An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Blocking Rice Shoot Gravitropism by Altering One Amino Acid in LAZY1.

Tiller angle is an important trait that determines plant architecture and yield in cereal crops. Tiller angle is partially controlled during gravistimulation by the dynamic re-allocation of LAZY1 (LA1) protein between the nucleus and plasma membrane, but the underlying mechanism remains unclear. In this study, we identified and characterized a new allele of LA1 based on analysis of a rice (Oryza sativa L.) spreading-tiller mutant la1G74V, which harbors a non-synonymous mutation in the predicted transmembrane (TM) domain-encoding region of this gene. The mutation causes complete loss of shoot gravitropism, leading to prostrate growth of plants. Our results showed that LA1 localizes not only to the nucleus and plasma membrane but also to the endoplasmic reticulum. Removal of the TM domain in LA1 showed spreading-tiller phenotype of plants similar to la1G74V but did not affect the plasma membrane localization; thus, making it distinct from its ortholog ZmLA1 in Zea mays. Therefore, we propose that the TM domain is indispensable for the biological function of LA1, but this domain does not determine the localization of the protein to the plasma membrane. Our study provides new insights into the LA1-mediated regulation of shoot gravitropism.

A lipid transfer protein variant with a mutant eight-cysteine motif causes photoperiod- and thermo-sensitive dwarfism in rice.

Plant height is an important trait for architecture patterning and crop yield improvement. Although the pathways involving gibberellins and brassinosteroids have been well studied, there are still many gaps in our knowledge of the networks that control plant height. In this study, we determined that a dominant photoperiod- and thermo-sensitive dwarf mutant is caused by the active role of a mutated gene Photoperiod-thermo-sensitive dwarfism 1 (Ptd1), the wild-type of which encodes a non-specific lipid transfer protein (nsLTP). Ptd1 plants showed severe dwarfism under long-day and low-temperature conditions, but grew almost normal under short-day and high-temperature conditions. These phenotypic variations were associated with Ptd1 mRNA levels and accumulation of the corresponding protein. Furthermore, we found that the growth inhibition in Ptd1 may result from the particular protein conformation of Ptd1 due to loss of two disulfide bonds in the eight-cysteine motif (8-CM) that is conserved among nsLTPs. These results contribute to our understanding of the novel function of disulfide bonds in the 8-CM, and provide a potential new strategy for regulation of cell development and plant height by modifying the amino acid residues involved in protein conformation patterning.

The far-upstream regulatory region of RFL is required for its precise spatial-temporal expression for floral development in rice.

KEY MESSAGE: A rice mutant aberrant floral organ 1 (afo1) was identified, showing increased floral organ number, aberrant floral organ identity and loss of floral meristem determinacy. A disruption of sequence integrity at 6292-bp upstream of RFL by a T-DNA insertion led to varied RFL expression patterns in floral meristem and floret in afo1 and caused the mutant phenotype. The LEAFY (LFY) transcription factor and its homologs affect many aspects of plant development, especially floral development. RICE FLORICAULA/LEAFY (RFL), the rice ortholog of LFY, has complicated expression patterns and different functions in floral development. However, the mechanisms regulating the spatial-temporal expression of RFL remain largely unknown. Here, we describe a rice aberrant floral organ 1 (afo1) mutant that was produced by a T-DNA insertion at 6292-bp upstream of the start codon of RFL. This insertion altered the expression of RFL in floral meristem (FM) and floret. The in situ hybridization result showed that, when florets appear, RFL was expressed almost exclusively at the palea/lemma adaxial base adjacent to lodicules in the wild-type panicle. However, in afo1 florets, RFL mRNA signals were detected in the region between lodicule and stamen, and strong signals persisted in FM. The altered pattern of RFL expression in afo1 resulted in enlarged FMs, more floral organs, aberrant floral organ identity, and loss of FM determinacy. Transformation of rice with an RFL construct driven by the 6292-bp upstream genomic sequence re-built the mutant phenotype similar to afo1. The results suggest that the far-upstream region of RFL may contain potential cis element(s) that are critical to define the precise spatial-temporal expression pattern of RFL for its function in floral development.

An asymmetric allelic interaction drives allele transmission bias in interspecific rice hybrids.

Hybrid sterility (HS) between Oryza sativa (Asian rice) and O. glaberrima (African rice) is mainly controlled by the S1 locus. However, our limited understanding of the HS mechanism hampers utilization of the strong interspecific heterosis. Here, we show that three closely linked genes (S1A4, S1TPR, and S1A6) in the African S1 allele (S1-g) constitute a killer-protector system that eliminates gametes carrying the Asian allele (S1-s). In Asian-African rice hybrids (S1-gS1-s), the S1TPR-S1A4-S1A6 interaction in sporophytic tissues generates an abortion signal to male and female gametes. However, S1TPR can rescue S1-g gametes, while the S1-s gametes selectively abort for lacking S1TPR. Knockout of any of the S1-g genes eliminates the HS. Evolutionary analysis suggests that S1 may have arisen from newly evolved genes, multi-step recombination, and nucleotide variations. Our findings will help to overcome the interspecific reproductive barrier and use Asian-African hybrids for increasing rice production.

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.