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Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

In plants, lipid transfer proteins (LTPs) transport pollen wall constituents from the tapetum to the exine, a process essential for pollen wall development. However, the functional cooperation of different LTPs in pollen wall development is not well understood. In this study, we have identified and characterized a grass-specific LTP gene, OsLTP47, an important regulator of pollen wall formation in rice (Oryza sativa). OsLTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays confirms that OsLTP47 has lipid-binding activity. Dysfunction of OsLTP47 causes disordered lipid metabolism and defective pollen walls, leading to male sterility. Yeast two-hybrid and pull-down assays reveal that OsLTP47 physically interacts with another LTP, OsC6. These findings suggest that the plasma membrane-localized OsLTP47 may function as a mediator in a lipid transfer relay through association with cytosolic and/or locular OsC6 for pollen wall development and that various LTPs may function in a coordinated manner to transport lipid molecules during pollen wall development.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

N6 -methyladenosine (m6 A) modification affects the post-transcriptional regulation of eukaryotic gene expression, but the underlying mechanisms and their effects in plants remain largely unknown. Here, we report that the N6 -adenine methyltransferase-like (MTL) domain-containing protein ENHANCED DOWNY MILDEW 2-LIKE (OsEDM2L) is essential for rice (Oryza sativa L.) anther development. The osedm2l knockout mutant showed delayed tapetal programmed cell death (PCD) and defective pollen development. OsEDM2L interacts with the transcription factors bHLH142 and TAPETUM DEGENERATION RETARDATION to regulate the expression of ETERNAL TAPETUM 1 (EAT1), a positive regulator of tapetal PCD. Mutation of OsEDM2L altered the transcriptomic m6 A landscape, and caused a distinct m6 A modification of the EAT1 transcript leading to dysregulation of its alternative splicing and polyadenylation, followed by suppression of the EAT1 target genes OsAP25 and OsAP37 for tapetal PCD. Therefore, OsEDM2L is indispensable for proper mRNA m6 A modification in rice anther development. This article is protected by copyright. All rights reserved.

The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9+ and ScCas9++ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9+ and ScCas9++ and a nickase variant ScCas9n++ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9++ has higher editing efficiency than ScCas9 and ScCas9+ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++ . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17 ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9++ represents a crucial new tool for improving plant editing.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Some progress has been made in understanding the pathways related to rice heading, but their applications to breeding japonica rice varieties adapted to grow in low-latitude areas (“indica to japonica”) are limited. We edited eight adaptation-related genes via a lab-established CRISPR/Cas9 system in a japonica variety, Shennong265 (SN265). All T0 plants and their progeny bearing random mutation permutations were planted in southern China and screened for changes in heading date. We found that the double mutant of Days to heading 2 (DTH2) and CONSTANS 3 (OsCO3) (dth2-osco3), two CONSTANS-like (COL) genes, showed significantly delayed heading under both short-day (SD) and long-day (LD) conditions in Guangzhou and manifested great yield increase under SD conditions. We further demonstrated that the heading-related Hd3a-OsMADS14 pathway was down-regulated in the dth2-osco3 mutant lines. The editing of the COL genes DTH2 and OsCO3 greatly improves the agronomic performance of japonica rice in Southern China.

CRISPR-dependent base editors enable direct nucleotide conversion without the introduction of double-strand DNA break or donor DNA template, thus expanding the CRISPR toolbox for genetic manipulation. However, designing guide RNAs (gRNAs) for base editors to enable gene correction or inactivation is more complicated than using the CRISPR system for gene disruption. Here, we present a user-friendly web tool named BEtarget dedicated to the design of gRNA for base editing. It is currently supported by 46 plant reference genomes and 5 genomes of non-plant model organisms. BEtarget supports the design of gRNAs with different types of protospacer adjacent motifs (PAM) and integrates various functions, including automatic identification of open reading frame, prediction of potential off-target sites, annotation of codon change, and assessment of gRNA quality. Moreover, the program provides an interactive interface for users to selectively display information about the desired target sites. In brief, we have developed a flexible and versatile web-based tool to simplify complications associated with the design of base editing technology. BEtarget is freely accessible at https://skl.scau.edu.cn/betarget/.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

