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Background  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.    Results  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 T 1  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 T 1  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).    Conclusions  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.

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