Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo

Abstract

Ppmar1 and Ppmar2 are two active mariner -like elements (MLEs) cloned from moso bamboo ( Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1 , 2 and 3 along with the wild types ( NES-0 ) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity. Electronic supplementary material The online version of this article (10.1186/s13100-019-0179-y) contains supplementary material, which is available to authorized users.

The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation

“…Insufficiency of enzymes encoded by TEs may explain the insufficient quantity for the transposition process. For instance, transposition of Ppmar1 and Ppmar2 (Mariner-like elements (MLEs) isolated from Moso bamboo) is determined by the quantity of transposases present inside the nucleus [156,157]. This suggests that MLEs generally have the potential to develop a self-regulatory strategy that can control their amplification and copy numbers by minimization of transposases.…”

Genetics and genomics of moso bamboo (Phyllostachys edulis): Current status, future challenges, and biotechnological opportunities toward a sustainable bamboo industry

“…Additionally, they identified transposon variations that depict adaptive responses during the evolutionary process of moso bamboo. Subsequently, through functional analyses of transposons, Ramakrishnan, Zhou, Pan, Hänninen, Yrjälä, et al (2019), Ramakrishnan, Zhou, Pan, Hänninen, Tang, et al (2019) identified activities of mariner‐like elements ( MLE ) of the moso bamboo, drawing evolutionary connotations.…”

“…Ramakrishnan, Zhou, Pan, Hänninen, Yrjälä, et al (2019) were able to alter the transposition frequency of Ppmar2 by 1.5–2.0 times more than that of the wild‐type yeast by increasing the affinities of terminal inverted repeats (TIRs) toward the DNA‐binding domain of TPase. Further, the nuclear export signal (NES) domains of both Ppmar1 and Ppmar2 elements regulate the nuclear export of TPases that ultimately influences the transposition activity of MLEs (Ramakrishnan, Zhou, Pan, Hänninen, Tang, et al, 2019). Altogether, TEs are potent gene regulators in the moso bamboo genome that vastly alter genetic and epigenetic architecture.…”

Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast

“…Two full-length MLEs named Ppmar1 and Ppmar2 were previously identified from the genome of moso bamboo (Phyllostachys edulis) (Zhou et al, 2015). Ppmar1 and Ppmar2 were shown to be transposable in Arabidopsis thaliana and yeast (Zhou et al, 2016;Zhou et al, 2017;Ramakrishnan et al, 2019a;Ramakrishnan et al, 2019b). Site-directed mutation boosted the activity of the Ppmar1 mutant (S171A) by more than 10-fold (Zhou et al, 2017).…”

“…expression vector, pAG415gal-ccdB, as described by Ramakrishnan et al (2019a;2019b). Ppmar2 non-autonomous transposon (Ppmar2NA) was cloned by amplifying its 5' and 3' TIRs as well as the adjacent sequence, followed by an overlap PCR.…”