Yeast Complementation Assay Research Articles

Functional identification of purine permeases reveals their roles in caffeine transport in tea plants (Camellia sinensis).

Caffeine is a characteristic secondary metabolite in tea plants. It confers tea beverage with unique flavor and excitation effect on human body. The pathway of caffeine biosynthesis has been generally established, but the mechanism of caffeine transport remains unclear. Here, eight members of purine permeases (PUPs) were identified in tea plants. They had diverse expression patterns in different tissues, suggesting their broad roles in caffeine metabolism. In this study, F1 strains of "Longjing43" ♂ × "Baihaozao" ♀ and different tea cultivars were used as materials to explore the correlation between caffeine content and gene expression. The heterologous expression systems of yeast and Arabidopsis were applied to explore the function of CsPUPs. Correlation analysis showed that the expressions of CsPUP1, CsPUP3.1, and CsPUP10.1 were significantly negatively correlated with caffeine content in tea leaves of eight strains and six cultivars. Furthermore, subcellular localization revealed that the three CsPUPs were not only located in plasma membrane but also widely distributed as circular organelles in cells. Functional complementation assays in yeast showed that the three CsPUPs could partly or completely rescue the defective function of fcy2 mutant in caffeine transport. Among them, transgenic yeast of CsPUP10.1 exhibited the strongest transport capacity for caffeine. Consistent phenotypes and functions were further identified in the CsPUP10.1-over-expression Arabidopsis lines. Taken together, it suggested that CsPUPs were involved in caffeine transport in tea plants. Potential roles of CsPUPs in the intracellular transport of caffeine among different subcellular organelles were proposed. This study provides a theoretical basis for further research on the PUP genes and new insights for caffeine metabolism in tea plants.

Proteomic Analysis of Roots Response to Potassium Deficiency and the Effect of TaHAK1-4A on K+ Uptake in Wheat.

Potassium (K+) is essential for plant growth and stress responses. A deficiency in soil K+ contents can result in decreased wheat quality and productivity. Thus, clarifying the molecular mechanism underlying wheat responses to low-K+ (LK) stress is critical. In this study, a tandem mass tag (TMT)-based quantitative proteomic analysis was performed to investigate the differentially abundant proteins (DAPs) in roots of the LK-tolerant wheat cultivar "KN9204" at the seedling stage after exposure to LK stress. A total of 104 DAPs were identified in the LK-treated roots. The DAPs related to carbohydrate and energy metabolism, transport, stress responses and defense, and post-translational modifications under LK conditions were highlighted. We identified a high-affinity potassium transporter (TaHAK1-4A) that was significantly up-regulated after the LK treatment. Additionally, TaHAK1-4A was mainly expressed in roots, and the encoded protein was localized in the plasma membrane. The complementation assay in yeast suggested that TaHAK1-4A mediates K+ uptake under extreme LK conditions. The overexpression of TaHAK1-4A increased the fresh weight and root length of Arabidopsis under LK conditions and improved the growth of Arabidopsis athak5 mutant seedlings, which grow poorly under LK conditions. Moreover, silencing of TaHAK1-4A in wheat roots treated with LK stress decreased the root length, dry weight, K+ concentration, and K+ influx. Accordingly, TaHAK1-4A is important for the uptake of K+ by roots exposed to LK stress. Our results reveal the protein metabolic changes in wheat induced by LK stress. Furthermore, we identified a candidate gene potentially relevant for developing wheat lines with increased K+ use efficiency.

CsGDH2.1 negatively regulates theanine accumulation in late-spring tea plants (Camellia sinensis var. sinensis).

Theanine, a unique and the most abundant non-proteinogenic amino acid in tea plants, endows tea infusion with the umami taste and anti-stress effects. Its content in tea correlates highly with green tea quality. Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring. However, how the decrease is regulated is largely unknown. In a genetic screening, we observed that a yeast mutant, glutamate dehydrolase 2 (gdh2), was hypersensitive to 40mM theanine and accumulated more theanine. This result implied a role of CsGDH2s in theanine accumulation in tea plants. Therefore, we identified the two homologs of GDH2, CsGDH2.1 and CsGDH2.2, in tea plants. Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant. Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues. Importantly, CsGDH2.1 expression was low in early spring, and increased significantly in late spring, in the new shoots of tea plants. These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots. Moreover, the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity, and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants. These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring, and provides a functional gene for improving late-spring green tea quality.

Identification and expression analysis of pineapple sugar transporters reveal their role in the development and environmental response.

