Pijx confers broad-spectrum seedling and panicle blast resistance by promoting the degradation of ATP ß subunit and OsRbohC-mediated ROS burst in rice.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most important diseases of rice. Utilization of blast-resistance genes is the most economical, effective, and environmentally friendly way to control the disease. However, genetic resources with broad-spectrum resistance (BSR) that is effective throughout the rice growth period are rare. In this work, using a genome-wide association study, we identify a new blast-resistance gene, Pijx, which encodes a typical CC-NBS-LRR protein. Pijx is derived from a wild rice species and confers BSR to M. oryzae at both the seedling and panicle stages. The functions of the resistant haplotypes of Pijx are confirmed by gene knockout and overexpression experiments. Mechanistically, the LRR domain in Pijx interacts with and promotes the degradation of the ATP synthase ß subunit (ATPb) via the 26S proteasome pathway. ATPb acts as a negative regulator of Pijx-mediated panicle blast resistance, and interacts with OsRbohC to promote its degradation. Consistently, loss of ATPb function causes an increase in NAPDH content and ROS burst. Remarkably, when Pijx is introgressed into two japonica rice varieties, the introgression lines show BSR and increased yields that are approximately 51.59% and 79.31% higher compared with those of their parents in a natural blast disease nursery. In addition, we generate PPLPijx Pigm and PPLPijx Piz-t pyramided lines and these lines also have higher BSR to panicle blast compared with Pigm- or Piz-t-containing rice plants. Collectively, this study demonstrates that Pijx not only confers BSR to M. oryzae but also maintains high and stable rice yield, providing new genetic resources and molecular targets for breeding rice varieties with broad-spectrum blast resistance.

CRISPR-Cas9-mediated editing of the OsHPPD 3' UTR confers enhanced resistance to HPPD-inhibiting herbicides in rice.

This study reports the creation of herbicide-resistant rice lines via CRISPR-Cas9-mediated editing of the 3' UTR of OsHPPD. Resistance index calculations revealed that two resistant lines, TS8-2#-10 and TS8-8#-6, exhibited 4.8-fold and 3.7-fold greater resistance to HPPD-inhibiting herbicides compared with the wild type, YG3012.

Substandard starch grain4 may function in amyloplast development by influencing starch and lipid metabolism in rice endosperm.

The amyloplast is a specialized plastid in rice endosperm cells where starch is synthesized and stored as starch granules (SGs). However, little is known about the molecular mechanism underlying amyloplast and SG development. In this study, a novel mutant (c134) demonstrating a floury endosperm with enlarged SGs and amyloplasts was identified. The floury endosperm was caused by rounder, loosely packed SG. Grain-quality profile and expression analysis showed reduced contents of total starch and amylose in the c134 mutant, as well as reduced expression of a number of genes involved in starch biosynthesis. Galactosyldiacylglycerol (GDG) content and fatty acid synthesis play important roles in plastid development, and in the c134 endosperm, an obvious decrease in GDG and various fatty acids was observed, with down-regulated expression of various genes involved in lipid biosynthesis. Furthermore, map-based cloning revealed an amino acid substitution (glycine to aspartic acid) in the substandard starch grain4 (SSG4) protein. The results of this study suggest that SSG4 influences the regulation of starch and lipid metabolism as well as amyloplast development, a finding that is useful for potential genetic improvement of rice grain quality in future starch and lipid breeding and biotechnology.

Genomic insight into balancing high yield, good quality, and blast resistance of japonica rice.

BACKGROUND: Balancing the yield, quality and resistance to disease is a daunting challenge in crop breeding due to the negative relationship among these traits. Large-scale genomic landscape analysis of germplasm resources is considered to be an efficient approach to dissect the genetic basis of the complex traits. Central China is one of the main regions where the japonica rice is produced. However, dozens of high-yield rice varieties in this region still exist with low quality or susceptibility to blast disease, severely limiting their application in rice production. RESULTS: Here, we re-sequence 200 japonica rice varieties grown in central China over the past 30 years and analyze the genetic structure of these cultivars using 2.4 million polymorphic SNP markers. Genome-wide association mapping and selection scans indicate that strong selection for high-yield and taste quality associated with low-amylose content may have led to the loss of resistance to the rice blast fungus Magnaporthe oryzae. By extensive bioinformatic analyses of yield components, resistance to rice blast, and taste quality, we identify several superior alleles for these traits in the population. Based on this information, we successfully introduce excellent taste quality and blast-resistant alleles into the background of two high-yield cultivars and develop two elite lines, XY99 and JXY1, with excellent taste, high yield, and broad-spectrum of blast resistance. CONCLUSIONS: This is the first large-scale genomic landscape analysis of japonica rice varieties grown in central China and we demonstrate a balancing of multiple agronomic traits by genomic-based strategy.

Identification and fine mapping of qPBR10-1, a novel locus controlling panicle blast resistance in Pigm-containing P/TGMS line.

