Abstract
BackgroundPentatricopeptide-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.ResultsHere, 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).ConclusionsOverexpression 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.
Highlights
Pentatricopeptide-repeat proteins (PPRs) characterized by tandem arrays of a degenerate 35-aminoacid 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
Overexpression of the mutated EMBRYO DEFECTIVE2279 (EMB2279)/SUPPRESSOR OF THF1 (SOT5) and EMBRYO DEFECTIVE2654 (EMB2654) coding sequence (CDS) in wild type (WT) leads to RNA-dependent RNA polymerase 6 (RDR6)‐dependent gene silencing We previously reported that a weak allele of emb2279-2/ sot5 mutant exhibits a virescent phenotype, which is caused by a point mutation that significantly reduces splicing efficiency of the seventh intron of SOT5 and generates two additional mRNA variants [8]
Our results showed that 80% of the 35S:SOT5 mutated protein 1 (SOT5-m1) transgenic lines exhibited severe leaf chlorosis at the early growth stage, and these leaves were gradually turned into pale green later; and 48% of 35S:SOT5 mutated protein 2 (SOT5-m2) transgenic lines displayed relatively mild leaf chlorosis and virescence (Table 1 and Fig. 2a)
Summary
Pentatricopeptide-repeat proteins (PPRs) characterized by tandem arrays of a degenerate 35-aminoacid 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. It is a common phenomenon that knockout mutants of plant essential genes display embryo or seedling lethal phenotype [1]. PPR proteins characterized by tandem arrays of a degenerate 35-amino-acid repeat (PPR motif ) are conserved in all eukaryotes and extremely expanded in higher plants.
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