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Abstract ATPase family AAA domain–containing 3 (ATAD3) proteins are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent in Fungi and Amoebozoa. These ∼600-amino acid proteins are anchored in the inner mitochondrial membrane and are essential in metazoans and Arabidopsis thaliana. ATAD3s comprise a C-terminal ATPases Associated with a variety of cellular Activities (AAA+) matrix domain and an ATAD3_N domain, which is located primarily in the inner membrane space but potentially extends to the cytosol to interact with the ER. Sequence and structural alignments indicate that ATAD3 proteins are most similar to classic chaperone unfoldases in the AAA+ family, suggesting that they operate in mitochondrial protein quality control. A. thaliana has four ATAD3 genes in two distinct clades that appear first in the seed plants, and both clades are essential for viability. The four genes are generally coordinately expressed, and transcripts are highest in growing apices and imbibed seeds. Plants with disrupted ATAD3 have reduced growth, aberrant mitochondrial morphology, diffuse nucleoids and reduced oxidative phosphorylation complex I. These and other pleiotropic phenotypes are also observed in ATAD3 mutants in metazoans. Here, we discuss the distribution of ATAD3 proteins as they have evolved in the plant kingdom, their unique structure, what we know about their function in plants and the challenges in determining their essential roles in mitochondria. 

Abstract Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template Alternate Protocol 2: Designing the plasmid‐based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts Basic Protocol 4: Identifying edited plants.