...due to the unique function and disease resistance mechanism of the xa13, this gene is increasingly favoured in practical breeding (Hajira et al., ). ...we evaluated editing efficiency through the ...website (http://crispr.hzau.edu.cn/cgi-bin/CRISPR/CRISPR) and designed two gRNAs targeting the promoter of the xa13 gene (Figure a,c). ...the deletion of this promoter sequence would cause the Xa13 gene to lose its ability to be induced by bacterial blight, thus making the rice lacking this fragment resistant to bacterial blight (Figure e,f). Since the Xa13 gene plays a key role in pollen development, only a partial sequence of the Xa13 gene promoter was edited to remove its ability to induce expression without affecting gene expression and function in the anthers (Figure g). ...this deletion strategy can improve rice disease resistance without affecting rice fertility.
Summary
Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles ...against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T2 generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T3 lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.
Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) ...genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a
papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.
Summary
Genetic transformation is a powerful means for the improvement of crop plants, but requires labor‐ and resource‐intensive methods. An efficient method for identifying single‐copy transgene ...insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time‐consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one‐ and two‐copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR‐based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence‐specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.
Significance Statement
An accurate and efficient method is presented for determining the transgene copy number in rice, citrus, potato, tomato, maize and wheat using droplet digital PCR. This technology simplifies and facilitates the quick and accurate measurement of transgene copy number for a large number of samples and is a significant improvement over existing methods.
SUMMARY
In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified ...87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co‐expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co‐expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)‐α‐bisabolene production of 7.95 mg L−1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high‐level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co‐expression from the algal nuclear genome.
Significance Statement
The designed expression concepts enable reliable and robust transgene expression from the nuclear genome of Chlamydomonas reinhardtii and will promote the use of this microalga as a green cell factory. This work is highly valuable to the greater synthetic biology research community and may have implications for other green microalgae as well.
Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The ...external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (
NPTII
) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic
Arabidopsis thaliana
plants. External application of the synthetic
NPTII
-encoding siRNAs down-regulated
NPTII
transcript levels in transgenic
A. thaliana
1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3′ ends. We also analyzed the effects of external
NPTII
-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the
NPTII
-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect.
Key Points•
Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis.
•
A more consistent effect was observed for methylated siRNAs.
•
The findings are important for development of plant gene regulation approaches.
Highly efficient and cost-effective transformation technologies are essential for studying gene function in the major cereal crops, wheat and barley. Demand for efficient transformation systems to ...allow over-expression, or RNAi-mediated silencing of target genes, is greatly increasing. This is due to technology advances, such as rapid genome sequencing, enhancing the rate of gene discovery and thus leading to a large number of genes requiring functional analysis through transformation pipelines. Barley can be transformed at very high efficiency but the methods are genotype-dependent. Wheat is more difficult to transform, however, recent advances are also allowing the development of high-throughput transformation systems in wheat. For many gene function studies, barley can be used as a model for wheat due to its highly efficient transformation rates and smaller, less complex genome. An ideal transformation system needs to be extremely efficient, simple to perform, inexpensive, genotype-independent, and give the required expression of the transgene. Considerable progress has been made in enhancing transformation efficiencies, controlling transgene expression and in understanding and manipulating transgene insertion. However, a number of challenges still remain, one of the key ones being the development of genotype-independent transformation systems for wheat and barley.
The evolutionary significance of introgression has been discussed for decades. Questions about potential impacts of transgene flow into wild and weedy populations brought renewed attention to the ...introgression of crop alíeles into those populations. In the past two decades, the field has advanced with considerable descriptive, experimental, and theoretical activity on the dynamics of crop gene introgression and its consequences. As illustrated by five case studies employing an array of different approaches, introgression of crop alleles has occurred for a wide array of species, sometimes without significant consequence, but on occasion leading to the evolution of increased weediness. A new theoretical context has emerged for analyzing empirical data, identifying factors that influence introgression, and predicting introgression's progress. With emerging molecular techniques and analyses, research on crop alíele introgression into wild and weedy populations is positioned to make contributions to both transgene risk assessment and reticulate evolution.
RNA thermometers offer straightforward, protein-independent methods to regulate gene expression at the post-transcriptional level. In this context, Chung and colleagues have discovered a ...revolutionary RNA thermometer in the chloroplast genome of Chlamydomonas reinhardtii. This will facilitate temperature-driven control of inducible transgene expression for biotechnology applications in plant and algal systems.
RNA thermometers offer straightforward, protein-independent methods to regulate gene expression at the post-transcriptional level. In this context, Chung and colleagues have discovered a revolutionary RNA thermometer in the chloroplast genome of Chlamydomonas reinhardtii. This will facilitate temperature-driven control of inducible transgene expression for biotechnology applications in plant and algal systems.
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