mRNA Translational Enhancers as a Tool for Plant Gene Engineering

Кочетов, А. В.; Филипенко, Е. А.; Nikulin, Pavel S.

doi:10.1007/978-4-431-55251-2_13

Cited by 2 publications

(2 citation statements)

References 57 publications

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“…The resulting fluorescence intensity was moderate compared with the transient expression in tobacco leaves. If a robust signal is desired, we suggest that split‐GFP complementation can be augmented by using a stronger driving promoter or including a translation enhancer in the inducible expression cassette (Kochetov et al ., ). For example, the SUPERR:sXVE expression cassette was shown to be fully inducible and stronger in Arabidopsis plants compared with the UBQ10:sXVE (Schlücking et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

et al. 2018

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Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta.

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Section: Discussionmentioning

confidence: 97%

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

et al. 2018

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“…Four synthetic 5′UTRs were generated (see Table 1 for a summary and Additional file 2 for sequences). These were designed based on the information available in the scientific literature about the properties of 5′UTRs associated with highly expressed genes and used as translational enhancers in transient expression systems [3, 4, 8, 13, 22]. Overall, desirable characteristics appeared to be relatively short sequences (about 60–70 bp) with low GC content, low secondary structure, repeats of an AAC motif, and a strong Kozak consensus sequence (UUAAAA immediately preceding the AUG start codon—the Kozak sequence elements downstream of the AUG are dependent on the protein coding sequence and therefore cannot be designed into a general-purpose expression system).…”

Section: Methodsmentioning

confidence: 99%

Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

2019

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Background: The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones. Results: In this work, we present the rational design of novel synthetic 5′ and 3′ untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5′UTRs behave as expression enhancers that outperform the HT 5′UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3′UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs. Conclusions: We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

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mRNA Translational Enhancers as a Tool for Plant Gene Engineering

Cited by 2 publications

References 57 publications

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

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