Genetic Assays Research Articles

Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization.

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.

TRY intron2 determined its expression in inflorescence activated by SPL9 and MADS-box genes in Arabidopsis

Plant trichomes are specialized epidermal cells that protect plants from insects and pathogens. In Arabidopsis, epidermal hairs decrease as internodes increase in height, with only few epidermal hairs produced on the sepals abaxial surface of the early flowers. TRIPTYCHON (TRY) is known to be a negative regulator of epidermal hair development in Arabidopsis, suppressing the formation of ectopic epidermal hairs in the inflorescence. Here, we reported that the second intron of TRY gene plays a critical role in trichome spatial distribution in Arabidopsis. The expression of TRY rises with the increasing stem nodes and reaches the peak in the inflorescence, while the trichomes distribution decrease. The transgenic plants showed that TRY promoter could only drive the genomic instead of coding sequences combined with GUS reporter gene, which indicates that the regulatory elements of TRY expression in inflorescence could be located in the intron regions. Multiple SPLs and MADS-box binding sites were found in the TRY intron2 sequence. Further genetic and biochemistry assays revealed that the flowering-related genes such as SPL9 could bind to these cis-elements directly, contributing to the TRY spatial expression. Since cotton fiber and Arabidopsis trichomes share similar regulatory mechanism, extended analysis showed that the intron2 of cotton TRY genes also contain the cis-elements. Thus, the introns harboring the transcription element may be the general way to regulate the gene expression in different plants and provides molecular clues for the related crops’ traits design.

SAUR15 interaction with BRI1 activates plasma membrane H+-ATPase to promote organ development of Arabidopsis.

Brassinosteroids (BRs) are an important group of plant steroid hormones that regulate growth and development. Several members of the SMALL AUXIN UP RNA (SAUR) family have roles in BR-regulated hypocotyl elongation and root growth. However, the mechanisms are unclear. Here, we show in Arabidopsis (Arabidopsis thaliana) that SAUR15 interacts with cell surface receptor-like kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) in BR-treated plants, resulting in enhanced BRI1 phosphorylation status and recruitment of the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1. Genetic and phenotypic assays indicated that the SAUR15 effect on BRI1 can be uncoupled from BRASSINOSTEROID INSENSITIVE 2 activity. Instead, we show that SAUR15 promotes BRI1 direct activation of plasma membrane H+-ATPase (PM H+-ATPase) via phosphorylation. Consequently, SAUR15–BRI1–PM H+-ATPase acts as a direct, PM-based mode of BR signaling that drives cell expansion to promote the growth and development of various organs. These data define an alternate mode of BR signaling in plants.

Eukaryotic RNA Polymerase ⍺‐like Subunits Heterodimerize by Distinct Interaction Mechanisms

DNA‐dependent RNA Polymerases (Pols) are present in every living cell and even encoded by some viruses. Pols are responsible for the process of transcription, and Pols from the three domains of life are constructed of a conserved core complex that includes a dimer of α or α‐like subunits that serve as a scaffold for Pol assembly. Bacteria and archaea each encode a single Pol that transcribes all forms of RNA, while eukaryotes encode three specialized Pols (I‐III) containing one of two distinct α‐like heterodimers. One α‐like heterodimer is shared between Pols I and III, while there is a paralogous Pol II heterodimer. The α‐like subunits are clinically relevant as mutations in the Pol I/III heterodimer are associated with Treacher Collins Syndrome and 4H Leukodystrophy, while mutations in the Pol II heterodimer are associated with Primary Ovarian Insufficiency. These mutations often result in defects in Pol assembly and/or activity, although the vast majority remain uncharacterized. It is currently unclear if heterodimer formation is functionally similar in α‐like subunit orthologs and/or paralogs and how these similarities and differences can be used to study the α‐like subunit associated diseases. To examine this, we mutated several regions of the yeast and human small α‐like subunits to test their contribution to heterodimer interaction using several types of biochemical and genetic assays. Here we show that different regions serve differential roles in heterodimerization, in a polymerase‐ and species‐specific manner, with both human subunits being more sensitive to mutations. This suggests that although the subunits are evolutionary conserved, they interact differently. More broadly, these findings help explain why some disease mutations have little to no effect in yeast and possibly other model systems, and may inform us how to make better disease models in the future.

