Phenotypic Defects Research Articles

Vesicle trafficking mediated by small GTPase CfRab6 in association with CfRic1 and CfRgp1 governs growth, conidiation, and pathogenicity of Colletotrichum fructicola.

Small GTPase of the Rab family functions as molecular switch in vesicle trafficking, regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In our ongoing efforts to study the pathogenesis of Colletotrichum fructicola, the causal agent of anthracnose in edible-oil plant Camellia oleifera, we identified CfRab6 as the Rab GTPase and characterized its roles in C. fructicola. Consistent with our hypothesis, targeted gene deletion revealed that the ΔCfrab6 mutant displays defects in vesicle trafficking, including endocytosis and autophagy. These combined effects led to the impairments in growth, conidia, and pathogenicity. Moreover, we demonstrated the critical importance of the GDP/GTP motifs are crucial for the normal function of CfRab6. Additionally, our findings demonstrated that CfRic1 and CfRgp1 act as conserved GEFs for CfRab6, supported by their interactions with CfRab6 and the partial restoration of the active GTP-bound CfRab6, which alleviated phenotypic defects in the ΔCfric1 and ΔCfrgp1 mutants. In conclusion, our study sheds new light on the significance of CfRab6-mediated vesicle trafficking in the physiology and pathogenicity of C. fructicola, which might offer new potential targets for the management of anthracnose disease.

Crosstalk between cyclic-di-guanosine monophosphate and the sensor kinase MtrB regulates MtrA-dependent genes, bacterial growth, biofilm formation and lysosomal trafficking of Mycobacterium tuberculosis.

Cyclic-di-guanosine monophosphate (c-di-GMP) plays an important role in bacterial signalling networks. C-di-GMP exerts a regulatory function through binding to diverse molecules that include transcription factors, riboswitches and sensor kinases (SKs), thereby regulating diverse processes. Here, we demonstrate the crosstalk between c-di-GMP and the SK MtrB of Mycobacterium tuberculosis. MtrB phosphorylates and regulates its cognate response regulator MtrA. C-di-GMP binds directly to the cytosolic domain of MtrB to inhibit its autophosphorylation. C-di-GMP levels in M. tuberculosis were manipulated by overexpressing a c-di-GMP synthesizing enzyme ydeH and a degrading enzyme rv1357c. We demonstrate that overexpression of ydeH lowers growth of the bacterium both in vitro and in M. tuberculosis grown in macrophages. This is in conformity with lowered expression of mtrA and selected genes of the mtrA regulon involved in cell wall turnover in the ydeH-overexpressing strain compared to the parent strain. We also demonstrate that overexpression of ydeH in M. tuberculosis hinders biofilm formation, whereas overexpression of rv1357c has the opposite effect. Neither of the two genes could rescue the biofilm defective phenotype of the MtrB knock out mutant (ΔmtrB), suggesting that c-di-GMP exerts its role on biofilm formation through MtrB. Finally, we show by fluorescence microscopy that the trafficking of M. tuberculosis overexpressing ydeH is significantly higher than that of the parent strain and that this is linked to reduced expression of the MtrB-dependent genes esxG and esxH, which play a role in subversion of lysosomal trafficking of M. tuberculosis. These results provide important new insight into the crosstalk between c-di-GMP and MtrB in M. tuberculosis.

The Identification of a Unique Gene MoUNG Required for Growth, Conidiation, and Pathogenicity in Magnaporthe oryzae Through T-DNA Insertion Mutagenesis