In plants, sugars are required for several essential functions, including growth, storage, signaling, defense and reproduction. Sugar transporters carry out the controlled movement of sugars from source (leaves) to sink (fruits and roots) tissues and determine the overall development of the plant. Various types of sugar transporter families have been described in plants, including sucrose transporters (SUC/SUT), monosaccharide transporter (MST) and SWEET (from "Sugar Will Eventually be Exported Transporters"). However, the information about pineapple sugar transporters is minimal. This study systematically identified and classified 45 MST and 4 SUC/SUT genes in the pineapple genome. We found that the expression patterns of sugar transporter genes have a spatiotemporal expression in reproductive and vegetative tissues indicating their pivotal role in reproductive growth and development. Besides, different families of sugar transporters have a diel expression pattern in photosynthetic and non-photosynthetic tissues displaying circadian rhythm associated participation of sugar transporters in the CAM pathway. Moreover, regulation of the stress-related sugar transporters during cold stress indicates their contribution to cold tolerance in pineapple. Heterologous expression (yeast complementation assays) of sugar transporters in a mutant yeast strain suggested that SUT1/2 have the ability to transport sucrose, and STP13, STP26, pGlcT-L2 and TMT4 are able to transport glucose, whereas SWEET11/13 transport both sucrose and fructose. The information provided here would help researchers further explore the underlying molecular mechanism involved in the sugar metabolism of pineapple.

Transcriptomic and functional characterization reveals CsHAK5;3 as a key player in K+ homeostasis in grafted cucumbers under saline conditions

Grafting can improve the salt tolerance of many crops. However, critical genes in scions responsive to rootstock under salt stress remain a mystery. We found that pumpkin rootstock decreased the content of Na+ by 70.24 %, increased the content of K+ by 25.9 %, and increased the K+/Na+ ratio by 366.0 % in cucumber scion leaves. RNA-seq analysis showed that ion transport-related genes were the key genes involved in salt stress tolerance in grafted cucumber. The identification and analysis of the expression of K+ transporter proteins in cucumber and pumpkin revealed six and five HAK5 members, respectively. The expression of CsHAK5;3 in cucumber was elevated in different graft combinations under salt stress and most notably in cucumber scion/pumpkin rootstock. CsHAK5;3 was localized to the plasma membrane, and a yeast complementation assay revealed that it can transport K+. CsHAK5;3 knockout in hairy root mutants decreased the K+ content of leaves (45.6 %) and roots (50.3 %), increased the Na+ content of leaves (29.3 %) and roots (34.8 %), and decreased the K+/Na+ ratio of the leaves (57.9 %) and roots (62.9 %) in cucumber. However, CsHAK5;3 overexpression in hairy roots increased the K+ content of the leaves (31.2 %) and roots (38.3 %), decreased the Na+ content of leaves (17.2 %) and roots (14.3 %), and increased the K+/Na+ ratio of leaves (58.9 %) and roots (61.6 %) in cucumber. In conclusion, CsHAK5;3 in cucumber can mediate K+ transport and is one of the key target pumpkin genes that enhance salt tolerance of cucumber grafted.

Glutathione degradation activity of γ-glutamyl peptidase 1 manifests its dual roles in primary and secondary sulfur metabolism in Arabidopsis.

Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

Identification and Characterization of Shaker K+ Channel Gene Family in Foxtail Millet (Setaria italica) and Their Role in Stress Response.

Potassium (K+) is one of the indispensable elements in plant growth and development. The Shaker K+ channel protein family is involved in plant K+ uptake and distribution. Foxtail millet (Setaria italica), as an important crop, has strong tolerance and adaptability to abiotic stresses. However, no systematic study focused on the Shaker K+ channel family in foxtail millet. Here, ten Shaker K+ channel genes in foxtail millet were identified and divided into five groups through phylogenetic analysis. Gene structures, chromosome locations, cis-acting regulatory elements in promoter, and post-translation modification sites of Shaker K+ channels were analyzed. In silico analysis of transcript level demonstrated that the expression of Shaker K+ channel genes was tissue or developmental stage specific. The transcription levels of Shaker K+ channel genes in foxtail millet under different abiotic stresses (cold, heat, NaCl, and PEG) and phytohormones (6-BA, BR, MJ, IAA, NAA, GA3, SA, and ABA) treatments at 0, 12, and 24 h were detected by qRT-PCR. The results showed that SiAKT1, SiKAT3, SiGORK, and SiSKOR were worth further research due to their significant responses after most treatments. The yeast complementation assay verified the inward K+ transport activities of detectable Shaker K+ channels. Finally, we found interactions between SiKAT2 and SiSNARE proteins. Compared to research in Arabidopsis, our results showed a difference in SYP121 related Shaker K+ channel regulation mechanism in foxtail millet. Our results indicate that Shaker K+ channels play important roles in foxtail millet and provide theoretical support for further exploring the K+ absorption mechanism of foxtail millet under abiotic stress.

