A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization.

To satisfy the need to develop highly sensitive methods for detecting the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and further enhance detection efficiency and capability, a new method was created for detecting SARS-CoV-2 of the open reading frames 1ab (ORF1ab) target gene by a electrochemiluminescence (ECL) biosensor based on dual-probe hybridization through the use of a detection model of "magnetic capture probes-targeted nucleic acids-Ru(bpy)32+ labeled signal probes". The detection model used magnetic particles coupled with a biotin-labeled complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab target gene as the magnetic capture probes and Ru(bpy)32+ labeled amino modified another complementary nucleic acid sequence as the signal probes, which combined the advantages of the highly specific dual-probe hybridization and highly sensitive ECL biosensor technology. In the range of 0.1 fM~10 µM, the method made possible rapid and sensitive detection of the ORF1ab gene of the SARS-CoV-2 within 30 min, and the limit of detection (LOD) was 0.1 fM. The method can also meet the analytical requirements for simulated samples such as saliva and urine with the definite advantages of a simple operation without nucleic acid amplification, high sensitivity, reasonable reproducibility, and anti-interference solid abilities, expounding a new way for efficient and sensitive detection of SARS-CoV-2.

Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants.

As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.

Target identification of hepatic fibrosis using Pien Tze Huang based on mRNA and lncRNA.

Hepatic fibrosis is a spontaneous wound-healing response triggered by chronic liver injury. Pien Tze Huang (PZH), a traditional Chinese herbal medicine, has been widely used to treat various hepatic diseases in Asia. We used a CCl4-induced mouse model to establish a PZH group of hepatic fibrosis mice treated with PZH and a control group of hepatic fibrosis mice without any treatment. We performed RNA-seq and mass spectrometry sequencing to investigate the mechanism of the PZH response in hepatic fibrosis and identified multiple differentially expressed transcripts (DETs) and proteins (DEPs) that may be drug targets of PZH. Liver functional indices, including serum albumin (ALB), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were significantly decreased in the PZH treatment group (P < 0.05) in the eighth week. Hematoxylin-eosin (HE), Masson and Sirius red staining demonstrated that PZH significantly inhibited infiltration of inflammatory cells and collagen deposition. A total of 928 transcripts and 138 proteins were differentially expressed in PZH-treated mice compared to the control group. Gene Ontology (GO) enrichment analysis suggested that PZH may alleviate liver injury and fibrosis by enhancing the immune process. Taken together, our results revealed that multiple DETs and DEPs may serve as drug targets of PZH in hepatic fibrosis patient in future clinical practice.

Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice.

Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in various crops to produce high-yielding F1 hybrid seeds. The gene open reading frame 352 (orf352) was reported to be an RT102-type CMS gene in rice (Oryza sativa), although the mechanism underlying its role in CMS is unknown. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs) to knockout orf352 from the mitochondrial genome in the CMS rice RT102A. We isolated 18 independent transformation events in RT102A that resulted in genome editing of orf352, including its complete removal from the mitochondrial genome in several plants. Sequence analysis around the mitoTALEN target sites revealed their induced double-strand breaks were repaired via homologous recombination. Near the 5'-target site, repair involved sequences identical to orf284, while repair of the 3'-target site yielded various new sequences that generated chimeric genes consisting of orf352 fragments. Plants with a chimeric mitochondrial gene encoding amino acids 179-352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a chimeric gene encoding amino acids 211-352 of ORF352 exhibited partial rescue of pollen viability and germination, although these plants failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179-210 from ORF352 may contribute to pollen abortion.

Mining of the water hyssop (Bacopa monnieri) transcriptome reveals genome sequences of two putative novel rhabdoviruses and a solendovirus.

The genomes of three putative novel viruses, tentatively named "Bacopa monnieri virus 1" (BmV1), "Bacopa monnieri virus 2" (BmV2), and "Bacopa monnieri virus 3" (BmV3) were identified in the transcriptome dataset of a medicinally important herb - water hyssop (Bacopa monnieri (L.) Wettst.). The BmV1 and BmV2 genomes resemble those of plant rhabdoviruses. The 13.3-kb-long BmV1 genome contains eight antisense ORFs in the order 3' l-N-P2'-P-P3-M-G-P6-L-t 5', with P2' ORF overlapping with P, while the 13.2-kb BmV2 genome contains six interspersed ORFs in the antisense orientation (3' l-N-P-P3-M-G-L-t 5'). The 8-kb BmV3 genome possesses five overlapping ORFs, with ORFs 2 to 5 being similar to those of solendoviruses. Based on genome organization, sequence similarity, and phylogeny, BmV1, BmV2, and BmV3 can be regarded as new members of the genera Cytorhabdovirus, Betanucleorhabdovirus, and Solendovirus, respectively.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

Complete genome sequence of a novel foveavirus isolated from Allium sativum L. in China.

