Antisense Orientation Research Articles

Syntenic lncRNA locus exhibits DNA regulatory functions with sequence evolution

Syntenic long non-coding RNAs (lncRNAs) often show limited sequence conservation across species, prompting concern in the field. This study delves into functional signatures of syntenic lncRNAs between humans and zebrafish. Syntenic lncRNAs are highly expressed in zebrafish, with ∼90 % located near protein-coding genes, either in sense or antisense orientation. During early zebrafish development and in human embryonic stem cells (H1-hESC), syntenic lncRNA loci are enriched with cis-regulatory repressor signatures, influencing the expression of development-associated genes. In later zebrafish developmental stages and specific human cell lines, these syntenic lncRNA loci function as enhancers or transcription start sites (TSS) for protein-coding genes. Analysis of transposable elements (TEs) in syntenic lncRNA sequences revealed intriguing patterns: human lncRNAs are enriched in simple repeat elements, while their zebrafish counterparts show enrichment in LTR elements. This sequence evolution likely arises from post-rearrangement mutations that enhance DNA elements or cis-regulatory functions. It may also contribute to vertebrate innovation by creating novel transcription factor binding sites within the locus. This study highlights the conserved functionality of syntenic lncRNA loci through DNA elements, emphasizing their conserved roles across species despite sequence divergence.

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Single-mode termination of phage transcriptions, disclosing bacterial adaptation for facilitated reinitiations.

Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation.

Antisense RNA regulates glutamine synthetase in a heterocyst-forming cyanobacterium.

Glutamine synthetase (GS) is a key enzyme involved in nitrogen assimilation and the maintenance of C/N balance, and it is strictly regulated in all bacteria. In cyanobacteria, GS expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, posttranslational regulation of GS is operated by protein-protein interaction with GS inactivating factors (IFs). In this study, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the gifA gene (encoding GS inactivating factor IF7) is transcribed downstream of the GS (glnA) gene, from the opposite strand, and the gifA mRNA extends into the glnA coding sequence in antisense orientation. Therefore, the dual RNA transcript that encodes gifA constitutes two functional regions: a 5' protein-coding region, encoding IF7, and a 3' untranslated region that acts as an antisense to glnA. By increasing the levels of such antisense RNA either in cis or in trans, we demonstrate that the amount of GS activity can be modulated by the presence of the antisense RNA. The tail-to-tail disposition of the glnA and gifA genes observed in many cyanobacterial strains from the Nostocales clade suggests the prevalence of such antisense RNA-mediated regulation of GS in this group of cyanobacteria.

Knockdown of orthotospovirus-derived silencing suppressor gene by plant-mediated RNAi approach induces viral resistance in tomato

RNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Tomato spotted wilt orthotospovirus (TSWV) species Orthotospovirus tomatomaculae, silencing suppressor (NSs) gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double-stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers (TSWV-NSs). Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT-NSs primer pairs to determine the relative gene expression of all constructs (TSWV-NSs both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941+sense construct, the expression of the NSs gene was noticeably reduced in the full clone (pFGC5941+sense + antisense) construct. For the validation of RNAi effectiveness, Nicotiana tabacum plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941+sense, pFGC5941 empty vector, and in wild-type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of NSs genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.

Targeting PknL as a novel therapeutic strategy against Mycobacterium tuberculosis

Abstract: INTRODUCTION: The bacterium “Mycobacterium tuberculosis (MTB)” causes tuberculosis, which is currently regarded as the most common cause of bacterial infection-related death globally. In addition to eukaryotes, MTB also encodes 11 “serine/threonine protein kinases (STPKs),” including PknA, B, C, D, E, F, G, H, J, K, and L that control cell division, cell development, metabolism, and dormancy. MATERIALS AND METHODS: The antisense and sense-orientated PknL have been cloned utilizing the shuttle mycobacterial vector-pAZI9018b, which is IPTG inducible. Utilizing the appropriate primers, the PknL (Rv2176) full-length gene from MTB has been amplified. The product which was amplified was then inoculated inside the vector at the “5’BamH1 and 3’Nde1” regions to produce PknL in the sense orientation (PknL-S) resembling PknL-S, and likewise, amplified substance was introduced into the vector at the 3’BamHI and 5’NdeI sites for producing PknL in the antisense orientation (PknL-AS). RESULTS: Regardless of the absence or presence of an inducer, PknL-AS had lower transcription levels than Wild type and PknL-S, but PknL-AS revealed a significant decrease in PknL expression during the end of 2nd week with 1 mM of IPTG and a further decrease on day 10 with 10 mM IPTG. In the presence of 10 mM IPTG, PknL expression has been close to, 1.8 log in Wild type, 1.4 log in PknL-AS, and 2.4 log in PknL-S. CONCLUSION: It has been clear that PknL may read environmental cues and respond appropriately, assisting bacteria to adapt to stressful situations in the host by lowering bacterial development, leading to a dormant state, and allowing bacteria to stay inside the host.