Unique genes refer to genes specific to a particular organism and play crucial roles in the biological functions, evolutionary processes, and adaptations to external environments. However, the roles of unique genes in plant pathogenic fungi remain largely unexplored. In this study, we identified a novel unique gene in the rice blast fungus Magnaporthe oryzae, named MoUNG (M. oryzae unique gene), through T-DNA insertion mutagenesis. The disruption of the MoUNG promoter region in the T-DNA insertion mutant (T30-104) led to an almost loss of MoUNG expression. MoUNG has no functional domains and lacks homologues in other organism. It is highly expressed during the early-infection stage between 16 and 32 h post-inoculation (HPI), in contrast to its expression in mycelia and at the later infection stage of 48 HPI. Notably, attempts to knock out MoUNG were unsuccessful, so we examined the T30-104 mutant and found it showed significantly reduced growth, conidiation, and pathogenicity. Introducing the full-length MoUNG with its promoter into T30-104 restored these phenotypic defects. Additionally, subcellular localization assays revealed that MoUNG exhibits a dot-like distribution within the cytoplasm of mycelium, conidium, appressorium, and invasive hypha. Furthermore, knock-down of MoUNG produced results similar to those observed with the insertion mutation. In conclusion, we identified a novel unique gene MoUNG in M. oryzae and demonstrated its involvement in growth, conidiation, and pathogenicity.

Treatment of intra-bony defect and periodontal phenotype modification therapy (PhMT): A one-year follow-up case report

Rationale: The treatment of a patient with an intrabony defect using periodontal phenotype modification therapy (PhMT) is alternative approach in correcting intrabony defects and changing periodontal phenotypes. Patient concerns: A 27-year-old male patient arrived with a severe intrabony defect and a poor periodontal phenotype. The patient received PhMT, which includes nonsurgical periodontal therapy, bone grafting, and orthodontic treatment. After 1 year, there were considerable changes in the intrabony defect, periodontal phenotype, and general oral health. Diagnoses: A 27-year-old male patient had a profound intrabony defect (10 mm) and a poor periodontal phenotype (thin gingiva, high frenum attachment). Interventions: The patient received PhMT, which includes nonsurgical periodontal therapy (scaling and root planing), bone grafting (allograft), and orthodontic treatment (correcting tooth placement). After 1 year, there were considerable improvements: Intrabony defect reduction (from 10 to 3 mm), improved periodontal phenotype (thickened gingiva, decreased frenum attachment), and improved dental health (lower pocket depths, increased attachment levels). PhMT successfully repaired the intrabony deficiency and altered the patient’s periodontal phenotype. Outcomes: Improved dental health and aesthetics were the result of the comprehensive treatment, which addressed the problem as well as the patient’s underlying periodontal features. Lessons: The efficacy of PhMT in correcting intrabony abnormalities and altering periodontal phenotypes is demonstrated in this case study. This strategy presents a viable remedy for patients with intricate periodontal requirements.

CALB1 and RPL23 Are Essential for Maintaining Oocyte Quality and Function During Aging

ABSTRACTWith advancing age, significant changes occur in the female reproductive system, the most notable of which is the decline in oocyte quality, a key factor affecting female fertility. However, the mechanisms underlying oocyte aging remain poorly understood. In this study, we obtained oocytes from aged and young female mice and performed single‐cell transcriptome sequencing, comparing our findings with existing proteomic analyses. Our analysis revealed that one of the primary characteristics of aging oocytes is the disruption of calcium ion homeostasis. Specifically, we identified two key genes involved in the oocyte aging process, Calb1 and Rpl23. Experimental validation demonstrated that knockdown of CALB1 in oocytes led to reduced calcium ion levels in the endoplasmic reticulum and mitochondria, resulting in mitochondrial dysfunction and meiotic defects. Further experiments suggested that RPL23 may function as a downstream gene of CALB1, and its knockdown caused mitochondrial dysfunction, excessive accumulation of reactive oxygen species (ROS), and spindle assembly defects. Notably, overexpression of these two genes in aging oocytes partially rescued the maternal age‐related defective phenotypes, underscoring their crucial roles in oocyte aging. This study provides a comprehensive understanding of the specific mechanisms underlying mouse oocyte aging at single‐cell resolution, supported by experimental validation, and offers new directions and potential targets for future research into age‐related reproductive health issues.

The GhANT-GoPGF module regulates pigment gland development in cotton leaves.

