Ribosomal Protein Genes Research Articles

Nutrient condition modulates the antibiotic tolerance of Pseudomonas aeruginosa

The variation in nutrient content across diverse environments has a significant impact on the survival and metabolism of microorganisms. In this study, we examined the influence of nutrients on the antibiotic tolerance of the PAO1 strain of Pseudomonas aeruginosa. Our findings indicate that under nutrient-rich conditions, this strain exhibited relatively high tolerance to ceftazidime, chloramphenicol, and tetracycline, but not aminoglycosides and fluoroquinolones. Transcriptome analysis revealed that genes associated with antibiotic tolerance were expressed more efficiently in nutrient-rich media, including ribosomal protein genes and multidrug efflux pump genes, which conferred higher tetracycline tolerance to the strain. Furthermore, the genes responsible for translation, biosynthesis, and oxidative phosphorylation were suppressed when nutrients were limited, resulting in decreased metabolic activity and lower sensitivity to ciprofloxacin. Artificial interference with ATP synthesis utilizing arsenate confirmed that the curtailment of energy provision bolstered the observed tolerance to ciprofloxacin. In general, our results indicate that this strain of P. aeruginosa tends to activate its intrinsic resistance mechanisms in nutrient-rich environments, thereby enhancing resistance to certain antibiotics. Conversely, in nutrient-limited environments, the strain is more likely to enter a dormant state, which enables it to tolerate antibiotics to which it would otherwise be sensitive. These findings further suggest that antibiotics released in environments with varying eutrophication levels may have divergent effects on the development of bacterial antibiotic resistance.

Horizontal Gene Transfer and Drug Resistance Involving Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) acquires drug resistance at a rate comparable to that of bacterial pathogens that replicate much faster and have a higher mutation rate. One explanation for this rapid acquisition of drug resistance in Mtb is that drug resistance may evolve in other fast-replicating mycobacteria and then be transferred to Mtb through horizontal gene transfer (HGT). This paper aims to address three questions. First, does HGT occur between Mtb and other mycobacterial species? Second, what genes after HGT tend to survive in the recipient genome? Third, does HGT contribute to antibiotic resistance in Mtb? I present a conceptual framework for detecting HGT and analyze 39 ribosomal protein genes, 23S and 16S ribosomal RNA genes, as well as several genes targeted by antibiotics against Mtb, from 43 genomes representing all major groups within Mycobacterium. I also included mgtC and the insertion sequence IS6110 that were previously reported to be involved in HGT. The insertion sequence IS6110 shows clearly that the Mtb complex participates in HGT. However, the horizontal transferability of genes depends on gene function, as was previously hypothesized. HGT is not observed in functionally important genes such as ribosomal protein genes, rRNA genes, and other genes chosen as drug targets. This pattern can be explained by differential selection against functionally important and unimportant genes after HGT. Functionally unimportant genes such as IS6110 are not strongly selected against, so HGT events involving such genes are visible. For functionally important genes, a horizontally transferred diverged homologue from a different species may not work as well as the native counterpart, so the HGT event involving such genes is strongly selected against and eliminated, rendering them invisible to us. In short, while HGT involving the Mtb complex occurs, antibiotic resistance in the Mtb complex arose from mutations in those drug-targeted genes within the Mtb complex and was not gained through HGT.

The transcriptomic landscape of normal and ineffective erythropoiesis at single-cell resolution.

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic and always reflect ineffective erythropoiesis yet result from diverse genetic mutations. To delineate shared mechanisms that lead to cell death, we studied the fate of single erythroid marrow cells from individuals with DBA or MDS-5q. We defined an unhealthy (vs healthy) differentiation trajectory using transcriptional pseudotime and cell surface proteins. The pseudotime trajectories diverge immediately after cells upregulate transferrin receptor (CD71), import iron, and initiate heme synthesis, although cell death occurs much later. Cells destined to die express high levels of heme-responsive genes, including ribosomal protein and globin genes, whereas surviving cells downregulate heme synthesis and upregulate DNA damage response, hypoxia, and HIF1 pathways. Surprisingly, 24%± 12% of cells from control subjects follow the unhealthy trajectory, implying that heme might serve as a rheostat directing cells to live or die. When heme synthesis was inhibited with succinylacetone, more DBA cells followed the healthy trajectory and survived. We also noted high numbers of messages with retained introns that increased as erythroid cells matured, confirmed the rapid cycling of colony forming unit-erythroid, and demonstrated that cell cycle timing is an invariant property of differentiation stage. Including unspliced RNA in pseudotime determinations allowed us to reliably align independent data sets and accurately query stage-specific transcriptomic changes. MDS-5q (unlike DBA) results from somatic mutation, so many normal (unmutated) erythroid cells persist. By independently tracking erythroid differentiation of cells with and without chromosome 5q deletions, we gained insight into why 5q+ cells cannot expand to prevent anemia.

