A KNOX â ¡ transcription factor suppresses the NLR immune receptor BRG8-mediated immunity in rice.

Nucleotide-binding site and leucine-rich repeat (NLR) proteins are activated by detecting pathogen effectors, which in turn trigger host defenses and cell death. Although many NLRs have been identified, the mechanism responsible for NLR-triggered defense responses are still poorly understood. In this study, through GWAS approach, we identified a novel NLR gene, Blast Resistance Gene 8 (BRG8), conferring resistance to rice blast and bacterial blight diseases. Consistently, the BRG8 overexpression and complementation lines exhibited enhanced resistance to both pathogens. Subcellular localization assays showed that BRG8 localized in both cytoplasm and nucleus. More evidence revealed that nuclear-localized BRG8 enhanced rice immunity without hypersensitive response (HR)-like phenotype. Furthermore, we also demonstrated the CC domain of BRG8 not only physically interacted with itself, but also interacted with the KNOX Ⅱ protein HOMEOBOX ORYZA SATIVA59 (HOS59). Knockout of HOS59 in BRG8 background showed enhanced resistance to M. oryzae strain CH171 and Xoo strain CR4, similar to BRG8 background. In contrast, overexpression of HOS59 in BRG8 background, compromised the HR-like phenotype and resistance response. Further analysis revealed that HOS59 promotes the degradation of BRG8 via the 26S proteasome pathway. Collectively, our study highlights HOS59 as NLR immune regulators, fine-tune BRG8-mediated immune responses against pathogens, and provides new insights into NLR association and function in plant immunity.

BTA2 regulates tiller angle and the shoot gravity response through controlling auxin content and distribution in rice.

Tiller angle is a key agricultural trait that establishes plant architecture, which in turn strongly affects grain yield by influencing planting density in rice. The shoot gravity response plays a crucial role in the regulation of tiller angle in rice, but the underlying molecular mechanism is largely unknown. Here, we report the identification of the BIG TILLER ANGLE2 (BTA2), which regulates tiller angle by controlling the shoot gravity response in rice. Loss-of-function mutation of BTA2 dramatically reduced auxin content and affected auxin distribution in rice shoot base, leading to impaired gravitropism and therefore a big tiller angle. BTA2 interacted with AUXIN RESPONSE FACTOR7 (ARF7) to modulate rice tiller angle through the gravity signaling pathway. The BTA2 protein was highly conserved during evolution. Sequence variation in the BTA2 promoter of indica cultivars harboring a less expressed BTA2 allele caused lower BTA2 expression in shoot base and thus wide tiller angle during rice domestication. Overexpression of BTA2 significantly increased grain yield in the elite rice cultivar Huanghuazhan under appropriate dense planting conditions. Our findings thus uncovered the BTA2-ARF7 module that regulates tiller angle by mediating the shoot gravity response. Our work offers a target for genetic manipulation of plant architecture and valuable information for crop improvement by producing the ideal plant type.

[Genetic dissection of rice resistance to bacterial blight].

Bacterial blight, a major disease in rice, poses a serious impact on rice production. In this study, a doubled haploid (DH) population derived from a cross between the introduced japonica cultivar 'Maybelle' and the indica landrace 'Baiyeqiu' was used to investigate the pathogenicity of four pathogen races causing bacterial blight. The results showed that the pathogenicity of all the pathogen races exhibited continuous, transgressive distribution in the DH population. Moreover, strong correlations existed between every two pathogen races, with the correlation coefficients ranging from 0.3 to 0.6. A total of 12 quantitative trait loci (QTLs) distributed on chromosomes 1, 2, 3, 5, 6, 7, 9, and 12 were detected for rice bacterial blight, explaining 4.95% to 16.05% of the phenotype. Among these QTLs, a major QTL located in the interval RM6024-RM163 on chromosome 5 was detected in three pathogen races. In addition, the pyramiding of the positive alleles can apparently improve the rice resistance to bacterial blight. This study is of great significance for broadening the genetic resources with resistance to bacterial blight in China.

Integrating genome-wide association study into genomic selection for the prediction of agronomic traits in rice (Oryza sativa L.).