Study of Trm7 Interactions With Binding Partners Trm732 and Trm734 for 2’‐O‐Methylation of the tRNA Anticodon Loop in Yeast

Transfer RNAs undergo post‐transcriptional modifications, including many which occur at or near the anticodon loop and are necessary for protein synthesis. In Saccharomyces cerevisiae, Trm7 is required for 2’‐O‐methylation of tRNA and requires interaction with Trm732 and Trm734 for modifications at positions 32 and 34, respectively. These proteins are widely conserved in eukaryotes, and mutations in the human ortholog of TRM7, FTSJ1, can cause non‐syndromic X‐linked intellectual disability by decreasing or eliminating methylation activity. Through a genetic assay, we have found Trm7 residues required for interaction with Trm732 or Trm734 and the subsequent methylation of tRNAPhe. These spot test assays showed that the Trm7 Y138R variant abrogates interaction with Trm734 and the Trm7 I144A variant abrogates interaction with Trm732. To verify that these genetic results are due to impaired interaction of Trm7 variants with its binding partners, we are performing immunoprecipitation assays. In addition, we have begun to study interactions of the individual proteins and complexes with tRNAPhe through biochemical methods such as electrophoretic mobility shift assays, to determine the role of each protein in tRNA binding. Through this study of Trm7 variants, we can better understand the interaction of Trm7 with Trm734 and Trm732, which will facilitate the study of conserved homologs in humans.

Investigating Disease‐Causing Mutations in SLC4 Transporter Homologs

The SLC4 family of transporters includes the archetypal transporter Band 3, or Anion Exchanger 1 (AE1), a bicarbonate and chloride ion exchanger essential for human respiration and the most abundant membrane protein in red blood cells. Mutations in SLC4 transporters such as AE1 can lead to diseases such as hereditary spherocytosis or renal tubular acidosis. The SLC4 family shares sequence and structural homology with borate transporters, which are found in plants and fungi and regulate cellular borate levels to protect against borate toxicity. Here we generated mutations in the S. cerevisiae borate transporter Bor1 homologous with known disease‐causing mutations of human AE1, and then tested for borate transport activity in a genetic complementation assay. Our results identify mutants with impaired function but wild‐type expression levels, as well as mutants with impaired function and depleted expression levels. These results are consistent with varying modes of deleterious impact of protein expression, sorting, and function that have been attributed to mutations in human AE1. These data show that mutations equivalent to disease‐causing mutations in human AE1 have major effects in ScBor1, strengthening the functional connection between SLC4 proteins and borate transporters. Additionally, we demonstrate that the ScBor1 D347 residue, which is homologous to the essential E681 of human AE1, can be complemented by the D347E but not D347N substitution, underscoring the necessity of having an acidic residue at this highly conserved functional location. Lastly, we identify several new amino acids critical for function through mutagenesis and complementation studies. These data collectively highlight similarities in the biochemical features of ScBor1 and HsAE1 and help shed light on the mechanisms of borate transporters and their SLC4 homologs.

PO-691-03 FUNCTIONAL ASSAYS IDENTIFY SPLICE-ALTERING VARIANTS IN MENDELIAN CHANNELOPATHIES

Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the Long QT Syndrome (LQTS). While splice-altering variants lying outside the 2 bp canonical splice sites can cause these diseases, their role remains poorly described in these arrhythmia syndromes. We implemented two functional assays to assess pathogenicity of 11 putative splice-altering variants of uncertain significance (VUS) in LQTS and BrS patients; 8/11 were intronic. We identified putative variants for study from a recent curation of rare variants detected in BrS and LQTS patients. We deployed the well established in vitro minigene assays to assess the splicing consequences of 9 VUS in the 3 genes. Two SCN5A variants were incompatible with the minigene approach, so we introduced each into a healthy control induced pluripotent stem cell line (iPSC) by CRISPR-Cas9 gene editing. We differentiated cells into iPSC cardiomyocytes (iPSC-CMs) and studied splicing by RT-PCR. We used the American College of Medical Genetics and Genomics (ACMG) functional assay criteria to reclassify VUS. We compared our assays to splicing predictions from the in silico tool SpliceAI. We identified aberrant splicing, with presumed disruption of protein sequence, in 7/9 VUS studied using the minigene assay and 1/2 in iPSC-CMs. We were able to reclassify 11 VUS, 10 to likely pathogenic, and 1 to likely benign. The computational tool SpliceAI had moderate sensitivity for the in vitro splicing results above a predictive threshold of 0.5 (7/11). VUS that are not located in canonical splice sites can nevertheless disrupt splicing and confer pathogenicity in BrS and LQTS.