The complete genome sequence of a novel foveavirus identified in garlic (Allium sativum L.) in China was determined using RNA-seq, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The entire genomic RNA (GenBank accession MT981417) is 8748 nucleotides long excluding the 3'-terminal poly(A) tail and contains five open reading frames (ORFs). These ORFs encode the viral replicase, a triple gene block, and a coat protein. The virus was tentatively named "garlic yellow stripe associated virus" (GarYSaV). Pairwise comparisons of protein sequences show that GarYSaV encodes proteins that share less than 47% identity with those of other foveaviruses, suggesting that it represents a new species in the genus. Phylogenetic analysis of amino acid sequences of the replicase and CP confirm that GarYSaV is a member of the genus Foveavirus. To our knowledge, this is the first report of a foveavirus in a monocot plant.

Identification of ancient viruses from metagenomic data of the Jomon people.

Ancient DNA studies provide genomic information about the origins, population structures, and physical characteristics of ancient humans that cannot be solely examined by archeological studies. The DNAs extracted from ancient human bones, teeth, or tissues are often contaminated with coexisting bacterial and viral genomes that contain DNA from ancient microbes infecting those of ancient humans. Information on ancient viral genomes is useful in making inferences about the viral evolution. Here, we have utilized metagenomic sequencing data from the dental pulp of five Jomon individuals, who lived on the Japanese archipelago more than 3000 years ago; this is to detect ancient viral genomes. We conducted de novo assembly of the non-human reads where we have obtained 277,387 contigs that were longer than 1000 bp. These contigs were subjected to homology searches against a collection of modern viral genome sequences. We were able to detect eleven putative ancient viral genomes. Among them, we reconstructed the complete sequence of the Siphovirus contig89 (CT89) viral genome. The Jomon CT89-like sequence was determined to contain 59 open reading frames, among which five genes known to encode phage proteins were under strong purifying selection. The host of CT89 was predicted to be Schaalia meyeri, a bacterium residing in the human oral cavity. Finally, the CT89 phylogenetic tree showed two clusters, from both of which the Jomon sequence was separated. Our results suggest that metagenomic information from the dental pulp of the Jomon people is essential in retrieving ancient viral genomes used to examine their evolution.

Molecular characterization and antiparasitic activity analysis of a novel piscidin 5-like type 4 from Larimichthys crocea.

Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture teleosts and cause high lethality and economic loss, especially Larimichthys crocea. Current methods of controlling or preventing this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. The antiparasitic activity of some antimicrobial peptides (AMPs) attracted extensive attention of scholars. In the study, a novel piscidin 5-like type 4 (termed Lc-P5L4) excavated from comparative transcriptome of C. irritans - immuned L. crocea was identified and characterized. Sequence analysis shows the full-length cDNA of Lc-P5L4 is 539 bp containing an open reading frame (ORF) of 198 bp which encodes a peptide of 65 amino acid residues. The genome consists of three exons and two introns which exist in its ORF, and all the exon-intron boundaries are in accordance with classical GT-AG rule (GT/intron/AG). Multiple alignments indicate the signal peptides share highly conserved identity, while mature peptides are more diverse. Phylogenetic analysis displays Lc-P5L4 clusters together with other members of piscidin 5-like family. Next, quantitative Real-time PCR (qRT-PCR) detection found C. irritans infection could upregulate Lc-P5L4 expression level in all tested tissues significantly, it appeared earliest upregulation in the theronts infection stage in the head kidney; the expression contents reached to maximum level in the intestine, gill and muscle during trophonts falling off stage; while it was just upregulated during secondary bacterial infection stage in the liver and spleen. The data showed Lc-P5L4 upregulation time points were in accordance with different infection stages. With recombinant Lc-P5L4 (rLc-P5L4) obtained through Escherichia coli system, in vitro assay showed rLc-P5L4 could cause cilia deactivation, cell bodiesclumping and sticking to each other, then cell membrane rupture and contents leakage. The data illustrated Lc-P5L4 played critical roles in the immune defense against C. irritans infection, and provided another proof that piscidins exhibit multiple anti- C. irritans features.

In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander.

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5'UTR and 3'UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg-1 of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and Vmax of 0.078 ± 0.008 mM and 1052.3 ± 33.59 units mg-1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4-9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and ß-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

Molecular cloning and oxidative-stress responses of a novel manganese superoxide dismutase (MnSOD) gene in the dinoflagellate Prorocentrum minimum.