Establishment and characterization of CSCRi006-A: an induced pluripotent stem cell line generated from a patient with Diamond-Blackfan Anemia (DBA) carrying ribosomal protein S19 (RPS19) mutation.

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes (Gadhiya and Wills in Diamond-Blackfan Anemia, https://www.statpearls.com/ ; 2023). A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Toward this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA-iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments.

Carriage of linezolid-resistant enterococci (LRE) among humans and animals in Nigeria: coexistence of the cfr, optrA, and poxtA genes in Enterococcus faecium of animal origin

In contrast to increasing reports of the emergence of linezolid-resistant enterococci (LRE) emanating from many countries in Europe, Asia, and North America, data on its status and dissemination from the African continent remain scarce, with the information available limited to countries in North Africa. This study investigated the carriage of LRE and the genetic mechanism of resistance among Enterococcus faecium and Enterococcus faecalis strains recovered from humans and animals in Makurdi, Nigeria. We conducted a cross-sectional study between June 2020 and July 2021 during which 630 non-duplicate human and animal faecal samples were collected and processed for the recovery of LRE. The genetic mechanisms for resistance were investigated using polymerase chain reaction (PCR) and Sanger sequencing. Linezolid-resistant enterococci were recovered from 5.87% (37/630; 95% CI: 4.17-8.00) of the samples, with the prevalence in animals and humans being 6.22% [(28/450); 95% CI: 4.17-8.87] and 5.00% [(9/180); 95% CI: 2.31-9.28], respectively. All isolates remained susceptible to vancomycin. No known point mutation mediating linezolid resistance was detected in the 23S rRNA and ribosomal protein genes; however, acquisition of one or more potentially transferable genes (cfr, optrA, and poxtA) was observed in 26 of the 37 LRE isolates. Co-existence of all three transferable genes in a single isolate was found in four E. faecium strains of animal origin. This study provides baseline evidence for the emergence and active circulation of LRE driven majorly by the acquisition of the optrA gene in Nigeria. To the best of our knowledge, our study is the first to report a co-carriage of all three transferable linezolid resistance determinants in E. faecium. Active LRE surveillance is urgently required to understand the extent of LRE spread across sub-Saharan Africa and to develop tailored mitigation strategies.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCRand western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

Identification of novel mutations in patients with Diamond-Blackfan anemia and literature review of RPS10 and RPS26 mutations.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia, physical malformation, and cancer predisposition. Twenty ribosomal protein genes and three non-ribosomal protein genes have been identified associated with DBA. To investigate the presence of novel mutations and gain a deeper understanding of the molecular mechanisms of disease, targeted next-generation sequencing was performed in 12 patients with clinically suspected DBA. Literatures were retrieved with complete clinical information published in English by November 2022. The clinical features, treatment, and RPS10/RPS26 mutations were analyzed. Among the 12 patients, 11 mutations were identified and 5 of them were novel (RPS19, p.W52S; RPS10, p.P106Qfs*11; RPS26, p.R28*; RPL5, p.R35*; RPL11, p.T44Lfs*40). Including 2 patients in this study, 13 patients with RPS10 mutations and 38 patients with RPS26 mutations were reported from 4 and 6 countries, respectively. The incidences of physical malformation in patients with RPS10 and RPS26 mutations (22% and 36%, respectively) were lower than the overall incidence in DBA patients (~50%). Patients with RPS26 mutations had a worse response rate of steroid therapy than RPS10 (47% vs. 87.5%), but preferred RBC transfusions (67% vs. 44%, p = 0.0253). Our findings add to the DBA pathogenic variant database and demonstrate the clinical presentations of the DBA patients with RPS10/RPS26 mutations. It shows that next-generation sequencing is a powerful tool for the diagnosis of genetic diseases such as DBA.