Targeting Pantothenate Kinase as an Effective Strategy for Antifungal Drug Development

Fungal infections affect over one billion people worldwide each year and are the leading cause of mortality by eukaryotic pathogens with an estimated 150 million severe life‐threatening cases and 1.7 million deaths reported annually. The rapid increase in the number of clinical cases and the emergence of multidrug resistant pathogens such as Candida auris emphasize the urgent need for new strategies to develop more effective, selective and safe antifungals. Coenzyme A (CoA) plays a central role in key cellular processes including the citric acid cycle, lipid metabolism, and ergosterol biosynthesis. The first step in the CoA biosynthesis pathway is catalyzed by a pantothenate kinase (PanK), which phosphorylates pantothenic acid (PA, or vitamin B5) to form phosphopantothenate. In fungi, this enzyme is encoded by a single copy PanK gene, which plays an essential role in cell viability. Here, we report the first crystal structure of the S. cerevisiae PanK enzyme, Cab1, at 1.8Å resolution. Cab1 was crystallized alone and in the presence of three lead high‐affinity inhibitors identified in a high‐throughput chemical screen of a library of 156,593 small molecules. Structure‐guided mutagenesis together with functional complementation assays in yeast identified key residues in Cab1 that are essential for catalysis, substrate binding and dimerization. Structure activity relationship and biological assays identified a lead inhibitor YU385599, with excellent activity against the human pathogens Candida albicans, C. glabrata, and C. parapsilosis. Together, our data validate PanK activity as a novel drug target and identify a novel class of small molecules for the treatment of fungal diseases.

PDAT regulates PE as transient carbon sink alternative to triacylglycerol in Nannochloropsis.

Triacylglycerols (TAGs) are the main storage lipids in photosynthetic organisms under stress. In the oleaginous alga Nannochloropsis oceanica, while multiple acyl CoA:diacylglycerol (DAG) acyltransferases (NoDGATs) are involved in TAG production, the role of the unique phospholipid:DAG acyltransferase (NoPDAT) remains unknown. Here, we performed a functional complementation assay in TAG-deficient yeast (Saccharomyces cerevisiae) and an in vitro assay to probe the acyltransferase activity of NoPDAT. Subcellular localization, overexpression, and knockdown (KD) experiments were also conducted to elucidate the role of NoPDAT in N. oceanica. NoPDAT, residing at the outermost plastid membrane, does not phylogenetically fall into the clades of algae or plants and uses phosphatidylethanolamine (PE) and phosphatidylglycerol with 16:0, 16:1, and 18:1 at position sn-2 as acyl-donors in vivo. NoPDAT KD, not triggering any compensatory mechanism via DGATs, led to an ∼30% decrease of TAG content, accompanied by a vast accumulation of PEs rich in 16:0, 16:1, and 18:1 fatty acids (referred to as "LU-PE") that was positively associated with CO2 availability. We conclude that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production. LU-PE can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels.

Role of OsCZMT1 in Na+ and Mg2+ transport and salinity insensitivity

Salinity stress is one of the most important abiotic stress factors affecting rice production worldwide. Using a forward genetics approach, a salt-insensitive TILLING line 1 (sitl1) rice mutant was isolated from a TILLING population. The sitl1 mutant exhibited reduced root growth and leaf chlorophyll content when grown under normal conditions, owing to a reduced ability to take up Mg2+; however, the defective phenotype could be restored by Mg2+ supplementation. Ionomic analysis revealed that the salinity insensitivity of sitl1 was the result of significantly reduced Mg2+ and Na+, and whole genome and RNA sequencing revealed that the sitl1 mutant caused a frameshift in the OsCZMT1 protein’s CorA-like ZntB cation transfer domain. Transient expression of an OsCZMT1-sGFP fusion protein revealed that OsCZMT1 was localized to the plasma membrane, and comprehensive expression analysis revealed that OsCZMT1 was mainly expressed in the roots and leaves of seedlings and highly upregulated in response to salinity stress. Complementation assays in yeast and CRISPR/Cas9-mediated knock-out mutants reveal that OsCZMT1 possesses both Mg2+ and Na+ transport activity. Taken together, these findings suggest that the identified OsCZMT1 frameshift mutation is responsible for the reduced Na+ and Mg2+ transport abilities, and that those reduced transport abilities confer salinity insensitivity. The study’s raw whole-genome sequencing and RNA-sequencing data have been accessioned in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under BioProject Accession Number PRJNA648465 (SRR12334077 – SRR12334084).

