Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum.

BACKGROUND: The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine-Dalgarno (SD) sequence augmented by an upstream translational "enhancer" (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. METHODS AND RESULTS: Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call "CON_G") that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. CONCLUSIONS: The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing.

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1.

The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central ß-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies.

Infectious clone construction and pathogenicity confirmation of Cotton leaf curl Multan virus (CLCuMuV), Ramie mosaic virus (RamV) and Corchorus yellow vein Vietnam virus (CoYVV) by southern blot analysis.

Geminiviruses are insect-transmissible, economically vital group of plant viruses, which cause significant losses to crop production and ornamental plants across the world. During this study, infectious clones of three devastating begomoviruses, i.e., Cotton leaf curl Multan virus (CLCuMuV), Ramie mosaic virus (RamV) and Corchorus yellow vein Vietnam virus (CoYVV) were constructed by following novel protocol. All infectious clones were confirmed by cloning and sequencing. All of the infectious clones were agro-inoculated in Agrobacterium. After the agro-infiltrations, all clones were injected into Nicotiana benthamiana and jute plants under controlled condition. After 28 days of inoculation, plants exhibited typical symptoms of their corresponding viruses. All the symptomatic and asymptomatic leaves were collected from inoculated plants for further analysis. The southern blot analysis was used to confirm the infection of studied begomoviruses. At the end, all the products were sequenced and analyzed.

Liquid-Cell Electron Tomography of Biological Systems.

Liquid-cell electron microscopy is a rapidly growing field in the imaging domain. While real-time observations are readily available to analyze materials and biological systems, these measurementshave been limited to the two-dimensional (2-D) image plane. Here, we introduce an exciting technical advance to image materials in 3-D while enclosed in liquid. The development of liquid-cell electron tomography permitted us to observe and quantify host-pathogen interactions in solution while contained in the vacuum system of the electron microscope. In doing so, we demonstrate new insights for the rules of engagement involving a unique bacteriophage and its host bacterium. A deeper analysis of the genetic content of the phage pathogens revealed structural features of the infectious units while introducing a new paradigm for host interactions. Overall, we demonstrate a technological opportunity to elevate research efforts for in situ imaging while providing a new level of dimensionality beyond the current state of the field.

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis.

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.

More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1.

Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.IMPORTANCE Flagellotropic bacteriophages belong to the tailed-phage order Caudovirales, the most abundant phages in the virome. While it is known that these viruses adhere to the bacterial flagellum and use flagellar rotation to reach the cell surface, their infection mechanisms are poorly understood. Characterizing flagellotropic-phage-host interactions is crucial to understanding how microbial communities are shaped. Using a transposon mutagenesis approach combined with a screen for motile, phage-resistant mutants, we identified lipopolysaccharides as the secondary cell surface receptor for phage 7-7-1. This is the first cell surface receptor identified for flagellotropic phages. One hypothetical membrane protein was also recognized as essential for infection. These new findings, together with previous results, culminated in an infection model for phage 7-7-1.

Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

Agrobacterium-mediated inoculation of chrysanthemum (Chrysanthemum morifolium) plants with chrysanthemum stunt viroid.

Agroinfiltration was tested as a method of inoculation of chrysanthemum plants with chrysanthemum stunt viroid (CSVd). Binary vectors harboring dimeric CSVd sequences in sense and antisense orientations were constructed, and Agrobacterium transfected with these binary vectors was infiltrated into chrysanthemum leaves. Northern blotting and reverse transcription polymerase chain reaction analysis showed that local infection was established within 7 days and systemic infection within 20 days. CSVd polarities showed no difference in infectivity. This study showed that agroinfiltration of chrysanthemum plants is an easy, rapid, and cost-effective method for CSVd inoculation.

Virus-induced gene silencing of fiber-related genes in cotton.

Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.

Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.

Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.

Virus-induced gene silencing as a scalable tool to study drought tolerance in plants.

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper.

The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny), whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1) to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(.) To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.

Minimum requirements of flagellation and motility for infection of Agrobacterium sp. strain H13-3 by flagellotropic bacteriophage 7-7-1.

The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. strain H13-3 (formerly Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 flagellum is complex and consists of three flagellin proteins: the primary flagellin FlaA, which is essential for motility, and the secondary flagellins FlaB and FlaD, which have minor functions in motility. Using quantitative infectivity assays, we showed that absence of FlaD had no effect on phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA deletion strain, which produces straight and severely truncated flagella, experienced a significantly reduced infectivity, similar to that of a flaB flaD strain, which produces a low number of straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to flagellation, flagellar rotation is required for infection. A strain that is nonmotile due to an in-frame deletion in the gene encoding the motor component MotA is resistant to phage infection. We also generated two strains with point mutations in the motA gene resulting in replacement of the conserved charged residue Glu98, which is important for modulation of rotary speed. A change to the neutral Gln caused the flagellar motor to rotate at a constant high speed, allowing a 2.2-fold-enhanced infectivity. A change to the positively charged Lys caused a jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar rotation, are important determinants for infection by phage 7-7-1.

Genome and proteome analysis of 7-7-1, a flagellotropic phage infecting Agrobacterium sp H13-3.

BACKGROUND: The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. METHODS: The sequence of the 7-7-1 genome was determined by pyro(454)sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS). RESULTS: Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100) and large (orf112) subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7), putative tail fibre (orf102), portal (orf113), major capsid (orf115) and tail sheath (orf126) proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114).

Sesbania mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication.

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.