Molecular cloning, expression and cellular localization of two long noncoding RNAs (mLINC-RBE and mLINC-RSAS) in the mouse testis

Long noncoding RNAs (lncRNAs) are transcribed in complex, overlapping, sense- and antisense orientations from intronic and intergenic regions of mammalian genomes. Transcription of genome in mammalian testis is more widespread compared to other organs. LncRNAs are involved in gene expression, chromatin regulation, mRNA stability and translation of proteins during diverse cellular functions. We report molecular cloning of two novel lncRNAs (mLINC-RBE and mLINC-RSAS) and their expression by RT-PCR as well as cellular localization by RNA in-situ hybridization in the mouse testes. mLINC-RBE is an intergenic lncRNA from chromosome 4, with 16.96 % repeat sequences, expressed as a sense transcript with piRNA sequences and its expression is localized into primary spermatocytes. mLINC-RSAS is an intergenic lncRNA from chromosome 2, with 49.7 % repeat sequences, expressed as both sense- and antisense transcripts with miRNA sequences and its expression is localized into different cell types, such as Sertoli cells, primary spermatocytes and round spermatids. The lncRNAs also contain sequences for some short peptides (micropeptides). This suggests that these two repeat sequence containing, intergenic genomic sense- and antisense transcripts expressed as lncRNAs with piRNAs, miRNAs, and showing cell-type specific, differential expression may regulate important functions in mammalian testes. Such functions may be regulated by RNA structures, RNA processing and RNA-protein complexes.

Response of sugarcane plants with modified cytokinin homeostasis under water deficit conditions

Cytokinins (CK) are one of the main phytohormones involved in plant growth and development and play a role in plant adaptation to stress. In this study, the response to water deficit stress was investigated in sugarcane (Saccharum spp. hybrids) plants after exogenous application of CK and partial silencing of endogenous cytokinin oxidase/dehydrogenase (CKX) gene expression in transgenic plants. The CKX enzyme is responsible for CK breakdown in plant cells. A foliar drench of N6-benzyladenin on leaves (100 μM) resulted in plants that survived more than 10 days of water deficit stress compared with the untreated control plants, which displayed senescence and leaf wilting after 10 days without water. Furthermore, after 10 days without water, the treated plants maintained a high efficiency of the photosystem II (PSII) and displayed higher levels of chlorophyll and relative water content, and after 20 days without water, high catalase activity and proline content when compared with the untreated controls. In addition, sugarcane calli were transformed with a recombinant vector containing a 237 bp sugarcane ShCKX target sequence which included a conserved CK binding domain cloned in sense and antisense orientations. Partial silencing of CKX expression in the transgenic plants resulted in significantly lower salicylic acid levels after 21 days of deficit stress compared with untransformed controls. Stressed transgenic plants accumulated less H2O2, more antioxidants (superoxide dismutase and catalase) and proline when compared with the wild type (WT) plants. Moreover, the transgenic plants were able to retain high levels of chlorophyll and most maintained the efficiency of PSII after watering was stopped. The transgenic genotype with the highest level of CKX silencing displayed delayed leaf senescence and maintained high leaf water content under water deficit stress compared with the WT plants. Both the exogenous application of CK and the partial silencing of CKX expression enabled the plants to resprout after re-watering following the stress period. The results indicate that the manipulation of CK levels, either by increasing CK exogenously or deceasing their break down by partial silencing of the CKX gene, in sugarcane positively affects the response to water deficit stress through a variety of physiological mechanisms.

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species.

Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

A Mycovirus VIGS Vector Confers Hypovirulence to a Plant Pathogenic Fungus to Control Wheat FHB.