The rice phosphate transporter OsPHT1;7 plays a dual role in phosphorus redistribution and anther development.

Inorganic phosphate (Pi) is the predominant form of phosphorus (P) readily accessible to plants, and Pi Transporter 1 (PHT1) genes are the major contributors to root Pi uptake. However, the mechanisms underlying the transport and recycling of Pi within plants, which are vital for optimizing P use efficiency, remain elusive. Here, we characterized a functionally unknown rice (Oryza sativa) PHT1 member barely expressed in roots, OsPHT1;7. Yeast complementation and Xenopus laevis oocyte assay demonstrated that OsPHT1;7 could mediate Pi transport. Reverse-transcription quantitative polymerase chain reaction and histochemical analyses showed that OsPHT1;7 was preferentially expressed in source leaves and nodes. A further fine-localization analysis by immunostaining showed that OsPHT1;7 expression was restricted in the vascular bundle (VB) sheath and phloem of source leaves as well as in the phloem of regular/diffuse- and enlarged-VBs of nodes. In accordance with this expression pattern, mutation of OsPHT1;7 led to increased and decreased P distribution in source (old leaves) and sink organs (new leaves/panicles), respectively, indicating that OsPHT1;7 is involved in P redistribution. Furthermore, OsPHT1;7 showed an overwhelmingly higher transcript abundance in anthers than other PHT1 members, and ospht1;7 mutants were impaired in P accumulation in anthers but not in pistils or husks. Moreover, the germination of pollen grains was significantly inhibited upon OsPHT1;7 mutation, leading to a >80% decrease in seed-setting rate and grain yield. Taken together, our results provide evidence that OsPHT1;7 is a crucial Pi transporter for Pi transport and recycling within rice plants, stimulating both vegetative and reproductive growth.

The Gene FvTST1 From Strawberry Modulates Endogenous Sugars Enhancing Plant Growth and Fruit Ripening.

Sugar is an important carbon source and contributes significantly to the improvement of plant growth and fruit flavor quality. Sugar transport through the tonoplast is important for intracellular homeostasis and metabolic balance in plant cells. There are four tonoplast sugar transporters (FvTST1-4) in strawberry genome. The qRT-PCR results indicated that FvTST1 has a differential expression pattern in different tissues and developmental stages, and exhibited highest expression level in mature fruits. The yeast complementation assay showed that FvTST1 can mediate the uptake of different sugars, such as fructose, glucose, sucrose, and mannose. Subcellular localization analyses revealed that FvTST1 was mainly targeted to the tonoplast. Transient expression of FvTST1 in strawberry fruits enhanced both fruit ripening and sugar accumulation. Furthermore, FvTST1-transformed tomato plants exhibited higher sucrose and auxin content, enhanced seed germination and vegetative growth, higher photosynthetic rate, early flowering, and bore fruit; fructose and glucose levels were higher in transgenic fruits than those in the control. Transcriptomic analysis indicated that the auxin signaling pathway was highly enriched pathway in up-regulated Gene-ontology terms. In transgenic plants, genes encoding transcription factors, such as phytochrome-interacting factors PIF1, -3, and -4, as well as their potential target genes, were also induced. Collectively, the results show that FvTST1 enhances plant growth and fruit ripening by modulating endogenous sugars, and highlight the biological significance of this gene for future breeding purposes.

A metal tolerance protein, MTP10, is required for the calcium and magnesium homeostasis in Arabidopsis

ABSTRACT Nutrient antagonism typically refers to the fact that too high a concentration of one nutrient inhibits the absorption of another nutrient. In plants, Ca2+ (Calcium) and Mg2+ (Magnesium) are the two most abundant divalent cations, which are known to have antagonistic interactions. Hence, maintaining their homeostasis is crucial for plant growth and development. In this study, we showed that MTP10 (Metal Tolerance Protein 10) is an important regulator for maintaining homeostasis of Mg and Ca in Arabidopsis. The mtp10 mutant displayed severe growth retardation in the presence of excess Mg2+, to which the addition of Ca2+ was able to rescue the phenotype of mtp10 mutant. Additionally, the deficiency of Ca2+ in the culture medium accelerated the high-Mg sensitivity of the mtp10 mutant. The yeast complementation assay suggested that AtMTP10 had no Ca2+ transport activity. And the ICP-MS data further confirmed the antagonistic relationship between Ca2+ and Mg2+, with the addition of Ca2+ reducing the excessive accumulation of Mg2+ and high-Mg inhibiting the uptake of Ca2+. We conclude that the Arabidopsis MTP10 is essential for the regulation of Mg and Ca homeostasis.