Accurately identifying varieties with targeted agronomic traits was thought to contribute to genetic selection and accelerate rice breeding progress. Genomic selection (GS) is a promising technique that uses markers covering the whole genome to predict the genomic-estimated breeding values (GEBV), with the ability to select before phenotypes are measured. To choose the appropriate GS models for breeding work, we analyzed the predictability of nine agronomic traits measured from a population of 459 diverse rice varieties. By the comparison of eight representative GS models, we found that the prediction accuracies ranged from 0.407 to 0.896, with reproducing kernel Hilbert space (RKHS) having the highest predictive ability in most traits. Further results demonstrated the predictivity of GS is altered by several factors. Moreover, we assessed the method of integrating genome-wide association study (GWAS) into various GS models. The predictabilities of GS combined peak-associated markers generated from six different GWAS models were significantly different; a recommendation of Mixed Linear Model (MLM)-RKHS was given for the GWAS-GS-integrated prediction. Finally, based on the above result, we experimented with applying the P-values obtained from optimal GWAS models into ridge regression best linear unbiased prediction (rrBLUP), which benefited the low predictive traits in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01423-y.

LG5, a Novel Allele of EUI1, Regulates Grain Size and Flag Leaf Angle in Rice.

Grain size and flag leaf angle are two important traits that determining grain yield in rice. However, the mechanisms regulating these two traits remain largely unknown. In this study, a rice long grain 5 (lg5) mutant with a large flag leaf angle was identified, and map-based cloning revealed that a single base substitution followed by a 2 bp insertion in the LOC_Os05g40384 gene resulted in larger grains, a larger flag leaf angle, and higher plant height than the wild type. Sequence analysis revealed that lg5 is a novel allele of elongated uppermost internode-1 (EUI1), which encodes a cytochrome P450 protein. Functional complementation and overexpression tests showed that LG5 can rescue the bigger grain size and larger flag leaf angle in the Xiushui11 (XS) background. Knockdown of the LG5 transcription level by RNA interference resulted in elevated grain size and flag leaf angle in the Nipponbare (NIP) background. Morphological and cellular analyses suggested that LG5 regulated grain size and flag leaf angle by promoting cell expansion and cell proliferation. Our results provided new insight into the functions of EUI1 in rice, especially in regulating grain size and flag leaf angle, indicating a potential target for the improvement of rice breeding.

Control of Grain Weight and Size in Rice (Oryza sativa L.) by OsPUB3 Encoding a U-Box E3 Ubiquitin Ligase.

Grain weight and size, mostly determined by grain length, width and thickness, are crucial traits affecting grain quality and yield in rice. A quantitative trait locus controlling grain length and width in rice, qGS1-35.2, was previously fine-mapped in a 57.7-kb region on the long arm of chromosome 1. In this study, OsPUB3, a gene encoding a U-box E3 ubiquitin ligase, was validated as the causal gene for qGS1-35.2. The effects were confirmed firstly by using CRISPR/Cas9-based mutagenesis and then through transgenic complementation of a Cas9-free knock-out (KO) mutant. Two homozygous KO lines were produced, each having a 1-bp insertion in OsPUB3 which caused frameshift mutation and premature termination. Compared with the recipient and a transgenic-negative control, both mutants showed significant decreases in grain weight and size. In transgenic complementation populations derived from four independent T0 plants, grain weight of transgenic-positive plants was significantly higher than transgenic-negative plants, coming with increased grain length and a less significant decrease in grain width. Based on data documented in RiceVarMap V2.0, eight haplotypes were classified according to six single-nucleotide polymorphisms (SNPs) found in the OsPUB3 coding region of 4695 rice accessions. Significant differences on grain size traits were detected between the three major haplotypes, Hap1, Hap2 and Hap3 that jointly occupy 98.6% of the accessions. Hap3 having the largest grain weight and grain length but intermediate grain width exhibits a potential for simultaneously improving grain yield and quality. In another set of 257 indica rice cultivars tested in our study, Hap1 and Hap2 remained to be the two largest groups. Their differences on grain weight and size were significant in the background of non-functional gse5, but non-significant in the background of functional GSE5, indicating a genetic interaction between OsPUB3 and GSE5. Cloning of OsPUB3 provides a new gene resource for investigating the regulation of grain weight and size.

Genomic insight into genetic changes and shaping of major inbred rice cultivars in China.

