TALE protein mediated overexpression of embryogenesis related marker genes in wheat microspores

Abstract

Stress treatment of microspore embryogenesis leads to the reprogramming of male gametophyte development pathways, which generates haploid green plants. However, low green plant production efficiency is a major hindrance in the adoption of double haploid (DH) production platform for wheat breeding. The aim of the current study was to induce the expression of microspore embryogenesis associated genes by over expressing the RKD transcription factor using Transcription activator-like effector (TALE) proteins. The TALE protein can modulate the gene expression by recognizing and binding host plant promoter sequences. Taking into consideration, we have cloned and analysed RKD promoter from wheat and triticale and used for the custom synthesis of TaRKD-TALE protein. The isolated and purified protein was conjugated with R9 (Cys (Npys)-(D-Arg)9) cargo peptides and successfully transduced into wheat microspores. The expression of early-culture abundant (ECA1), RWP-RK domain-containing proteins (RKD1) and Tapetum determinant 1 (TPD1) marker genes in wheat linked to microspore embryogenesis was studied. The expression of all three (ECA1, TPD1 and RKD1) embryogenesis related marker genes in the TALE transduced microspore was significantly higher than the control. Microspore culture transduced by R9-TaRKD-TALE protein yielded a significantly higher number of embryo-like structures (ELS) and the total green plants in wheat cultivars AC Fielder. Conversely, the microspore cell death has not been significantly affected by the transduction of R9-TaRKD-TALE, while showing a higher recovery at 120 h of microspore culture. This work will be helpful to exploit the TALE protein to improve our understanding of DH production and other pathways related genes.