Figure 6. HAP2 protein expression in L. tropica promastigotes is associated with in vitro hybridization capacity.

Schematic representation of the mNeonGreen (mNG)-HAP2 fusion reporter construct generated in the L747 strain (A) and in the MA37 strain (F). Stationary phase promastigotes of either L747 mNG-HAP2 or MA37 mNG-HAP2 parental lines were used to inoculate fresh cultures that were immediately divided into two flasks, with one flask remaining untreated and the other exposed to 6.5 Gy of γ-radiation or supplemented with 250 μM of H₂O₂ or 0.005% of methyl methanesulfonate (MMS). Flow cytometry histogram plots showing the fluorescence intensity of mNG-HAP2 in L747 (B) and in MA37 (G) 1 day post-inoculation, with or without irradiation. Proportion of mNG-HAP2⁺ cells over time in L747 mNG-HAP2 cultures with or without exposure to γ-radiation (C), to H₂O₂ (D) or to MMS (E), n = 3 replicates. Proportion of mNG-HAP2⁺ cells in MA37 transfectants after exposure to γ-radiation (H), to H₂O₂ (I), or to MMS (J), n = 4 replicates. (K) Proportion of culture wells exhibiting growth of LMA hybrids after in vitro crosses using the different indicated pairwise combinations of FACS-sorted mNG-HAP2⁺ and mNG-HAP2^- cells 1 day after irradiation; data are presented as mean values (black line) and individual measurements. (L) Minimum frequency of hybridization-competent cells for both L747 and MA37 parental strains in in vitro crosses using different combinations of FACS-sorted mNG-HAP2⁺ and mNG-HAP2^- cells (n = 2 independent experiments). Frequencies collected from each single experiment are linked by a line. Red lines and green lines represent data from L747 and MA37 parental strains, respectively.

Figure 6—figure supplement 1. Representative flow cytometry analysis used to validate hybrids generated by crossing different combinations of mNG-HAP2⁺ and mNG-HAP2^- sorted cells from parental strains expressing either tdTomato or mCherry in the small ribosomal subunit rRNA locus (SSU).