Summary Rice (Oryza sativa) is a short‐day (SD) plant originally having strong photoperiod sensitivity (PS), with SDs promoting and long days (LDs) suppressing flowering. Although the evolution of PS in rice has been extensively studied, there are few studies that combine the genetic effects and underlying mechanism of different PS gene combinations with variations in PS.We created a set of isogenic lines among the core PS‐flowering genes Hd1, Ghd7 and DTH8 using CRISPR mutagenesis, to systematically dissect their genetic relationships under different day‐lengths. We investigated their monogenic, digenic, and trigenic effects on target gene regulation and PS variation.We found that Hd1 and Ghd7 have the primary functions for promoting and repressing flowering, respectively, regardless of day‐length. However, under LD conditions, Hd1 promotes Ghd7 expression and is recruited by Ghd7 and/or DTH8 to form repressive complexes that collaboratively suppress the Ehd1‐Hd3a/RFT1 pathway to block heading, but under SD conditions Hd1 competes with the complexes to promote Hd3a/RFT1 expression, playing a tradeoff relationship with PS flowering. Natural allelic variations of Hd1, Ghd7 and DTH8 in rice populations have resulted in various PS performances.Our findings reveal that rice PS flowering is controlled by crosstalk of two modules – Hd1–Hd3a/RFT1 in SD conditions and (Hd1/Ghd7/DTH8)–Ehd1–Hd3a/RFT1 in LD conditions – and the divergences of these genes provide the basis for rice adaptation to broad regions.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

Rice mitochondrial aldehyde dehydrogenase OsALDH2b contributes to tapetal degradation, which is essential for male fertility., Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Although several genes involved in tapetum development have been identified, the molecular basis of tapetum degeneration regulation remains poorly understood. In this study, we identified and characterized the nucleus-encoded, conserved mitochondrial aldehyde dehydrogenase OsALDH2b as a key regulator of tapetum degeneration in rice (Oryza sativa). OsALDH2b was highly expressed in anthers from meiosis to the early microspore stage. Mutation of OsALDH2b resulted in excess malonaldehyde accumulation and earlier programmed cell death in the tapetum, leading to premature tapetum degeneration and abnormal microspore development. These results demonstrate that OsALDH2b negatively regulates tapetal programmed cell death and is required for male reproductive development, providing insights into the regulation of tapetum development in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.

Heading date determines the seasonal and regional adaptation of rice (Oryza sativa L.) varieties and is mainly controlled by photoperiod sensitivity (PS). The core heading date genes Hd1, Ghd7, DTH8, and PRR37 act synergistically in regulating the PS. In this study, we systematically analyze the heading date, PS, and agronomic traits of eight homozygous lines with various combinations of Hd1, Ghd7, and DTH8 alleles in the prr37 background under long-day (LD) and short-day (SD) conditions, respectively. We find that Hd1 alone promotes heading, regardless of the day length. However, under LDs, Hd1 suppresses flowering, in coordination with functional Ghd7 or with Ghd7 and DTH8. These loci cooperate to negatively regulate the Ehd1-Hd3a/RFT1 pathway and delay heading. Under SDs, Hd1 competes with various heading suppressors to promote heading. Therefore, the dual function of Hd1 is vital for PS. The lines carrying Hd1 alone show reduced plant height with fewer primary and secondary branches in panicles. Lines carrying Ghd7 and DTH8 (with hd1) show delayed heading and improve agronomic traits. Overall, our results reveal the regulation of rice PS flowering by the core heading date genes and their effects on agronomic traits, providing valuable information for the selection of rice varieties for adaptation to different light and temperature conditions.