MicroRNA-1 targets ribosomal protein genes to regulate the growth, development and reproduction of Schistosoma japonicum

Eggs laid by mature female schistosomes are primarily responsible for the pathogenesis of schistosomiasis and critical for transmission. Consequently, elucidating the mechanism of sexual maturation as well as egg production may lead to new strategies for the control of schistosomiasis. MicroRNAs (miRNAs) are involved in multiple biological processes including reproduction in many organisms, yet their roles have not been well characterized in schistosomes. Here, we investigated microRNA-1 (miR-1), which was downregulated gradually in both male and female Schistosoma japonicum after they reached sexually maturity. The expression of miR-1, as shown with quantitative reverse transcription PCR (qRT-PCR), was lower in the reproductive organs of adult females compared with the somatic tissues. Overexpression of miR-1 in adult worms destroyed the morphological architecture of reproductive organs and reduced the subsequent oviposition, which may be due to the activation of apoptosis pathways. Through in silico analysis, 34 potential target genes of miR-1 were identified, including five ribosomal protein genes, called rp-s13, rp-l7ae, rp-l14, rp-l11 and rp-s24e. In vitro dual-luciferase reporter gene assays and miRNA overexpression experiments further validated that these ribosomal protein genes were directly regulated by miR-1. In contrast to the gene expression of miR-1, qRT-PCR and in situ hybridization experiments demonstrated these ribosomal protein genes were enriched in the sexual organs of adult females. Using RNA interference to silence the ribosomal protein genes in different developmental stages in a mouse model system, we demonstrated that these miR-1 target genes not only participated in the reproductive development of S. japonicum, but also were required for the growth and survival of the parasite in the early developmental stages. Taken together, our data suggested that miR-1 may affect the growth, reproduction and oviposition of S. japonicum by targeting the ribosomal protein genes, which provides insights for exploration of new anti-schistosome strategies.

Haploinsufficiency of the essential gene Rps12 causes defects in erythropoiesis and hematopoietic stem cell maintenance.

Ribosomal protein (Rp) gene haploinsufficiency can result in Diamond-Blackfan Anemia (DBA), characterized by defective erythropoiesis and skeletal defects. Some mouse Rp mutations recapitulate DBA phenotypes, although others lack erythropoietic or skeletal defects. We generated a conditional knockout mouse to partially delete Rps12. Homozygous Rps12 deletion resulted in embryonic lethality. Mice inheriting the Rps12KO/+ genotype had growth and morphological defects, pancytopenia, and impaired erythropoiesis. A striking reduction in hematopoietic stem cells (HSCs) and progenitors in the bone marrow (BM) was associated with decreased ability to repopulate the blood system after competitive and non-competitive BM transplantation. Rps12KO/+ lost HSC quiescence, experienced ERK and MTOR activation, and increased global translation in HSC and progenitors. Post-natal heterozygous deletion of Rps12 in hematopoietic cells using Tal1-Cre-ERT also resulted in pancytopenia with decreased HSC numbers. However, post-natal Cre-ERT induction led to reduced translation in HSCs and progenitors, suggesting that this is the most direct consequence of Rps12 haploinsufficiency in hematopoietic cells. Thus, RpS12 has a strong requirement in HSC function, in addition to erythropoiesis.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Cinnamomum camphora in China.

