Tetratricopeptide Repeat Protein Research Articles

Insight into the genetic basis of ammonia tolerance in razor clam Sinonovacula constricta by genome-wide association study

With the development of high-density integrated farming model, the razor clam Sinonovacula constricta faces severe pressures from environmental factors, especially ammonia nitrogen, which is the top factor threaten the growth and survival of aquatic animals. Hence, ammonia tolerance has been one of the most important economic traits for razor clam. To cultivate new strains with ammonia tolerance trait, it is necessary to dissect genetic basis comprehensively underlying ammonia tolerance of S. constricta. In this study, we performed a genome wide association study (GWAS) to identify the single nucleotide polymorphisms (SNPs) and genes associated with ammonia tolerance through whole genome resequencing of 231 individuals. As a result, 56 associated SNPs (-log10P = 5) which were embedded in or adjacent to 236 genes were identified among a total of 4,577,102 SNPs. Most of these genes were related to various processes of metabolism, homeostasis, response to stress and apoptosis, suggesting that the polygenic architecture in the trait of ammonia tolerance was complex in razor clam. Among these sites, 11 genome-wide significant associated SNPs (-log10P = 6.66) adjacent to five candidate genes, including beta-galactosidase (glb1), tetratricopeptide repeat protein 28 (ttc28), Na+/K+-ATPase alpha-1 (nka), tubulin beta (tubb) and elongation factor 1-alpha (eef1a). In particular, NKA has been demonstrated to be involved in NH4+ excretion to reduce ammonia accumulation in mollusca. Our results provide insight into genetic basis of ammonia tolerance, and the SNPs would be valuable markers for selective breeding of ammonia-tolerant strain in S. constricta.

Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato.

Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

Genetic mapping of a single nuclear locus determines the white flesh color in watermelon (Citrullus lanatus L.).

Flesh color is an important trait in watermelon (Citrullus lanatus L.). Several flesh color genes have been identified in watermelon; however, the inheritance of and the molecular basis underlying the white flesh trait remain largely unknown. In this study, segregation populations were constructed by crossing the canary yellow flesh line HSH-F with the white flesh line Sanbai to fine-map the white flesh gene in watermelon. Genetic analysis indicated that the white flesh trait is controlled by a single recessive locus, termed Clwf2. Map-based cloning delimited the Clwf2 locus to a 132.3-kb region on chromosome 6. The candidate region contains 13 putative genes, and four of them-Cla97C06G121860, Cla97C06G121880, Cla97C06G121890, and Cla97C06G121900-were significantly downregulated in the white flesh compared to the canary yellow flesh watermelon fruits. The Cla97C06G121890 gene, which encodes a tetratricopeptide repeat protein, showed almost no expression in the white flesh fruit before maturity, whereas it had a very high expression in the canary yellow flesh fruit at 18 days after pollination. Transmission electron microscopy revealed rounded and regularly shaped chromoplasts in both the canary yellow and white flesh fruits. Further quantitative real-time PCR analysis showed that the expression levels of several key plastid division genes and almost the entire carotenoid biosynthesis pathway genes were downregulated in the white flesh compared to the canary yellow flesh fruits. This study suggests that the proliferation inhibition of chromoplasts and downregulation of the CBP genes block the accumulation of carotenoids in watermelon and lead to white flesh. These findings advance and extend the understanding of the molecular mechanisms underlying white flesh trait formation and carotenoid biosynthesis in watermelon.

Recruiting the autophagy-lysosome pathway to engineered condensate-forming proteins.