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5' tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Plant height is an important agronomic trait for lodging resistance and yield. Here, we report a new plant-height-related gene, OsUBR7 in rice (Oryza sativa L.); knockout of OsUBR7 caused fewer cells in internodes, resulting in a semi-dwarf phenotype. OsUBR7 encodes a putative E3 ligase containing a plant homeodomain finger and a ubiquitin protein ligase E3 component N-recognin 7 (UBR7) domain. OsUBR7 interacts with histones and monoubiquitinates H2B (H2Bub1) at lysine148 in coordination with the E2 conjugase OsUBC18. OsUBR7 mediates H2Bub1 at a number of chromatin loci for the normal expression of target genes, including cell-cycle-related and pleiotropic genes, consistent with the observation that cell-cycle progression was suppressed in the osubr7 mutant owing to reductions in H2Bub1 and expression levels at these loci. The genetic divergence of OsUBR7 alleles among japonica and indica cultivars affects their transcriptional activity, and these alleles may have undergone selection during rice domestication. Overall, our results reveal a novel mechanism that mediates H2Bub1 in plants, and UBR7 orthologs could be utilized as an untapped epigenetic resource for crop improvement.

A gain-of-function rice mutant Photoperiod-thermo-sensitive dwarfism 1 is caused by a mutated gene, Ptd1, that encodes a nsLTP variant with an altered protein conformation due to disruption of the conserved eight-cysteine-motif., 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.

Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (

Salt stress is one of the major factors limiting crop production globally, including rice (Oryza sativa). Although a number of genes involved in salt tolerance have been functionally identified, the mechanism underlying salt tolerance in rice is still poorly understood. Here, we reported a novel C2 domain-containing protein, OsC2DP required for salt tolerance in rice. OsC2DP was predominately expressed in the roots and its expression was repressed by salt stress. Transient expression of OsC2DP in rice protoplast cells showed that it was localized in the cytosol. Immunostaining further showed that OsC2DP was able to translocate from the cytosol to plasma membrane under salt conditions. Knockout of OsC2DP did not affect Na+ concentration in the roots, but increased shoot Na+ concentration, resulting in a significant sensitivity of rice to salt stress. Furthermore, the quantitative Real-time PCR and transcriptomic analysis showed that the expression level of some genes related to salt tolerance were indirectly regulated by OsC2DP, especially OsSOS1 and OsNHX4. These results indicate that OsC2DP has an important role in salt tolerance and these findings provide new insights into the regulation of OsC2DP gene for rice breeding with high salt tolerance.

Hybrid sterility presents a major bottleneck in hybrid crop breeding and causes postzygotic reproductive isolation in speciation. Here, we summarize the current understanding of the genetics of rice hybrid sterility and highlight new advances in deciphering the molecular basis of the major genetic loci for hybrid sterility in rice. We also discuss practical strategies for overcoming reproductive barriers to utilize hybrid vigor in inter-specific and inter-subspecific hybrid rice breeding.

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A 20-year-old young woman with cauliflower-shaped lesions on her vulva was misdiagnosed as condyloma acuminatum. Further evaluation revealed positive serological tests for syphilis. On histological examination, and numerous plasma cells were seen to infiltrate the dermis heavily. The cauliflower-shaped lesions on vulva completely disappeared after benzathine penicillin treatment. Cauliflower-shaped lesions are a rare manifestation of secondary syphilis.

Efficient stacking of multiple genes is a critical element in metabolic engineering of complex pathways, synthetic biology, and genetic improvement of complex agronomic traits in plants. Here we present a high-efficiency multigene assembly and transformation vector system, TransGene Stacking II (TGS II), for these purposes. The operation process is described in detail, and the successful operation mainly depends on effective reagents, special Escherichia coli strains, and basic molecular biological means without other specific equipments.

