Crop breeding aims to generate pure inbred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.
The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.The actin cytoskeleton of plant cells is temporally and spatially regulated in response to external stimuli and plays an important role in many physiological processes, like cytoplasmic streaming, division plane coordination, and tip growth (Volkmann and Baluska, 1999;Staiger et al., 2000). A host of accessory proteins are required to regulate the equilibrium between monomeric G-actin and F-actin, to bundle and crosslink actin filaments to one another, or to promote nucleation of new actin filaments (Ayscough, 1998). The formation of actin trimers, the smallest nuclei for polymerization, is the rate-limiting step during spontaneous filament assembly. Thus, cellular activities that either block or stimulate trimer formation have a large influence on actin polymerization (Cooper et al., 1983;Winter et al., 1997). Relatively recent studies have provided important new insights into how the assembly and disassembly of the actin cytoskeleton is regulated. The Arp2/3 complex has been demonstrated as a nucleation factor in the stimulation of branched filament formation (for review, see Pollard and Beltzner, 2002). Recently, a conserved family of formin homology proteins has emerged as a new class of actin nucleator that directs assembly of straight filaments independently of the Arp2/3 complex (for review, see Zigmond, 2004).Formins are large multidomain proteins that have been found in all eukaryotes examined and are required for multiple actin-related processes, such as cytokinesis and maintenance of cell polarity, in many organisms (Wasserman, 1998;Sawin, 2002). The mouse limb deformity gene encodes the first formin identified. Subse...
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