Biomolecular condensates (BCs) are membraneless, subcellular compartments segregated from the cytoplasm by liquid-liquid phase separation, a thermodynamic process driven by the chemical properties of the constituent biomolecules. BCs are involved in a range of cellular processes such as signalling and transcriptional regulation, as well as the autophagy-lysosome pathway, one of the cell's main degradation mechanisms. Selective autophagy can recognise BCs containing cargo proteins through direct interactions with LC3, a protein present on the inner membrane of phagophores. Consensus tetratricopeptide repeat proteins (CTPRs) are highly stable proteins composed of repeated helix-turn-helix subunits that are useful in protein engineering as a result of their modular architecture and amenability to rational design. In this project, the CTPR scaffold that has been engineered to bind LC3 through grafted LIR motifs is fused to low complexity domains (LCDs) to induce BC formation. Here we investigate the effects of different LIR motifs within the engineered BCs to understand how LC3 interactions can change the physico-chemical properties of the condensates. Using these BCs we hope to gain a better understanding of how and why the autophagy-lysosome pathway utilises BCs for client degradation, with the potential for adapting the engineered proteins as targeted protein degradation therapeutics.

Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

How Different Pathologies Are Affected by IFIT Expression.

The type-I interferon (IFN) system represents the first line of defense against viral pathogens. Recognition of the virus initiates complex signaling pathways that result in the transcriptional induction of IFNs, which are then secreted. Secreted IFNs stimulate nearby cells and result in the production of numerous proinflammatory cytokines and antiviral factors. Of particular note, IFN-induced tetratricopeptide repeat (IFIT) proteins have been thoroughly studied because of their antiviral activity against different viral pathogens. Although classically studied as an antiviral protein, IFIT expression has recently been investigated in the context of nonviral pathologies, such as cancer and sepsis. In oral squamous cell carcinoma (OSCC), IFIT1 and IFIT3 promote metastasis, while IFIT2 exhibits the opposite effect. The role of IFIT proteins during bacterial/fungal sepsis is still under investigation, with studies showing conflicting roles for IFIT2 in disease severity. In the setting of viral sepsis, IFIT proteins play a key role in clearing viral infection. As a result, many viral pathogens, such as SARS-CoV-2, employ mechanisms to inhibit the type-I IFN system and promote viral replication. In cancers that are characterized by upregulated IFIT proteins, medications that decrease IFIT expression may reduce metastasis and improve survival rates. Likewise, in cases of viral sepsis, therapeutics that increase IFIT expression may improve viral clearance and reduce the risk of septic shock. By understanding the effect of IFIT proteins in different pathologies, novel therapeutics can be developed to halt disease progression.

A mechanism for red coloration in vertebrates

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

The tetratricopeptide repeat protein OsTPR075 promotes heading by regulating florigen transport in rice.

Rice (Oryza sativa) is one of the most important crops worldwide. Heading date is a vital agronomic trait that influences rice yield and adaption to local conditions. Hd3a, a proposed florigen that primarily functions under short-day (SD) conditions, is a mobile flowering signal that promotes the floral transition in rice. Nonetheless, how Hd3a is transported from leaves to the shoot apical meristem (SAM) under SDs remains elusive. Here, we report that FT-INTERACTING PROTEIN9 (OsFTIP9) specifically regulates rice flowering time under SDs by facilitating Hd3a transport from companion cells (CCs) to sieve elements (SEs). Furthermore, we show that the tetratricopeptide repeat (TPR) protein OsTPR075 interacts with both OsFTIP9 and OsFTIP1 and strengthens their respective interactions with Hd3a and the florigen RICE FLOWERING LOCUS T1 (RFT1). This in turn affects the trafficking of Hd3a and RFT1 to the SAM, thus regulating flowering time under SDs and long-day conditions, respectively. Our findings suggest that florigen transport in rice is mediated by different OsFTIPs under different photoperiods and those interactions between OsTPR075 and OsFTIPs are essential for mediating florigen movement from leaves to the SAM.

Two Pex5 Proteins With Different Cargo Specificity Are Critical for Peroxisome Function in Ustilago maydis.

Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.

Identification and analysis of Rap-Phr system in Bacillus cereus 0-9.