Ca2+-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families have been characterized, we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants. Here, we demonstrate that two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, are key regulators of plant Mn homeostasis. Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we show that sequential phosphorylation of MTP8, initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26, provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport. Collectively, our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.

Identification and molecular characterization of the high-affinity copper transporters family in Solanum lycopersicum

Copper (Cu) plays a key role as cofactor in the plant proteins participating in essential cellular processes, such as electron transport and free radical scavenging. Despite high-affinity Cu transporters (COPTs) being key participants in Cu homeostasis maintenance, very little is known about COPTs in tomato (Solanum lycopersicum) even though it is the most consumed fruit worldwide and this crop is susceptible to suboptimal Cu conditions. In this study, a six-member family of COPT (SlCOPT1-6) was identified and characterized. SlCOPTs have a conserved architecture consisting of three transmembrane domains and β-strains. However, the presence of essential methionine residues, a methionine-enriched amino-terminal region, an Mx3Mx12Gx3G Cu-binding motif and a cysteine rich carboxy-terminal region, all required for their functionality, is more variable among members. Accordingly, functional complementation assays in yeast indicate that SlCOPT1 and SlCOPT2 are able to transport Cu inside the cell, while SlCOPT3 and SlCOPT5 are only partially functional. In addition, protein interaction network analyses reveal the connection between SlCOPTs and Cu PIB-type ATPases, other metal transporters, and proteins related to the peroxisome. Gene expression analyses uncover organ-dependency, fruit vasculature tissue specialization and ripening-dependent gene expression profiles, as well as different response to Cu deficiency or toxicity in an organ-dependent manner.

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus.

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

Mutation in ZDHHC15 Leads to Hypotonic Cerebral Palsy, Autism, Epilepsy, and Intellectual Disability.

ObjectiveTo determine whether mutations reported for ZDHHC15 can cause mixed neurodevelopmental disorders, we performed both functional studies on variant pathogenicity and ZDHHC15 function in animal models.MethodsWe examined protein function of 4 identified variants in ZDHHC15 in a yeast complementation assay and locomotor defects of loss-of-function genotypes in a Drosophila model.ResultsAlthough we assessed multiple patient variants, only 1 (p.H158R) affected protein function. We report a patient with a diagnosis of hypotonic cerebral palsy, autism, epilepsy, and intellectual disability associated with this bona fide damaging X-linked variant. Features include tall forehead with mild brachycephaly, down-slanting palpebral fissures, large ears, long face, facial muscle hypotonia, high-arched palate with dental crowding, and arachnodactyly. The patient had mild diminished cerebral volume, with left-sided T2/FLAIR hyperintense periatrial ovoid lesion. We found that loss-of-function mutations in orthologs of this gene cause flight and coordinated movement defects in Drosophila.ConclusionsOur findings support a functional expansion of this gene to a role in motor dysfunction. Although ZDHHC15 mutations represent a rare cause of neurodevelopmental disability, candidate variants need to be carefully assessed before pathogenicity can be determined.

Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum

BackgroundPlasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT.MethodsUsing the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter’s substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron.ResultsThe combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein.ConclusionThe structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

Medicago truncatula Yellow Stripe‐Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

TaARPC5 is required for wheat defense signaling in response to infection by the stripe rust fungus

Numerous studies using a combination of confocal microscopic- and pharmacological-based approaches have demonstrated that the actin cytoskeleton dynamically responds to pathogen infection. Here, we observed that phalloidin treatment induced actin nucleation, resulting in enhanced resistance of wheat against the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). To define the mechanism underpinning this process, we characterized a family of conserved actin-binding proteins, the actin related protein (ARP) family, which controls actin polymerization. Specifically, we identified and characterized a wheat ARPC gene (TaARPC5), which encodes a 136-amino acid protein containing a P16-Arc domain, the smallest subunit of the ARP2/3 complex. TaARPC5 mRNA accumulation was induced following the infection of plants with the avirulent Pst strain, and following the elicitation with flagellin (e.g., flg22) as well. Subcellular localization analysis revealed that TaARPC5 is primarily localized to the cortical actin cytoskeleton, and its precise cellular localizations suggest the proximity to processes correlated with the actin-organelle interface. Upon treatment with virulent Pst, TaARPC5-knockdown plants exhibited a significant reduction in the expression of PTI-specific mRNAs. Conversely, we observed enhanced induction of reactive oxygen species (ROS) accumulation and a decrease in TaCAT1 expression following infection with an incompatible Pst isolate. Together with yeast complementation assays, the current study demonstrates a role for TaARPC5 in resistance signaling in wheat against Pst infection by regulating the host actin cytoskeleton.