Random RNAi screening for negative regulators of drought resistance in Arabidposis

Many regions of the world, including the United States, are facing long-term droughts that threaten agricultural productivity and sustainability. The genes that are responsible for drought resistance can be divided into two general families: a family of genes that enhances drought resistance and a family of negative regulating genes whose expression will reduce drought resistance. Over-expressing positive and suppressing negative regulators will improve drought resistance. Currently, T-DNA disruption and enhanced tagging are the two major bioengineering approaches used to dissect these drought-related genes. However, the T-DNA disruption approach cannot screen for lethal genes and the screening is time consuming. Enhanced tagging has similar drawbacks for screening lethal or toxic genes. In contrast, the RNAi approach will offer a new strategy to overcome these problems and has clear advantages. First, the RNAi method will not completely shut off gene expression, which will allow lethal genes to be recovered during screening. Second, RNAi has a dominant downregulation of the target genes and can be used to screen the TOgeneration, thus shortening the screening time. The question is how to establish a population that covers all of the ORFs of the entire genome with downregulated gene expression. Currently RNAi vectors have been developed for silencing specific genes with known sequences. Although Arabidopsis whole genome sequences are readily available, targeting each hypothetical ORF is labor intensive and time consuming. In the proposed studies, we will generate a single-strand 21 nucleotide (nt) small RNA expression library that will cover the entire Arabidopsis genome. This small RNA expression library will be used for the large-scale transformation of Arabidopsis plants. The TOtransformants will be directly used to screen for the negative regulators of drought resistance under drought conditions. Specifically, our objectives are: (1) to generate a single-stranded 21 nt RNA expression library using a binary vector that contains a pol-III promoter and TTTTT as a terminator; (2) to achieve large-scale transformation of Arabidopsis plants to produce TOtransgenic seeds using the Agrobacterium-mediated flower dipping approach; (3) to perform large-scale screening for negative regulators of drought resistant mutants during seed germination on a high osmotic stress medium, and subsequent different plant developmental stages under drought conditions; and (4) to isolate the integrated 21 nt RNA transcription region for sequences and identify the small RNA targeting gene. The proposed experiments are expected to establish a better approach for screening negative regulators of abiotic resistance in plants, and discover new genes that regulate drought resistance. These results will have implications for the improvement of agricultural plants growing under drought conditions.