Efficient stacking of multiple genes is a critical element in metabolic engineering of complex pathways, synthetic biology, and genetic improvement of complex agronomic traits in plants. Here we present a high-efficiency multigene assembly and transformation vector system, TransGene Stacking II (TGS II), for these purposes. The operation process is described in detail, and the successful operation mainly depends on effective reagents, special Escherichia coli strains, and basic molecular biological means without other specific equipments.

Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) is involved in regulating male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, swollen anther wall 1 (SAW1), for anther development in rice (Oryza sativa L.). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

Plasma membrane-localized OsLecRK5 enhances the activity of UGP1 by phosphorylation for callose biosynthesis during microsporogenesis in rice., The temporary callose layer surrounding the tetrads of microspores is critical for male gametophyte development in flowering plants, as abnormal callose deposition can lead to microspore abortion. A sophisticated signaling network regulates callose biosynthesis but these pathways are poorly understood. In this study, we characterized a rice male-sterile mutant, oslecrk5, which showed defective callose deposition during meiosis. OsLecRK5 encodes a plasma membrane-localized lectin receptor-like kinase, which can form a dimer with itself. Moreover, normal anther development requires the K-phosphorylation site (a conserved residue at the ATP-binding site) of OsLecRK5. In vitro assay showed that OsLecRK5 phosphorylates the callose synthesis enzyme UGP1, enhancing callose biosynthesis during anther development. Together, our results demonstrate that plasma membrane-localized OsLecRK5 phosphorylates UGP1 and promotes its activity in callose biosynthesis in rice. This is the first evidence that a receptor-like kinase positively regulates callose biosynthesis.

Genome editing with CRISPR/Cas9 has become an effective tool for gene functionality dissection and crop improvement. Accordingly, we have developed a CRISPR/Cas9 vector system for robust multi-gene editing in plants. Our inventory includes six versatile binary vectors and 12 intermediary sgRNA vectors with different U3 and U6 promoters, to achieve high efficient multi-gene editing, under specific antibiotic selections in both monocot and dicot plants. Additionally, we also have developed an integrated web-based software toolkit, CRISPR-GE, to facilitate the utilization CRISPR/Cas9 in plants. In this protocol, we provide detailed procedures for using the CRISPR/Cas9 system, including target site selection, construction of CRISPR/Cas9 binary vectors, Sanger-sequencing-based genotyping of mutant sites, and troubleshooting for common problems.

Use of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) and Cpf1 systems in plants (Ma et al., 2016; Wang et al., 2017) involves many steps, including the selection of appropriate specific target site(s) that should have no highly homologous sequences as the potential off-target sites in the genome, the design and synthesis of oligonucleotides involving the target sequences, the preparation of expression cassette(s) for the target single guide RNAs (sgRNAs) that provide target sequence specificity, the construction of plant-transformation/expression vector(s), and the transformation of plants, followed by the detection and determination of the targeted mutations in the transgenic plants.

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA samples. Here we describe protocols for preparation of rice plant gDNAs for various applications including: (1) maxipreps and (2) minipreps of gDNAs for restriction analysis, gene cloning, PCR amplification, genomic sequencing, and genotyping; (3) preparation of megabase-sized nuclear DNA for construction of large-insert genomic libraries and long-range physical mapping; and (4) 96-well-format high-throughput gDNA preparation for PCR-based genotyping. The methods are also suitable for other plant species including dicots. © 2016 by John Wiley & Sons, Inc.

Hybrid rice has contributed greatly to global food security. Cytoplasmic male sterility (CMS) and photo/ thermo sensitive genic male sterility (P/TGMS) are genetic bases for three-line and two-line hybrid rice production, respectively. In contrast, (sub-) specific hybrid sterility (HS) is a major barrier for utilization of hybrid vigor of distant hybrid rice. Therefore, understanding the molecular regulatory mechanism of rice fertility is a key technical issue for hybrid rice industry, and a long-standing basic scientific issue for nuclear-cytoplasmic interaction and reproductive isolation. Chinese geneticists of plant sciences have made tremendous contributions on the molecular genetic basis of rice fertility related to hybrid rice production. Here, we review the development of hybrid rice production systems in China and summarize current advance on genetic basis and molecular mechanism of CMS, P/TGMS, and HS involved in hybrid rice. We also discuss problems of hybrid rice production in China and point out new direction for future utilization of heterosis in rice.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

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.