Mycovirus-mediated hypovirulence has the potential to control fungal diseases. However, the availability of hypovirulence-conferring mycoviruses for plant fungal disease control is limited as most fungal viruses are asymptomatic. In this study, the virus-induced gene silencing (VIGS) vector p26-D4 of Fusarium graminearum gemytripvirus 1 (FgGMTV1), a tripartite circular single-stranded DNA mycovirus, is successfully constructed to convert the causal fungus of cereal Fusarium head blight (FHB) into a hypovirulent strain. p26-D4, with an insert of a 75-150bp fragment of the target reporter transgene transcript in both sense and antisense orientations, efficiently triggered gene silencing in Fusarium graminearum. Notably, the two hypovirulent strains, p26-D4-Tri101, and p26-D4-FgPP1, obtained by silencing the virulence-related genes Tri101 and FgPP1 with p26-D4, can be used as biocontrol agents to protect wheat from a fungal disease FHB and mycotoxin contamination at the field level. This study not only describes the first mycovirus-derived VIGS system but also proves that the VIGS vector can be used to establish multiple hypovirulent strains to control pathogenic fungi.

Nitrogen-regulated antisense transcription in the adaptation to nitrogen deficiency in Nostoc sp. PCC 7120.

Transcriptomic analyses using high-throughput methods have revealed abundant antisense transcription in bacteria. Antisense transcription is often due to the overlap of mRNAs with long 5' or 3' regions that extend beyond the coding sequence. In addition, antisense RNAs that do not contain any coding sequence are also observed. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that, under nitrogen limitation, behaves as a multicellular organism with division of labor among two different cell types that depend on each other, the vegetative CO2-fixing cells and the nitrogen-fixing heterocysts. The differentiation of heterocysts depends on the global nitrogen regulator NtcA and requires the specific regulator HetR. To identify antisense RNAs potentially involved in heterocyst differentiation, we assembled the Nostoc transcriptome using RNA-seq analysis of cells subjected to nitrogen limitation (9 or 24 h after nitrogen removal) in combination with a genome-wide set of transcriptional start sites and a prediction of transcriptional terminators. Our analysis resulted in the definition of a transcriptional map that includes >4,000 transcripts, 65% of which contain regions in antisense orientation to other transcripts. In addition to overlapping mRNAs, we identified nitrogen-regulated noncoding antisense RNAs transcribed from NtcA- or HetR-dependent promoters. As an example of this last category, we further analyzed an antisense (as_gltA) of the gene-encoding citrate synthase and showed that transcription of as_gltA takes place specifically in heterocysts. Since the overexpression of as_gltA reduces citrate synthase activity, this antisense RNA could eventually contribute to the metabolic remodeling that occurs during the differentiation of vegetative cells into heterocysts.

Expanding the Repertoire of the Plant-Infecting Ophioviruses through Metatranscriptomics Data.

Ophioviruses (genus Ophiovirus, family Aspiviridae) are plant-infecting viruses with non-enveloped, filamentous, naked nucleocapsid virions. Members of the genus Ophiovirus have a segmented single-stranded negative-sense RNA genome (ca. 11.3-12.5 kb), encompassing three or four linear segments. In total, these segments encode four to seven proteins in the sense and antisense orientation, both in the viral and complementary strands. The genus Ophiovirus includes seven species with viruses infecting both monocots and dicots, mostly trees, shrubs and some ornamentals. From a genomic perspective, as of today, there are complete genomes available for only four species. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and molecular characterization of 33 novel viruses with genetic and evolutionary cues of ophioviruses. Genetic distance and evolutionary insights suggest that all the detected viruses could correspond to members of novel species, which expand the current diversity of ophioviruses ca. 4.5-fold. The detected viruses increase the tentative host range of ophioviruses for the first time to mosses, liverwort and ferns. In addition, the viruses were linked to several Asteraceae, Orchidaceae and Poaceae crops/ornamental plants. Phylogenetic analyses showed a novel clade of mosses, liverworts and fern ophioviruses, characterized by long branches, suggesting that there is still plenty of unsampled hidden diversity within the genus. This study represents a significant expansion of the genomics of ophioviruses, opening the door to future works on the molecular and evolutionary peculiarity of this virus genus.

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis in mosquitoes

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depend on key biogenesis factors, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identify transcriptional readthrough as a conserved mechanism for cluster-derived piRNA biogenesis in the vector mosquitoes Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between the two Aedes species suggest that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution, and piRNA function in these important vector species.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Redox homeostasis at SAM: a new role of HINT protein.

ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Development and application of a virus-induced gene silencing system for functional genomics in pecan (Carya illinoinensis)

Pecan is a high-value nut tree that cultivated globally. Nowadays, in-depth studies of this species are often hampered by lacking of efficient molecular genetic tools. To facilitate functional genomics in pecan, a virus-induced gene silencing (VIGS) system based on tobacco rattle virus (TRV) was explored in this study. We used pecan magnesium chelatase subunit H (CiChlH) gene as a visual indicator to evaluate the effect of several factors including infiltration method, insert size, insert orientation, inoculation concentration, host genotype, and culture temperature on VIGS. Our results showed that the optimized conditions for pecan VIGS system were as followed: construction of TRV-based vector harboring ∼300 bp target sequence in sense or antisense orientation, adjustment of inoculation solution to a final OD600 of 0.7, use of syringe infiltration method, inoculation of ‘Pawnee’ or ‘Shaoxing’ seedlings at 2∼4 true-leaf stage with obvious shoot apical growth, and subjecting plants to a temperature cycle of 24 °C (day, 12 h)/16 °C (night, 12 h). Using the optimized system, we obtained an expected photobleaching at 4 weeks post infiltration. To further verify the stability of the system, a pecan phenylalanine ammonialyase (CiPAL) gene was used for silencing and we detected a significant reduction in total phenolics in silenced plants with respect to control seedlings, as anticipated. Altogether, the TRV-based VIGS system has been successfully established in pecan and could be used for future reverse genetic studies.

Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase.

We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni-NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.

Mutated Pkhd1 alone is sufficient to cause autoimmune biliary disease on the nonobese diabetic (NOD) genetic background

We previously reported that nonobese diabetic (NOD) congenic mice (NOD.c3c4 mice) developed an autoimmune biliary disease (ABD) with similarities to human primary biliary cholangitis (PBC), including anti-mitochondrial antibodies and organ-specific biliary lymphocytic infiltrates. We narrowed the possible contributory regions in a novel NOD.Abd3 congenic mouse to a B10 congenic region on chromosome 1 (“Abd3”) and a mutated Pkhd1 gene (Pkhd1del36−67) upstream from Abd3, and we showed via backcrossing studies that the NOD genetic background was necessary for disease. Here, we show that NOD.Abd3 mice develop anti-PDC-E2 autoantibodies at high levels, and that placing the chromosome 1 interval onto a scid background eliminates disease, demonstrating the critical role of the adaptive immune system in pathogenesis. While the NOD genetic background is essential for disease, it was still unclear which of the two regions in the Abd3 locus were necessary and sufficient for disease. Here, using a classic recombinant breeding approach, we prove that the mutated Pkhd1del36−67 alone, on the NOD background, causes ABD. Further characterization of the mutant sequence demonstrated that the Pkhd1 gene is disrupted by an ETnII-beta retrotransposon inserted in intron 35 in an anti-sense orientation. Homozygous Pkhd1 mutations significantly affect viability, with the offspring skewed away from a Mendelian distribution towards NOD Pkhd1 homozygous or heterozygous genotypes. Cell-specific abnormalities, on a susceptible genetic background, can therefore induce an organ-specific autoimmunity directed to the affected cells. Future work will aim to characterize how mutant Pkhd1 can cause such an autoimmune response.

Silencing of multiple target genes via ingestion of dsRNA and PMRi affects development and survival in Helicoverpa armigera

RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA) is a powerful tool to knockdown genetic targets crucial for the growth and development of agriculturally important insect pests. Helicoverpa armigera is a pest feeding on more than 30 economically important crops worldwide and a major threat. Resistance to insecticides and Bt toxins has been gradually increasing in the field. RNAi-mediated knockdown of H. armigera genes by producing dsRNAs homologous to genetic targets in bacteria and plants has a high potential for insect management to decrease agricultural loss. The acetylcholinesterase (AChE), ecdysone receptor (EcR) and v-ATPase-A (vAA) genes were selected as genetic targets. Fragments comprising a coding sequence of < 500 bp were cloned into the L4440 vector for dsRNA production in bacteria and in a TRV-VIGS vector in antisense orientation for transient expression of dsRNA in Solanum tuberosum leaves. After ingesting bacterial-expressed dsRNA, the mRNA levels of the target genes were significantly reduced, leading to mortality and abnormal development in larva of H. armigera. Furthermore, the S. tuberosum plants transformed with TRV-VIGS expressing AChE exhibited higher mortality > 68% than the control plants 17%, recorded ten days post-feeding and significant resistance in transgenic (transient) plants was observed. Moreover, larval lethality and molting defects were observed in larva fed on potato plants expressing dsRNA specific to EcR. Analysis of transcript levels by quantitative RT–PCR revealed that larval mortality was attributable to the knockdown of genetic targets by RNAi. The results demonstrated that down-regulation of H. armigera genes involved in ATP hydrolysis, transcriptional stimulation of development genes and neural conduction has aptitude as a bioinsecticide to control H. armigera population sizes and therefore decreases crop loss.