Nipponbare Research Articles

Methyltransferase 1 (OsMTS1) interacts with hydroxycinnamoyltransferase 1 (OsHCT1) and promotes heading by upregulating heading date 1 (Hd1)

Heading date determines the distribution and yield potentials of rice, and is an ideal target for crop improvement using CRISPR/Cas9 genome editing system. In this study, we reported the loss-of-function of Methyltransferase 1 (MTS1), which promotes heading in rice. Here, we constructed knockouts and overexpression transgenic plants of OsMTS1 in ZH8015 and Nipponbare (NIP) for the first time to validate its heading date function in rice subspecies Oryza sativa ssp. Indica and O. Sativa ssp. Japonica, respectively. The OsMTS1 knockouts in ZH8015 and NIP rice significantly promoted heading date under both natural short days (NSD) and natural long days (NLD) conditions, while the overexpression of OsMTS1 significantly delayed heading date in ZH8015 and NIP rice under both NSD and NLD conditions. Likewise, the complementation transgenic plants displayed late heading date phenotype. OsMTS1 repressed heading through up-regulating Heading date 1 (Hd1) and down-regulating Early heading date 1 (Ehd1) and Heading date 3a (Hd3a). The OsMTS1 protein interacted with OsHCT1 proteins using a yeast two-hybrid (Y2H) assay. The Y2H and overexpression confirmed that OsMTS1 interacted with OsHCT1, which delayed heading by 4.7 days under NLD. Taken together, CRISPR/Cas9, genetic complementation, and overexpression results validated that OsMTS1 represses heading in Indica and Japonica rice under both NLD and NSD conditions. These results demonstrated that OsMTS1 is a useful target for breeding early maturing rice varieties by CRISPR/Cas9 gene editing of the functional allele.

Genetic Dissection of Major Rice QTLs for Strong Culms and Fine Mapping of qWS5 for Breeding Application in Transplanted System

BackgroundRice is one of the major staples that feeds about one half of the global populations, and it is important to identify the genetic loci for the traits related to yield improvement. Lodging will cause severe yield loss when it happens, and stem diameter has been characterized as an important trait for lodging resistance. However, most QTLs for stem diameter have not been finely dissected due to their sensitivity to environmental fluctuation.ResultIn this study, we performed QTL analysis for stem diameter using populations derived from Nipponbare (NIP) and strong culm variety YYP1, and confirmed the single and combined effect of three major QTLs by recombinant inbred lines (RILs). Based on the QTL location, we found that qWS5 is a novel QTL not well characterized before. To finely dissect the novel locus, several recombinant heterogeneous inbred families (HIFs) were selected from the RILs for linkage analysis and their derived nearly isogenic lines (NILs) were subjected to detailed trait investigation throughout different years. The HIF-NILs strategy confined the QTL to about 380 kb region supported by repeated genotype and phenotype data, and it lays the foundation for QTL cloning in the future. In addition, introgression of the QTL to an elite japonica variety SD785 was performed by successive backcrossing, and it confirmed the value of qWS5 in increasing stem diameter and other agronomic traits during rice breeding.ConclusionsWe prove that qWS5 is a novel QTL with relatively stable effect for stem diameter and the QTL can be finely mapped to small region by the HIF-NILs strategy. The result will facilitate the improvement of rice lodging resistance by molecular marker assisted selection breeding.

OsJRL negatively regulates rice cold tolerance via interfering phenylalanine metabolism and flavonoid biosynthesis.

The identification of new genes involved in regulating cold tolerance in rice is urgent because low temperatures repress plant growth and reduce yields. Cold tolerance is controlled by multiple loci and involves a complex regulatory network. Here, we show that rice jacalin-related lectin (OsJRL) modulates cold tolerance in rice. The loss of OsJRL gene functions increased phenylalanine metabolism and flavonoid biosynthesis under cold stress. The OsJRL knock-out (KO) lines had higher phenylalanine ammonia-lyase (PAL) activity and greater flavonoid accumulation than the wild-type rice, Nipponbare (NIP), under cold stress. The leaves had lower levels of reactive oxygen species (ROS) and showed significantly enhanced cold tolerance compared to NIP. In contrast, the OsJRL overexpression (OE) lines had higher levels of ROS accumulation and showed lower cold tolerance than NIP. Additionally, the OsJRL KO lines accumulated more abscisic acid (ABA) and jasmonic acid (JA) under cold stress than NIP. The OsJRL OE lines showed increased sensitivity to ABA compared to NIP. We conclude that OsJRL negatively regulates the cold tolerance of rice via modulation of phenylalanine metabolism and flavonoid biosynthesis.

Pleiotropic Effects of miR5504 Underlying Plant Height, Grain Yield and Quality in Rice.

