In planta ORFeome analysis by large-scale over-expression of GATEWAY®-compatible cDNA clones:: screening of ERF transcription factors involved in abiotic stress defense

Genomic approaches have generated large Arabidopsis thaliana open reading frame (ORF) collections. However, tools are required to functionally characterize this ORFeome. Here we describe a batch procedure to simultaneously recombine a population of GATEWAY((R))-tagged full-length cDNAs into a plant expression vector. A pool of agrobacteria carrying these constructs has been used in flower-dip transformation experiments to obtain a collection of transgenic lines that over-express HA-tagged ORFs. This AtTORF-Ex library can be used in various screening approaches to identify particular gene family members involved in plant development or stress responses. The feasibility of the approach was studied using a near-complete collection of the Arabidopsis ethylene response factor (ERF) transcription factor (TF) family. Quality control performed at each step of the procedure revealed that the complexity of the population is maintained, and that almost all members of the ORF collection are covered by the plant library. The frequency of multiple transformation events has been determined as approximately 4%. Significant transgene expression was detected at the RNA and protein level in more than 60% and 30% of the transgenic plants, respectively. Striking phenotypic alterations were observed in approximately 4% of the plants. Many ERF TFs have been shown to participate in plant stress responses. As a proof of principle, the AtTORF-Ex library has been used in a selection procedure to isolate TFs involved in enhanced abiotic stress tolerance. The corresponding TF gene can be easily polymerase chain reaction-amplified using GATEWAY((R))att site-specific primers. In summary, we describe here a method that can be generally applied for functional analysis of ORFeomes in planta.