A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Precision genome editing heralds rapid de novo domestication for new crops.

The de novo domestication has the potential to rapidly capitalize on desirable traits of wild plants. In this issue of Cell, Yu et al. report a route of de novo domestication of an allotetraploid rice, heralding the creation of a novel staple food crop to support global food security.

Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

A route to de novo domestication of wild allotetraploid rice.

Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.

Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector.

Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.

A phosphate starvation response-centered network regulates mycorrhizal symbiosis.

To secure phosphorus (P) from soil, most land plants use a direct phosphate uptake pathway via root hairs and epidermis and an indirect phosphate uptake pathway via mycorrhizal symbiosis. The interaction between these two pathways is unclear. Here, we mapped a network between transcription factors and mycorrhizal symbiosis-related genes using Y1H. Intriguingly, this gene regulatory network is governed by the conserved P-sensing pathway, centered on phosphate starvation response (PHR) transcription factors. PHRs are required for mycorrhizal symbiosis and regulate symbiosis-related genes via the P1BS motif. SPX-domain proteins suppress OsPHR2-mediated induction of symbiosis-related genes and inhibit mycorrhizal infection. In contrast, plants overexpressing OsPHR2 show improved mycorrhizal infection and are partially resistant to P-mediated inhibition of symbiosis. Functional analyses of network nodes revealed co-regulation of hormonal signaling and mycorrhizal symbiosis. This network deciphers extensive regulation of mycorrhizal symbiosis by endogenous and exogenous signals and highlights co-option of the P-sensing pathway for mycorrhizal symbiosis.

A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance.

Rice feeds half the world's population, and rice blast is often a destructive disease that results in significant crop loss. Non-race-specific resistance has been more effective in controlling crop diseases than race-specific resistance because of its broad spectrum and durability. Through a genome-wide association study, we report the identification of a natural allele of a C2H2-type transcription factor in rice that confers non-race-specific resistance to blast. A survey of 3,000 sequenced rice genomes reveals that this allele exists in 10% of rice, suggesting that this favorable trait has been selected through breeding. This allele causes a single nucleotide change in the promoter of the bsr-d1 gene, which results in reduced expression of the gene through the binding of the repressive MYB transcription factor and, consequently, an inhibition of H2O2 degradation and enhanced disease resistance. Our discovery highlights this novel allele as a strategy for breeding durable resistance in rice.

COLD1 confers chilling tolerance in rice.

Rice is sensitive to cold and can be grown only in certain climate zones. Human selection of japonica rice has extended its growth zone to regions with lower temperature, while the molecular basis of this adaptation remains unknown. Here, we identify the quantitative trait locus COLD1 that confers chilling tolerance in japonica rice. Overexpression of COLD1(jap) significantly enhances chilling tolerance, whereas rice lines with deficiency or downregulation of COLD1(jap) are sensitive to cold. COLD1 encodes a regulator of G-protein signaling that localizes on plasma membrane and endoplasmic reticulum (ER). It interacts with the G-protein α subunit to activate the Ca(2+) channel for sensing low temperature and to accelerate G-protein GTPase activity. We further identify that a SNP in COLD1, SNP2, originated from Chinese Oryza rufipogon, is responsible for the ability of COLD(jap/ind) to confer chilling tolerance, supporting the importance of COLD1 in plant adaptation.

Transposase-assisted target-site integration for efficient plant genome engineering.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

NLRs guard metabolism to coordinate pattern- and effector-triggered immunity.

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants enable them to respond to pathogens by activating the production of defence metabolites that orchestrate immune responses1-4. How the production of defence metabolites is promoted by immune receptors and coordinated with broad-spectrum resistance remains elusive. Here we identify the deubiquitinase PICI1 as an immunity hub for PTI and ETI in rice (Oryza sativa). PICI1 deubiquitinates and stabilizes methionine synthetases to activate methionine-mediated immunity principally through biosynthesis of the phytohormone ethylene. PICI1 is targeted for degradation by blast fungal effectors, including AvrPi9, to dampen PTI. Nucleotide-binding domain, leucine-rich-repeat-containing receptors (NLRs) in the plant immune system, such as PigmR, protect PICI1 from effector-mediated degradation to reboot the methionine-ethylene cascade. Natural variation in the PICI1 gene contributes to divergence in basal blast resistance between the rice subspecies indica and japonica. Thus, NLRs govern an arms race with effectors, using a competitive mode that hinges on a critical defence metabolic pathway to synchronize PTI with ETI and ensure broad-spectrum resistance.

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

DELLA-mediated gene repression is maintained by chromatin modification in rice.