The discovery and exploitation of the Wild Abortive type cytoplasmic male sterility (CMS-WA) is a key issue in hybrid rice breeding for utilization of heterosis, which is the best successful case for exploitation of wild plant resources in agriculture. In this review, we traced the history of discovery and research of CMS-WA, and introduced the latest progresses on study of the molecular basis of CMS-WA and its restoration as well as the evolution of the related genes. The Chinese scientist Yuan LongPing pioneered the study of male sterility in rice since 1964. In 1970s, Yuan’s group discovered a male sterile wild rice ( Oryza rufipogon Griff.) plant in Hainan Island of China, and designated this material as “Wild Abortive (WA)”. Genetic study showed that this male sterility is conferred by the cytoplasm, thus is called CMS-WA. Chinese breeders made great efforts to breed CMS lines by introgression of the CMS-WA cytoplasm into a number of rice cultivars via backcrossing. Accordingly, related maintainer lines and restorer lines were bred to successfully develop the “three-line” system for hybrid rice production. The commercial hybrid rice was released in 1976 in China, and has occupied about 50% of the total rice planting area since the late 1980s, which increased grain yield by 20%–30%. In recent years, scientists have made great progress on the molecular studies of CMS-WA and its restoration, and the evolution of the genes for this system. As representative in this field, LIU YaoGuang’s group isolated the CMS-WA gene WA352 from the mitochondrial genome, which is a new gene consisting of multiple mitochondrial genomic segments of unknown function. WA352 is expressed constitutively into three transcripts, but the WA352 protein accumulates specifically in the anther tapetal cells at the microspore mother cell stage, and interacts directly with a nucleus-encoded protein COX11, which is the assembly factor for cytochrome c oxidase and also has a role in scavenge of reactive oxygen species (ROS) and inhibition of programmed cell death (PCD). When WA352 arrests COX11 by the interaction, the metabolism of ROS is affected, leading to ROS burst and cytochrome c release to the cytosol and triggering premature PCD in the tapetum. This abnormal tapetal degeneration eventually causes pollen abortion. Two loci, Rf3 and Rf4 , were mapped on chromosome 1 and 10, respectively, as the restorer genes for CMS-WA. Rf4 was firstly cloned recently by Liu’s group. Rf4 encodes a pentatricopeptide repeat (PPR) protein (PPR9-782-M) possessing a mitochondrial transit signal and 18 PPR motifs, with high similarity to PPR3-791-M encoded by Rf1a of the CMS-BT system. The Rf4 protein decreases the mRNA level of WA352 , thereby restoring WA352 -mediated male sterility probably in a post-transcriptional mechanism. However, Rf3 does not affect the WA352 transcript abundance but impairs the production of WA352 protein. Thus, the studies indicate that plant CMS/restoration systems comprises multiple layers of cytoplasmic-nuclear gene interactions in the molecular level. Recently, LIU’s group further studied the evolutionary trajectory of WA352 by identification and characterization of a number of mitochondrial genomic recombinant structures related to WA352 in the wild rice. These structures originated and evolved through complex evolutionary routes by multiple rearrangements in the mitochondrial genome of O. rufipogon. The study revealed that functional CMS genes originated from non-CMS protogenes by sequence variation-based functionalization as well as substoichiometric shifting (i.e., copy number variation). Finally, this review discussed the key issues and future directions of the hybrid rice breeding programs.

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a “multi-recombination/protogene formation/functionalization” mechanism involving gradual variations in the structure, sequence, copy number, and function.