Figure 8.

Cotransformation with GSP1 and FUS1 Rescues the bp31 Phenotype.

(A) A schematic drawing of the construct used to express FUS1 mRNA. The HSP70+RbcS promoter is connected to the FUS1 cDNA (FUS1c). UTR, untranslated region.

(B) DIC ([a] and [c]) and fluorescence ([b] and [d]) images of SYBR Green I–stained young zygotes. N, cell nucleus; WT, the wild type; arrows, cp nucleoids; arrowheads, mt nucleoids. The preferential digestion of the mt− cp nucleoids was observed in bp31 transformed with the GSP1gu and FUS1c.

(C) The frequencies of gametes (blue), zygotes with mt− cp nucleoids (red), and zygotes lacking mt− cp nucleoids (green) at 30, 60, 90, and 120 min after mating (bp31+GSP1gu+FUS1c mt+ × wild-type mt−).

(D) Pellicle formation in wild-type mt+ × wild-type mt−, bp31 mt+ × wild-type mt−, and bp31+GSP1gu+FUS1c × wild-type mt−.

(E) The expression profiles of gamete-specific (GSP1, FUS1, and SAG1) and zygote-specific (EZY1, ZSP1, and ZYS3) genes in bp31+GSP1gu+FUS1c × wild-type mt−. Gametes (G) and zygotes (30, 60, 90 min after mating) were analyzed.

(F) Tetrad analysis of bp31+GSP1gu+FUS1c × myx mt− to examine the mtDNA inheritance and bp31+GSP1gu+FUS1c × CL218 mt− to examine the cpDNA inheritance. The typical results of two complete tetrads are shown. The total DNA was extracted, and the cytb, aadA, and atpB sequences (as a control chloroplast gene) were amplified using PCR. The cytb PCR product was digested with MnlI. The bottom panel shows the result of plating tetrads on TAP agar plates containing spectinomycin and paromomycin to determine the transmission of AphVIII and aadA, respectively. The uniparental inheritance of the cp/mtDNA was restored in bp31+GSP1gu+FUS1c, whereas a 1:1 Mendelian transmission was present for the nuclear AphVIII transgene.