Relocation of chloroplast proteins from cytosols into chloroplasts.

The chloroplasts in terrestrial plants play a functional role as a major sensor for perceiving physiological changes under normal and stressful conditions. Despite the fact that the plant chloroplast genome encodes around 120 genes, which are mainly essential for photosynthesis and chloroplast biogenesis, the functional roles of the genes remain to be determined in plant's response to environmental stresses. Photosynthetic electron transfer D (PETD) is a key component of the chloroplast cytochrome b6f complex. Chloroplast ndhA (NADH dehydrogenase A) and ndhB (NADH dehydrogenase B) interact with photosystem I (PSI), forming NDH-PSI supercomplex. Notably, artificial targeting of chloroplasts-encoded proteins, PETD, NDHA, or NDHB, was successfully relocated from cytosols into chloroplasts. The result suggests that artificial targeting of proteins to chloroplasts is potentially open to the possibility of chloroplast biotechnology in engineering of plant tolerance against biotic and abiotic stresses.

Molecular Bases of Heat Stress Responses in Vegetable Crops With Focusing on Heat Shock Factors and Heat Shock Proteins.

The effects of the climate change including an increase in the average global temperatures, and abnormal weather events such as frequent and severe heatwaves are emerging as a worldwide ecological concern due to their impacts on plant vegetation and crop productivity. In this review, the molecular processes of plants in response to heat stress-from the sensing of heat stress, the subsequent molecular cascades associated with the activation of heat shock factors and their primary targets (heat shock proteins), to the cellular responses-have been summarized with an emphasis on the classification and functions of heat shock proteins. Vegetables contain many essential vitamins, minerals, antioxidants, and fibers that provide many critical health benefits to humans. The adverse effects of heat stress on vegetable growth can be alleviated by developing vegetable crops with enhanced thermotolerance with the aid of various genetic tools. To achieve this goal, a solid understanding of the molecular and/or cellular mechanisms underlying various responses of vegetables to high temperature is imperative. Therefore, efforts to identify heat stress-responsive genes including those that code for heat shock factors and heat shock proteins, their functional roles in vegetable crops, and also their application to developing vegetables tolerant to heat stress are discussed.

RsmD, a Chloroplast rRNA m2G Methyltransferase, Plays a Role in Cold Stress Tolerance by Possibly Affecting Chloroplast Translation in Arabidopsis.

Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized ArabidopsisRsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD- green fluorescent protein fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e. had shorter roots, lower fresh weight and pale-green leaves, compared with wild-type (WT) plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the WT, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered WT phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsisto cold stress.

N6 -Methyladenosine mRNA methylation is important for salt stress tolerance in Arabidopsis.

As the most abundant internal modification of mRNA, N6 -methyladenosine (m6 A) methylation of RNA is emerging as a new layer of epitranscriptomic gene regulation in cellular processes, including embryo development, flowering-time control, microspore generation and fruit ripening, in plants. However, the cellular role of m6 A in plant responses to environmental stimuli remains largely unexplored. In this study, we show that m6 A methylation plays an important role in salt stress tolerance in Arabidopsis. All mutants of m6 A writer components, including MTA, MTB, VIRILIZER (VIR) and HAKAI, displayed salt-sensitive phenotypes in an m6 A-dependent manner. The vir mutant, in which the level of m6 A was most highly reduced, exhibited salt-hypersensitive phenotypes. Analysis of the m6 A methylome in the vir mutant revealed a transcriptome-wide loss of m6 A modification in the 3' untranslated region (3'-UTR). We demonstrated further that VIR-mediated m6 A methylation modulates reactive oxygen species homeostasis by negatively regulating the mRNA stability of several salt stress negative regulators, including ATAF1, GI and GSTU17, through affecting 3'-UTR lengthening linked to alternative polyadenylation. Our results highlight the important role played by epitranscriptomic mRNA methylation in the salt stress response of Arabidopsis and indicate a strong link between m6 A methylation and 3'-UTR length and mRNA stability during stress adaptation.

Arabidopsis Mitochondrial Transcription Termination Factor mTERF2 Promotes Splicing of Group IIB Introns.

Plastid gene expression (PGE) is essential for chloroplast biogenesis and function and, hence, for plant development. However, many aspects of PGE remain obscure due to the complexity of the process. A hallmark of nuclear-organellar coordination of gene expression is the emergence of nucleus-encoded protein families, including nucleic-acid binding proteins, during the evolution of the green plant lineage. One of these is the mitochondrial transcription termination factor (mTERF) family, the members of which regulate various steps in gene expression in chloroplasts and/or mitochondria. Here, we describe the molecular function of the chloroplast-localized mTERF2 in Arabidopsis thaliana. The complete loss of mTERF2 function results in embryo lethality, whereas directed, microRNA (amiR)-mediated knockdown of MTERF2 is associated with perturbed plant development and reduced chlorophyll content. Moreover, photosynthesis is impaired in amiR-mterf2 plants, as indicated by reduced levels of photosystem subunits, although the levels of the corresponding messenger RNAs are not affected. RNA immunoprecipitation followed by RNA sequencing (RIP-Seq) experiments, combined with whole-genome RNA-Seq, RNA gel-blot, and quantitative RT-PCR analyses, revealed that mTERF2 is required for the splicing of the group IIB introns of ycf3 (intron 1) and rps12.

Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses.

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

Lack of FIBRILLIN6 in Arabidopsis thaliana affects light acclimation and sulfate metabolism.

Arabidopsis thaliana contains 13 fibrillins (FBNs), which are all localized to chloroplasts. FBN1 and FBN2 are involved in photoprotection of photosystem II, and FBN4 and FBN5 are thought to be involved in plastoquinone transport and biosynthesis, respectively. The functions of the other FBNs remain largely unknown. To gain insight into the function of FBN6, we performed coexpression and Western analyses, conducted fluorescence and transmission electron microscopy, stained reactive oxygen species (ROS), measured photosynthetic parameters and glutathione levels, and applied transcriptomics and metabolomics. Using coexpression analyses, FBN6 was identified as a photosynthesis-associated gene. FBN6 is localized to thylakoid and envelope membranes, and its knockout results in stunted plants. The delayed-growth phenotype cannot be attributed to altered basic photosynthesis parameters or a reduced CO2 assimilation rate. Under moderate light stress, primary leaves of fbn6 plants begin to bleach and contain enlarged plastoglobules. RNA sequencing and metabolomics analyses point to an alteration in sulfate reduction in fbn6. Indeed, glutathione content is higher in fbn6, which in turn confers cadmium tolerance of fbn6 seedlings. We conclude that loss of FBN6 leads to perturbation of ROS homeostasis. FBN6 enables plants to cope with moderate light stress and affects cadmium tolerance.

The coordinated action of PPR4 and EMB2654 on each intron half mediates trans-splicing of rps12 transcripts in plant chloroplasts.

The pentatricopeptide repeat proteins PPR4 and EMB2654 have been shown to be required for the trans-splicing of plastid rps12 transcripts in Zea mays (maize) and Arabidopsis, respectively, but their roles in this process are not well understood. We investigated the functions of the Arabidopsis and Oryza sativa (rice) orthologs of PPR4, designated AtPPR4 (At5g04810) and OsPPR4 (Os4g58780). Arabidopsis atppr4 and rice osppr4 mutants are embryo-lethal and seedling-lethal 3 weeks after germination, respectively, showing that PPR4 is essential in the development of both dicot and monocot plants. Artificial microRNA-mediated mutants of AtPPR4 displayed a specific defect in rps12 trans-splicing, with pale-green, yellowish or albino phenotypes, according to the degree of knock-down of AtPPR4 expression. Comparison of RNA footprints in atppr4 and emb2654 mutants showed a similar concordant loss of extensive footprints at the 3' end of intron 1a and at the 5' end of intron 1b in both cases. EMB2654 is known to bind within the footprint region in intron 1a and we show that AtPPR4 binds to the footprint region in intron 1b, via its PPR motifs. Binding of both PPR4 and EMB2654 is essential to juxtapose the two intron halves and to maintain the RNAs in a splicing-competent structure for the efficient trans-splicing of rps12 intron 1, which is crucial for chloroplast biogenesis and plant development. The similarity of EMB2654 and PPR4 orthologs and their respective binding sites across land plant phylogeny indicates that their coordinate function in rps12 trans-splicing has probably been conserved for 500 million years.

CFM9, a Mitochondrial CRM Protein, Is Crucial for Mitochondrial Intron Splicing, Mitochondria Function and Arabidopsis Growth and Stress Responses.

Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.

A chloroplast-targeted pentatricopeptide repeat protein PPR287 is crucial for chloroplast function and Arabidopsis development.

BACKGROUND: Even though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis ( http://ppr.plantenergy.uwa.edu.au ). RESULTS: The homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants. CONCLUSIONS: These results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.

A chloroplast-targeted cabbage DEAD-box RNA helicase BrRH22 confers abiotic stress tolerance to transgenic Arabidopsis plants by affecting translation of chloroplast transcripts.