Camphor tree (Cinnamomum camphora) has a wide distribution in the world and is mainly distributed in the South and southwest in China. It can be used as both a wood and a medicine, with high value in industry, medicine, and ecology. In May 2022, it was observed that the approximately ten to fifteen years old Camphor trees were exhibiting witches'-broom, small leaf morphology and chlorosis, and leaf drop in Panzhihua City, Sichuan Province, China. The witches'-broom symptoms consisted of many small branches with little leaves at the top of branches. It was named C. camphora witches'-broom disease (CCWB) and was found in some areas of Miyi, Yanbian, Renhe Xiqu and Dongqu counties. More than 28% of the plants were infected on the five areas surveyed. Total 100 samples were collected from five areas, with 15 symptomatic plants and 5 asymptomatic plants each area. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The nested PCR was employed to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions OP662614; OP662615; OP662616). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et al. 2003) (GenBank accessions OP649592; OP649593; OP649594). The fragment from 25 symptomatic samples was consistent with the positive control, and asymptomatic plants were negative, confirming an association of a phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CCWB phytoplasma showed that it has a 99.44% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.59% identity with rapeseed phyllody phytoplasma (GenBank accession CP055264). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of CCWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CCWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the plants were positive for the phytoplasma using nested PCR after grafting for 30 d in nursery conditions. It is noteworthy that the plants were seriously damaged by aphid, Psyllidae and Ceroplastes. It is speculated that the insects of Homoptera typically transmit phytoplasmas by feeding on plant sap, thus it is necessary to control aphids in order to control the C. camphora witches'-broom disease. To the best of our knowledge, Camphor tree is a new host plant of 'Ca. Phytoplasma asteris' in China. The newly emerged disease is a threat to Camphor tree production.

The Product of the Fission Yeast fhl1 Gene Binds to the HomolE Box and Activates In Vitro Transcription of Ribosomal Protein Genes.

Fission yeast ribosomal protein genes (RPGs) contain a HomolD box as a core promoter element required for transcription. Some of the RPGs also contain a consensus sequence named HomolE, located upstream of the HomolD box. The HomolE box acts as an upstream activating sequence (UAS), and it is able to activate transcription in RPG promoters containing a HomolD box. In this work, we identified a HomolE-binding protein (HEBP) as a polypeptide of 100 kDa, which was able to bind to the HomolE box in a Southwestern blot assay. The features of this polypeptide were similar to the product of the fhl1 gene of fission yeast. The Fhl1 protein is the homolog of the FHL1 protein of budding yeast and possesses fork-head-associated (FHA) and fork-head (FH) domains. The product of the fhl1 gene was expressed and purified from bacteria, and it was demonstrated that is able to bind the HomolE box in an electrophoretic mobility assay (EMSA), as well as being able to activate in vitro transcription from an RPG gene promoter containing HomolE boxes upstream of the HomolD box. These results indicate that the product of the fhl1 gene of fission yeast can bind to the HomolE box, and it activates the transcription of RPGs.

Overexpressing Ribosomal Protein L16D Affects Leaf Development but Confers Pathogen Resistance in Arabidopsis.

In plant cells, multiple paralogs from ribosomal protein (RP) families are always synchronously expressed, which is likely contributing to ribosome heterogeneity or functional specialization. However, previous studies have shown that most RP mutants share common phenotypes. Consequently, it is difficult to distinguish whether the phenotypes of the mutants have resulted from the loss of specific genes or a global ribosome deficiency. Here, to investigate the role of a specific RP gene, we employed a gene overexpression strategy. We found that Arabidopsis lines overexpressing RPL16D (L16D-OEs) display short and curled rosette leaves. Microscopic observations reveal that both the cell size and cell arrangement are affected in L16D-OEs. The severity of the defect is positively correlated with RPL16D dosage. By combining transcriptomic and proteomic profiling, we found that overexpressing RPL16D decreases the expression of genes involved in plant growth, but increases the expression of genes involved in immune response. Overall, our results suggest that RPL16D is involved in the balance between plant growth and immune response.

Targeting of Calbindin 1 rescues erythropoiesis in a human model of Diamond Blackfan anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by congenital anomalies, cancer predisposition and a severe hypo-proliferative anemia. It was the first disease linked to ribosomal dysfunction and >70 % of patients have been identified to have a haploinsufficiency of a ribosomal protein (RP) gene, with RPS19 being the most common mutation. There is significant variability within the disease in terms of phenotype as well as response to therapy suggesting that other genes contribute to the pathophysiology and potential management of this disease. To explore these questions, we performed a genome-wide CRISPR screen in a cellular model of DBA and identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily, as a potential modifier of the disordered erythropoiesis in DBA. We used human derived CD34+ cells cultured in erythroid stimulating media with knockdown of RPS19 as a model for DBA to study the effects of CALB1. We found that knockdown of CALB1 in this DBA model promoted erythroid maturation. We also noted effects of CALB1 knockdown on cell cycle. Taken together, our results reveal CALB1 is a novel regulator of human erythropoiesis and has implications for using CALB1 as a novel therapeutic target in DBA.

