Figure 2.

_DNDMC1 Fusion Transcript Transport Induces Aberrant Pollen Formation.

(A) Flowers of grafted wild-type stock plants supported by 35S_pro:YFP-_DNDMC1 transgenic scions and reciprocal grafts are fertile.

(B) Upper panel: Grafted wild-type/wild-type or wild-type/_DNDMC1:tRNA^Met plants showed normal pollen production when mock treated. Lower panel: Estradiol-induced expression of _DNDMC1:tRNA^Met in scion or stock plant parts resulted in partially sterile anthers in both transgenic and wild-type flowers. The latter suggests _DNDMC1:tRNA^Met mRNA transport and expression of the truncated DMC1 protein in wild-type male meiocytes.

(C) Flowers of grafted _DNDMC1:BEL5 transgenic plants. Upper panel: Mock-treated wild-type/estradiol>>_DNDMC1:BEL5 grafts showed weak male sterility. Lower panel: Flowers of grafted plants treated with estradiol exhibit partial male sterility.

(D) RT-PCR assays on RNAs samples from grafted wild-type tissues revealed that the YFP-_DNDMC1 control transcript is not allocated over graft junctions into wild-type stock leaves (n = 6) or scion flowers (n = 8). ACTIN2 (ACT2) specific RT-PCR was used as a positive control.

(E) RT-PCR assays on RNA samples from grafted plants. _DNDMC1:tRNA^Met and _DNDMC1:BEL5 is detected in transgenic and in wild-type scion flowers. Appearance of a specific PCR product in samples from grafted wild-type stock leaves and wild-type flowers (red asterisks) suggests mobility of the _DNDMC1:tRNA^Met fusion transcript. Number of tested grafted plants is shown on the right.

(F) CLSM images of sepals formed on YFP-_DNDMC1 producing scions. Upper panel: YFP-_DNDMC1/YFP-_DNDMC1 control graft with expected high green fluorescence emitted by YFP-_DNDMC1. Middle panel: Control graft with siRNA-producing stock plants (hpDMC1) with expected low YFP fluorescence and distribution in YFP-_DNDMC1 flowers (Zhang et al., 2014). Lower panel: YFP-_DNDMC1 scion grafted onto _DNDMC1:tRNA^Met transgenic stock shows similar YFP fluorescence levels as YFP-_DNDMC1/YFP-_DNDMC1 control grafts. Note that YFP-_DNDMC1 fusion protein is detected in all epidermal leaf cells except when grafted with hpDMC1 producing DMC1 siRNA lines. Green, presence of YFP-_DNDMC1; blue, plastid autofluorescence. Bar = 300 µm.

(G) Statistical analysis of data from automated detection of misshaped pollen appearing on grafted plants. Misshapen pollen formation was significantly higher on wild-type scions supported by _DNDMC1:tRNA^Met and _DNDMC1:BEL5 stock plants than in control grafts. Asterisks indicate highly significant differences against controls using χ² test for independence of variables in a contingency table. Biological replicates: n > 8. Error bars indicate sd. For details, see Supplemental Table 1.