Rice blast is one of the most widespread and devastating diseases in rice production. Tremendous success has been achieved in the identification and characterization of genes and quantitative trait loci (QTLs) conferring seedling blast resistance, however, genetic studies on panicle blast resistance have lagged far behind. In this study, two advanced backcross inbred sister lines (MSJ13 and MSJ18) were obtained in the process of introducing Pigm into C134S and showed significant differences in the panicle blast resistance. One F2 population derived from the crossing MSJ13/MSJ18 was used to QTL mapping for panicle blast resistance using genotyping by sequencing (GBS) method. A total of seven QTLs were identified, including a major QTL qPBR10-1 on chromosome 10 that explains 24.21% of phenotypic variance with LOD scores of 6.62. Furthermore, qPBR10-1 was verified using the BC1F2 and BC1F3 population and narrowed to a 60.6-kb region with six candidate genes predicted, including two genes encoding exonuclease family protein, two genes encoding hypothetical protein, and two genes encoding transposon protein. The nucleotide variations and the expression patterns of the candidate genes were identified and analyzed between MSJ13 and MSJ18 through sequence comparison and RT-PCR approach, and results indicated that ORF1 and ORF2 encoding exonuclease family protein might be the causal candidate genes for panicle blast resistance in the qPBR10-1 locus. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01268-3.

Transcriptomic and Co-Expression Network Profiling of Shoot Apical Meristem Reveal Contrasting Response to Nitrogen Rate between Indica and Japonica Rice Subspecies.

Reducing nitrogen (N) input is a key measure to achieve a sustainable rice production in China, especially in Jiangsu Province. Tiller is the basis for achieving panicle number that plays as a major factor in the yield determination. In actual production, excessive N is often applied in order to produce enough tillers in the early stages. Understanding how N regulates tillering in rice plants is critical to generate an integrative management to reduce N use and reaching tiller number target. Aiming at this objective, we utilized RNA sequencing and weighted gene co-expression network analysis (WGCNA) to compare the transcriptomes surrounding the shoot apical meristem of indica (Yangdao6, YD6) and japonica (Nipponbare, NPB) rice subspecies. Our results showed that N rate influenced tiller number in a different pattern between the two varieties, with NPB being more sensitive to N enrichment, and YD6 being more tolerant to high N rate. Tiller number was positively related to N content in leaf, culm and root tissue, but negatively related to the soluble carbohydrate content, regardless of variety. Transcriptomic comparisons revealed that for YD6 when N rate enrichment from low (LN) to medium (MN), it caused 115 DEGs (LN vs. MN), from MN to high level (HN) triggered 162 DEGs (MN vs. HN), but direct comparison of low with high N rate showed a 511 DEGs (LN vs. HN). These numbers of DEG in NPB were 87 (LN vs. MN), 40 (MN vs. HN), and 148 (LN vs. HN). These differences indicate that continual N enrichment led to a bumpy change at the transcription level. For the reported sixty-five genes which affect tillering, thirty-six showed decent expression in SAM at tiller starting phase, among them only nineteen being significantly influenced by N level, and two genes showed significant interaction between N rate and variety. Gene ontology analysis revealed that the majority of the common DEGs are involved in general stress responses, stimulus responses, and hormonal signaling process. WGCNA network identified twenty-two co-expressing gene modules and ten candidate hubgenes for each module. Several genes associated with tillering and N rate fall on the related modules. These indicate that there are more genes participating in tillering regulation in response to N enrichment.

Global Transcriptome and Co-Expression Network Analysis Reveal Contrasting Response of Japonica and Indica Rice Cultivar to γ Radiation.

Japonica and indica are two important subspecies in cultivated Asian rice. Irradiation is a classical approach to induce mutations and create novel germplasm. However, little is known about the differential response between japonica and indica rice after γ radiation. Here, we utilized the RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA) to compare the transcriptome differences between japonica Nipponbare (NPB) and indica Yangdao6 (YD6) in response to irradiation. Japonica subspecies are more sensitive to irradiation than the indica subspecies. Indica showed a higher seedling survival rate than japonica. Irradiation caused more extensive DNA damage in shoots than in roots, and the severity was higher in NPB than in YD6. GO and KEGG pathway analyses indicate that the core genes related to DNA repair and replication and cell proliferation are similarly regulated between the varieties, however the universal stress responsive genes show contrasting differential response patterns in japonica and indica. WGCNA identifies 37 co-expressing gene modules and ten candidate hub genes for each module. This provides novel evidence indicating that certain peripheral pathways may dominate the molecular networks in irradiation survival and suggests more potential target genes in breeding for universal stress tolerance in rice.

Comprehensive evaluation of resistance effects of pyramiding lines with different broad-spectrum resistance genes against Magnaporthe oryzae in rice (Oryza sativa L.).

