Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte
The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.
KeywordsPhyscomitrella patens Moss Forward genetic screen Tnt1 retrotransposon LTR Initiator methionyl-tRNA Gametophyte development
Critical GC value
- P. patens
Thermal asymmetric interlaced PCR
We sincerely thank Prof. Pascal Ratet (CNRS, France) for providing us tobacco Tnt1 retrotransposon (pCAMBIA-1391Xc-Tnt1) construct. BM acknowledges fellowship support from CSIR, New Delhi. All authors thank IISER Pune and DST Govt. of India for core funding support for this investigation. We are thankful to Prof. Meenu Kapoor for her timely help in moss establishment at IISER Pune. Thanks to Mr. Nitish Lahigude for his support in moss maintenance and Ms. Kavya Mohan for her technical help in cryopreservation of moss mutants. We also thank Prof. David Hannapel, Iowa State University for critical reading of our manuscript.
BM and AKB have conceived and designed the experiments. BM, VR, SJ, and AB have carried out all experiments. BM and AKB have written the manuscript.
The present study was supported by a grant (Grant No. EMR/2016/004852) from Department of Science and Technology (DST), Government of India to AKB. Core funding and infrastructure was provided by Indian Institute of Science Education and Research (IISER) Pune, India.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Doyle JJ (1990) Isolation of plant DNA from fresh tissue. Focus (Madison) 12:13–15Google Scholar
- Feuerbach F, Drouaud J, Lucas H (1997) Retrovirus-like end processing of the tobacco Tnt1 retrotransposon linear intermediates of replication. J Virol 71:4005–4015Google Scholar
- Hori K, Maruyama F, Fujisawa T et al (2014) Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation. Nat Commun 5Google Scholar
- Kartha KK, Engelmann F (1994) Cryopreservation and germplasm storage. In: Vasil IK, Thorpe TA (eds) Plant cell and tissue culture. Springer, Dordrecht, pp 195–230Google Scholar
- Sambrook J, Fritsch EF, Maniatis T et al (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Schaefer DG, Zrÿd J-P (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206. https://doi.org/10.1046/j.1365-313X.1997.11061195.x CrossRefGoogle Scholar
- Stevenson SR, Kamisugi Y, Trinh CH et al (2016) Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE (ANR), a regulator of ABA responses unique to basal land plants and required for desiccation tolerance. Plant Cell 28:1310–1327Google Scholar