In this study, eight rap-related genes were found in the Bacillus cereus 0-9 genome; five rap genes were located on chromosomes and three on large plasmids. A total of five Rap proteins in B. cereus 0-9 were annotated as 'tetratricopeptide repeat proteins'. SMART Server analysis showed that the eight Rap proteins had typical tetrapeptide repeat sequence (TPR) domains. Biofilm assays and crystal violet staining showed that overexpression of the rapp1 and rap5 genes affected the biofilm formation of B. cereus 0-9, and the activities of Rapp1 and Rap5 proteins were inhibited by their corresponding cognate Phr, suggesting that the Rap-Phr quorum sensing (QS) system might also exist in the B. cereus 0-9 strain. In addition, overexpression of rap1 genes inhibited in the extracellular amylase decomposition capacity of B. cereus 0-9. The results of the sporulation assay indicated that overexpression of the eight rap genes inhibited the spore formation of B. cereus 0-9 to varying degrees. These results provide a reference for research on the regulation of the Rap-Phr QS system in B. cereus.

Genome-wide CRISPR-Cas9 screening identifies the CYTH2 host gene as a potential therapeutic target of influenza viral infection.

Host genes critical for viral infection are effective antiviral drug targets with tremendous potential due to their universal characteristics against different subtypes of viruses and minimization of drug resistance. Accordingly, we execute a genome-wide CRISPR-Cas9 screen with multiple rounds of survival selection. Enriched in this screen are several genes critical for host sialic acid biosynthesis and transportation, including the cytohesin 2 (CYTH2), tetratricopeptide repeat protein 24 (TTC24), and N-acetylneuraminate synthase (NANS), which we confirm are responsible for efficient influenza viral infection. Moreover, we reveal that CYTH2 is required for the early stage of influenza virus infection by mediating endosomal trafficking. Furthermore, CYTH2 antagonist SecinH3 blunts influenza virus infection invivo. In summary, these data suggest that CYTH2 is an attractive target for developing host-directed antiviral drugs and therapeutics against influenza virus infection.

Electronic Transport in Molecular Wires of Precisely Controlled Length Built from Modular Proteins.

DNA molecular wires have been studied extensively because of the ease with which molecules of controlled length and composition can be synthesized. The same has not been true for proteins. Here, we have synthesized and studied a series of consensus tetratricopeptide repeat (CTPR) proteins, spanning 4 to 20 nm in length, in increments of 4 nm. For lengths in excess of 6 nm, their conductance exceeds that of the canonical molecular wire, oligo(phenylene-ethylenene), because of the more gradual decay of conductance with length in the protein. We show that, while the conductance decay fits an exponential (characteristic of quantum tunneling) and not a linear increase of resistance with length (characteristic of hopping transport), it is also accounted for by a square-law dependence on length (characteristic of weakly driven hopping). Measurements of the energy dependence of the decay length rule out the quantum tunneling case. A resonance in the carrier injection energy shows that allowed states in the protein align with the Fermi energy of the electrodes. Both the energy of these states and the long-range of hopping suggest that the reorganization induced by hole formation is greatly reduced inside the protein. We outline a model for calculating the molecular-electronic properties of proteins.

Inhibition of H3K27me3 Demethylases Promotes Plasmablast Formation.

B cell differentiation into Ab-secreting plasma cells requires transcriptional, metabolic, and epigenetic remodeling. Histone H3 lysine 27 trimethylation (H3K27me3), a histone modification associated with gene silencing, is dynamically regulated during B cell differentiation. Although several studies have focused on mechanisms involving the gain of this modification in plasmablasts (PB), the role of active demethylation of H3K27me3 by ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji domain-containing protein 3 (JMDJ3) during B cell differentiation has not been examined. In this study, this process was assessed using a pharmacological inhibitor of UTX and JMJD3, GSK-J4. Treatment of ex vivo stimulated mouse B cells with GSK-J4 led to an increase in PB frequency without affecting the ability of the newly formed PB to secrete Abs. Consistent with the role of UTX and JMJD3 in promoting gene expression, the majority of differentially expressed were downregulated upon GSK-J4 treatment. GSK-J4-treated cells downregulated genes associated with signaling and P53 pathways. Inhibitor treated cells upregulated genes associated with cell cycle and proliferation, which correlated with an increase in actively proliferating cells. Unexpectedly, a majority of the downregulated transcripts corresponded to genes that in the wild-type setting were genes that gain H3K27me3 and downregulated in PB. Together, our results show that UTX and JMDJ3 are required to restrain B cell differentiation and suggest that they function as a rheostat for H3K27me3 to control this process.

Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools.

ConspectusThe last decades have witnessed unprecedented scientific breakthroughs in all the fields of knowledge, from basic sciences to translational research, resulting in the drastic improvement of the lifespan and overall quality of life. However, despite these great advances, the treatment and diagnosis of some diseases remain a challenge. Inspired by nature, scientists have been exploring biomolecules and their derivatives as novel therapeutic/diagnostic agents. Among biomolecules, proteins raise much interest due to their high versatility, biocompatibility, and biodegradability.Protein binders (binders) are proteins that bind other proteins, in certain cases, inhibiting or modulating their action. Given their therapeutic potential, binders are emerging as the next generation of biopharmaceuticals. The most well-known example of binders are antibodies, and inspired by them researchers have developed alternative binders using protein design approaches. Protein design can be based on naturally occurring proteins in which, by means of rational design or combinatorial approaches, new binding interfaces can be engineered to obtain specific functions or based on de novo proteins emerging from state-of-the-art computational methodologies.Among the novel designed proteins, a class of engineered repeat proteins, the consensus tetratricopeptide repeat (CTPR) proteins, stand out due to their stability and robustness. The CTPR unit is a helix-turn-helix motif constituted of 34 amino acids, of which only 8 are essential to ensure correct folding of the structure. The small number of conserved residues of CTPR proteins leaves plenty of freedom for functional mutations, making them a base scaffold that can be easily and reproducibly tailored to endow desired functions to the protein. For example, the introduction of metal-binding residues (e.g., histidines, cysteines) drives the coordination of metal ions and the subsequent formation of nanomaterials. Additionally, the CTPR unit can be conjugated with other peptides/proteins or repeated in tandem to encode larger CTPR proteins with superhelical structures. These properties allow for the design of both binder and nanomaterial-coordination modules as well as their combination within the same molecule, making the CTPR proteins, as we have demonstrated in several recent examples, the ideal platform to develop protein–nanomaterial hybrids. Generally, the fusion of two distinct materials exploits the best properties of each; however, in protein–nanomaterial hybrids, the fusion takes on a new dimension as new properties arise.These hybrids have ushered the use of protein-based nanomaterials as biopharmaceuticals beyond their original therapeutic scope and paved the way for their use as theranostic agents. Despite several reports of protein-stabilized nanomaterials found in the literature, these systems offer limited control in the synthesis and properties of the grown nanomaterials, as the protein acts just as a stabilizing agent with no significant functional contribution. Therefore, the rational design of protein-based nanomaterials as true theranostic agents is still incipient. In this context, CTPR proteins have emerged as promising scaffolds to hold simultaneously therapeutic and diagnostic functions through protein engineering, as it has been recently demonstrated in pioneering in vitro and in vivo examples.

BnaA02.YTG1, encoding a tetratricopeptide repeat protein, is required for early chloroplast biogenesis in Brassica napus

Chloroplasts are essential for plant growth and development, as they play a key role in photosynthesis. The chloroplast biogenesis process is complex and its regulatory mechanism remains elusive. We characterized a spontaneous Brassica napus (rapeseed) mutant, ytg, that showed a delayed greening phenotype in all green organs and retarded growth. We identified BnaA02.YTG1 encoding a chloroplast-localized tetratricopeptide repeat protein widely expressed in rapeseed tissues. We speculated that the ytg phenotype was caused by the deletion of BnaA02.YTG1 based on sequence comparison of 4608 (with normal green leaves, isolated from the elite Chinese rapeseed cultivar ZS11) and ytg combined with transcriptome data and CRISPR/Cas9 gene editing results. The homologous gene (BnaC02.YTG1) restored the phenotype of the mutant. BnaA02.YTG1 interacted with MORF2, MORF8, and OZ1. RNA editing of the ndhD-2, ndhF-290, petL-5, and ndhG-50 plastid transcripts was affected in ytg. These findings suggested that BnaA02.YTG1 participates in RNA editing events. We predicted 29 RNA editing sites in the chloroplast of Brassica napus by comparison with the Arabidopsis chloroplast genome. We conclude that BnaA02.YTG1 affects the posttranscriptional regulation of plastid gene expression and suggest that a tetratricopeptide repeat protein is involved in the chloroplast RNA editing in rapeseed.