The Genetic and Molecular Analyses of RAD51C and RAD51D Identifies Rare Variants Implicated in Hereditary Ovarian Cancer from a Genetically Unique Population.

Simple SummaryWe have investigated RAD51C and RAD51D, hereditary ovarian cancer risk genes, in French Canadians of Quebec, Canada. This population of Western European origins exhibits a unique genetic landscape as shown by the frequency of carriers of specific rare pathogenic variants. As studying French Canadians could facilitate the identification and interpretation of clinically relevant variants, we performed genetic analyses of RAD51C and RAD51D in this population comprised of cases with a family history of ovarian cancer or those who developed it at a young age. We identified candidate variants and then investigated them in other French Canadian study groups. We performed biological assays and revealed possible mechanisms that would affect gene function. Using engineered cells expressing one of our protein variants, we also showed that they were more sensitive to a recently approved treatment for ovarian cancer. Our findings support the role of inherited variants in RAD51C and RAD51D in ovarian cancer.To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

Antimicrobial activity, molecular typing and in vitro safety assessment of Lactococcus garvieae isolates from healthy cultured rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment

Lactococcus garvieae is the aetiological agent of lactococcosis, an ichthyopathology affecting rainbow trout (Oncorhynchus mykiss, Walbaum) farming and having recently been considered as a potential food-borne zoonotic agent. This work aims to characterise a population of 53 L. garvieae isolates from healthy cultured rainbow trout and rearing environment by using microbiological, biochemical and genetic assays to unveil the potential impact that their presence may have, not only for fish, but also for consumers. The lactococci exhibited a broad antimicrobial spectrum against Gram-negative ichthyopathogens, likely due to production of organic acids. The 53 L. garvieae isolates were grouped into two and five genetic clusters by PFGE and ERIC-PCR, respectively. All the lactococcal isolates produce α-haemolysis and harbour, at least, 10 putative virulence factors (hly-1, hly-2, hly-3, NADH oxidase, sod, pgm, adhPsaA, eno, LPxTG-3 and adhCI). While all the lactococci were resistant to streptomycin and clindamycin, no resistance genes were found. The manuscript concludes that the non-existence of active lactococcosis does not guarantee the absence of potentially virulent L. garvieae, which also exhibit a great capacity for adaptation and persistence in the environment. Furthermore, the antibiotic resistance profile shown may lead to re-evaluate the treatments of choice in human patients.

Genetic analysis of DAF-18/PTEN missense mutants for the ability to maintain quiescence of the somatic gonad and germ line in Caenorhabditis elegans dauer larvae.

The mammalian tumor suppressor PTEN has well-established lipid phosphatase and protein phosphatase activities. DAF-18, the Caenorhabditis elegans ortholog of PTEN, has a high degree of conservation in the catalytic domain, and human PTEN complements a null allele of daf-18, suggesting conserved protein function. Insights gleaned from studies of mammalian PTEN have been applied to studies of DAF-18 in C. elegans, including predicted enzymatic properties of mutants. Here, we characterize DAF-18 missense mutants previously treated as selectively disrupting either protein or lipid phosphatase activity in genetic assays to connect distinct phenotypes to specific enzymatic activities of DAF-18/PTEN. We analyze the ability of these mutants to maintain quiescence of the somatic gonad and germ line in dauer larvae, a state of diapause during which development is suspended. We show that transgenes expressing either the putative lipid phosphatase-deficient or putative protein phosphatase-deficient form fail to complement a daf-18 null allele, and that the corresponding homozygous endogenous missense mutant alleles fail to maintain developmental quiescence. We also show that the endogenous daf-18 missense alleles fail to complement each other, suggesting that one or both of the missense forms are not activity-selective. Furthermore, homozygous daf-18 missense mutants have a more severe phenotype than a daf-18 null mutant, suggesting the presence of functionally compromised mutant DAF-18 is more deleterious than the absence of DAF-18. We discuss how these genetic properties complicate the interpretation of genetic assays to associate specific enzymatic activities with specific phenotypes.

Lactic Acid Bacteria and Bioactive Amines Identified during Manipueira Fermentation for Tucupi Production.