DELLA proteins are master regulators of gibberellic acid (GA) signaling through their effects on gene expression. Enhanced DELLA accumulation in rice and wheat varieties has greatly contributed to grain yield increases during the green revolution. However, the molecular basis of DELLA-mediated gene repression remains elusive. In this work, we show that the rice DELLA protein SLENDER RICE1 (SLR1) forms a tripartite complex with Polycomb-repressive complex 2 (PRC2) and the histone deacetylase HDA702 to repress downstream genes by establishing a silent chromatin state. The slr1 mutation and GA signaling resulted in dissociation of PRC2 and HDA702 from GA-inducible genes. Loss-of-function or downregulation of the chromatin regulators impaired SLR1-dependent histone modification and gene repression. Time-resolved analysis of GA signaling revealed that GA-induced transcriptional activation was associated with a rapid increase of H3K9ac followed by H3K27me3 removal. Collectively, these results establish a general epigenetic mechanism for DELLA-mediated gene repression and reveal details of the chromatin dynamics during transcriptional activation stimulated by GA signaling.

Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice.

Unusually low temperatures caused by global climate change adversely affect rice production. Sensing cold to trigger signal network is a key base for improvement of chilling tolerance trait.  Here, we report that Oryza sativa Calreticulin 3 (OsCRT3) localized at the endoplasmic reticulum (ER) exhibits conformational changes under cold stress, thereby enhancing its interaction with CBL-interacting protein kinase 7 (OsCIPK7) to sense cold. Phenotypic analyses of OsCRT3 knock-out mutants and transgenic overexpression lines demonstrate that OsCRT3 is a positive regulator in chilling tolerance. OsCRT3 localizes at the ER and mediates increases in cytosolic calcium levels under cold stress. Notably, cold stress triggers secondary structural changes of OsCRT3 and enhances its binding affinity with OsCIPK7, which finally boosts its kinase activity. Moreover, Calcineurin B-like protein 7 (OsCBL7) and OsCBL8 interact with OsCIPK7 specifically on the plasma membrane. Taken together, our results thus identify a cold-sensing mechanism that simultaneously conveys cold-induced protein conformational change, enhances kinase activity, and Ca2+ signal generation to facilitate chilling tolerance in rice.

Adaxial-abaxial bipolar leaf genes encode a putative cytokinin receptor and HD-Zip III, and control the formation of ectopic shoot meristems in rice.

Shoot apical meristems (SAMs) continuously initiate organ formation and maintain pluripotency through dynamic genetic regulations and cell-to-cell communications. The activity of meristems directly affects the plant's structure by determining the number and arrangement of organs and tissues. We have taken a forward genetic approach to dissect the genetic pathway that controls cell differentiation around the SAM. The rice mutants, adaxial-abaxial bipolar leaf 1 and 2 (abl1 and abl2), produce an ectopic leaf that is fused back-to-back with the fourth leaf, the first leaf produced after embryogenesis. The abaxial-abaxial fusion is associated with the formation of an ectopic shoot meristem at the adaxial base of the fourth leaf primordium. We cloned the ABL1 and ABL2 genes of rice by mapping their chromosomal positions. ABL1 encodes OsHK6, a histidine kinase, and ABL2 encodes a transcription factor, OSHB3 (Class III homeodomain leucine zipper). Expression analyses of these mutant genes as well as OSH1, a rice ortholog of the Arabidopsis STM gene, unveiled a regulatory circuit that controls the formation of an ectopic meristem near the SAM at germination.

Small peptide signaling via OsCIF1/2 mediates Casparian strip formation at the root endodermal and nonendodermal cell layers in rice.

The Casparian strip (CS) is a ring-like lignin structure deposited between endodermal cells that forms an apoplastic barrier to control the selective uptake of nutrients in vascular plants. However, the molecular mechanism of CS formation in rice (Oryza sativa), which possesses one CS each in the endodermis and exodermis, is relatively unknown. Here, we functionally characterized CS INTEGRITY FACTOR1 (OsCIF1a, OsCIF1b), OsCIF2, and SCHENGEN3 (OsSGN3a, OsSGN3b) in rice. OsCIF1s and OsCIF2 were mainly expressed in the stele, while OsSGN3s localized around the CS at the endodermis. Knockout of all three OsCIFs or both OsSGN3s resulted in a discontinuous CS and a dramatic reduction in compensatory (less localized) lignification and suberization at the endodermis. By contrast, ectopic overexpression of OsCIF1 or OsCIF2 induced CS formation as well as overlignification and oversuberization at single or double cortical cell layers adjacent to the endodermis. Ectopic co-overexpression of OsCIF1 and SHORTROOT1 (OsSHR1) induced the formation of more CS-like structures at multiple cortical cell layers. Transcriptome analysis identified 112 downstream genes modulated by the OsCIF1/2-OsSGN3 signaling pathway, which is involved in CS formation and activation of the compensatory machinery in native endodermis and nonnative endodermis-like cell layers. Our results provide important insights into the molecular mechanism of CIF-mediated CS formation at the root endodermal and nonendodermal cell layers.

Population-level exploration of alternative splicing and its unique role in controlling agronomic traits of rice.

