Screening Proteins Interacting With DELLA Or PIPE Using Arabidopsis Thaliana CDNA Library

Gibberellins (GAs) are large family of plant hormones that induce a wide range of plant growth responses, including seed germination, stem elongation, leaf expansion, induction of flowering, and pollen maturation. DELLA proteins are important factors in GA signaling, which act as a repressors. Relative work of our lab demonstrated that PIPE, a protein phosphatase, was involved in GA signal transduction.We used RGA and GAI as the baits, which belong to DELLA family to screen the cDNA library. As putative transcription factors, RGA and GAI may have transcription activation activity which would cause false positive results during yeast two-hybrid screening. To exclude the transcription activation activity, six different fragments of RGA and GAI were amplified, i.e., RGA(amino acid residues 1-587), RGAΔN(amino acid residues 200-587), RGAΔC(amino acid residues 1-408), GAI(amino acid residues 1-532), GAIΔN(amino acid residues 156-532) and GAlΔC(amino acid residues 1-264). Six baits were constructed by in-frame fusion in bait vector, and transcription activation activity of them was tested. Because the AN of RGA and GAI had not strong activation activity, we use them for subsequent cDNA library screening. About 17 positive clones of RGAΔN and 114 positive clones of GAI AN were obtained. By analysis of these positive-colons, we find that these genes were involved in the regulation of chloroplast development, cytoskeleton organization and proteins degradation.Because DELLA proteins were proposed to be regulated by protein phosphorylation/dephosphorylation, we focused interested in two genes of these clones, which were AT3G21222 and AT5G22750. To deeply identify the function of AT3G21222 and AT5G22750, we observed their T-DNA insertion alleles. Morphological analyses revealed that none of them exhibit obvious mutant phenotype. In addition, we examined the responses of SALK-038064 to GA3. As a result, it was less sensitive to GA3 compared to wild type.We used PIPE as the bait to screen the cDNA library, and about 104 positive clones were obtained. We were interested in 1 clone, ADFx, which is important for actin cytoskeleton organization.