BACKGROUND: Broad-spectrum resistance gene pyramiding helps the development of varieties with broad-spectrum and durable resistance to M. oryzae. However, detailed information about how these different sources of broad-spectrum resistance genes act together or what are the best combinations to achieve broad-spectrum and durable resistance is limited. RESULTS: Here a set of fifteen different polygene pyramiding lines (PPLs) were constructed using marker-assisted selection (MAS). Using artificial inoculation assays at seedling and heading stage, combined with natural induction identification under multiple field environments, we evaluated systematically the resistance effects of different alleles of Piz locus (Pigm, Pi40, Pi9, Pi2 and Piz) combined with Pi1, Pi33 and Pi54, respectively, and the interaction effects between different R genes. The results showed that the seedling blast and panicle blast resistance levels of PPLs were significantly higher than that of monogenic lines. The main reason was that most of the gene combinations produced transgressive heterosis, and the transgressive heterosis for panicle blast resistance produced by most of PPLs was higher than that of seedling blast resistance. Different gene pyramiding with broad-spectrum R gene produced different interaction effects, among them, the overlapping effect (OE) between R genes could significantly improve the seedling blast resistance level of PPLs, while the panicle blast resistance of PPLs were remarkably correlated with OE and complementary effect (CE). In addition, we found that gene combinations, Pigm/Pi1, Pigm/Pi54 and Pigm/Pi33 displayed broad-spectrum resistance in artificial inoculation at seedling and heading stage, and displayed stable broad-spectrum resistance under different disease nursery. Besides, agronomic traits evaluation also showed PPLs with these three gene combinations were at par to the recurrent parent. Therefore, it would provide elite gene combination model and germplasms for rice blast resistance breeding program. CONCLUSIONS: The development of PPLs and interaction effect analysis in this study provides valuable theoretical foundation and innovative resources for breeding broad-spectrum and durable resistant varieties.

[Effect of miR-21 down-regulated by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells].

Objective: To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 µmol/L) of H 2O 2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H 2O 2 was obtained. To analyze the effect of H 2O 2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H 2O 2 group (group B), osteogenic induction group (group C), and H 2O 2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H 2O 2 group (group A1), H 2O 2+osteogenic induction group (group B1), H 2O 2+osteogenic induction+miR-21 inhibitor group (group C1), and H 2O 2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H 2O 2 group (group A2), H 2O 2+osteogenic induction group (group B2), H 2O 2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H 2O 2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining. Results: The results of MTS and RT-PCR showed that the appropriate concentration of H 2O 2 was 160 µmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks ( P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D ( P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 ( P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks ( P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks ( P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week ( P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2. Conclusion: H 2O 2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

Identification of Genes Related to Cold Tolerance and a Functional Allele That Confers Cold Tolerance.

Cold stress is a major factor limiting rice (Oryza sativa) production worldwide, especially at the seedling and booting stages. The identification of genes associated with cold tolerance (CT) in rice is important for sustainable food production. Here, we report the results of a genome-wide association study to identify the genetic loci associated with CT by using a 1,033-accession diversity panel. We identified five CT-related genetic loci at the booting stage. Accessions carrying multiple cold-tolerant alleles displayed a higher seed-setting rate than did accessions that had no cold-tolerant alleles or carried a single allele. At the seedling stage, eight genetic loci related to CT have been identified. Among these, LOC_Os10g34840 was identified as the candidate gene for the qPSR10 genetic locus that is associated with CT in rice seedlings. A single-nucleotide polymorphism (SNP), SNP2G, at position 343 in LOC_Os10g34840 is responsible for conferring CT at the seedling stage in rice. Further analysis of the haplotype network revealed that SNP2G was present in 80.08% of the temperate japonica accessions but only 3.8% of the indica ones. We used marker-assisted selection to construct a series of BC4F3 near-isogenic lines possessing the cold-tolerant allele SNP2G When subjected to cold stress, plants carrying SNP2G survived better as seedlings and showed higher grain weight than plants carrying the SNP2A allele. The CT-related loci identified here and the functional verification of LOC_Os10g34840 will provide genetic resources for breeding cold-tolerant varieties and for studying the molecular basis of CT in rice.

Effect of long-term fertilisation on the weed community of a winter wheat field.

Effects of fertilisation and other management techniques on a weed community were evaluated during wheat growth in a rice-wheat cropping system. Fertiliser treatments were C0 (C means chemical, C0 means zero chemical fertiliser.), CN (N fertiliser), CNK (N plus K fertiliser), CNPK (N plus P and K fertiliser), CNP (N plus P fertiliser), and CPK (P plus K fertiliser). Weed density, biomass, and bio-diversity were determined. Redundancy analysis (RDA) was used to investigate the relationship between fertiliser management, weed species, and weed density. The overall weed densities in the C0 and CPK treatments were the greatest during wheat seeding and ripening periods and were significantly greater than densities in the other treatments. N, P and organic matter in soil were highly correlated with weed species and density, whereas K in soil was not significantly correlated with weed species and weed density. N fertiliser significantly reduced weed density. Balanced fertilisation maintained weed species richness and resulting in a high yield of wheat. CNPK application reduced weed damage and improved the productivity and stability of the farmland ecosystem.