Identification And Functional Analysis Of DELLA-Interacting Proteins In Cotton Fibers

Cotton(Gossypium hirsutum) is a widely grown textile crop which is cultivated mainly for its fibers and occupies an important position in the national economy. Growth and development of the fibers is controlled by a multi-channel signal network, including several plant hormone. DELLA protein is a key negative regulatory factors in the gibberellin(GA) signaling pathways. DELLA proteins have a N-terminal response to GA signal, and a conserved C-terminal GRAS domain involved in transcriptional regulation. Due to lack of typical DNA binding domain, DELLAs usually function interacting with other regulatory proteins, and influencing their function.Ethylene as an important plant hormone can promote the fibers elongation in upland cotton. EIN3(Ethylene insensitive 3) is an essential nuclear transcription factor in ethylene signaling pathway, and is an integrating point between ethylene and other signal.This experiment aimed to elucidate DELLA proteins’ roles in fiber growth and development via DELLA-interacting proteins. Firstly, on the basis of comprehensive identification of cotton EIN3 homologs, we analyze their evolution, classification, expression and interactions with DELLAs. The effects of protein interations on downstream genes expression were futher analyzed via tobacco transient expression. Secondly, we generated two cDNA libraries of 10 DPA and 20 DPA cotton fibers. The DELLA interacting proteins were screened from these libraries, and verified by transient expression. The main results are as follows:1. Construction and screening of the DELLA bait proteinFour pairs DELLA genes, including each 4 A-subgenome(GhGAI1A ~ 4A) and D subgenome(GhGAI1D ~ 4D), were identified from upland cotton. The full length of or truncated sequences of GhGAI1D~4D were used to construct yeast two hybrid(Y2H) bait, and to detect self-activation and cell toxicity in yeast. It was showed that GRAS domain of GhGAI1 D and 3D, and N region of GhGAI1 D had no self-activation and cell toxicity in Y2 H system. Further tests indicated that none of the GRAS regions of 8 cotton DELLA proteins had self-activation and cell toxicity. Therefore, we used the GRAS domains of cotton DELLA proteins as a bait for library screening and detecting protein-protein interactions.2. Comprehensive analysis of interaction between DELLAs and GhEIN3sinteraction from upland cottonIn tetraploid upland cotton, 9 pairs of EIN3 homologs from A and D subgenomes(GhEIN3-1A~9A and 1D~9D, respectively) were all conserved. The expression analysis revealed that four pairs GhEIN3 s genes(GhEIN3-1A~4A, GhEIN3-1D~4D) obviously expressed in cotton fibers and had the similar expression patterns in different tissues and different fiber developmental stages. Ethylene treatment significantly increased GhEIN3-1A, 1D, 2D and 3D in vitro-cultuvated fibers. Transient expression assay showed that all GhEIN3 s significantly activated the EBS(EIN3-binding site)-containing promoters, but with probably different activation effeciencies on different promoters. These results indicated that GhEIN3 s may play an important role in regulating fiber development.The GRAS of each Gh GAI gene was cloned into pGBKT7 which contains the GAL4-binding domain, and GhEIN3-1~4A/D were constructed into pGADT7 which contains the GAL4-activation domain. The pairwise Y2 H assay showed that GhEIN3-1A interacted with all GhGAIs, while other GhEIN3 s did not interacted with GhGAIs. The transient expression result indicated that interaction with DELLA protein inhibited GhEIN3-1A activation of target genes.DELLA, interacting with GhHOX3, inhibited GhHOX3 binding to and activating the expression of pEXPA1 and pRDL1. At the same time, we found that DELLA-GhEIN3-1A interaction can not restore GhHOX3 activation on pEXPA1 and pRDL1, indicating that DELLA-EIN3-1A interactions may have no effects on DELLA downstream genes.3. Screening DELLA interaction proteinsWe made GhGAI1D-GRAS bait and screened potential DELLA-interacting proteins in the cDNA libraries. By co-transformation, and tobacco transient expression verification,we identified two positive interacting proteins: the C4 zinc finger protein FYVE-D and CCCH type zinc finger protein ZinCX8-D.Further, in tobacco transient expression, we confirmed that GhHOX3 activated pEXPA1 and pRDL1 expression, and interaction DELLA protein(GhGAI1D) inhibited GhHOX3 activation of pEXPA1 and pRDL1. While FYVE-D and ZinCX8-D interacted with DELLA, Gh HOX3 activations on target genes were restored. Therefore, FYVE-D and ZinCX8-D may affect the expression of the DELLA downstream genes, by interacting with DELLAs.