Gossypium spp. pigment glands are a good model for studying plant secretory cavity structures. GoPGF (GOSSYPIUM PIGMENT GLAND FORMATION) is a well-characterized master transcription factor that controls gland formation in cotton; however, little is known about its transcriptional regulation. This study integrates yeast one-hybrid sequencing data and the previously reported single-cell RNA sequencing data to identify upstream GoPGF binding proteins. Several transcription factors preferentially expressed in pigment gland cells (PGCs) are identified, including the cotton AINTEGUMENTA ortholog GhANT. Silencing of GhANT produces a defective leaf-specific PGC phenotype. Knockdown of GhANT reduces mesophyll gland number and gossypol production, while CRISPR-mediated GhANT knockout suppresses mesophyll development. Overexpression of GhANT increases organ size but not cell size. GhANT binds to two CCG boxes in the GoPGF promoter to trigger GoPGF-GhJUB1-regulated gland formation. Our study dissects the subtle regulation of tissue-specific gland morphogenesis in cotton and provides molecular mechanisms to study secretory cavity structures widespread among vascular plants.

Mycobacterium tuberculosis short mutant H37Rv-S with reduced growth adaptability is more readily recognized by the host immune system.

Mycobacterium tuberculosis (Mtb) is the bacterium responsible for causing Tuberculosis (TB) and understanding its mechanisms of virulence, persistence, and pathogenesis is a global research priority. Attenuated strains of Mtb are valuable tools for investigating the genes and proteins involved in these processes. In this study, we identified an Mtb mutant, H37Rv-S, which exhibits a shorter mycelium, smoother colony, slower growth, and reduced antibiotics resistance compared to the wild-type strain H37Rv. Genomic sequencing revealed 34 mutation events in the coding regions of H37Rv-S, affecting 31 genes. We conducted TMT-labeling quantitative proteomics to compare the expression differences between H37Rv-S and H37Rv, as well as their infected bone marrow derived macrophages (BMDMs). The results showed that 716 protein groups (23.96%) in H37Rv-S and 115 protein groups (2.99%) in the infected BMDMs were differentially expressed. The dysregulated proteins in H37Rv-S correspond with its phenotype characteristics. Among the 31 affected genes in H37Rv-S, 10 showed upregulation and 1 showed downregulation at the protein level. Notable, 16 associated network proteins in the phoP/phoR system were significantly dysregulated due to a frameshift mutation in phoP, altering its protein sequence after Phe45. The dysregulated host proteins in H37Rv-S were associated with immune response, necroptosis, and ferroptosis. Additionally, H37Rv-S demonstrated reduced survival capability in strain fluorescence labeling and colony-forming unit (CFU) counting post-infection of BMDM cells. These findings suggest that H37Rv-S is an attenuated strain exhibiting defective phenotype characteristics and is more readily recognized and eliminated by the host. This enhanced understanding of the differences between virulent and attenuated strains could facilitate the development of new targets and therapeutics for TB prevention and treatment.

Identification and characterization of two APETALA2 homolog genes in lotus (Nelumbo nucifera) involved in sepal and petal development

BackgroundLotus (Nelumbo nucifera) is a significant aquatic ornamental genus widely utilized in horticulture for its decorative, culinary, medicinal, and other practical uses. It presents a variety of flower shapes, including few-petalled, semi-double-petalled, double-petalled and thousand-petalled flowers, making it an ideal candidate for studying the flower development of aquatic plants. However, the molecular mechanism of floral development in lotus remains elusive.ResultsIn this study, two APETALA2 (AP2) homologues, NnAP2a and NnAP2b, were identified in lotus. Interestingly, both NnAP2a and NnAP2b proteins contained two conserved AP2 domains and were verified to be located primarily in the nucleus. Both NnAP2a and NnAP2b showed high expression levels in the floral buds and petals. Ectopic expression of NnAP2a and NnAP2b in Arabidopsis led to an increase in the number of petals and sepals compared to the wild type (WT). Meanwhile, each of the two NnAP2 genes was able to rescue the sepal and petal defective phenotype of the ap2-6 mutant in Arabidopsis. Furthermore, protein–protein interaction assays indicated that NnAP2s could form a protein complex with other proteins involved in floral organ development, such as AP3, PISTILLATA (PI), and SEPALLATA3 (SEP3).ConclusionsThese results suggest that NnAP2s could influence sepal and petal development in N. nucifera. Our findings not only provide some insights into molecular mechanism underlying sepal and petal development and formation of lotus, but also might help its breeding in improving flower morphology.

