Identification and functional analysis of αKNL2 genes in cowpea

Although the KINETOCHORE NULL2 (KNL2) protein is an essential inner centromeri c protein that is crucially important for assembly and functioning of kinetochores, our understanding of its organization, dynamics and function of distinct isoforms in the cells of plant species undergoing mitosis/meiosis is far from complete. In this study, we identified and characterized two KNL2.1/KNL2.2 genes in cowpea. GUS reporter constructs and qRT-PCR revealed that the expression profiles of both genes were variable across organs, with the highest expression in leaves and roots. Using an EYFP gene fusion coupled with immunostaining, it was demonstrated that both KNL2 variants colocalized at centromeres in a cell-cycle-dependent manner. The CRISPR/Cas9 technique was used to generate various in-frame deletion and out-of-frame knock-out knl2 mutants. Single- and double-gene knock-out mutants were generated, and the effects of mutations on plant development and seed setting were analyzed. The results are discussed both with respect to the roles of these proteins in kinetochore assembly and in the context of using KNL2 genes for in vivo production of haploids in cowpea. Significance statementThis study identifies two paralogous KNL2 genes in cowpea and reveals their functional redundancy during centromere assembly and essential role in seed development. These findings expand our knowledge of kinetochore dynamics and provide a basis for exploring the evolutionary diversification of centromeric proteins in legumes.