Transcripts of plant organelle genes are modified by cytidine-touridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virusinduced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.nucleoid | RNA editosome | dual targeting P osttranscriptional C-to-U RNA editing occurs in plastid and plant mitochondrial transcripts. In a typical vascular plant, ∼30 C targets in chloroplasts and over 500 C targets in mitochondria are targeted for editing (1, 2). The majority of the editing events results in encoding of a different amino acid than the one predicted from the genomic sequence. The editing-encoded amino acid is usually more conserved relative to residues present in homologous proteins in other organisms than the genomically encoded amino acid. Because there is presently no known case in which useful genetic variation results from partial editing of a transcript population, the current concept is that editing is a correction mechanism for thymidine-to-cytidine (T-to-C) mutations that have arisen in plant organelle genomes (1,3,4).Little is known about the molecular apparatus that is responsible for recognizing the correct C target for editing and converting it to U, although plant mitochondrial RNA editing was discovered over 20 y ago (5-7). cis-Elements for recognition of editing sites have been identified proximal and 5′ to the nucleotide to be modified (8-10). As few as 22 nt in sequence surrounding the C target is sufficient to specify RNA editing (9). In 2005, a pentatricopeptide repeat (PPR) motif-containing protein termed CRR4 was discovered to ...

Several nuclear-encoded proteins containing pentatricopeptide repeat (PPR) motifs have previously been identified to be transfactors essential for particular chloroplast RNA editing events through analysis of mutants affected in chloroplast biogenesis or function. Other PPR genes are known to encode proteins involved in other aspects of organelle RNA metabolism. A function has not been assigned to most members of the large plant PPR gene family. Arabidopsis and rice each contain over 400 PPR genes, of which about a fifth exhibit a C-terminal DYW domain. We describe here a comparative genomics approach that will facilitate identification of the role of RNA-binding proteins in organelle RNA metabolism. We have implemented this strategy to identify an Arabidopsis nuclear-encoded gene RARE1 that is required for editing of the chloroplast accD transcript. RARE1 carries 15 PPR motifs, an E/E+ and a DYW domain, whereas previously reported editing factors CRR4, CRR21, and CLB19 lack a DYW domain. The accD gene encodes the b carboxyltransferase subunit of acetyl coA carboxylase, which catalyzes the first step in fatty acid biosynthesis in chloroplasts. Despite a lack of accD C794 editing and lack of restoration of an evolutionarily conserved leucine residue in the b carboxyltransferase protein, rare1 mutants are unexpectedly robust and reproduce under growth room conditions. Previously the serine-to-leucine alteration caused by editing was deemed essential in the light of the finding that a recombinantly expressed ''unedited'' form of the pea acetyl coA carboxylase was catalytically inactive.Keywords: chloroplast; RNA editing; accD; RARE1; pentatricopeptide; trans-factor INTRODUCTION Vascular plant organelle transcripts undergo C-to-U RNA editing (for review, see Hanson et al. 1996;Bock 2000;Tillich et al. 2005;Shikanai 2006). In Arabidopsis, 34 editing events are known to occur in chloroplast transcripts (Tillich et al. 2005;Chateigner-Boutin and Small 2007), while 508 Cs are known to be modified to Us in Arabidopsis mitochondria (Bentolila et al. 2008). The amino acid encoded by edited transcripts often differs from the one predicted from unedited transcripts, usually resulting in increased evolutionary conservation of the amino acid sequence from the one predicted from genomic sequence (Gualberto et al. 1989), although start and stop codons in organelle transcripts are also sometimes created by C-to-U editing (Wintz and Hanson 1991;Kudla et al. 1992). The residues modified by RNA editing are often important for the three-dimensional structure of the protein (Yura and Go 2008). RNA editing appears to be a mechanism to correct defective organelle genes at the transcript level.The suite of particular C-to-U editing events varies from one plant species to another, even though RNA editing probably arose in an ancestor common to the land plants (Tillich et al. 2006). Between divergent species, such as between dicots and monocots, RNA editing C targets vary considerably. In species that do not contain a particular C-...

The native ‘ōhi’a lehua (Metrosideros polymorpha) has cultural, biological and ecological significance to Hawai’i, but it is seriously threatened by a disease commonly referred to as rapid ‘ōhi’a death (ROD). Preliminary investigations showed that a Ceratocystis species similar to C. fimbriata s.lat. was the cause of the disease. In this study, we used a combination of the phylogenetic, morphological and biological species concepts, as well as pathogenicity tests and microsatellite analyses, to characterise isolates collected from diseased ‘ōhi’a trees across Hawai’i Island. Two distinct lineages, representing new species of Ceratocystis, were evident based on multigene phylogenetic analyses. These are described here as C. lukuohia and C. huliohia. Ceratocystis lukuohia forms part of the Latin American clade (LAC) and was most closely associated with isolates from Syngonium and Xanthosoma from the Caribbean and elsewhere, including Hawai’i, and C. platani, which is native to eastern USA. Ceratocystis huliohia resides in the Asian-Australian clade (AAC) and is most closely related to C. uchidae, C. changhui and C. cercfabiensis, which are thought to be native to Asia. Morphology and interfertility tests support the delineation of these two new species and pathogenicity tests show that both species are aggressive pathogens on seedlings of M. polymorpha. Characterisation of isolates using microsatellite markers suggest that both species are clonal and likely represent recently-introduced strains. Intensive research is underway to develop rapid screening protocols for early detection of the pathogens and management strategies in an attempt to prevent the spread of the pathogens to the other islands of Hawai’i, which are currently disease free.

Myrtaceae (myrtle family) 'ōhi'a, 'ōhi'a lehua, lehua (Hawai'i)