Sec24C Participates in Cuticular Wax Transport by Facilitating Plasma Membrane Localization of ABCG5.

Cuticular waxes synthesised in the endoplasmic reticulum of epidermal cells must be exported to the outer surface of the epidermis to fulfil their barrier function. Beyond transmembrane trafficking mediated by ABC transporters, little is known about the movement of wax molecules. In this study, we characterise a mutant named sugar-associated vitrified 1 (sav1), which exhibits a vitrified phenotype and displays a reduced root length when cultivated on sugar-free medium. The mutation in SAV1, which encodes the protein Sec. 24C, leads to ultrastructural alterations in cuticle membranes, decreased deposition of epicuticular wax crystals, and modifications in the chemical composition of very-long-chain fatty acids in cuticular waxes. SAV1 is a membrane protein and expressed during the early stages of seedling development. The defective phenotype of sav1-1 in sugar-free medium resembles that of abcg5, which encodes an ATP-BINDING CASSETTE TRANSPORTER subfamily G 5 (ABCG5) protein involved in cuticle layer formation. Further investigations reveal that SAV1 interacts with ABCG5, influencing the membrane localisation of ABCG5. Collectively, our results suggest that SAV1 plays a critical role in wax transport by altering the subcellular localisation of ABCG5.

The rice microRNA159-SPOROCYTELESS EAR2 module regulates starch biosynthesis during pollen development and maintains male fertility.

Starch is an indispensable energy reserve for pollen and failure of starch biosynthesis in pollen leads to male sterility in flowering crops. Nonetheless, the regulatory mechanisms underlying starch biosynthesis in rice (Oryza sativa) pollen remain unclear. Here, we identified a target of the microRNA OsmiR159, SPOROCYTELESS ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-ASSOCIATED AMPHIPHILIC-REPRESSION 2 (OsSPEAR2). OsSPEAR2 is predominantly expressed in mature pollen and OsSPEAR2 possesses transcriptional repressor activity and localizes in the nucleus. Disruption of OsSPEAR2 results in severely shrunken pollen grains and male sterility. OsSPEAR2 interacts with multiple OsTCPs, including OsTCP14. OsTCP14 is a target of OsmiR319 and a knockout mutation in OsTCP14 partially rescues the defective pollen phenotype of Osspear2. In addition, transcriptome analyses revealed significant downregulation of numerous genes associated with carbohydrate metabolism specifically in Osspear2 anthers, including several genes critical for starch biosynthesis. Moreover, OsTCP14 directly represses the expression of the essential starch biosynthesis gene OsUGP2; however, this repression could be alleviated by OsSPEAR2. Noteworthily, embryophyte-specific SPEAR2 and SPOROCYTELESS were also identified as miR159 targets involved in regulating plant growth and development in Arabidopsis (Arabidopsis thaliana), indicating that the miR159-SPEAR regulatory module may be conserved among embryophytes. Collectively, our findings reveal OsmiR159-OsSPEAR2-OsTCP14-OsUGP2 as a regulatory cascade that modulates starch biosynthesis during pollen development in rice.

THE FAM53C/DYRK1A axis regulates the G1/S transition of the cell cycle.