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions.

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP-kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air-water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

Versatile allosteric properties in Pex5-like tetratricopeptide repeat proteins to induce diverse downstream function.

Proteins composed of tetratricopeptide repeat (TPR) arrays belong to the α-solenoid tandem-repeat family that have unique properties in terms of their overall conformational flexibility and ability to bind to multiple protein ligands. The peroxisomal matrix protein import receptor Pex5 comprises two TPR triplets that recognize protein cargos with a specific C-terminal Peroxisomal Targeting Signal (PTS) 1 motif. Import of PTS1-containing protein cargos into peroxisomes through a transient pore is mainly driven by allosteric binding, coupling and release mechanisms, without a need for external energy. A very similar TPR architecture is found in the functionally unrelated TRIP8b, a regulator of the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. TRIP8b binds to the HCN ion channel via a C-terminal sequence motif that is nearly identical to the PTS1 motif of Pex5 receptor cargos. Pex5, Pex5-related Pex9, and TRIP8b also share a less conserved N-terminal domain. This domain provides a second protein cargo-binding site and plays a distinct role in allosteric coupling of initial cargo loading by PTS1 motif-mediated interactions and different downstream functional readouts. The data reviewed here highlight the overarching role of molecular allostery in driving the diverse functions of TPR array proteins, which could form a model for other α-solenoid tandem-repeat proteins involved in translocation processes across membranes.

Exploring the binding of rationally engineered tandem-repeat proteins to E3 ubiquitin ligase Keap1.

The process of displaying functional peptides by ‘grafting’ them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display.

Chromosome-scale genome assembly provides insights into the evolution and flavor synthesis of passion fruit (Passiflora edulis Sims)

Passion fruit (Passiflora edulis Sims) is an economically valuable fruit that is cultivated in tropical and subtropical regions of the world. Here, we report an ~1341.7 Mb chromosome-scale genome assembly of passion fruit, with 98.91% (~1327.18 Mb) of the assembly assigned to nine pseudochromosomes. The genome includes 23,171 protein-coding genes, and most of the assembled sequences are repetitive sequences, with long-terminal repeats (LTRs) being the most abundant. Phylogenetic analysis revealed that passion fruit diverged after Brassicaceae and before Euphorbiaceae. Ks analysis showed that two whole-genome duplication events occurred in passion fruit at 65 MYA and 12 MYA, which may have contributed to its large genome size. An integrated analysis of genomic, transcriptomic, and metabolomic data showed that ‘alpha-linolenic acid metabolism’, ‘metabolic pathways’, and ‘secondary metabolic pathways’ were the main pathways involved in the synthesis of important volatile organic compounds (VOCs) in passion fruit, and this analysis identified some candidate genes, including GDP-fucose Transporter 1-like, Tetratricopeptide repeat protein 33, protein NETWORKED 4B isoform X1, and Golgin Subfamily A member 6-like protein 22. In addition, we identified 13 important gene families in fatty acid pathways and eight important gene families in terpene pathways. Gene family analysis showed that the ACX, ADH, ALDH, and HPL gene families, especially ACX13/14/15/20, ADH13/26/33, ALDH1/4/21, and HPL4/6, were the key genes for ester synthesis, while the TPS gene family, especially PeTPS2/3/4/24, was the key gene family for terpene synthesis. This work provides insights into genome evolution and flavor trait biology and offers valuable resources for the improved cultivation of passion fruit.

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways, is an essential component of the antiviral innate immune response.

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.