An ATP-binding cassette transporter, OsABCB24, is involved in female gametophyte development and early seed growth in rice.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP-binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%-100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two-nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane-localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5- to 2-fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

CRISPR enables sustainable cereal production for a greener future.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.

Recent Molecular Aspects and Integrated Omics Strategies for Understanding the Abiotic Stress Tolerance of Rice.

Rice is an important staple food crop for over half of the world's population. However, abiotic stresses seriously threaten rice yield improvement and sustainable production. Breeding and planting rice varieties with high environmental stress tolerance are the most cost-effective, safe, healthy, and environmentally friendly strategies. In-depth research on the molecular mechanism of rice plants in response to different stresses can provide an important theoretical basis for breeding rice varieties with higher stress resistance. This review presents the molecular mechanisms and the effects of various abiotic stresses on rice growth and development and explains the signal perception mode and transduction pathways. Meanwhile, the regulatory mechanisms of critical transcription factors in regulating gene expression and important downstream factors in coordinating stress tolerance are outlined. Finally, the utilization of omics approaches to retrieve hub genes and an outlook on future research are prospected, focusing on the regulatory mechanisms of multi-signaling network modules and sustainable rice production.

Detection of QTLs Regulating Six Agronomic Traits of Rice Based on Chromosome Segment Substitution Lines of Common Wild Rice (Oryza rufipogon Griff.) and Mapping of qPH1.1 and qLMC6.1.

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide.

CRISPR/Cas-mediated editing of cis-regulatory elements for crop improvement.

To improve future agricultural production, major technological advances are required to increase crop production and yield. Targeting the coding region of genes via the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has been well established and has enabled the rapid generation of transgene-free plants, which can lead to crop improvement. The emergence of the CRISPR/Cas system has also enabled scientists to achieve cis-regulatory element (CRE) editing and, consequently, engineering endogenous critical CREs to modulate the expression of target genes. Recent genome-wide association studies have identified the domestication of natural CRE variants to regulate complex agronomic quantitative traits and have allowed for their engineering via the CRISPR/Cas system. Although engineering plant CREs can be advantageous to drive gene expression, there are still many limitations to its practical application. Here, we review the current progress in CRE editing and propose future strategies to effectively target CREs for transcriptional regulation for crop improvement.

Epitranscriptomics: An Additional Regulatory Layer in Plants' Development and Stress Response.

Epitranscriptomics has added a new layer of regulatory machinery to eukaryotes, and the advancement of sequencing technology has revealed more than 170 post-transcriptional modifications in various types of RNAs, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and long non-coding RNA (lncRNA). Among these, N6-methyladenosine (m6A) and N5-methylcytidine (m5C) are the most prevalent internal mRNA modifications. These regulate various aspects of RNA metabolism, mainly mRNA degradation and translation. Recent advances have shown that regulation of RNA fate mediated by these epitranscriptomic marks has pervasive effects on a plant's development and responses to various biotic and abiotic stresses. Recently, it was demonstrated that the removal of human-FTO-mediated m6A from transcripts in transgenic rice and potatoes caused a dramatic increase in their yield, and that the m6A reader protein mediates stress responses in wheat and apple, indicating that regulation of m6A levels could be an efficient strategy for crop improvement. However, changing the overall m6A levels might have unpredictable effects; therefore, the identification of precise m6A levels at a single-base resolution is essential. In this review, we emphasize the roles of epitranscriptomic modifications in modulating molecular, physiological, and stress responses in plants, and provide an outlook on epitranscriptome engineering as a promising tool to ensure food security by editing specific m6A and m5C sites through robust genome-editing technology.

Recent Insights into Anthocyanin Pigmentation, Synthesis, Trafficking, and Regulatory Mechanisms in Rice (Oryza sativa L.) Caryopsis.

Anthocyanins are antioxidants used as natural colorants and are beneficial to human health. Anthocyanins contribute to reactive oxygen species detoxification and sustain plant growth and development under different environmental stresses. They are phenolic compounds that are broadly distributed in nature and are responsible for a wide range of attractive coloration in many plant organs. Anthocyanins are found in various parts of plants such as flowers, leaves, stems, shoots, and grains. Considering their nutritional and health attributes, anthocyanin-enriched rice or pigmented rice cultivars are a possible alternative to reduce malnutrition around the globe. Anthocyanin biosynthesis and storage in rice are complex processes in which several structural and regulatory genes are involved. In recent years, significant progress has been achieved in the molecular and genetic mechanism of anthocyanins, and their synthesis is of great interest to researchers and the scientific community. However, limited studies have reported anthocyanin synthesis, transportation, and environmental conditions that can hinder anthocyanin production in rice. Rice is a staple food around the globe, and further research on anthocyanin in rice warrants more attention. In this review, metabolic and pre-biotic activities, the underlying transportation, and storage mechanisms of anthocyanins in rice are discussed in detail. This review provides potential information for the food industry and clues for rice breeding and genetic engineering of rice.

