Haploid induction by nanobody-targeted ubiquitin-proteasome-based degradation of EYFP-tagged CENH3 in Arabidopsis thaliana

Abstract

The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous 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 (SPOP) proteins can recognize maternally derived EYFP-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the gCENH3-EYFP complemented cenh3.1 mother with plants expression 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 gCENH3 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.

Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis

“…To assess the effect of temperature on centromeres, we analyzed the CENH3 signal on pachytene chromosomes in anthers of the Arabidopsis eYFP:CENH3 reporter line (Le Goff et al, 2020; Demidov et al, 2022) grown at 21, 26 and 30°C. While eYFP:CENH3 was readily detectable on pachytene chromosomes at 21°C, the signal decreased significantly at 26°C and declined further at 30°C (Figure 3A,B).…”

“…Plant material and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0), cenh3-4 (Capitao et al, 2021) and spo11-2-3 (Hartung et al, 2007) were generated by crossing HTA10:RFP (Valuchova et al, 2020) and pRPS5A::TagRFP:TUB4 (Prusicki et al, 2019) reporter lines with cenh3-4 mutant and BMF3::BMF3:GFP reporter line, respectively. eYFP:CENH3 reporter line was described and characterized in previous studies (Le Goff et al, 2020;Demidov et al, 2022).…”

“…CENH3 is present on meiotic chromosomes from early prophase I (25, 26) and its signal is most visible in pachytene when homologous chromosomes are fully synapsed. To evaluate the effect of temperature on centromeres, we analyzed the CENH3 signal on pachytene chromosomes in anthers of the Arabidopsis eYFP:CENH3 reporter line (35,36) grown at 21, 26 and 30°C. While eYFP:CENH3 was easily detectable on pachytene chromosomes at 21°C, the signal decreased significantly at 26°C and declined further at 30°C (Figure 3A,B).…”

Creating novel ornamentals via new strategies in the era of genome editing

“…In addition to haploids, diploids, and aneuploids are produced as a result of the recruitment of some chromosomes into the main nucleus and the degradation of other chromosomes (Tan et al, 2015). Recent studies showed that direct degradation of CENH3 via a nanobody-based method produced haploids even though the prior study showed that RNAi-mediated suppression of CENH3 expression did not produce any haploids (Demidov et al, 2022) (Figure 2C).…”

Plant chromosome engineering – past, present and future

“…In addition to genetic modification, a parental genome has been eliminated using targeted in vivo degradation of the CENH3 protein. Arabidopsis thaliana haploids were produced by using degraded Green Fluorescent Protein (deGradFP) (Demidov et al ., 2022), a system that uses the ubiquitin‐proteasome pathway in conjunction with a green fluorescent protein (GFP)‐specific nanobody (VHHGFP4) fused to an E3 ligase (Caussinus & Affolter, 2016).…”