The annual planting area of major inbred rice (Oryza sativa) cultivars reach more than half of the total annual planting area of inbred rice cultivars in China. However, how the major inbred rice cultivars changed during decades of genetic improvement and why they can be prevalently cultivated in China remains unclear. Here, we investigated the underlying genetic changes of major inbred cultivars and the contributions of landraces and introduced cultivars during the improvement by resequencing a collection of 439 rice accessions including major inbred cultivars, landraces, and introduced cultivars. The results showed that landraces were the main genetic contribution sources of major inbred Xian (Indica) cultivars, while introduced cultivars were that of major inbred Geng (Japonica) cultivars. Selection scans and haplotype frequency analysis shed light on the reflections of some well-known genes in rice improvement, and breeders had different preferences for the Xian's and Geng's breeding. Six candidate regions associated with agronomic traits were identified by genome-wide association mapping, five of which were under positive selection in rice improvement. Our study provides a comprehensive insight into the development of major inbred rice cultivars and lays the foundation for genomics-based breeding in rice.

Identification of SMG3, a QTL Coordinately Controls Grain Size, Grain Number per Panicle, and Grain Weight in Rice.

Grain size, grain number per panicle, and grain weight are key agronomic traits that determine grain yield in rice. However, the molecular mechanisms coordinately controlling these traits remain largely unknown. In this study, we identified a major QTL, SMG3, that is responsible for grain size, grain number per panicle, and grain weight in rice, which encodes a MYB-like protein. The SMG3 allele from M494 causes an increase in the number of grains per panicle but produces smaller grain size and thousand grain weight. The SMG3 is constitutively expressed in various organs in rice, and the SMG3 protein is located in the nucleus. Microscopy analysis shows that SMG3 mainly produces long grains by increasing in both cell length and cell number in the length direction, which thus enhances grain weight by promoting cell expansion and cell proliferation. Overexpression of SMG3 in rice produces a phenotype with more grains but reduces grain length and weight. Our results reveal that SMG3 plays an important role in the coordinated regulation of grain size, grain number per panicle, and grain weight, providing a new insight into synergistical modification on the grain appearance quality, grain number per panicle, and grain weight in rice.

Validation of a QTL for Grain Size and Weight Using an Introgression Line from a Cross between Oryza sativa and Oryza minuta.

BACKGROUND: Grain size and weight are important target traits determining grain yield and quality in rice. Wild rice species possess substantial elite genes that can be served as an important resource for genetic improvement of rice. In this study, we identify and validate a novel QTL on chromosome 7 affecting the grain size and weight using introgression lines from cross of Oryza sativa and Oryza minuta. RESULTS: An introgression line 'IL188' has been achieved from a wild species Oryza minuta (2n = 48, BBCC, W303) into O. sativa japonica Nipponbare. The F2 and F2:3 populations derived from a cross between IL188 and Nipponbare were used to map QTLs for five grain size traits, including grain length (GL), grain width (GW), grain length to width ratio (LWR), grain thickness (GT) and thousand grain weight (TGW). A total of 12 QTLs for the five grain traits were identified on chromosomes 1, 2, 3, 6, 7, and 8. The QTL-qGL7 controlling GL on chromosome 7 was detected stably in the F2 and F2:3 populations, and explained 15.09-16.30% of the phenotypic variance. To validate the effect of qGL7, eight residual heterozygous line (RHL) populations were developed through selfing four F2:3 and four F2:4 plants with different heterozygous segments for the target region. By further developing SSR and Indel markers in the target interval, qGL7 was delimited to a ~ 261 kb region between Indel marker Y7-12 and SSR marker Y7-38, which also showed significant effects on grain width and thousand grain weight. Comparing with the reference genome of Nipponbare, stop or frameshift mutations in the exon of the three putative genes LOC_Os07g36830, LOC_Os07g36900 and LOC_Os07g36910 encoding F-box domain-containing proteins may be the candidate genes for qGL7. Scanning electron microscopy analysis of the glume's epidermal cells showed that the cell length and width of NIL-qGL7IL188 was higher than NIL-qGL7Nip, indicating that qGL7 increases grain size and weight by regulating cell expansion. CONCLUSIONS: In this study, we detected 12 QTLs regulating grain size and weight using an introgression line from a cross between Oryza sativa and Oryza minuta. Of these loci, we confirmed and delimited the qGL7 to a ~ 261 kb region. Three putative genes, LOC_Os07g36830, LOC_Os07g36900 and LOC_Os07g36910 encoding F-box domain-containing proteins may be the candidate genes for qGL7. These results provide a basis for map-based cloning of the qGL7 gene and useful information for marker assisted selection in rice grain quality improvement.

