A new method for functional analysis of plastid EMBRYO-DEFECTIVE PPR genes by efficiently constructing cosuppression...

Pentatricopeptide-repeat proteins (PPRs) characterized by tandem arrays of a degenerate 35-amino-acid repeat (PPR motif) can bind a single strand RNA and regulate organelle gene expression at the post-transcriptional level, including RNA cleavage, splicing, editing and stability etc. PPRs are conserved in all eukaryotes and extremely expanded in higher plants. Many knockout mutants of PPR genes are embryonically lethal. These genes are named EMB PPRs and functional analysis of them is hindered by the difficulty in obtaining their knockout mutants.

Here, we report a new method for functional analysis of plastid EMB PPRs by efficiently constructing their cosuppression lines in Arabidopsis. When we overexpressed a mutated full length or truncated coding sequence (CDS) of EMB PPRs, such as EMB2279, EMB2654 and EMB976 (all belong to the P family PPRs) in the wild-type (WT) background, a large portion of T₁ plants displayed chlorosis phenotypes, which are similar to those of the weak allele mutants, knockdown lines or partially complementary lines. RT-PCR analysis showed that overexpression of the truncated EMB PPRs led to significant and specific downregulation of their corresponding endogenous mRNAs. However, when these EMB PPRs were overexpressed in the Post transcriptional Gene Silencing (PTGS) deficient mutant, RNA-dependent RNA polymerase 6 (rdr6), none of the T₁ plants displayed chlorosis phenotypes. These results indicate that the chlorosis phenotype results from post transcriptional silencing of the corresponding endogenous gene (also known as sense cosuppression).

Overexpression of an appropriately truncated EMB PPR CDS in WT leads to gene silencing in a RDR6-dependent manner, and this method can be employed to study the unknown function of EMB PPR genes. By this method, we showed that EMB976 is required for splicing of chloroplast clpP1 intron 2 and ycf3 intron 1.