There is scarce information regarding lactic acid bacteria (LAB) and the production of biogenic amines during manipueira fermentation for tucupi. Thus, the objective of this study was to isolate and identify LAB, and to determine their impact on bioactive amine formation. Spontaneous fermentation of manipueira was carried out at laboratory scale and selected LAB colonies were isolated and identified by sequencing techniques and comparison with sequences from a virtual database. Only two LAB species of the genus Lactobacillus were identified during fermentation: Lactobacillus fermentum and Lactobacillus plantarum. L. fermentum was the predominant, whereas L. plantarum was only detected in manipueira prior to fermentation. Spermidine and putrescine were detected throughout fermentation, whereas histamine was produced at the final stage. There was positive correlation between LAB counts and putrescine and histamine levels, suggesting that the identified LAB are responsible for the synthesis of these amines during manipueira fermentation. Genetic assays are needed to verify whether the LAB identified have the genes responsible for decarboxylation of amino acids.

OsMAPK6 phosphorylates a zinc finger protein OsLIC to promote downstream OsWRKY30 for rice resistance to bacterial blight and leaf streak.

Rice OsLIC encoding a CCCH zinc finger transcription factor plays an important role in immunity. However, the immune signaling pathways that OsLIC-involved and the underlying mechanisms that OsLIC-conferred resistance against pathogens are largely unclear. Here, we show that OsLIC, as a substrate for OsMAPK6, negatively regulates resistance to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) by directly suppressing OsWRKY30 transcription. Biochemical assays showed that OsLIC bound to OsWRKY30 promoter and suppressed its transcription. Genetic assays confirmed that the osilc knockout mutants and OsWRKY30-overexpressing plants exhibited enhanced resistance to Xoo and Xoc, knocking out OsWRKY30 in the oslic mutants attenuated the resistance against bacterial pathogens. OsMAPK6 physically interacted with and phosphorylated OsLIC leading to decreased OsLIC DNA-binding activity, therefore, overexpression of OsLIC partially suppressed OsMAPK6-mediated rice resistance. In addition, both OsMAPK6-phosphorylated activation of OsLIC and phosphorylation-mimic OsLIC5D had reduced DNA-binding activity towards OsWRKY30 promoter, thereby promoting OsWRKY30 transcription. Collectively, these results reveal that OsMAPK6-mediated phosphorylation of OsLIC positively regulates rice resistance to Xoo and Xoc by modulating OsWRKY30 transcription, suggesting that OsMAPK6-OsLIC-OsWRKY30 module is an immune signaling pathway in response to the bacterial pathogens.

ITRAQ-based proteomic analysis of Bacillus subtilis strain NCD-2 regulated by PhoPR two-component system: A comparative analysis with transcriptomics revealed the regulation for fengycin production by branched chain amino acids

The PhoPR two-component system (TCS) is a signal transduction pathway to regulate the phosphate starvation response in Bacillus subtilis and regulated fengycin production in strain NCD-2 under low phosphate condition. The purpose of this study was to characterize the proteome level responses in the phoP-null mutant (MP) and the phoR-null mutant (MR), and to integrate the proteomics with the transcriptomic data obtained previously. The metabolic pathway for fengycin was predicted based on omics analysis as well as molecular genetics assay. Results showed the proteins and genes associated with biosynthesis of branched chain amino acids (BCAAs) were regulated by PhoPR TCS, and liquid chromatography mass spectrometry (LC-MS) analysis also confirmed that the production of BCAAs was down-regulated in the MP and MR mutants, when compared to wild-type strain NCD-2. Protein network analysis showed that the BCAA metabolism was linked to the biosynthesis of lipopeptides. The MP and MR strains decreased the fengycin production when cultured in modified Landy medium supplied with 0.42 mM phosphate, however, the fengycin production could be restored when the glutamic acid was replaced with BCAAs that were added to modified Landy medium. The lpdV gene, which is responsible for the BCAA degradation process, was deleted in strain NCD-2. Compared with the wild-type strain, the lpdV mutant produced significantly less fengycin in the medium supplied with BCAAs. Considered together, the results of this study indicate that the PhoPR TCS regulates fengycin production by affecting BCAA biosynthesis.