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

Computational and Transcriptomic Analysis Unraveled OsMATE34 as a Putative Anthocyanin Transporter in Black Rice (Oryza sativa L.) Caryopsis.

Anthocyanin is a flavonoid compound with potential antioxidant properties beneficial to human health and sustains plant growth and development under different environmental stresses. In black rice, anthocyanin can be found in the stems, leaves, stigmas, and caryopsis. Although the anthocyanin biosynthesis in rice has been extensively studied, limited knowledge underlying the storage mechanism and transporters is available. This study undertook the complementation of computational and transcriptome analysis to decipher a potential multidrug and toxic compound extrusion (MATE) gene candidate for anthocyanin transportation in black rice caryopsis. The phylogenetic analysis showed that OsMATE34 has the same evolutionary history and high similarities with VvAM1, VvAM3, MtMATE2, SlMATE/MTP77, RsMATE8, AtFFT, and AtTT12 involved in anthocyanin transportation. RNA sequencing analysis in black caryopsis (Bc; Bc11, Bc18, Bc25) and white caryopsis (Wc; Wc11, Wc18, Wc25), respectively, at 11 days after flowering (DAF), 18 DAF, and 25 DAF revealed a total of 36,079 expressed genes, including 33,157 known genes and 2922 new genes. The differentially expressed genes (DEGs) showed 15,573 genes commonly expressed, with 1804 and 1412 genes uniquely expressed in Bc and Wc, respectively. Pairwise comparisons showed 821 uniquely expressed genes out of 15,272 DEGs for Wc11 vs. Bc11, 201 uniquely expressed genes out of 16,240 DEGs for Wc18 vs. Bc18, and 2263 uniquely expressed genes out of 16,240 DEGs for Wc25 vs. Bc25. Along with anthocyanin biosynthesis genes (OsPAL, OsCHS, OsCHI, OsF3H, OsDFR, OsANS, and OsUFGT/Os3GT), OsMATE34 expression was significantly upregulated in all Bc but not in Wc. OsMATE34 expression was similar to OsGSTU34, a transporter of anthocyanin in rice leaves. Taken together, our results highlighted OsMATE34 (Os08g0562800) as a candidate anthocyanin transporter in rice caryopsis. This study provides a new finding and a clue to enhance the accumulation of anthocyanin in rice caryopsis.

Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins.

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein-protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance.

Development of Chromosome Segment Substitution Lines (CSSLs) Derived from Guangxi Wild Rice (Oryza rufipogon Griff.) under Rice (Oryza sativa L.) Background and the Identification of QTLs for Plant Architecture, Agronomic Traits and Cold Tolerance.

Common wild rice contains valuable resources of novel alleles for rice improvement. It is well known that genetic populations provide the basis for a wide range of genetic and genomic studies. In particular, chromosome segment substitution lines (CSSLs) ais a powerful tool for fine mapping of quantitative traits, new gene discovery and marker-assisted breeding. In this study, 132 CSSLs were developed from a cultivated rice (Oryza sativa) cultivar (93-11) and common wild rice (Oryza rufipogon Griff. DP30) by selfing-crossing, backcrossing and marker-assisted selection (MAS). Based on the high-throughput sequencing of the 93-11 and DP30, 285 pairs of Insertion-deletions (InDel) markers were selected with an average distance of 1.23 Mb. The length of this DP30-CSSLs library was 536.4 cM. The coverage rate of substitution lines cumulatively overlapping the whole genome of DP30 was about 91.55%. DP30-CSSLs were used to analyze the variation for 17 traits leading to the detection of 36 quantitative trait loci (QTLs) with significant phenotypic effects. A cold-tolerant line (RZ) was selected to construct a secondary mapping F2 population, which revealed that qCT2.1 is in the 1.7 Mb region of chromosome 2. These CSSLs may, therefore, provide powerful tools for genome wide large-scale gene discovery in wild rice. This research will also facilitate fine mapping and cloning of QTLs and genome-wide study of wild rice. Moreover, these CSSLs will provide a foundation for rice variety improvement.

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line.

Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.

Generation of High Yielding and Fragrant Rice (Oryza sativa L.) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations.

