Advancing organelle genome transformation and editing for crop improvement

Abstract

Plant cells contain three organelles that harbor DNA: the nucleus, plastids, and mitochondria. Plastid transformation has emerged as an attractive platform for the generation of transgenic plants, also referred to as transplastomic plants. Plastid genomes have been genetically engineered to improve crop yield, nutritional quality, and resistance to abiotic and biotic stresses, as well as for recombinant protein production. Despite many promising proof-of-concept applications, transplastomic plants have not been commercialized to date. Sequence-specific nuclease technologies are widely used to precisely modify nuclear genomes, but these tools have not been applied to edit organelle genomes because the efficient homologous recombination system in plastids facilitates plastid genome editing. Unlike plastid transformation, successful genetic transformation of higher plant mitochondrial genome transformation was tested in several research group, but not successful to date. However, stepwise progress has been made in modifying mitochondrial genes and their transcripts, thus enabling the study of their functions. Here, we provide an overview of advances in organelle transformation and genome editing for crop improvement, and we discuss the bottlenecks and future development of these technologies.

Keywords: crop improvement; genome editing; homologous recombination; organelle; transformation.

Figure 1

Basic application of transplastomic technology to gene expression, genome modification, and in vivo analysis of RNA editing. (A) Targeting of a gene of interest (GOI) to a neutral insertion site in the plastid genome. Integration of GOI and selectable marker gene (SMG) cassettes into the plastid genome occurs through homologous recombination. A typical cassette comprises a promoter (green boxes), 5′ UTR (white boxes), coding region, and 3′ UTR (red boxes). Possible recombination events leading to successful plastid transformation are indicated by dashed arrows. (B) Targeted knockout of a plastid gene can be achieved by insertion of or replacement with the SMG cassette. (C) Introduction of a heterologous RNA editing site into an endogenous plastid gene by gene replacement. L(R)HRR, left (right) homologous recombination region; ptDNA, plastid DNA; X, endogenous gene; X∗, gene X that contains an RNA editing site (E) from a different species.

Figure 2

PPR proteins involved in organelle editing and their application in plastid engineering. (A) A model for the association of a single PLS-type PPR editing factor with its target RNA substrates in organelles. A presumptive RNA target is recognized by the PPR–RNA binding code proposed by Barkan et al. (2012) based on amino acid identities at the 6 and 1' amino acids positions of the S and P repeats (T/S + N: A; T/S + D: G; N + N: C/U; N + S: C > U; N + D: U > C). The role of the L repeat is not yet clear. The three carboxy-terminal PPRs of PLS-type PPR proteins generally differ in their amino acid conservation and are labeled P2, L2, and S2 (Cheng et al., 2016). The DYW domain with cytidine deaminase activity catalyzes the C-to-U conversion in plant organelles. (B) Expression of a gene of interest controlled by a synthetic 5′ UTR that is specifically stabilized by a designed PPR protein. The nuclear-encoded PPR protein is regulated by a tissue-specific or inducible promoter (Rojas et al., 2019; Yu et al., 2019). cTP, chloroplast transit peptide; GOI, gene of interest; L(R)B, left (right) border of transfer DNA; PPR, pentatricopeptide repeat; SMG, selectable marker gene; UTR, untranslated region.

Figure 3

Gene editing tools that are potentially able to modify mitochondrial genomes. (A) MitoTALEN used to precisely knock out two cytoplasmic male sterility-associated genes (Kazama et al., 2019). (B) In the mitoCRISPR-Cas system, a stem–loop-added single guide RNA could be imported to mitochondria and functionally interact with Cas to mediate sequence-specific mitochondrial DNA cleavage. (C) Two DddA_tox halves of DdCBE can reconstitute deamination activity only when assembled adjacently on the target DNA, analogous to the assembly of FokI monomers to reconstitute dsDNA nuclease activity in TALENs. The RNA-free DdCBE is a promising option that can be tested in crop mitochondria. DdCBE, DddA (an interbacterial toxin)-derived cytosine base editor; mitoTS, mitochondrial targeting sequence; PNPase, polynucleotide phosphorylase; UGI, uracil glycosylase inhibitor.