Identification And Characterization Of TALE Superfamily Transcription Factors And Functional Verification Of GhBLH7-D06

Cotton(Gossypium spp.)is the most important natural fiber crop in the world.Cotton fiber length and strength are both key traits of fiber quality,and fiber strength(FS)is tightly correlated with secondary cell wall(SCW)biosynthesis.The three-amino-acid-loop-extension(TALE)superclass homeoproteins are involved in regulating diverse biological processes in plants,and some TALE members has been identified to play a key role in regulating SCW formation.However,little is known about the functions of TALE members in cotton(Gossypium spp.).In this study,we focused on TALE superclass genes and carried out the following works:1.For the first time,based on gene homology,46,47,88 and 94 TALE superfamily genes were identified in G.arboreum,G.raimondii,G.barbadense and G.hirsutum,respectively.Phylogenetic and evolutionary analysis showed the evolutionary conservation of two cotton TALE families(including BEL1-like and KNOX families).Gene structure analysis also indicated the conservation of Gh TALE members under selection.The analysis of promoter cis-elements and expression patterns suggested potential transcriptional regulation functions in fiber SCW biosynthesis and responses to some phytohormones for Gh TALE proteins.Genome-wide analysis of colocalization of TALE transcription factors with SCW-related QTLs revealed that some BEL1-like genes and KNAT7 homologs may participate in the regulation of cotton fiber strength formation.Overexpression of Gh KNAT7-A03 and Gh BLH6-A13 significantly inhibited the synthesis of lignocellulose in interfascicular fibers of Arabidopsis.Yeast two-hybrid(Y2H)experiments showed extensive heteromeric interactions between Gh KNAT7 homologs and some GhBEL1-like proteins.Yeast one-hybrid(Y1H)experiments identified the upstream Gh MYB46 binding sites in the promoter region of Gh TALE members and defined the downstream genes that can be directly bound and regulated by Gh TALE heterodimers.2.In addition to the interaction between GhBEL1-Like protein and Gh KNAT7 subgroup homologous proteins,we studied the Gh BLH7-D06 protein which could not interact with Gh KNAT7.Tissue expression pattern indicated that Gh BLH7-D06 was also involved in the regulation of SCW synthesis in cotton fiber and stem.The expression of Gh BLH7-D06 in response to Verticillium dahliae infection and phytohormone treatment indicated that Gh BLH7-D06 was a negative regulator of Cotton Verticillium wilt resistance,and the response to Me JA also indicated that Gh BLH7-D06 might be a bridge between them.Through viral-induced gene silencing(VIGS)technique,we found that reducing the expression of Gh BLH7-D06 could enhance the resistance of cotton plants to Verticillium wilt.The acquisition of resistance may be mainly due to the significant overexpression of genes related to lignin synthesis and metabolism,which also proves that Gh BLH7-D06 negatively regulates the resistance of cotton to Verticillium wilt.Through screening of yeast two-hybrid libraries and confirmation of bimolecular fluorescence complementary technology,we found that an OFP transcription factor Gh OFP3-D13 could interact with Gh BLH7-D06,and Gh OFP3-D13 was a stem-specific negative regulator of Cotton Verticillium wilt resistance.Furthermore,we found that Gh BLH7-D06 could target the promoter region of Gh PAL-A06,inhibit its expression and inhibit lignin biosynthesis through Y1 H experiment and Dual-Luciferase reporting system.Gh OFP3-D13 is a cofactor involved in regulating SCW deposition and Verticillium Wilt Resistance in cotton plants.The discovery of these regulatory mechanisms not only enriched the regulatory network of TALE transcription factors,but also provided theoretical basis for further understanding the formation of cotton fiber strength and acquisition of plant Verticillium wilt resistance,as well as exploring new core gene resources for efficient and rapid improvement of cotton germplasm.