Although the roles of many DEAD-box RNA helicases (RHs) have been determined in the nucleus as well as in cytoplasm during stress responses, the importance of chloroplast-targeted DEAD-box RHs in stress response remains largely unknown. In this study, we determined the function of BrRH22, a chloroplast-targeted DEAD-box RH in cabbage (Brassica rapa), in abiotic stress responses. The expression of BrRH22 was markedly increased by drought, heat, salt, or cold stress and by ABA treatment, but was largely decreased by UV stress. Expression of BrRH22 in Arabidopsis enhanced germination and plantlet growth under high salinity or drought stress. BrRH22-expressing plants displayed a higher cotyledon greening and better plantlet growth upon ABA treatment due to decreases in the levels of ABI3, ABI4, and ABI5. Further, BrRH22 affected translation of several chloroplast transcripts under stress. Notably, BrRH22 had RNA chaperone function. These results altogether suggest that chloroplast-transported BrRH22 contributes positively to the response of transgenic Arabidopsis to abiotic stress by affecting translation of chloroplast genes via its RNA chaperone activity.

quatre-quart1 is an indispensable U12 intron-containing gene that plays a crucial role in Arabidopsis development.

Despite increasing understanding of the importance of the splicing of U12-type introns in plant development, the key question of which U12 intron-containing genes are essential for plant development has not yet been explored. Here, we assessed the functional role of the quatre-quart1 (QQT1) gene, one of the ~230 U12 intron-containing genes in Arabidopsis thaliana. Expression of QQT1 in the U11/U12-31K small nuclear ribonucleoprotein mutant (31k) rescued the developmental-defect phenotypes of the 31k mutant, whereas the miRNA-mediated qqt1 knockdown mutants displayed severe defects in growth and development, including severely arrested stem growth, small size, and the formation of serrated leaves. The structures of the shoot apical meristems in the qqt1 mutants were abnormal and disordered. Identification of QQT1-interacting proteins via a yeast two-hybrid screening and a firefly luciferase complementation-imaging assay revealed that a variety of proteins, including many chloroplast-targeted proteins, interacted with QQT1. Importantly, the levels of chloroplast-targeted proteins in the chloroplast were reduced, and the chloroplast structure was abnormal in the qqt1 mutant. Collectively, these results provide clear evidence that QQT1 is an indispensable U12 intron-containing gene whose correct splicing is crucial for the normal development of Arabidopsis.

The mitochondrial pentatricopeptide repeat protein PPR19 is involved in the stabilization of NADH dehydrogenase 1 transcripts and is crucial for mitochondrial function and Arabidopsis thaliana development.

Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana. The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3'-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed. Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes. Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.

Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa).

Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes.

Emerging Roles of RNA-Binding Proteins in Plant Growth, Development, and Stress Responses.

Posttranscriptional regulation of RNA metabolism, including RNA processing, intron splicing, editing, RNA export, and decay, is increasingly regarded as an essential step for fine-tuning the regulation of gene expression in eukaryotes. RNA-binding proteins (RBPs) are central regulatory factors controlling posttranscriptional RNA metabolism during plant growth, development, and stress responses. Although functional roles of diverse RBPs in living organisms have been determined during the last decades, our understanding of the functional roles of RBPs in plants is lagging far behind our understanding of those in other organisms, including animals, bacteria, and viruses. However, recent functional analysis of multiple RBP family members involved in plant RNA metabolism and elucidation of the mechanistic roles of RBPs shed light on the cellular roles of diverse RBPs in growth, development, and stress responses of plants. In this review, we will discuss recent studies demonstrating the emerging roles of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development, and stress responses.

A chloroplast-localized S1 domain-containing protein SRRP1 plays a role in Arabidopsis seedling growth in the presence of ABA.

Although the roles of S1 domain-containing proteins have been characterized in diverse cellular processes in the cytoplasm, the functional roles of a majority of S1 domain-containing proteins targeted to the chloroplast are largely unknown. Here, we characterized the function of a nuclear-encoded chloroplast-targeted protein harboring two S1 domains, designated SRRP1 (for S1 RNA-binding ribosomal protein 1), in Arabidopsis thaliana. Subcellular localization analysis of SRRP1-GFP fusion proteins revealed that SRRP1 is localized to the chloroplast. The T-DNA tagged loss-of-function srrp1 mutants displayed poorer seedling growth and less cotyledon greening than the wild-type plants on MS medium supplemented with abscisic acid (ABA), suggesting that SRRP1 plays a role in seedling growth in the presence of ABA. Splicing of the trnL intron and processing of 5S rRNA in chloroplasts were altered in the mutant plants. Importantly, SRRP1 complemented the growth-defective phenotypes of an RNA chaperone-deficient Escherichia coli mutant at low temperatures and had nucleic acid-melting ability, indicating that SRRP1 possesses RNA chaperone activity. Taken together, these results suggest that SRRP1, the chloroplast-localized S1 domain-containing protein, harboring RNA chaperone activity affects the splicing and processing of chloroplast transcripts and plays a role in Arabidopsis seedling growth in the presence of ABA.

