Figure 4. The dTALE-activating GhSWEET10 induces water-soaking in cotton.

(a) RVDs of dTALEs and the corresponding targeted EBE sequences in the G. hirsutum genome. (b) Xcm HM2.2S expressing a dTALE uniquely targeting GhSWEET10D induces water-soaking in cotton. Cotyledons from 2-week-old Ac44E plants were syringe-inoculated with different Xcm strains at OD₆₀₀=0.1 and photographed at 4 dpi. The table displays the presence (+) or absence (−) of water-soaking. (c) RT–PCR analysis of GhSWEET10, GhKBS1 and GhMDR1 targeted by their respective dTALEs. Two-week-old cotyledons of Ac44E were syringe-inoculated with Xcm strains HM2.2S (OD₆₀₀=0.1) containing different dTALEs. Inoculated tissues were collected at 24 hpi for RT–PCR analysis. (d) GaSWEET10 is induced by Avrb6 in G. arboreum protoplasts. Protoplasts isolated from G. arboreum were transfected with Avrb6 or an empty vector control (Ctrl) and samples were collected 12 h after transfection for RT–PCR analysis. (e) Transactivation assay of GaSWEET10 and GhSWEET10D promoters in response to Avrb6 in cotton protoplasts. Alignment of Avrb6 EBEs reveals a polymorphism between GhSWEET10D and GaSWEET10 promoters, which were highlighted in blue. Protoplasts isolated from commercial cotton variety FM706V were co-transfected with pGaSWEET10::LUC or pGhSWEET10D::LUC and Avrb6 or a vector control (Ctrl). The data are shown as mean±s.d. (n=3) from three independent repeats. (f) The dTALE matching the GaSWEET10 promoter causes water-soaking on G. arboreum. The EBE sequences of two dTALEs targeted to different regions of the GaSWEET10 promoter are shown. dTALE2 with an EBE sequence perfectly matching the GaSWEET10 promoter but not dTALE1 with a partially matching EBE sequence restores Xcm-mediated water-soaking on cotton. Cotyledons from 2-week-old G. arboreum were syringe-inoculated with different Xcm strains at OD₆₀₀=0.1 and photographed at 4 dpi. The above experiments were repeated three times with similar results.