Alternative splicing (AS) plays crucial roles in regulating various biological processes in plants. However, the genetic mechanisms underlying AS and its role in controlling important agronomic traits in rice (Oryza sativa) remain poorly understood. In this study, we explored AS in rice leaves and panicles using the rice minicore collection. Our analysis revealed a high level of transcript isoform diversity, with approximately one-fifth of the potential isoforms acting as major transcripts in both tissues. Regarding the genetic mechanism of AS, we found that the splicing of 833 genes in the leaf and 1,230 genes in the panicle was affected by cis-genetic variation. Twenty-one percent of these AS events could only be explained by large structural variations. Approximately 77.5% of genes with significant splicing quantitative trait loci (sGenes) exhibited tissue-specific regulation, and AS can cause 26.9% (leaf) and 23.6% (panicle) of sGenes to have altered, lost, or gained functional domains. Additionally, through splicing-phenotype association analysis, we identified phosphate-starvation-induced RING-type E3 ligase (OsPIE1; LOC_Os01g72480), whose splicing ratio was significantly associated with plant height. In summary, this study provides an understanding of AS in rice and its contribution to the regulation of important agronomic traits.

Pan-transcriptomic analysis reveals alternative splicing control of cold tolerance in rice.

Plants have evolved complex mechanisms to adapt to harsh environmental conditions. Rice (Oryza sativa) is a staple food crop that is sensitive to low temperatures. However, its cold stress responses remain poorly understood, thus limiting possibilities for crop engineering to achieve greater cold tolerance. In this study, we constructed a rice pan-transcriptome and characterized its transcriptional regulatory landscape in response to cold stress. We performed Iso-Seq and RNA-Seq of 11 rice cultivars subjected to a time-course cold treatment. Our analyses revealed that alternative splicing-regulated gene expression plays a significant role in the cold stress response. Moreover, we identified CATALASE C (OsCATC) and Os03g0701200 as candidate genes for engineering enhanced cold tolerance. Importantly, we uncovered central roles for the 2 serine-arginine-rich proteins OsRS33 and OsRS2Z38 in cold tolerance. Our analysis of cold tolerance and resequencing data from a diverse collection of 165 rice cultivars suggested that OsRS2Z38 may be a key selection gene in japonica domestication for cold adaptation, associated with the adaptive evolution of rice. This study systematically investigated the distribution, dynamic changes, and regulatory mechanisms of alternative splicing in rice under cold stress. Overall, our work generates a rich resource with broad implications for understanding the genetic basis of cold response mechanisms in plants.

Transcription factors WOX11 and LBD16 function with histone demethylase JMJ706 to control crown root development in rice.

Crown roots are the main components of root systems in cereals. Elucidating the mechanisms of crown root formation is instrumental for improving nutrient absorption, stress tolerance, and yield in cereal crops. Several members of the WUSCHEL-related homeobox (WOX) and lateral organ boundaries domain (LBD) transcription factor families play essential roles in controlling crown root development in rice (Oryza sativa). However, the functional relationships among these transcription factors in regulating genes involved in crown root development remain unclear. Here, we identified LBD16 as an additional regulator of rice crown root development. We showed that LBD16 is a direct downstream target of WOX11, a key crown root development regulator in rice. Our results indicated that WOX11 enhances LBD16 transcription by binding to its promoter and recruiting its interaction partner JMJ706, a demethylase that removes histone H3 lysine 9 dimethylation (H3K9me2) from the LBD16 locus. In addition, we established that LBD16 interacts with WOX11, thereby impairing JMJ706-WOX11 complex formation and repressing its own transcriptional activity. Together, our results reveal a feedback system regulating genes that orchestrate crown root development in rice, in which LBD16 acts as a molecular rheostat.

Histone deacetylase OsHDA716 represses rice chilling tolerance by deacetylating OsbZIP46 to reduce its transactivation function and protein stability.

Low temperature is a major environmental factor limiting plant growth and crop production. Epigenetic regulation of gene expression is important for plant adaptation to environmental changes, whereas the epigenetic mechanism of cold signaling in rice (Oryza sativa) remains largely elusive. Here, we report that the histone deacetylase (HDAC) OsHDA716 represses rice cold tolerance by interacting with and deacetylating the transcription factor OsbZIP46. The loss-of-function mutants of OsHDA716 exhibit enhanced chilling tolerance, compared with the wild-type plants, while OsHDA716 overexpression plants show chilling hypersensitivity. On the contrary, OsbZIP46 confers chilling tolerance in rice through transcriptionally activating OsDREB1A and COLD1 to regulate cold-induced calcium influx and cytoplasmic calcium elevation. Mechanistic investigation showed that OsHDA716-mediated OsbZIP46 deacetylation in the DNA-binding domain reduces the DNA-binding ability and transcriptional activity as well as decreasing OsbZIP46 protein stability. Genetic evidence indicated that OsbZIP46 deacetylation mediated by OsHDA716 reduces rice chilling tolerance. Collectively, these findings reveal that the functional interplay between the chromatin regulator and transcription factor fine-tunes the cold response in plant and uncover a mechanism by which HDACs repress gene transcription through deacetylating nonhistone proteins and regulating their biochemical functions.