Dinoflagellate algae are microeukaryotes that have distinct genomes and gene regulation systems, making them an interesting model for studying protist evolution and genomics. In the present study, we discovered a novel manganese superoxide dismutase (PmMnSOD) gene from the marine dinoflagellate Prorocentrum minimum, examined its molecular characteristics, and evaluated its transcriptional responses to the oxidative stress-inducing contaminants, CuSO4 and NaOCl. Its cDNA was 1238 bp and contained a dinoflagellate spliced leader sequence, a 906 bp open reading frame (301 amino acids), and a poly (A) tail. The gene was coded on the nuclear genome with one 174 bp intron; signal peptide analysis showed that it might be localized to the mitochondria. Real-time PCR analysis revealed an increase in gene expression of MnSOD and SOD activity when P. minimum cells were separately exposed to CuSO4 and NaOCl. In addition, both contaminants considerably decreased chlorophyll autofluorescence, and increased intracellular reactive oxygen species. These results suggest that dinoflagellate MnSOD may be involved in protecting cells against oxidative damage.

Transcriptome Analysis Reveals the Accumulation Mechanism of Anthocyanins in Buckwheat (Fagopyrum esculentum Moench) Cotyledons and Flowers.

Buckwheat (Fagopyrum esculentum) is a valuable crop which can produce multiple human beneficial secondary metabolites, for example, the anthocyanins in sprouts and flowers. However, as the predominant group of visible polyphenols in pigmentation, little is known about the molecular mechanisms underlying the anthocyanin biosynthesis within buckwheat. In this study, a comparative transcriptome analysis of green and red common buckwheat cultivars was carried out through RNA sequencing. Overall, 3727 and 5323 differently expressed genes (DEGs) were identified in flowers and cotyledons, respectively. Through GO and KEGG analysis, we revealed that DEGs in flowers and cotyledons are predominately involved in biosynthesis of anthocyanin. A total of 42 unigenes encoding 11 structural enzymes of the anthocyanin biosynthesis were identified as DEGs. We also identified some transcription factor families involved in the regulation of anthocyanin biosynthesis. Real-time qPCR validation of candidate genes was performed in flowers and cotyledons, and the results suggested that the high expression level of structural genes involved in anthocyanin biosynthetic pathway promotes anthocyanin accumulation. Our results provide the insight understanding for coloration of red common buckwheat.

Characterization of the transcriptional regulator CsbHLH62 that negatively regulates EGCG3"Me biosynthesis in Camellia sinensis.

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) in tea (Camellia sinensis (L.) O. Kuntze) is a major source of O-methylated catechin and renowned for a wide range of health effects. However, the transcriptional regulation mechanisms of EGCG3"Me biosynthesis remain unclear. In the present work, the basic Helix-Loop-Helix (bHLH) transcription factor, designated as CsbHLH62, belonging to GBOF group of bHLH families, was isolated and characterized from Camellia sinensis. CsbHLH62 contains an Open Reading Frame of 1662 bp and encodes a polypeptide of 553 amino acids. Subcellular location and transcriptional activity analysis showed it as a nucleus protein and possessed transcriptional inhibition activity. Furthermore, the expression of CsbHLH62 was decreased during EGCG3"Me accumulation. More importantly, E-box motifs (5'-CANNTG-3') were found in the promoters of CCoAOMT, CsLAR, and CsDFR, and further transient expression assays showed that CsbHLH62 repressed the transcription of CCoAOMT, CsLAR, and CsDFR. Collectively, these results suggest that CsbHLH62 acts as a transcriptional repressor that might be negatively affecting the accumulation of EGCG3"Me. These findings provide novel insights into the regulatory mechanism of EGCG3"Me biosynthesis, which might help to breed high EGCG3"Me-content tea plants.

De Novo assembly, characterization and development of EST-SSRs from Bletilla striata transcriptomes profiled throughout the whole growing period.

Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding.

Complete chloroplast genome sequence of Fagopyrum dibotrys: genome features, comparative analysis and phylogenetic relationships.

