A new method for functional analysis of plastid EMBRYO-DEFECTIVE PPR genes by efficiently constructing cosuppression lin
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(2020) 16:154 Chen et al. Plant Methods https://doi.org/10.1186/s13007-020-00696-0
Open Access
RESEARCH
A new method for functional analysis of plastid EMBRYO‑DEFECTIVE PPR genes by efficiently constructing cosuppression lines in Arabidopsis Jingli Chen, Haojie Zhu, Jirong Huang and Weihua Huang*
Abstract Background: 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. 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 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, RNAdependent 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). Conclusions: Overexpression of an appropriately truncated EMB PPR CDS in WT leads to gene silencing in a RDR6dependent 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. Keywords: Arabidopsis, EMB PPR genes, Cosuppression, PTGS, RDR6 Background It is a common phenomenon that knockout mutants of plant essential genes display embryo or seedling lethal phenotype [1]. In the Arabidopsis genome, there are about 510 EMBRYO-DEFECTIVE (EMB) genes. *Correspondence: [email protected] Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Among them, 34 genes belong to the pentatricopeptiderepeat (PPR) gene family [1, 2]. 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. It has been known that over 450 PPR proteins are present in Arabidopsis and divided into two subfamilies, designated P
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