A GoldenBraid-Compatible Virus-Based Vector System for Transient Expression of Heterologous Proteins in Plants.

We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This vector system consists of three domestication vectors carrying three GB parts-the cauliflower mosaic virus (CaMV) 35S promoter with PVX upstream of the second subgenomic promoter of the PVX coat protein (PVX CP SGP), nopaline synthase (NOS) terminator with PVX downstream of the first PVX CP SGP and the gene of interest (GOI). The full-length PVX clone carrying the sequence encoding a green fluorescent protein (GFP) as GOI was incorporated into the binary GB vector in a one-step reaction of three GB parts using the four-nucleotide GB standard syntax. We investigated whether the obtained vector named GFP/pGBX enables systemic PVX infection and expression of GFP in Nicotiana benthamiana plants. We show that this GB-compatible vector system can be used for simple and efficient assembly of PVX-based expression constructs and that it meets the current need for interchange of standard biological parts used in different expression systems.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors.

Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors.

KEY MESSAGE: This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

Efficient expression of Human papillomavirus 16 E7 oncoprotein fused to C-terminus of Tobacco mosaic virus (TMV) coat protein using molecular chaperones in Escherichia coli.

The Human papillomavirus 16 (HPV16) E7 oncoprotein is a promising candidate for development of anti-cancer therapeutic vaccine. We have prepared the expression construct carrying mutagenized E7 oncoprotein fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein via 15 amino acids ß-sheet linker. The fusion protein was expressed in Escherichia coli MC 1061 cells. We have obtained high level expression, but most of the protein remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular chaperones (TF, DnaK-DnaJ-GrpE, GroEL-GroES) were used. The immunological reactivity of expressed recombinant protein was evaluated with anti-E7 and anti-TMV antibodies. The distribution of expressed product during ultracentrifugation on sucrose gradient was studied.

Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants.

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108-120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2 108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2 108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2 108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2 108-120 epitope were found after both methods of vaccine delivery.

Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells.

The E7 oncoprotein from Human papillomavirus type 16 (HPV16) is an attractive candidate for anti-cancer therapeutical vaccine development. In this study, we engineered different fusions of mutagenized coding sequence of E7 oncoprotein (E7ggg) with coat protein of Potato virus X (PVX CP) both on 5'- and 3'-terminus of PVX CP and evaluated the influence of the length of linker (no linker, 4, 15aa) connecting PVX CP and E7ggg on their production. At first the expression in Escherichia coli was conducted to assess the characteristics of the recombinant protein prior to be further produced in plants, that is, resultant proteins were used for screening of their immunological reactivity with antibodies against PVX CP and E7. Fusion proteins successfully expressed in bacteria and plants were partially purified and their reactivity and ability to form virus-like particles were evaluated with anti-E7 antibodies.

Short communication: Molecular analysis of Potato leafroll virus isolates from the Czech Republic.

The complete genomes of three Czech isolates VIRUBRA 1/045, VIRUBRA 1/046, and VIRUBRA 1/047 of Potato leafroll virus (PLRV) were sequenced and compared with 13 complete sequences of PLRV isolates available in GenBank. Among the Czech isolates, VIRUBRA 1/046 and 1/047 showed the highest nucleotide (nt) identity (98.7%). PLRV was the most conserved virus in both open reading frames (ORFs) 3 and 4. The most variable regions were ORFs 0 and Rap1. Interestingly, isolate VIRUBRA 1/045 significantly differed from the other two Czech isolates in ORFs 0 and 1. Moreover, we identified mutations in the amino acid (aa) sequences, which were specific for the Czech isolates. Phylogenetic analysis based on ORF0 showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on sequence analysis of the genome sequences of PLRV isolates from the Czech Republic.

Transient expression of HPV16 E7 peptide (aa 44-60) and HPV16 L2 peptide (aa 108-120) on chimeric potyvirus-like particles using Potato virus X-based vector.

The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein.

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.