Identification of ribosomal protein family as immune-cell-related biomarkers of NAFLD by bioinformatics and experimental analyses.

Immune cells play an integral role in the development and progression of non-alcoholic fatty liver disease (NAFLD). This study was to identify immune-cell-related biomarkers for the diagnosis and treatment of NAFLD. First, we introduced human liver transcriptome data from the GEO database (GSE48452 and GSE126848) and performed a weighted gene co-expression network analysis (WGCNA) to screen out the modules related to immune cell infiltration and to identify immune-cell-related differentially expressed genes (ICR-DEGs) associated with NAFLD progression. Further, the protein-protein interaction (PPI) network of ICR-DEGs was established to obtain hub genes and subsequently, the expression trend analysis was conducted to identify immune-cell-related biomarkers of NAFLD. Finally, the mRNA expression of biomarkers was validated in a NAFLD mouse model induced by high-fat diet (HFD) feeding. In total, we identified 66 ICR-DEGs and 13 hub genes associated with NAFLD. Among them, 9 hub genes (CD247, CD74, FCGR2B, IL2RB, INPP5D, MRPL16, RPL35, RPS3A, RPS8) were correlated with the infiltrating immune cells by the Pearson correlation analysis. Subsequently, 4 immune-cell-related biomarkers (RPL35, RPS3A, RPS8, and MRPL16) with the same expression trends in GSE48452 and GSE126848 datasets were identified. These biomarkers were enriched in immune-related pathways and had a good ability to distinguish between NASH and healthy samples. Moreover, we constructed a competing endogenous RNA (ceRNA) network of biomarkers and predicted twenty potential therapeutic drugs targeting RPS3A such as taxifolin and sitagliptin. Finally, experimental validation indicated that the hepatic mRNA expression of Rpl35, Rps3A, and Rps8 was significantly decreased in NAFLD mice. This study identified four ribosomal protein genes (RPL35, RPS3A, RPS8, and MRPL16) as immune-cell-related biomarkers of NAFLD, which may actively participate in the immune processes during NAFLD progression and could serve as potential targets for the diagnosis and treatment of NAFLD.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Transcriptome and physiological determination reveal the effects of selenite on the growth and selenium metabolism in mung bean sprouts

Selenium (Se) biofortification of crops has been studied to substantially improve the Se content in human dietary food intake. In the present study, Vigna radiata (mung bean) seeds were soaked in different concentrations of sodium selenite (Na2SeO3). Low concentration of selenite is conducive to seed germination and growth, and can increase the fresh weight (FW) and dry weight (DW) of sprouts. The concentration of Na2SeO3 lower than 50 mg/kg resulted in noticeable elongation in the stem and marginal elongation in root. Mung bean seeds soaked with 80 mg/kg Na2SeO3 accounted for 93.77% of organic Se after growing for about 5 days. Transcriptome data revealed that Se treatment enhances starch and sugar metabolism, along with the up-regulation of ribosomal protein and DNA synthesis related genes. Further analysis indicated that the mung bean seeds absorbed Na2SeO3 through PHT1.1 and NIP2. Se (IV) was transformed into Se (VI) and transported to stems, leaves and roots through cotyledons during the germination of bean sprouts. SULTR3;3 may play an important role in the transit process. Se (VI) or Se (IV) transported to the leaves was catalytically transformed into SeCys through SiR and CS, and SeCys is further converted to MeSeCys through SMT. Most SeCys were transformed into SeHCys through CBL, transported to plastids, and finally transformed into SeMet through Met Synthase.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Gene Losses and Homology of the Chloroplast Genomes of Taxillus and Phacellaria Species.