Genome-Wide Association Study of Rice Rooting Ability at the Seedling Stage.

BACKGROUND: Rice rooting ability is a complex agronomical trait that displays heterosis and plays an important role in rice growth and production. Only a few quantitative trait loci (QTLs) have been identified by bi-parental population. More genes or QTLs are required to dissect the genetic architecture of rice rooting ability. RESULTS: To characterize the genetic basis for rice rooting ability, we used a natural rice population, genotyped by a 90 K single nucleotide polymorphism (SNP) array, to identify the loci associated with rooting-related traits through the genome-wide association study (GWAS). Population structure analysis divided the natural population into two subgroups: indica and japonica. We measured four traits for evaluating rice rooting ability, namely root growth ability (RGA), maximum root length (MRL), root length (RL), and root number (RN). Using the association study in three panels consisting of one for the full population, one for indica, and one for japonica, 24 SNPs associated with rooting ability-related traits were identified. Through comparison of the relative expression levels and DNA sequences between germplasm with extreme phenotypes, results showed that LOC_Os05g11810 had non-synonymous variations at the coding region, which may cause differences in root number, and that the expression levels of LOC_Os04g09900 and LOC_Os04g10060 are closely associated with root length variation. CONCLUSIONS: Through evaluation of the rice rooting ability-related traits and the association mapping, we provided useful information for understanding the genetic basis of rice rooting ability and also identified some candidate genes and molecular markers for rice root breeding.

OsPLS4 Is Involved in Cuticular Wax Biosynthesis and Affects Leaf Senescence in Rice.

Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.

Using Heading date 1 preponderant alleles from indica cultivars to breed high-yield, high-quality japonica rice varieties for cultivation in south China.

Heading date 1 (Hd1) is an important gene for the regulation of flowering in rice, but its variation in major cultivated rice varieties, and the effect of this variation on yield and quality, remains unknown. In this study, we selected 123 major rice varieties cultivated in China from 1936 to 2009 to analyse the relationship between the Hd1 alleles and yield-related traits. Among these varieties, 19 haplotypes were detected in Hd1, including two major haplotypes (H8 and H13) in the japonica group and three major haplotypes (H14, H15 and H16) in the indica group. Analysis of allele frequencies showed that the secondary branch number was the major aimed for Chinese indica breeding. In the five major haplotypes, SNP316 (C-T) was the only difference between the two major japonica haplotypes, and SNP495 (C-G) and SNP614 (G-A) are the major SNPs in the three indica haplotypes. Association analysis showed that H16 is the most preponderant allele in modern cultivated Chinese indica varieties. Backcrossing this allele into the japonica variety Chunjiang06 improved yield without decreasing grain quality. Therefore, our analysis offers a new strategy for utilizing these preponderant alleles to improve yield and quality of japonica varieties for cultivation in the southern areas of China.

A Nck-associated protein 1-like protein affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice.

Water deficit is a major environmental threat affecting crop yields worldwide. In this study, a drought stress-sensitive mutant drought sensitive 8 (ds8) was identified in rice (Oryza sativa L.). The DS8 gene was cloned using a map-based approach. Further analysis revealed that DS8 encoded a Nck-associated protein 1 (NAP1)-like protein, a component of the SCAR/WAVE complex, which played a vital role in actin filament nucleation activity. The mutant exhibited changes in leaf cuticle development. Functional analysis revealed that the mutation of DS8 increased stomatal density and impaired stomatal closure activity. The distorted actin filaments in the mutant led to a defect in abscisic acid (ABA)-mediated stomatal closure and increased ABA accumulation. All these resulted in excessive water loss in ds8 leaves. Notably, antisense transgenic lines also exhibited increased drought sensitivity, along with impaired stomatal closure and elevated ABA levels. These findings suggest that DS8 affects drought sensitivity by influencing actin filament activity.

PGL3 is required for chlorophyll synthesis and impacts leaf senescence in rice.

