Extended Data Fig. 8: Ectopic expression of ECS1 and ECS2 in synergid cells leads to a decrease of LURE1.2 protein levels and strongly reduced pollen tube attraction rate.
From: Fertilized egg cells secrete endopeptidases to avoid polytubey
a, Ectopically expressed ECS1–GFP fusion protein in synergid cells is secreted to the filiform apparatus. b, Immunofluorescence revealed that LURE1 levels were significant decreased in ovules ectopically expressing ECS1-GFP in synergid cells using the DD31 promoter. c, Quantification of LURE1 fluorescence intensity in WT and ECS1-ectopically expressed ovules as shown in (b) (n = 10). d, Ectopic expression of ECS1 in synergid cells resulted in pollen tube attraction defects 6 HAP. Pollen tube growth analysis was performed using a Lat52::GUS reporter line. e, Percentages of ovules attracting pollen tubes observed in WT pistils and those ectopically expressing ECS1 in synergid cells of three independent lines (L1–L3) at 6 HAP (n = 50 for WT and DD31::ECS1 L1; 60 for DD31::ECS1 L2 and DD31::ECS1 L3). f, Immunofluorescence revealed that LURE1 levels were significantly decreased in ovules ectopically expressing ECS2 in synergid cells using the DD31 promoter. g, Quantification of LURE1 fluorescence intensity in WT ovules and those ectopically expressing ECS2 (n = 10). h, Similarly, LURE1.2–GFP signals were significantly decreased in ovules ectopically expressing ECS2 in synergid cells. i, Quantification of LURE1.2–GFP fluorescence intensity as described in b (n = 101). j, k, Ectopic expression of ECS2 in synergid cells resulted in reduction of pollen tube attraction 6 HAP. Lat52::DsRed (j) and Lat52::GUS reporter line (k) were used in this analysis. l, Proportions of ovules attracting pollen tubes observed in WT plants and those ectopically expressing ECS2 in synergid cells at 6 HAP (n = 50 for WT, DD31::ECS2 L2 and DD31::ECS2 L3; 80 for DD31::ECS2 L1). Data in c, g, i are presented in box-and-whisker plots. Bottom and top of the box, 25th and 75th percentiles; centre line, 50th percentile; whiskers, minimum and maximum data. ** indicates statistical difference compared to WT (Two-tailed Student’s t-test; P < 0.01). Same letters (in e and l) indicate lack of significant differences according to the Tukey–Kramer multiple comparison test (one-way ANOVA between groups; P = 1.94 × 10−7; F = 32.11 in e; P = 3.24 × 10−8, F = 37.88 in l). P < 0.05 was considered as significant. Scale bars, 20 μm (a, b, f, h), 100 μm (j). ec, egg cell; sy, synergid; pt, pollen tube; ov, ovule.