Research on the chloroplast genome of parasitic plants is limited. In particular, the homology between the chloroplast genomes of parasitic and hyperparasitic plants has not been reported yet. In this study, three chloroplast genomes of Taxillus (Taxillus chinensis, Taxillus delavayi, and Taxillus thibetensis) and one chloroplast genome of Phacellaria (Phacellaria rigidula) were sequenced and analyzed, among which T. chinensis is the host of P. rigidula. The chloroplast genomes of the four species were 119,941-138,492 bp in length. Compared with the chloroplast genome of the autotrophic plant Nicotiana tabacum, all of the ndh genes, three ribosomal protein genes, three tRNA genes and the infA gene were lost in the three Taxillus species. Meanwhile, in P. rigidula, the trnV-UAC gene and the ycf15 gene were lost, and only one ndh gene (ndhB) existed. The results of homology analysis showed that the homology between P. rigidula and its host T. chinensis was low, indicating that P. rigidula grows on its host T. chinensis but they do not share the chloroplast genome. In addition, horizontal gene transfer was not found between P. rigidula and its host T. chinensis. Several candidate highly variable regions in the chloroplast genomes of Taxillus and Phacellaria species were selected for species identification study. Phylogenetic analysis revealed that the species of Taxillus and Scurrula were closely related and supported that Scurrula and Taxillus should be treated as congeneric, while species in Phacellaria had a close relationship with that in Viscum.

Genome-wide gene expression responses to experimental manipulation of Saccharomyces cerevisiae repressor activator protein 1 (Rap1) expression level

Precise regulation of transcription in gene expression is critical for all aspects of normal organism form, fitness, and function and even minor alterations in the level, location, and timing of gene expression can result in phenotypic variation within and between species including evolutionary innovations and human disease states. Eukaryotic transcription is regulated by a complex interplay of multiple factors working both at a physical and molecular levels influencing this process. In Saccharomyces cerevisiae, the TF with the greatest number of putative regulatory targets is the essential gene Repressor Activator Protein 1 (RAP1). While much is known about the roles of Rap1 in gene regulation and numerous cellular processes, the response of Rap1 target genes to systematic titration of RAP1 expression level remains unknown. To fill this knowledge gap, we used a strain with a tetracycline-titratable promoter replacing wild-type regulatory sequences of RAP1 to systematically reduce the expression level of RAP1 and followed this with RNA sequencing (RNA-seq) to measure genome-wide gene expression responses. Previous research indicated that Rap1 plays a significant regulatory role in particular groups of genes including telomere-proximal genes, homothallic mating (HM) loci, glycolytic genes, DNA repair genes, and ribosomal protein genes; therefore, we focused our analyses on these groups and downstream targets to determine how they respond to reductions in RAP1 expression level. Overall, despite being known as both an activator and as a repressor of its target genes, we found that Rap1 acts as an activator for more target genes than as a repressor. Additionally, we found that Rap1 functions as an activator of ribosomal protein genes and a repressor for HM loci genes consistent with predictions from the literature. Unexpectedly, we found that Rap1 functions as a repressor of glycolytic enzyme genes contrary to prior reports of it having the opposite effect. We also compared the expression of RAP1 to five different genes related to DNA repair pathway and found that decreasing RAP1 downregulated four of those five genes. Finally, we found no effect of RAP1 depletion on telomere-proximal genes despite its functioning to silence telomeric repeat-containing RNAs. Together our results enrich our understanding of this important transcriptional regulator.

Integrative analysis of transcriptome and yeast screening system identified heat stress-responding genes in ryegrass

Heat stress threatens plant survival and productivity as global warming continues. Thus, it is urgent to understand heat tolerance genetically, especially in cool-season plants. Herein, ryegrass, a cool season grass, was subjected to two heat-temperature treatments (acquired and basal thermotolerances), followed by heat response gene identification using a transcriptome and yeast screening system. A total of 2730 and 8087 differentially expressed genes (DEGs) were identified by transcriptome in AT (acquired thermotolerance) and BT (basal thermotolerance) treatments, while 124 potential heat tolerance genes were identified by yeast screening. This study is the first to reveal the continuous changes in gene expression levels in ryegrass at the whole-genome level during the process of high temperature response. Our findings indicate that, in both the AT and BT experiments, there was a general inhibition of photosynthesis-related gene expression levels over time, whereas the expression levels of endoplasmic reticulum protein processing-related genes were primarily induced. The analysis of the yeast screening system indicated that the ryegrass ribosomal protein genes may play an essential role in high temperature response. Our study identified some genes and proteins related to heat tolerance in ryegrass and provided new and crucial clues to understand the resistance of ryegrass to heat stress.