A nuclear-encoded chloroplast-targeted S1 RNA-binding domain protein affects chloroplast rRNA processing and is crucial for the normal growth of Arabidopsis thaliana.

Despite the fact that a variety of nuclear-encoded RNA-binding proteins (RBPs) are targeted to the chloroplast and play essential roles during post-transcriptional RNA metabolism in the chloroplast, the physiological roles of the majority of chloroplast-targeted RBPs remain elusive. Here, we investigated the functional role of a nuclear-encoded S1 domain-containing RBP, designated SDP, in the growth and development of Arabidopsis thaliana. Confocal analysis of the SDP-green fluorescent protein revealed that SDP was localized to the chloroplast. The loss-of-function sdp mutant displayed retarded seed germination and pale-green phenotypes, and grew smaller than the wild-type plants. Chlorophyll a content and photosynthetic activity of the sdp mutant were much lower than those of wild-type plants, and the structures of the chloroplast and the prolamellar body were abnormal in the sdp mutant. The processing of rRNAs in the chloroplast was defective in the sdp mutant, and SDP was able to bind chloroplast 23S, 16S, 5S and 4.5S rRNAs. Notably, SDP possesses RNA chaperone activity. Transcript levels of the nuclear genes involved in chlorophyll biosynthesis were altered in the sdp mutant. Collectively, these results suggest that chloroplast-targeted SDP harboring RNA chaperone activity affects rRNA processing, chloroplast biogenesis and photosynthetic activity, which is crucial for normal growth of Arabidopsis.

A chloroplast-localized DEAD-box RNA helicaseAtRH3 is essential for intron splicing and plays an important role in the growth and stress response in Arabidopsis thaliana.

Although many DEAD-box RNA helicases (RHs) are targeted to chloroplasts, the functional roles of the majority of RHs are still unknown. Recently, the chloroplast-localized Arabidopsis thaliana AtRH3 has been demonstrated to play important roles in intron splicing, ribosome biogenesis, and seedling growth. To further understand the functional role of AtRH3 in intron splicing and growth and the stress response in Arabidopsis, the newly-generated artificial microRNA-mediated knockdown plants as well as the previously characterized T-DNA tagged rh3-4 mutant were analyzed under normal and stress conditions. The rh3 mutants displayed retarded growth and pale-green phenotypes, and the growth of mutant plants was inhibited severely under salt or cold stress but marginally under dehydration stress conditions. Splicing of several intron-containing chloroplast genes was defective in the mutant plants. Importantly, splicing of ndhA and ndhB genes was severely inhibited in the mutant plants compared with the wild-type plants under salt or cold stress but not under dehydration stress conditions. Moreover, AtRH3 complemented the growth-defect phenotype of the RNA chaperone-deficient Escherichia coli mutant and had the ability to disrupt RNA and DNA base pairs, indicating that AtRH3 possesses RNA chaperone activity. Taken together, these results demonstrate that AtRH3 plays a prominent role in the growth and stress response of Arabidopsis, and suggest that proper splicing of introns governed by RNA chaperone activity of AtRH3 is crucial for chloroplast function and the growth and stress response of plants.

MicroRNA400-guided cleavage of Pentatricopeptide repeat protein mRNAs Renders Arabidopsis thaliana more susceptible to pathogenic bacteria and fungi.

Although a large number of microRNAs (miRNAs) have been identified in different plant species, the functional roles and targets of the majority of miRNAs have not yet been determined. Here, Arabidopsis thaliana miRNA400 (miR400) was investigated for its functional role in the defense response to diverse pathogens. Transgenic Arabidopsis plants that overexpress MIR400 (35S::MIR400) displayed much more severe disease symptoms than the wild-type plants when infected with the bacterium Pseudomonas syringae pv. tomato DC3000 or the fungus Botrytis cinerea. MiR400 guided the cleavage of two genes (At1g06580 and At1g62720) encoding pentatricopeptide repeat (PPR) proteins. To confirm further that the miR400-mediated defense response was due to the cleavage of PPR mRNAs, loss-of-function mutant and artificial miRNA-mediated knockdown mutants of PPR were generated, and their disease responses were analyzed upon pathogen challenge. Similar to the 35S::MIR400 plants, the ppr mutants displayed much more severe disease symptoms than the wild-type plants when challenged with the pathogens, indicating that miR400 affects the defense response by cleaving PPR mRNAs. Expression of miR400 was down-regulated, whereas the PPR1 and PPR2 transcripts increased upon pathogen challenge. Collectively, the present study reveals that miR400-mediated dysfunction of PPR proteins renders Arabidopsis more susceptible to pathogenic bacteria and fungi, which emphasizes the importance of PPR proteins in plant defense against diverse pathogens.