Paper-based genetic assays with bioconjugated gold nanorods and an automated readout pipeline

Paper-based biosensors featuring immunoconjugated gold nanoparticles have gained extraordinary momentum in recent times as the platform of choice in key cases of field applications, including the so-called rapid antigen tests for SARS-CoV-2. Here, we propose a revision of this format, one that may leverage on the most recent advances in materials science and data processing. In particular, we target an amplifiable DNA rather than a protein analyte, and we replace gold nanospheres with anisotropic nanorods, which are intrinsically brighter by a factor of ~ 10, and multiplexable. By comparison with a gold-standard method for dot-blot readout with digoxigenin, we show that gold nanorods entail much faster and easier processing, at the cost of a higher limit of detection (from below 1 to 10 ppm in the case of plasmid DNA containing a target transgene, in our current setup). In addition, we test a complete workflow to acquire and process photographs of dot-blot membranes with custom-made hardware and regression tools, as a strategy to gain more analytical sensitivity and potential for quantification. A leave-one-out approach for training and validation with as few as 36 sample instances already improves the limit of detection reached by the naked eye by a factor around 2. Taken together, we conjecture that the synergistic combination of new materials and innovative tools for data processing may bring the analytical sensitivity of paper-based biosensors to approach the level of lab-grade molecular tests.

Cytochrome P450 and Glutathione S-Transferase Confer Metabolic Resistance to SYP-14288 and Multi-Drug Resistance in Rhizoctonia solani.

SYP-14288 is a fungicide as an uncoupler of oxidative phosphorylation, which is effective in controlling fungal pathogens like Rhizoctonia solani. To determine whether R. solani can develop SYP-14288 resistance and possibly multi-drug resistance (MDR), an SYP-14288-resistant mutant of R. solani X19-7 was generated from wild-type strain X19, and the mechanism of resistance was studied through metabolic and genetic assays. From metabolites of R. solani treated with SYP-14288, three compounds including M1, M2, and M3 were identified according to UPLC-MS/MS analysis, and M1 accumulated faster than M2 and M3 in X19-7. When X19-7 was treated by glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) and SYP-14288 together, or by DEM plus one of tested fungicides that have different modes of action, a synergistic activity of resistance occurred, implying that GSTs promoted metabolic resistance against SYP-14288 and therefore led to MDR. By comparing RNA sequences between X19-7 and X19, six cytochrome P450s (P450s) and two GST genes were selected as a target, which showed a higher expression in X19-7 than X19 both before and after the exposure to SYP-14288. Furthermore, heterologous expression of P450 and GST genes in yeast was conducted to confirm genes involved in metabolic resistance. In results, the P450 gene AG1IA_05136 and GST gene AG1IA_07383 were related to fungal resistance to multiple fungicides including SYP-14288, fluazinam, chlorothalonil, and difenoconazole. It was the first report that metabolic resistance of R. solani to uncouplers was associated with P450 and GST genes.

YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri.

The intracellular pathogen Shigella flexneri, which is the causative agent of bacillary dysentery, significantly influences the worldwide implication of diarrheal infections, consequentially causing about 1.1 million deaths each year. Due to a nonavailability of an authorized vaccine and the upsurge of multidrug resistance amongst Shigella strains, there has been a huge demand for further genetic analyses which could help in the advancement of new/improved drugs, and finding vaccine candidates against the pathogen. The present study aims to illustrate the role of the yfiB gene in Shigella virulence, part of the periplasmic YfiBNR tripartite signalling system. This system is involved in the regulation of cyclic-di-GMP levels inside the bacterial cells, a vital messenger molecule impacting varied cellular processes such as biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms such as adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect the native c-di-GMP levels. We found that this subsequently causes a negative effect on the biofilm formation, bacterial invasion, host–surface attachment, and the overall virulence of Shigella. This study also carried out a structural and functional assessment of the YfiB protein and determined critical amino acid residues, essential for proper functioning of this signalling system. The present work improves our understanding of the in vivo persistence and survival of Shigella, brings light to the c-di-GMP led regulation of Shigella virulence, and provides a prospective new target to design anti-infection drugs and vaccines against S. flexneri and other bacterial pathogens.

Characterisation of two novel genes encoding Δ9 fatty acid desaturases (CeSADs) for oleic acid accumulation in the oil-rich tuber of Cyperus esculentus