Rice leaf color mutants play a great role in research about the formation and development of chloroplasts and the genetic mechanism of the chlorophyll (Chl) metabolism pathway. pgl3 is a rice leaf color mutant derived from Xiushui11 (Oryza sativa L. spp. japonica), treated with ethyl methane sulfonate (EMS). The mutant exhibited a pale-green leaf (pgl) phenotype throughout the whole development as well as reduced grain quality. Map-based cloning of PGL3 revealed that it encodes the chloroplast signal recognition particle 43 kDa protein (cpSRP43). PGL3 affected the Chl synthesis by regulating the expression levels of the Chl synthesis-associated genes. Considerable reactive oxygen species were accumulated in the leaves of pgl3, and the transcription levels of its scavenging genes were down-regulated, indicating that pgl3 can accelerate senescence. In addition, high temperatures could inhibit the plant's growth and facilitate the process of senescence in pgl3.

Divergent Hd1, Ghd7, and DTH7 Alleles Control Heading Date and Yield Potential of Japonica Rice in Northeast China.

The heading date is a vital factor in achieving a full rice yield. Cultivars with particular flowering behaviors have been artificially selected to survive in the long-day and low-temperature conditions of Northeast China. To dissect the genetic mechanism responsible for heading date in rice populations from Northeast China, association mapping was performed to identify major controlling loci. A genome-wide association study (GWAS) identified three genetic loci, Hd1, Ghd7, and DTH7, using general and mixed linear models. The three genes were sequenced to analyze natural variations and identify their functions. Loss-of-function alleles of these genes contributed to early rice heading dates in the northern regions of Northeast China, while functional alleles promoted late rice heading dates in the southern regions of Northeast China. Selecting environmentally appropriate allele combinations in new varieties is recommended during breeding. Introducing the early indica rice's genetic background into Northeast japonica rice is a reasonable strategy for improving genetic diversity.

Genome-wide association study of cold tolerance of Chinese indica rice varieties at the bud burst stage.

KEY MESSAGE: A region containing three genes on chromosome 1 of indica rice was associated with cold tolerance at the bud burst stage; these results may be useful for breeding cold-tolerant lines. Low temperature at the bud burst stage is one of the major abiotic stresses limiting rice growth, especially in regions where rice seeds are sown directly. In this study, we investigated cold tolerance of rice at the bud burst stage and conducted a genome-wide association study (GWAS) based on the 5K rice array of 249 indica rice varieties widely distributed in China. We improved the method to assess cold tolerance at the bud burst stage in indica rice, and used severity of damage (SD) and seed survival rate (SR) as the cold-tolerant indices. Population structure analysis demonstrated that the Chinese indica panel was divided into three subgroups. In total, 47 significant single-nucleotide polymorphism (SNP) loci associated with SD and SR, were detected by association mapping based on mixed linear model. Because some loci overlapped between SD and SR, the loci contained 13 genome intervals and most of them have been reported previously. A major QTL for cold tolerance on chromosome 1 at the position of 31.6 Mb, explaining 13.2% of phenotypic variation, was selected for further analysis. Through LD decay, GO enrichment, RNA-seq data, and gene expression pattern analyses, we identified three genes (LOC_Os01g55510, LOC_Os01g55350 and LOC_Os01g55560) that were differentially expressed between cold-tolerant and cold-sensitive varieties, suggesting they may be candidate genes for cold tolerance. Together, our results provide a new method to assess cold tolerance in indica rice, and establish the foundation for isolating genes related to cold tolerance that could be used in rice breeding.

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence.

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes.

Rational design of high-yield and superior-quality rice.

Rice (Oryza sativa L.) is a staple food for more than half of the world's population. To meet the ever-increasing demand for food, because of population growth and improved living standards, world rice production needs to double by 20301. The development of new elite rice varieties with high yield and superior quality is challenging for traditional breeding approaches, and new strategies need to be developed. Here, we report the successful development of new elite varieties by pyramiding major genes that significantly contribute to grain quality and yield from three parents over five years. The new varieties exhibit higher yield potential and better grain quality than their parental varieties and the China's leading super-hybrid rice, Liang-you-pai-jiu (LYP9 or Pei-ai 64S/93-11). Our results demonstrate that rational design is a powerful strategy for meeting the challenges of future crop breeding, particularly in pyramiding multiple complex traits.