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Summary Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Summary Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system‐wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant‐based expression system that utilizes viral‐based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%. A transgenic N. benthamiana line (Sm5) engineered to facilitate cell‐to‐cell movement enhances the local and systemic spread of a fluorescently tagged TMV vector.

Summary Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Baculoviruses are insect-specific pathogens widely used in biotechnology. In particular, the Autographa californica nucleopolyhedrovirus (AcMNPV) has been exploited as a platform for bio-inputs production. This is why the improvement of the technologies used for the production of recombinant baculoviruses takes on particular relevance. To achieve this goal, we developed a highly versatile baculoviral transfer vector generation system called PluriBAC. The PluriBAC system consists of three insert entry levels using Golden Gate assembly technology. The wide availability of vectors and sticky ends allows enough versatility to combine more than four different promoters, genes of interest, and terminator sequences. Here, we report not only the rational design of the PluriBAC system but also its use for the generation of baculoviral reporter vectors applied to different fields of biotechnology. We demonstrated that recombinant AcMNPV baculoviruses generated with the PluriBAC system were capable of infecting Spodoptera frugiperda larvae. On the other hand, we found that the recombinant budded virions (BV) generated using our system were capable of transducing different types of tumor and normal cells both in vitro and in vivo. Our findings suggest that the PluriBAC system could constitute a versatile tool for the generation of insecticide and gene therapy vectors.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative‐strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize‐infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full‐length infectious clone was established using agrobacterium‐mediated delivery of full‐length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA‐directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST‐LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV‐GFP. Complementary DNA (cDNA) clones of MMV‐wild type (WT) and MMV‐GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV‐WT and MMV‐GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross‐kingdom studies of virus replication in plant and insect hosts. The maize mosaic virus infectious clone enables stable and robust expression of heterologous genes in maize and planthoppers, and is a versatile tool for cross‐kingdom studies of virus replication in plant and insect hosts.

Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) infect rice, causing bacterial blight and bacterial leaf streak, respectively, which are two economically important bacterial diseases in paddy fields. The interactions of Xoo and Xoc with rice can be used as models for studying fundamental aspects of bacterial pathogenesis and host tissue specificity. However, an improved vector system for gene expression analysis is desired for Xoo and Xoc because some broad host range vectors that can replicate stably in X. oryzae pathovars are low‐copy number plasmids. To overcome this limitation, we developed a modular plasmid assembly system to transfer the functional DNA modules from the entry vectors into the pHM1‐derived backbone vectors on a high‐copy number basis. We demonstrated the feasibility of our vector system for protein detection, and quantification of virulence gene expression under laboratory conditions and in association with host rice and nonhost tobacco cells. This system also allows execution of a mutant complementation equivalent to the single‐copy chromosomal integration system and tracing of pathogens in rice leaf. Based on this assembly system, we constructed a series of protein expression and promoter‐probe vectors suitable for classical double restriction enzyme cloning. These vector systems enable cloning of all genes or promoters of interest from Xoo and Xoc strains. Our modular assembly system represents a versatile and highly efficient toolkit for gene expression analysis that will accelerate studies on interactions of X. oryzae with rice. We developed a modular plasmid assembly system to transfer DNA modules from entry vectors into high‐copy number pHM1‐derived vectors and use it for protein detection, mutant complementation, and tracing pathogens in leaves.

Virus-based gene expression is a simple and powerful approach for functional genetic studies in plants. Here, sugarcane mosaic virus (SCMV, Potyvirus sacchari) was engineered as a dual gene expression vector for simultaneous expression of two heterologous proteins in maize plants. Inoculation of the full-length cDNA clone of SCMV from agro-infiltrated Nicotiana benthamiana resulted in a rapid systemic infection in maize. To assess the possibility of SCMV as the gene expression vector, the marker gene GFP or GUS was inserted into either the NIb/CP or P1/HC-Pro junction site of SCMV to produce single-gene expression vectors. The results showed that these engineered SCMV vectors permitted efficient gene expression in systemically infected leaves and had the genetic capacity of inserts of more than 1800 bp, suggesting that both junction sites are suitable for heterologous gene insertion and expression. Furthermore, two different genes GFP and mCherry could be expressed simultaneously by engineering them into either NIb/CP or P1/HC-Pro junction sites of the same vector. These results clearly demonstrate the suitability of SCMV as a transient dual gene expression vector for maize plants.

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The β-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.

Corynebacterium glutamicum has been safely used in white biotechnology for the last 60 years and the portfolio of new pathways and products is increasing rapidly. Hence, expression vectors play a central role in discovering endogenous gene functions and in establishing heterologous gene expression. In this work, new expression vectors were designed based on two strategies: (i) a library screening of constitutive native and synthetic promoters and (ii) an increase of the plasmid copy number. Both strategies were combined and resulted in a very strong expression and overproduction of the fluorescence protein GfpUV. As a second test case, the improved vector for constitutive expression was used to overexpress the endogenous xylulokinase gene xylB in a synthetic operon with xylose isomerase gene xylA from Xanthomonas campestris. The xylose isomerase activity in crude extracts was increased by about three-fold as compared to that of the parental vector. In terms of application, the improved vector for constitutive xylA and xylB expression was used for production of the N-methylated amino acid sarcosine from monomethylamine, acetate, and xylose. As a consequence, the volumetric productivity of sarcosine production was 50% higher as compared to that of the strain carrying the parental vector.

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.