Fagopyrum dibotrys, belongs to Polygonaceae family, is one of national key conserved wild plants of China with important medicinal and economic values. Here, the complete chloroplast (cp) genome sequence of F. dibotrys is reported. The cp genome size is 159,919 bp with a typical quadripartite structure and consisting of a pair of inverted repeat regions (30,738 bp) separated by large single copy region (85,134 bp) and small single copy region (13,309 bp). Sequencing analyses indicated that the cp genome encodes 131 genes, including 80 protein-coding genes, 28 tRNA genes and 4 rRNA genes. The genome structure, gene order and codon usage are typical of angiosperm cp genomes. We also identified 48 simple sequence repeats (SSR) loci, fewer of them are distributed in the protein-coding sequences compared to the noncoding regions. Comparison of F. dibotrys cp genome to other Polygonaceae cp genomes indicated the inverted repeats (IRs) and coding regions were more conserved than single copy and noncoding regions, and several variation hotspots were detected. Coding gene sequence divergence analyses indicated that five genes (ndhK, petL rpoC2, ycf1, ycf2) were subject to positive selection. Phylogenetic analysis among 42 species based on cp genomes and 50 protein-coding genes indicated a close relationship between F. dibotrys and F. tataricum. In summary, the complete cp genome sequence of F. dibotrys reported in this study will provide useful plastid genomic resources for population genetics and pave the way for resolving phylogenetic relationships of order Caryophyllales.

Characterization of genes specific to sua-CMS in Nicotiana tabacum.

KEY MESSAGE: Six unique ORFs were characterized in tobacco plants with sua-CMS sterile cytoplasm, identifying the mtDNA basis for pollen sterility. sua-CMS (cytoplasmic male sterility), the most widely used sterile system in tobacco hybrids, is the only CMS type identified as having no negative effects on agronomic or quality traits in tobacco (Nicotiana tabacum) and as being fully male sterile. CMS is often associated with alterations of mitochondrial DNA (mtDNA), including novel chimeric open reading frames (ORFs), which result from rearrangement and recombination. Here, we obtained 34 mitochondrial ORFs in the sua-CMS line msZhongyan100 (sZY) by BLAST analysis. When we amplified these mitochondrial ORFs in seven tobacco CMS lines including sua-, glu-, rep-, rus-, tab1-, tab2-, and tab3-CMS types and in fertile tobacco, we found that six ORFs-orf82, orf103, orf115a, orf91, orf115b, and orf100-were located in three small regions (m-sr) of the mitochondrial genome of sZY and were unique to the sua-CMS line. We further amplified the m-sr fragments in three different backcross populations of the seven types of CMS, three F1 hybrids with sua-CMS sterile cytoplasm, two sua-CMS lines, and 284 fertile tobacco accessions. The ORFs were specific to plants with the sua-CMS background. All six unique ORFs were chimeric and had no homology with the mitochondrial genomes of fertile tobacco. Transcript analysis revealed that the ORFs were highly expressed in the anthers and floral buds of sZY. These six ORFs were specific to sua-CMS and could be used as molecular markers to identify sua-CMS lines, which is useful for improving breeding for heterosis in tobacco.

A selfish genetic element confers non-Mendelian inheritance in rice.

Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2 Furthermore, this toxin-antidote system may have promoted the differentiation and/or maintained the genome stability of wild and cultivated rice.

Comparative chloroplast genomics between the invasive weed Mikania micrantha and its indigenous congener Mikania cordata: Structure variation, identification of highly divergent regions, divergence time estimation, and phylogenetic analysis.

Mikania micrantha and Mikania cordata are the only two species in genus Mikania (Asteraceae) in China. They share very similar morphological and life-history characteristics but occupy quite different habitats. Most importantly, they generate totally different ecological consequences. While M. micrantha has become an exotic invasive weed, M. cordata exists as an indigenous species with no harmful effects on native plants or habitats. As a continuous study of our previously reported M. micrantha chloroplast (cp) genome, in this study we have further sequenced the M. cordata cp genome to (1) conduct a comparative genome analysis to gain insights into the mechanism of invasiveness; (2) develop cp markers to examine the population genetic adaptation of M. micrantha; and (3) screen variable genome regions of phylogenetic utility. The M. cordata chloroplast genome is 151,984 bp in length and displays a typical quadripartite structure. The number and distribution of protein coding genes, tRNA genes, and rRNA genes of M. cordata are identical to those of M. micrantha. The main difference lays in that the pseudogenization of ndhF and a 118-bp palindromic repeat only arises in M. cordata. Fourteen highly divergent regions, 235 base substitutions, and 58 indels were identified between the two cp genomes. Phylogenetic inferences revealed a sister relationship between M. micrantha and M. cordata whose divergence was estimated to occur around 1.78 million years ago (MYA). Twelve cpSSR loci were detected to be polymorphic and adopted to survey the genetic adaptation of M. micrantha populations. No cpSSR loci were found to undergo selection. Our results build a foundation to examine the invasive mechanism of Mikania weed.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.