Transmembrane Protein GbTMEM209 Inhibits Fibre Elongation via Competitive Interaction With GbHOX3 in Gossypium barbadense

Sea Island cotton (Gossypium barbadense) produces premium-quality fibres, yet the genetic basis underlying its fibre development remains elusive. Here, we identify two key non-synonymous single nucleotide polymorphisms (SNPs, G/C and G/A) in the gene Gbar_D13G024080, which encodes the TRANSMEMBRANE PROTEIN 209 (TMEM209). These SNPs resulted in amino acid changes (V/L and R/K), and are significantly correlated with the fibre length in Sea Island cotton. CRISPR-Cas9-mediated knockout of GbTMEM209 significantly enhanced fibre length and fibre strength in both G. hirsutum and G. barbadense. Conversely, overexpression of GbTMEM209 in G. hirsutum led to reduced fibre length. Further mechanistic investigation revealed that GbTMEM209 competitively interacts with GbHOX3 to impair its transcriptional activation on cell wall-loosening genes GbEXPA1 and GbRDL1. Moreover, during the elongation stage of the fibres, GbTMEM209 and GbHOX3 exhibit an antagonistic relationship, which jointly regulate the development of cotton fibres. Virus-induced gene silencing (VIGS) of GbHOX3, GbEXPA1, or GbRDL1 consistently resulted in shortened fibres in Sea Island cotton, validating their critical roles in fibre development. Our findings establish GbTMEM209 as a novel negative regulator of fibre elongation and uncover a protein competition-mediated transcriptional control mechanism in cotton fibre morphogenesis. These findings provide valuable genetic targets and conceptual insights for molecular breeding programs aimed at improving cotton fibre quality.