A growing number of therapies are being developed to target the cell cycle machinery for the treatment of cancer and other human diseases. Consequently, a greater understanding of the factors regulating cell cycle progression becomes essential to help enhance the response to these new therapies. Here, using data from the Cancer Dependency Map, we identified the poorly-studied factor FAM53C as a new regulator of cell cycle progression. We found that FAM53C is critical for this cell cycle transition and that it acts upstream of the CyclinD-CDK4/6-RB axis in the regulation of the G1/S transition. By mass spectrometry, biochemical, and cellular assays, we identified and validated DYRK1A as a cell cycle kinase that is inhibited by and directly interacts with FAM53C. DYRK1A kinase inhibition rescues the G1 arrest induced by FAM53C knock-down. Consistent with the role for FAM53C identified in cells in culture, FAM53C knockout human cortical organoids display increased cell cycle arrest and growth defects. In addition, Fam53C knockout mice show defects in body growth and behavioral phenotypes. Because DYRK1A dysregulation contributes to developmental disorders such as Down syndrome as well as tumorigenesis, future strategies aiming at regulating FAM53C activity may benefit a broad range of patients.

MamF-like proteins are distant Tic20 homologs involved in organelle assembly in bacteria

Organelle-specific protein translocation systems are essential for organelle biogenesis and maintenance in eukaryotes but thought to be absent from prokaryotic organelles. Here, we demonstrate that MamF-like proteins are crucial for the formation and functionality of bacterial magnetosome organelles. Deletion of mamF-like genes in the Alphaproteobacterium Magnetospirillum gryphiswaldense results in severe defects in organelle positioning, biomineralization, and magnetic navigation. These phenotypic defects result from the disrupted targeting of a subset of magnetosomal proteins that contain C-terminal glycine-rich integral membrane domains. Phylogenetic analyses reveal an ancient evolutionary link between MamF-like proteins and plastidial Tic20. Our findings redefine the molecular roles of MamF-like proteins and suggest that organelle-specific protein targeting systems also play a role in bacterial organelle formation.

Phosphoenolpyruvate carboxylase (PEPC) is essential for the glycolytic pathway and parasite proliferation in Babesia gibsoni

Apicomplexan parasites predominantly generate ATP and lactic acid through glycolysis and anaerobic glucose metabolism, incorporating CO2 into glycolysis via a stage-dependent phosphoenolpyruvate carboxylase (PEPC) mechanism. Although the role of PEPC in plant and bacterial carbon fixation is well documented, its function within Babesia remains largely unexplored. This study employs reverse genetics to probe the biological role of PEPC in Babesia gibsoni, noting its conservation across similar protozoa, suggesting a pivotal and conserved biological function. Western blotting and immunofluorescence (IFA) experiments using the BgPEPC-3 × Flag strain revealed that the BgPEPC protein has a molecular weight of 105 kDa and localizes predominantly to the cytoplasm. Attempts to knock out the PEPC gene in BgPEPC-3 × Flag strains failed under standard media conditions, succeeded only with the addition of 5 mM malate, an upstream metabolite of oxaloacetic acid (OAA). In addition to malate, the downstream metabolite of OAA can also partially compensate for the phenotypic defects caused by PEPC deficiency. This intervention alleviated severe growth deficits, underscoring the critical role of aspartate in the parasite lifecycle. Moreover, metabolic inhibitors such as L-cycloserine and triazamidine, which target aspartate aminotransferase and mitochondrial functions, respectively, demonstrated increased efficacy against BgPEPC knockout strains. The lack of a compensatory response to malic acid supplementation underscores the integral role of BgPEPC in intermediary carbon metabolism and its necessity in providing aspartate as a precursor to pyrimidine synthesis. Collectively, these findings suggest that PEPC could be a potential target for future drug development against B. gibsoni infections.Graphical

GPATCH11 variants cause mis-splicing and early-onset retinal dystrophy with neurological impairment