The significant increase in grain yield and quality are often antagonistic but a constant demand for breeders and consumers. Some genes related to cytochrome P450 family are known for rice organ growth but their role in controlling grain yield is still unknown. Here, we generated new rice mutants with high yield and improved aroma by simultaneously editing three cytochrome P450 homoeologs (Os03g0603100, Os03g0568400, and GL3.2) and OsBADH2 with the CRISPR/Cas9 system, and RNA-sequencing and proteomic analysis were performed to unveil the subsequent changes. High mutation efficiency was achieved in both target sites of each gene and the mutations were predominantly only deletions, while insertions were rare, and no mutations were detected in the five most likely off-target sites against each sgRNA. Mutants exhibited increased grain size, 2-acetyl-1-pyrroline (2AP) content, and grain cell numbers while there was no change in other agronomic traits. Transgene-DNA-free mutant lines appeared with a frequency of 44.44% and homozygous mutations were stably transmitted, and bi-allelic and heterozygous mutations followed Mendelian inheritance, while the inheritance of chimeric mutations was unpredictable. Deep RNA sequencing and proteomic results revealed the regulation of genes and proteins related to cytochrome P450 family, grain size and development, and cell cycle. The KEGG and hub-gene and protein network analysis showed that the gene and proteins related to ribosomal and photosynthesis pathways were mainly enriched, respectively. Our findings provide a broad and detailed basis to understand the role of CRISPR/Cas9 in rice yield and quality improvement.

Programmed Editing of Rice (Oryza sativa L.) OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of Pyruvate Enzymes and Cell Cycle Proteins.

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.

Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations.

Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the yield potential and lodging resistance. Here, semi-dwarf mutant lines were developed through CRISPR/Cas9-based editing of OsGA20ox2 in an indica rice cultivar. Total 24 independent lines were obtained in T0 generation with the mean mutation rate of 73.5% including biallelic (29.16%), homozygous (47.91%) and heterozygous (16.66%) mutations, and 16 T-DNA-free lines (50%) were obtained in T1 generation without off-target effect in four most likely sites. Mutations resulted in a changed amino acid sequence of mutant plants and reduced gibberellins (GA) level and PH (22.2%), flag leaf length (FLL) and increased yield per plant (YPP) (6.0%), while there was no effect on other agronomic traits. Mutants restored their PH to normal by exogenous GA3 treatment. The expression of the OsGA20ox2 gene was significantly suppressed in mutant plants, while the expression level was not affected for other GA biosynthesis (OsGA2ox3 and OsGA3ox2) and signaling (D1, GIDI and SLR1) genes. The mutant lines showed decreased cell length and width, abnormal cell elongation, while increased cell numbers in the second internode sections at mature stage. Total 30 protein spots were exercised, and 24 proteins were identified, and results showed that OsGA20ox2 editing altered protein expression. Five proteins including, glyceraldehyde-3-phosphate dehydrogenase, putative ATP synthase, fructose-bisphosphate aldolase 1, S-adenosyl methionine synthetase 1 and gibberellin 20 oxidase 2, were downregulated in dwarf mutant lines which may affect the plant growth. Collectively, our results provide the insights into the role of OsGA20ox2 in PH and confirmed that CRISPR-Cas9 is a powerful tool to understand the gene functions.

High-resolution mapping and breeding application of a novel brown planthopper resistance gene derived from wild rice (Oryza. rufipogon Griff).

BACKGROUND: The brown planthopper (Nilaparvata lugens Stål; BPH), one of the most destructive pests of rice, has proven to be a substantial threat, conferring enormous production losses in Asia and becoming a difficult challenge to manipulate and control under field conditions. The continuous use of insecticides promotes the resurgence of BPH, which results in resistant varieties adapting through the upgrading of new BPH biotypes. To overcome resistance acquired by BPH against resistance varieties, different forms of novel resistant gene fusions act as functional domains for breeding to enhance insect resistance. RESULTS: The current study reports on the novel BPH resistance gene Bph36 derived from two introgression lines (RBPH16 and RBPH17) developed from wild rice GX2183 which was previously reported to be resistant to BPH. Using two F2 crossing populations (Kangwenqizhan × RBPH16 and Huanghuazhan × RBPH17) in a bulked segregant analysis (BSA) for identification of resistant genes and QTL analysis, two QTLs for BPH resistance were generated on the long and short arms of chromosome 4, which was further confirmed by developing BC1F2:3 populations by backcrossing via marker assisted selection (MAS) approach. One BPH resistance locus on the short arm of chromosome 4 was mapped to a 38-kb interval flanked by InDel markers S13 and X48, and then was named Bph36, whereas another locus on the long arm of chromosome 4 was also detected in an interval flanked by RM16766 and RM17033, which was the same as that of Bph27. An evaluation analysis based on four parameters (BPH host selection, honeydew weight, BPH survival rate and BPH population growth rate) shows that Bph36 conferred high levels antibiosis and antixenosis to BPH. Moreover, Bph36 pyramided with Bph3, Bph27, and Bph29 through MAS into elite cultivars 9311 and MH511 (harbored Xa23), creating different background breeding lines that also exhibited strong resistance to BPH in the seedling or tillering stage. CONCLUSION: Bph36 can be utilized in BPH resistance breeding programs to develop high resistant rice lines and the high-resolution fine mapping will facilitate further map-based cloning and marker-assisted gene pyramiding of resistant gene. MAS exploited to pyramid with Bph3, Bph27, Bph29, and Xa23 was confirmed the effectiveness for BPH resistance breeding in rice and provided insights into the molecular mechanism of defense to control this devastating insect.

Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage.

Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.