*4.6. Characteristics and Reasoning for Selection of a Verification Method(s)*

It should be emphasized that the selection of a method for assembling reporter constructs is of highest importance, since it is necessary to clone the target regulatory sequences with a reporter gene without the introduction of additional nucleotides, which may influence the translation modulation. The classical restriction–ligation cloning method does not allow generation of such constructs and requires several cloning stages. Several more economical and efficient technologies facilitating and accelerating the design of such constructs have been recently proposed. These technologies make it possible to produce seamless fusions of a "regulatory sequence–reporter gene". Most of them utilize the recombination between homologous sequences residing at the ends of the DNA fragments to be assembled [63]. For example, the Gateway®cloning system is based on the well-characterized site-specific recombinase system of the lambda phage for recombination of DNA segments. The DNA segments are flanked by the ATT recombination sites, which provide seamless assembly of almost any sequence [64]. However, this system also has certain limitations, namely, the need that the sequences overlap for at least 15 nucleotides at their ends. Correspondingly, the assembly of nonoverlapping DNA fragments requires additional terminal extensions or the use of bridge oligonucleotides [65]. Moreover, in our view, the approaches, such as Golden Gate system [66,67] or CPEC (circular polymerase extension cloning) strategy are more purposeful for designing the regulatory sequence–reporter gene constructs [68]. The Golden Gate method utilizes the IIS type restriction fragments to generate 4-nucleotide sticky ends, flanking each DNA portion, which then can be effectively ligated using T4 ligase. The assembly is performed in a single reaction and gives a seamless or nearly seamless target construct. This is determined by that the IIS type recognition sites are removed during ligation leaving only four nucleotide which positions may be determined by researcher [69,70].

The principle underlying the CPEC method utilizes the polymerase extension mechanism and one DNA polymerase for the in vitro assembly and cloning of sequences in any vector in a single-stage reaction. CPEC allows for an integrated, combinatorial, or multifragment assembly of sequences as well as for routine cloning procedures [68]. Thus, the Golden Gate and CPEC technologies have important advantages suggesting their utility in designing of the reporter constructs intended for studying and experimentally verifying the role of the regulatory regions in transcripts during translation.

As for the functional assessment of the constructs carrying a target regulatory sequence fused with a reporter gene, two basic experimental approaches are used: they utilized (i) a transient (temporary) and/or (ii) stable (constant) expression of reporter constructs in plants [4,71]. In the case of the transient expression of reporter constructs, transfection of the protoplasts derived from leaves of *N. tabacum* L. cv. BY2, lettuce, or *A. thaliana* are used as well as agroinfiltration of leaves (*N. benthamiana*, *N. excelsior*, *N. tabacum* var. xanth, or *A. thaliana*) or plant cell suspension culture (*N. tabacum* BY2 or *A. thaliana* T87) [4,71,72] (Supplement Table S1). In the current view, the transient expression of reporter constructs is regarded as a more efficient approach because of lower material and time expenditures. The protocols for generation of protoplasts and agroinfiltration have been elaborated for many model and non-model plants, widening the range of the used plant objects. However, there are some limitations in the application of transient expression associated with the variation of protoplasts in the transformation efficiency and with the delivery of constructs to plant cells in agroinfiltration [4,73].

A stable expression of reporter constructs in plants requires production of transgenic plants or transgenic plant cell suspension cultures. This makes it possible to solve increasingly more complex problems of translational control, such as translation regulation under different growth and stress conditions or long-term physiological effects of certain changes in a sequence that modulate translation. In particular, polysome fractionation of control and transgenic plants makes it possible to confirm that the transcript of a reporter gene controlled by a tested regulatory sequence is actually associated with the polysome fraction. Thus, it is possible to assess the translational status of the mRNA of a reporter gene fused with the tested regulatory sequence, including under the effect of stress factors [8].

When using the methods involving both stable and transient expressions, the choice of an adequate control is of a paramount importance to ascertain that the change in expression of the reporter protein is actually associated with the change in translation (rather than with transcription, protein stability, and so on) [4,51].

According to the available experimental data, the strategy of reporter systems is in demand for a wide range of studies into individual regulatory sequences or their nucleotide contexts. The use of this strategy gives the unique data on the functional role of target sequences in translation efficiency; however, this strategy is, as a rule, supplemented with other methods. The choice of a particular method depends on the regulatory sequence or its context to be studied be it a full-sized 5'UTR or its regulatory elements, such as RNA G-quadruplex, IRES, uORFs, and so on, which is in part summarized above and is comprehensively described in the corresponding publications.
