones on tapetum cells of anther in susceptible cultivar (Dongxuan 3). (a) hyphae were located on the tapetum cells in control anthers; (b) There were no hyphae on tapetum cells of anther in MeJa treated samples; (c) There were no hyphae on tapetum cells of anther in SA treated samples; (d) There were no hyphae on tapetum cells of anther in ABA treated samples. Scale bar = 25 µm.

Figure 6

Effect of T. controversa on pollen germination (%). (a) pollen germination in control samples of resistant cultivar (Yinong 18); (b) pollen germination in control samples of moderately resistant cultivar (Pin 9928); (c) pollen germination in control samples of susceptible cultivar (Dongxuan 3); (d) pollen germination in T. controversa infected samples of resistant cultivar (Yinong 18); (e) pollen germination in T. controversa infected samples of moderately resistant cultivar (Pin 9928); (f) pollen germination in T. controversa infected samples of susceptible cultivar (Dongxuan 3) Scale bar = 100 µm.

Figure 7

Level of disease incidence in resistant (Yinong 18), moderately resistant (Pin 9928) and susceptible (Dongxuan 3) wheat cultivars to T. controversa infection. Yinong 18 showed 8.89%, Pin 9928 showed 26.67%, and Dongxuan 3 showed 62.2% disease incidence.