As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction.

Members of the pentatricopeptide repeat (PPR) protein family are sequence-specific RNA-binding proteins that play crucial roles in organelle RNA metabolism. Each PPR protein consists of a tandem array of PPR motifs, each of which aligns to one nucleotide of the RNA target. The di-residues in the PPR motif, which are referred to as the PPR codes, determine nucleotide specificity. Numerous PPR codes are distributed among the vast number of PPR motifs, but the correlation between PPR codes and RNA bases is poorly understood, which hinders target RNA prediction and functional investigation of PPR proteins. To address this issue, we developed a modular assembly method for high-throughput construction of designer PPRs, and by using this method, 62 designer PPR proteins containing various PPR codes were assembled. Then, the correlation between these PPR codes and RNA bases was systematically explored and delineated. Based on this correlation, the web server PPRCODE ( http://yinlab.hzau.edu.cn/pprcode ) was developed. Our study will not only serve as a platform for facilitating target RNA prediction and functional investigation of the large number of PPR family proteins but also provide an alternative strategy for the assembly of custom PPRs that can potentially be used for plant organelle RNA manipulation.

Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

No abstract

No abstract

Dear Editor, Mitochondria are essential organelles in cellular metabolism, homeostasis, and apoptosis. 1,2 Most mitochondrial proteins are synthesized as precursors in the cytosol and then imported into mitochondria by specific protein translocase complexes, including the translocase of the outer membrane complex (TOM complex), the carrier translocase of the inner membrane complex (TIM22 complex), the presequence translocase of the inner membrane complex (TIM23 complex), the sorting and assembly machinery (SAM complex), and the mitochondrial import complex (MIM complex). 3 The TIM22 complex is responsible for the translocation and insertion of hydrophobic membrane proteins, including mitochondrial carrier proteins and translocase subunits (Tim17, Tim22 and Tim23). 3 In humans, TIM22 is a 440-kDa complex comprising at least six components: the hypothetical channelforming protein Tim22, three small Tim proteins (Tim9, Tim10a and Tim10b), Tim29 and acylglycerol kinase (AGK). 1 Considering the functional importance of mitochondrial protein import, the TIM22 complex has been linked to many diseases. For example, mutations in the TIM22 gene have been reported to cause earlyonset mitochondrial myopathy. 4 AGK participates in lipid biosynthesis, and mutations in the AGK gene lead to Sengers syndrome. 2 Mutations in the TIMM8A gene (also called DDP1) cause deafness dystonia syndrome. 2 Despite advances in our knowledge of the function and pathophysiology of the TIM22 complex, reports of its structural characterization are scarce. The structural studies of the TIM22 complex are restricted to the investigation of the structures of Tim9/10a 5,6 and Tim9/10/12 hexameric chaperone 7 and a nuclear magnetic resonance (NMR) analysis of carrier precursors associated with the Tim9/Tim10 complex. 8 Here, we report the cryo-EM structure of the human TIM22 complex at an overall resolution of 3.7 Å. We coexpressed all six known components of the TIM22 complex in human embryonic kidney (HEK) 293 F cells (Fig. 1a). After Flag tag affinity purification followed by gel filtration, the resultant TIM22 complex displayed good resolution behavior (Fig. 1b). The apparent molecular weight was assessed by blue native PAGE to be approximate 440 kDa (Supplementary information, Fig. S1a), consistent with previous findings. 9-12 Mass spectrometry (MS) analysis of the purified complex confirmed the presence of all known components of the TIM22 complex. Furthermore, mitochondria from cells overexpressing the TIM22 complex exhibited more efficient carrier-importing activity than those from wild-type cells (Supplementary information,

CCT (CONSTANS, CONSTANS-LIKE and TOC1) domain-containing proteins are a large family unique to plants. They transcriptionally regulate photoperiodic flowering, circadian rhythms, vernalization, and other related processes. Through their CCT domains, CONSTANS (CO) and HEADING DATE 1 (HD1) coordinate with the NUCLEAR FACTOR Y (NF-Y) B/C dimer to specifically target a conserved 'CCACA' motif within the promotors of their target genes. However, the mechanism underlying DNA recognition by the CCT domain remains unclear. Here we determined the crystal structures of the rice (Oryza sativa) NF-YB/YC dimer and the florigen gene Heading date 3a (Hd3a)-bound HD1 CCT /NF-YB/YC trimer with resolutions of 2.0 Å and 2.55 Å, respectively. The CCT domain of HD1 displays an elongated structure containing two -helices and two loops, tethering Hd3a to the NF-YB/YC dimer. Helix 2 and loop 2 are anchored into the minor groove of the