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. 2017 Apr 19;8:526. doi: 10.3389/fpls.2017.00526

Figure 5.

Figure 5

Md-miR156ab affects ALT1 susceptibility by suppressing the expression of MdWRKYN1, which regulates various PR genes. (A) Schematic of constructs used for overexpressing MdWRKYN1 via Agrobacterium tumefaciens-mediated transient expression. (B) MdWRKYN1 content at 24 hpi with ALT1, as revealed by real-time PCR. (C) The infection symptoms of WT, EV, and OE-MdWRKYN1-infiltrated Golden Delicious (GD) plants. (D) Disease rates in GD leaves overexpressing MdWRKYN1 at 24 hpi with ALT1. (E) Expression levels of 12 PR genes in GD leaves overexpressing MdWRKYN1 with or without ALT1 inoculation. In (B–E) WT, non-infiltrated GD plants; EV, empty-vector (pFGC5941)-infiltrated plants; OE-MdWRKYN1, MdWRKYN1-overexpressing GD plants. Spore inoculum concentration: 2 × 105 CFU/ml. Spore growth was measured at 6 dpi. Error bars = SD; **P < 0.01; *P < 0.05 (Student's t-test). Real-time PCR data were calculated based on three biological and three technical replicates. (F) Schematic of Short Tandem Target Mimic (STTM) constructs used for silencing Md-miR156ab via Agrobacterium tumefaciens-mediated transient expression. Red indicates the spacer region and the spacer sequence. Blue indicates the bulge sequences in the miRNA binding sites. (G) Real-time PCR confirming knockdown of Md-miR156ab activity. (H) MdWRKYN1 expression 24 hpi with ALT1, as revealed by real-time PCR analysis. (I) Disease rate in GD leaves following the reduction in Md-miR156ab activity at 24 hpi with ALT1. (J) Infection symptoms of STTM-miR156ab-infiltrated ALT1-inoculated GD plants in comparison with wild-type and empty-vector controls. (K) Expression of 12 PR genes in GD leaves in STTM-miR156ab-infiltrated plants, along with wild-type and empty-vector controls, at 24 hpi with ALT1. In (G–K) WT, non-infiltrated GD plants; EV, empty-vector (pFGC5941)-infiltrated plants; STTM-miR156ab, GD plants with silenced Md-miR156ab activity. Spore inoculum concentration: 2 × 105 CFU/ml. Spore growth was measured at 6 dpi. Error bars = SD; **P < 0.01; *P < 0.05 (Student's t-test). Real-time PCR data were calculated based on three biological and three technical replicates.