Here we conduct a study involving 12 individuals with retinal dystrophy, neurological impairment, and skeletal abnormalities, with special focus on GPATCH11, a lesser-known G-patch domain-containing protein, regulator of RNA metabolism. To elucidate its role, we study fibroblasts from unaffected individuals and patients carrying the recurring c.328+1 G > T mutation, which specifically removes the main part of the G-patch domain while preserving the other domains. Additionally, we generate a mouse model replicating the patients’ phenotypic defects, including retinal dystrophy and behavioral abnormalities. Our results reveal a subcellular localization of GPATCH11 characterized by a diffuse presence in the nucleoplasm, as well as centrosomal localization, suggesting potential functions in RNA and cilia metabolism. Transcriptomic analysis performed on mouse retina detect dysregulation in both gene expression and splicing activity, impacting key processes such as photoreceptor light responses, RNA regulation, and primary cilia-associated metabolism. Proteomic analysis of mouse retina confirms the roles GPATCH11 plays in RNA processing, splicing, and transcription regulation, while also suggesting additional functions in synaptic plasticity and nuclear stress response. Our research provides insights into the diverse roles of GPATCH11 and identifies that the mutations affecting this protein are responsible for a recently characterized described syndrome.

Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis.

Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension (CE) during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar CE defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis (gpc4), we identified dact1/2-specific roles during early development. Comparative whole transcriptome analysis between wildtype and gpc4 and wildtype and dact1/2 compound mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8. Overexpression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.

Molecular hydrogen positively influences root gravitropism involving auxin signaling and starch accumulation.

Although geoscience of natural hydrogen (H2), hydrogen-producing soil bacteria, and especially plant-based H2, has been observed, it is not clear whether or how above H2 resources influence root gravitropic responses. Here, pharmacological, genetic, molecular, and cell biological tools were applied to investigate how plant-based H2 coordinates gravity responses in Arabidopsis roots. Since roots show higher H2 production than shoots, exogenous H2 supply was used to mimic this function. After H2 supplementation, the asymmetric expression of the auxin-response reporter DR5 driven by auxin influx and efflux carriers, and thereafter positive root gravitropism were observed. These positive responses in root gravitropism were sensitive to auxin polar transport inhibitors, and importantly, the defective phenotypes observed in aux1-7, pin1, and pin2 mutants were not significantly altered by exogenous H2. The observed starch accumulation was matched with the reprogramming gene expression linked to starch synthesis and degradation. Transgenic plants expressing hydrogenase1 (CrHYD1) from Chlamydomonas reinhardtii not only displayed higher endogenous H2 concentrations, the inducible AUX1 gene expression and starch accumulation, but also showed pronounced root gravitropism. Collectively, above evidence preliminarily provides a framework for understanding the molecular basis of the possible functions of both plant/soil-based and nature H2 in root architecture.

Maternally expressed FERTILIZATION-INDEPENDENT ENDOSPERM1 regulates seed dormancy and aleurone development in rice.

Seed dormancy, an essential trait for plant adaptation, is determined by the embryo itself and the surrounding tissues. Here, we found that rice (Oryza sativa) FERTILIZATION-INDEPENDENT ENDOSPERM1 (OsFIE1) regulates endosperm-imposed dormancy and the dorsal aleurone thickness in a manner dependent on the parent of origin. Maternally expressed OsFIE1 suppresses gibberellin (GA) biosynthesis in the endosperm by depositing trimethylation of lysine 27 on histone H3 (H3K27me3) marks on GA biosynthesis-related genes, thus inhibiting germination and aleurone differentiation. Knockout of rice GA 20-oxidase1 (OsGA20ox1) alleviated the phenotypic defects in osfie1. The aleurone-positive determinant Crinkly 4 (OsCR4) is another target of the OsFIE1-containing Polycomb repressive complex 2 (PRC2). We found that OsFIE1 plays an important role in genomic imprinting in the endosperm of germinating seeds, particularly for paternally expressed genes associated with H3K27me3. The increased aleurone thickness of osfie1 substantially improved grain nutritional quality, indicating that the osfie1 gene may be utilized for breeding nutrient-enriched rice. The findings provide insights into the essential roles of PRC2-mediated H3K27me3 methylation in the acquisition of seed dormancy and endosperm cell differentiation in rice.

