ARS | Publication request: Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 10, 2011
Publication Date: February 17, 2012
Citation: Velten, J.P., Cakir, C., Youn, E., Chen, J., Cazzonelli, C. 2012. Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct. PLoS One. 7(2):e30141. DOI:10.1371/journal.pone.0030141.

Interpretive Summary: Dramatic variability in the expression of new genetic material introduced into plants has been a factor impacting the success of plant genetic engineering efforts since its inception. We have developed a transgenic plant line in which an added gene that controls plant pigment production can be reproducibly silenced, with the process of silencing being easily monitored by visible changes in the plant's color. The induced silencing has been found to be associated with the production of small-interfering RNA targeting the transgene. We are exploring the molecular mechanisms responsible for initiation, maintenance and spread of this silencing phenomenon. A better understanding of these processes will allow more efficient plant genetic engineering and will provide a better understanding of how plants make use of silencing to regulate gene expression.

Technical Abstract: Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines Myb27 and Myb237 accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After a single self-fertilization, progeny of the Myb27 line that were homozygous for the myb transgene (27Hmo) displayed an unexpected pigmentation pattern, with most leaves containing sectors of dramatically reduced anthocyanin production. A similar leaf pigmentation pattern was observed in hemizygous Myb27 plants (27Hmi) in which systemic silencing (27Hmi-SysSil) of the AtMYB90 transgene was initiated by localized Agrobacteria-mediated transient expression of a hairpin construct producing double-stranded RNA (dsRNA) targeted to the AtMYB90 coding region.