Cyperus esculentus is considered one of the most promising oil crops due to its oil-rich tuber, wide adaptability and large biomass production. Preferable triacylglycerol (TAG) composition, especially high oleic acid content, makes tuber oil suitable for human consumption and biodiesel production. However, the mechanism underlying oleic acid enrichment in the tuber remains unknown. Plastidial stearoyl-ACP desaturase (SAD) catalyses the formation of monounsaturated fatty acids (MUFAs), which may function crucially for high accumulation of oleic acid in C. esculentus tubers. In this study, two full-length cDNAs encoding SAD were isolated from the developing tubers of C. esculentus, namely, CeSAD1 and CeSAD2, with ORFs of 1194 bp and 1161 bp, respectively. Quantitative RT-PCR analysis showed that CeSAD genes were highly expressed in tubers. The expression pattern during tuber formation was also significantly correlated with fatty acid and oil accumulation dynamics. Overexpression of each CeSAD gene could restore the normal growth of the defective yeast BY4389, indicating that both CeSADs had fatty acid desaturase activity to catalyse MUFA biosynthesis. A tobacco genetic transformation assay demonstrated that both CeSAD enzymes had high enzyme activity. Exogenous addition of exogenous fatty acids to feed yeast revealed that CeSAD1 has a more substantial substrate preference ratio for C18:0 than CeSAD2 did. Moreover, the overexpression of CeSAD1 significantly increased host tolerance against low-temperature stress. Our data add new insights into the deep elucidation of oleic acid-enriched oils in Cyperus esculentus tubers, showing CeSAD, especially CeSAD1, as the target gene in genetic modification to increase oil and oleic yields in oil crops as well as stress tolerance.

ABI5 binding protein2 inhibits ABA responses during germination without ABA-INSENSITIVE5 degradation.

Overexpression of ABA-INSENSITIVE5 binding proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. We tested the hypothesis that AFPs promote germination in Arabidopsis (Arabidopsis thaliana) by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING, were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although weak direct interactions were detected between AFPs and E3 ligases, loss of function for these E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 overexpression reduces ABA sensitivity, thereby permitting germination prior to ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance. Our quantitative proteomics analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of wild-type or dwa1 dwa2 mutants with or without AFP2 overexpression allowed us to separate and refine the changes in protein accumulation patterns associated with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Dual roles for nuclear RNAi Argonautes in Caenorhabditis elegans dosage compensation.

Dosage compensation involves chromosome-wide gene regulatory mechanisms which impact higher order chromatin structure and are crucial for organismal health. Using a genetic approach, we identified Argonaute genes which promote dosage compensation in Caenorhabditis elegans. Dosage compensation in C. elegans hermaphrodites is initiated by the silencing of xol-1 and subsequent activation of the dosage compensation complex which binds to both hermaphrodite X chromosomes and reduces transcriptional output by half. A hallmark phenotype of dosage compensation mutants is decondensation of the X chromosomes. We characterized this phenotype in Argonaute mutants using X chromosome paint probes and fluorescence microscopy. We found that while nuclear Argonaute mutants hrde-1 and nrde-3, as well as mutants for the piRNA Argonaute prg-1, exhibit derepression of xol-1 transcripts, they also affect X chromosome condensation in a xol-1-independent manner. We also characterized the physiological contribution of Argonaute genes to dosage compensation using genetic assays and found that hrde-1 and nrde-3 contribute to healthy dosage compensation both upstream and downstream of xol-1.

Delivery of an Rhs‐family nuclease effector reveals direct penetration of the gram‐positive cell envelope by a type VI secretion system in Acidovorax citrulli

AbstractThe type VI secretion system (T6SS) is a double‐tubular nanomachine widely found in gram‐negative bacteria. Its spear‐like Hcp tube is capable of penetrating a neighboring cell for cytosol‐to‐cytosol protein delivery. However, gram‐positive bacteria have been considered impenetrable to such T6SS action. Here we report that the T6SS of a plant pathogen, Acidovorax citrulli (AC), could deliver an Rhs‐family nuclease effector RhsB to kill not only gram‐negative but also gram‐positive bacteria. Using bioinformatic, biochemical, and genetic assays, we systematically identified T6SS‐secreted effectors and determined that RhsB is a crucial antibacterial effector. RhsB contains an N‐terminal PAAR domain, a middle Rhs domain, and an unknown C‐terminal domain. RhsB is subject to self‐cleavage at both its N‐ and C‐terminal domains and its secretion requires the upstream‐encoded chaperone EagT2 and VgrG3. The toxic C‐terminus of RhsB exhibits DNase activities and such toxicity is neutralized by either of the two downstream immunity proteins, RimB1 and RimB2. Deletion of rhsB significantly impairs the ability of killing Bacillus subtilis while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete Escherichia coli and B. subtilis in planta but also is highly potent in killing other bacterial and fungal species. Collectively, these findings highlight the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.