Small RNAs from Natural Antisense Transcripts of GhMIXTAMYB-Like3_A12 Causes the Naked Seed Phenotype in Cotton N1 mutant

Cotton fiber is a unique plant epidermal cell type derived form cotton seed epidermis. Each of the cottonseed fiber is a single cell organ which facilitates the plant cell fate determination. Therefore, cottonseed fiber is a model system for plant cell differentiation study. The seed fiber patterning may share some similar components such as transcriptional factors in cell fate determination with leaf hair. Given the complexity of cotton genome and physiological feature of seed fibers, we propose a unique regulation machine developed in cotton fiber control. Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene (N₁) encoding a MYBMIXTA-like transcription factor (MML) in chromosome A12, GhMML3_A12, for seed trichome development, revealing a unique transcriptional regulatory network for cotton fiber development different from leaf trichomes. The extremely low expression of GhMML3_A12 in N₁ is associated with the natural antisense transcript (NAT) production on the 3’ end of gene, driven by its antisense promoter. Therefore, bidirectional transcriptions of GhMML3_A12 form double strand RNAs and generate 21-22nt small RNAs, which, in a fiber-specific manner, mediate GhMML3_A12 mRNA cleavage and result in the production of naked seeds, and further as trans-NATs inhibit lint fiber development in N₁ plants. However, NAT production of GhMML3_A12 in the wild type is greatly suppressed by its CHH methylation at the 3’ proximal region, which is associated with the chromatin state alteration. (NATs) are commonly observed in both animal and plant kingdoms, but only a limited number of such genes have been identified as being involved in gene regulation in plants. The present research reports the first observation of gene-mediated NATs and siRNA directly controlling cottonseed fiber development.