Human CFTR deficient iPSC-macrophages reveal impaired functional and transcriptomic response upon Pseudomonas aeruginosa infection.

Cystic fibrosis (CF) is a hereditary autosomal recessive disease driven by deleterious variants of the CFTR gene, leading, among other symptoms, to increased lung infection susceptibility. Mucus accumulation in the CF lung is, as of yet, considered as one important factor contributing to its colonization by opportunistic pathogens such as Pseudomonas aeruginosa. However, in recent years evidence was provided that alveolar macrophages, which form the first line of defense against airborne pathogens, seem to be intrinsically defective with regard to bactericidal functionality in the CF lung. To assess the impact of CFTR deficiency in human macrophages only insufficient systems are available. To address this problem and to evaluate the role of CFTR in human macrophages, we successfully differentiated human induced pluripotent stem cells (iPSC) from a CF p.Phe508del homozygous individual and a healthy donor into primitive macrophages (iMacΔF508 and iMacWT), respectively, and compared the bactericidal functionality in the relevant cell type. iMacΔF508 showed impaired P. aeruginosa clearance and intracellular killing capacity in comparison to iMacWT. Furthermore, iMacΔF508 exhibited a less acidic lysosomal pH, and upon P. aeruginosa infection, there were signs of mitochondrial fragmentation and autophagosome formation together with a hyperinflammatory phenotype and deficient type I interferon response. In summary, we present a defective phenotype in iMacΔF508 upon P. aeruginosa infection, which will constitute an ideal platform to further study the role of macrophages in the context of CF.

Transfer RNA supplementation rescues HARS deficiency in a humanized yeast model of Charcot-Marie-Tooth disease.

Aminoacyl-tRNA synthetases are indispensable enzymes in all cells, ensuring the correct pairing of amino acids to their cognate tRNAs to maintain translation fidelity. Autosomal dominant mutations V133F and Y330C in histidyl-tRNA synthetase (HARS) cause the genetic disorder Charcot-Marie-Tooth type 2W (CMT2W). Treatments are currently restricted to symptom relief, with no therapeutic available that targets the cause of disease. We previously found that histidine supplementation alleviated phenotypic defects in a humanized yeast model of CMT2W caused by HARS V155G and S356N that also unexpectedly exacerbated the phenotype of the two HARS mutants V133F and Y330C. Here, we show that V133F destabilizes recombinant HARS protein, which is rescued in the presence of tRNAHis. HARS V133F and Y330C cause mistranslation and cause changes to the proteome without activating the integrated stress response as validated by mass spectrometry and growth defects that persist with histidine supplementation. The growth defects and reduced translation fidelity caused by V133F and Y330C mutants were rescued by supplementation with human tRNAHis in a humanized yeast model. Our results demonstrate the feasibility of cognate tRNA as a therapeutic that rescues HARS deficiency and ameliorates toxic mistranslation generated by causative alleles for CMT.

A RhoGAP controls apical actin polymerization by inhibiting formin in Arabidopsis pollen tubes

Formin is an important player in promoting apical actin polymerization in pollen tubes, but the mechanism regulating its activity remains unknown. We here identify REN1, a Rho GTPase-activating protein, as a negative regulator of formins in Arabidopsis pollen tubes. Specifically, we found that depletion of REN1 promotes apical actin polymerization and increases the amount of filamentous actin in pollen tubes. Interestingly, the effect of REN1 loss of function phenocopies the effect of formin gain of function, as it causes the formation of supernumerary membrane-derived actin bundles, which leads to tube swelling and membrane deformation. Importantly, inhibition of formins suppresses the phenotypic defects in ren1 mutant pollen tubes. We further demonstrate that REN1 physically interacts with the Arabidopsis formin protein AtFH5, predominantly with the C terminus, and inhibits the ability of AtFH5 to nucleate and assemble actin invitro. Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes, demonstrating that REN1 regulates apical actin polymerization at least partially through inhibiting AtFH5. We thus uncover a novel mechanism regulating formins and actin polymerization in plants.