Haploid induction by nanobody-targeted ubiquitin-proteasome-based degradation of EYFP-tagged CENH3 in Arabidopsis thaliana. (6th September 2022)

• Record Type: Journal Article
• Authors: Demidov, Dmitri; Lermontova, Inna; Moebes, Michael; Kochevenko, Andriy; Fuchs, Jörg; Weiss, Oda; Rutten, Twan; Sorge, Eberhard; Zuljan, Erika; Giehl, Ricardo Fabiano Hettwer; Mascher, Martin; Somasundaram, Saravanakumar; Conrad, Udo; Houben, Andreas
• Editors: Costa, Fabrizio
• Is Part Of: Journal of experimental botany. Volume 73:Number 22(2022)
• Page Start: 7243
• Page End: 7254
• Publication Date: 2022-09-06
• Subjects: Arabidopsis -- CENH3 -- deGradFP -- GFP degradation -- haploid induction -- nanobody; Botany -- Periodicals; Botany, Experimental -- Periodicals; Plant physiology -- Periodicals; 580
• DOI: 10.1093/jxb/erac359
• Languages: English
• ISSNs: 0022-0957

Abstract

Abstract : Targeted CENH3 degradation enables haploid production in Arabidopsis thaliana and, compared with other strategies, has no floral abnormalities or reduced seed setting to obtain high haploid recovery per cross. Abstract: The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana . We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the uniparental degradation of EYFP-fused genomic CENH3 during early embryogenesis leads to a decrease in its level at centromeres and subsequently weakens the centromeres. The male-derived wild type CENH3 containing centromere outcompetes the CENH3-EYFP depleted centromere. Consequently, maternal chromosomes undergo elimination, resulting in haploids.