(A)
Erysium cheiranthoides (3 HAP), Cardamine flexuosa (3 HAP), Rorippa indica (6 HAP) or Descurainia sophia (6 HAP) pollen tubes on WT (A. thaliana) stigmas pre-treated with/without 10 μM of synthetic pR26 peptide. The percentage values, shown as average percentage (s.d.), indicate average pollen tube penetration ratio of the corresponding pollen in A. thaliana pistils. Scale bars, 200 μm.
(B-E) Treatment of stigmas with the “key” pRALF26 and opening the stigmatic “lock” by knocking out sRALFs, respectively, broke the intergeneric reproductive barrier. In (B) and (D), arrowheads indicate enlarged ovules. Values in (B) and (D), shown as average (s.d.), indicate the number of enlarged ovules in the pod. (C) and (E) show DIC images of hybrid embryos. Dashed lines highlight the hybrid embryos formed 9 and 7 days after pollination (DAP), respectively. Scale bars, 1 mm (siliques) and 50 μm (embryos). Each experiment was repeated at least three times with consistent results.
(F) CSEM images of A. thaliana stigma (At) pollinated with Cardamine flexuosa (left), C. flexuosa and A. thaliana pollen (middle), as well as C. flexuosa pollen after pretreatment with synthetic pRALF26 peptide at 3 HAP (right). Black arrows point to C. flexuosa pollen tubes and white arrows point to A. thaliana pollen tubes penetrating papilla cells, whereas black arrow heads point to C. flexuosa pollen tubes failing to penetrate papilla cells. Scale bars, 25 μm.
(G) Graphic diagram describing the pollen mentor effect (allowing incompatible pollen tubes (blue) to penetrate and grow towards the transmitting tract. This can be induced by paracrine pRALF peptides, which are either derived from compatible pollen tubes (orange) or after treatment of synthetic peptides. External application of pRALFs enhances the efficiency of the pollen mentor effect, allowing distantly related intergeneric species to hybridize with A. thaliana and generate conventionally unattainable hybrids. See also Figure S6.