Research Article: Mediated Plastid RNA Editing in Plant Immunity

Date Published: October 31, 2013

Publisher: Public Library of Science

Author(s): Javier García-Andrade, Vicente Ramírez, Ana López, Pablo Vera, David Mackey.


Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.

Partial Text

Plastid function relies on nuclear gene expression, and the import of nuclear gene products into plastids [1]. In fact, the plastid genome of current land plants encodes 75–80 proteins [2], whereas nuclear-encoded chloroplast proteins are estimated between 3500 and 4000 [3]. Current data approximates several hundred nuclear-encoded proteins are involved in post-transcriptional regulation of plastid gene expression [4], [5]. One such regulation is mediated through RNA editing, a post-transcriptional process that alters specific cytidine residues to uridine (C-to-U) in different plastid RNAs [6]. Thirty-four sites are edited in 18 transcripts of Arabidopsis plastids [7]. Among the nuclear-encoded proteins regulating RNA editing, the pentatricopeptide repeat (PPR) protein family has attracted notable interest [8]. This family comprises 450 members defined by a tandem array of PPR motifs. PPRs are also involved in almost all stages of plastid gene expression, including splicing, RNA cleavage, translation, and RNA stabilization [9]. The pioneer work of Kotera et al. [10], [11] revealed the Arabidopsis PPR protein CHLORORESPIRATORY REDUCTION4 (CRR4) acts as a site-specific recognition factor for RNA editing of the site 1 (ndhD-1) in the plastid ndhD transcript. ndhD encodes the D subunit of the chloroplast NADH dehydrogenase-like complex (NDH), involved in cyclic electron flow (CEF) around photosystem I (PSI) [11], [12]. Consequently, crr4 mutants are defective in ndhD transcript editing at the ndhD-1 site, and CEF is compromised [10], [11]. Subsequently, the number of PPR-encoding genes participating in editing control in the chloroplast has enlarged [9]. Although empirical evidence has been demonstrated for only a few PPR proteins, it is currently accepted that PPR proteins act as sequence-specific RNA binding adaptors, and hypothetical inferences suggest PPRs recruit effector enzymes or proteins to the target RNAs [13], [14]. While the mechanism by which specific PPR proteins recognize specific editing sites is becoming understood, questions still remain to be completely solved including the characterization of the molecular components that conform the RNA editing apparatus (editosome) or the still unsolved identification of editing enzyme itself. Therefore, identification of additional components modulating editing activities in plastids, and ascertaining how control of the post-transcriptional mechanism of chloroplast function influences other biological processes, in particular immune responses, is of great importance.

This study provides new insights into the control of disease resistance in plants, and reinforces the importance of the chloroplasts in plant immunity. Our data identified OCP3 targeted to chloroplast, a finding that conceptually changed the previous assumption that OCP3 could function as a nuclear transcription factor. Furthermore, confocal microscopy revealed that OCP3 accumulated in plastids matching several PPR proteins. Moreover, OCP3 was found to be closely co-expressed with a cluster of 9 genes encoding PPR proteins including CRR21. CRR21 is responsible for site 2 editing at ndhD transcript, and ndhD encodes the D subunit of the chloroplast NDH complex [13], a crucial component of the CEF machinery around PSI [11]. In crr21 plants, NDH complex activity is impaired and CEF activity compromised [13], supporting a predominant post-transcriptional level of control. All these observations prompted us to hypothesize that OCP3 was involved in RNA editing in plastids. Therefore, we performed a comparative systematic study of the editing status of chloroplast transcripts between Col-0 and different ocp3 mutants. This study revealed that OCP3-defective plants carry specific editing defects at ndhB-6, ndhB-4, ndhB-3, and ndhB-2 sites. The observation that OCP3 associates in vivo with the ndhB transcript, as revealed by RIP assays, reinforce the consideration that OCP3 contributes to control over the extent of ndhB transcript editing. However, OCP3 appears not to carry any structural motif resembling the conserved RNA recognition motif (RRM), not even the motifs characteristic of other proteins functioning as trans-factors essential for editing, such as those present in the large subclasses of the pentatricopeptide repeat (PPR)-containing family proteins [13], [14]. This may suggest that the association of OCP3 with the ndhB RNA molecule may be likely indirect, presumably though the interaction with canonical RNA binding proteins recognizing appropriate cis-elements present in the ndhB RNA molecule such as those RNA-binding proteins mentioned above. Therefore, OCP3 may serve a regulatory role on the editing apparatus by regulating and/or adjusting the editing extent of the ndhB transcript according to external environmental cues. This appears to be the case also for other described editing accessory proteins such as the recently identified multiple organellar RNA editing factor (MORF) and members of the RNA-editing interacting protein (RIP) family [39], [40].




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