MicroRNAs (miRNAs) are known to play critical roles in regulating rice agronomic traits through mRNA cleavage or translational repression. Our previous study indicated that miR5504 regulates plant height by affecting cell proliferation and expansion. Here, the two independent homozygous mir5504 mutants (CR1 and CR2) and overexpression lines (OE1 and OE2) were further used to investigate the functions of miR5504. The panicle length, 1000-grain weight and grain yield per plant of miR5504-OE lines were identical to those of Nipponbare (NIP), but the 1000-grain weight of mir5504 mutants was reduced by about 10% and 9%, respectively. Meanwhile, the grain width and thickness of mir5504 mutants decreased significantly by approximately 10% and 11%, respectively. Moreover, the cytological results revealed a significant decrease in cell number along grain width direction and cell width in spikelet in mir5504, compared with those in NIP. In addition, several major storage substances of the rice seeds were measured. Compared to NIP, the amylose content of the mir5504 seeds was noticeably decreased, leading to an increase of nearly 10 mm in gel consistency (GC) in mir5504 lines. Further investigation confirmed that LOC_Os08g16914 was the genuine target of miR5504: LOC_Os08g16914 over-expression plants phenocopied the mir5504 mutants. This study provides insights into the role of miR5504 in rice seed development.

OsJAZ4 Fine-Tunes Rice Blast Resistance and Yield Traits

JAZ proteins function as transcriptional regulators that form a jasmonic acid–isoleucine (JA-Ile) receptor complex with coronatine insensitive 1 (COI1) and regulate plant growth and development. These proteins also act as key mediators in signal transduction pathways that activate the defense-related genes. Herein, the role of OsJAZ4 in rice blast resistance, a severe disease, was examined. The mutation of OsJAZ4 revealed its significance in Magnaporthe oryzae (M. oryzae) resistance and the seed setting rate in rice. In addition, weaker M. oryzae-induced ROS production and expression of the defense genes OsO4g10010, OsWRKY45, OsNAC4, and OsPR3 was observed in osjaz4 compared to Nipponbare (NPB); also, the jasmonic acid (JA) and gibberellin4 (GA4) content was significantly lower in osjaz4 than in NPB. Moreover, osjaz4 exhibited a phenotype featuring a reduced seed setting rate. These observations highlight the involvement of OsJAZ4 in the regulation of JA and GA4 content, playing a positive role in regulating the rice blast resistance and seed setting rate.

Identification of Increased Grain Length 1 (IGL1), a novel gene encoded by a major QTL for modulating grain length in rice.

A novel quantitative trait locus qIGL1, which performed a positive function in regulating grain length in rice, was cloned by the map-based cloning approach; further studies revealed that it corresponded to LOC_Os03g30530, and the IGL1 appeared to contribute to lengthening and widening of the cells on the surface of grain hulls. Grain length is a prominent determinant for grain weight and appearance quality of rice. In this study, we conducted quantitative trait locus mapping to determine a genomic interval responsible for a long-grain phenotype observed in a japonica cultivar HD385. This led to the identification of a novel QTL for grain length on chromosome 3, named qIGL1 (for Increased Grain Length 1); the HD385 (Handao 385)-derived allele showed enhancement effects on grain length, and such an allele as well as NIP (Nipponbare)-derived allele was designated qigl1 HD385 and qIGL1NIP, respectively. Genetic analysis revealed that the qigl1HD385 allele displayed semidominant effects on grain length. Fine mapping further narrowed down the qIGL1 to an ~ 70.8-kb region containing 9 open reading frames (ORFs). A comprehensive analysis indicated that LOC_Os03g30530, which corresponded to ORF6 and carried base substitutions and deletions in HD385 relative to NIP, thereby causing changes or losses of amino-acid residues, was the true gene for qIGL1. Comparison of grain traits between a pair of near-isogenic lines (NILs), termed NIL-igl1HD385 and NIL-IGL1NIP, discovered that introduction of the igl1HD385 into the NIP background significantly resulted in the elevations of grain length and 1000-grain weight. Closer inspection of grain surfaces revealed that the cell length and width in the longitudinal direction were significantly longer and greater, respectively, in NIL-igl1HD385 line compared with in NIL-IGL1NIP line. Hence, our studies identified a new semidominant natural allele contributing to the increase of grain length and further shed light on the regulatory mechanisms of grain length.

A Structure Variation in qPH8.2 Detrimentally Affects Plant Architecture and Yield in Rice.

Plant height is an important agronomic trait associated with plant architecture and grain yield in rice (Oryza sativa L.). In this study, we report the identification of quantitative trait loci (QTL) for plant height using a chromosomal segment substitution line (CSSL) population with substituted segments from japonica variety Nipponbare (NIP) in the background of the indica variety 9311. Eight stable QTLs for plant height were identified in three environments. Among them, six loci were co-localized with known genes such as semidwarf-1 (sd1) and Grain Number per Panicle1 (GNP1) involved in gibberellin biosynthesis. A minor QTL qPH8.2 on chromosome 8 was verified and fine-mapped to a 74 kb region. Sequence comparison of the genomic region revealed the presence/absence of a 42 kb insertion between NIP and 9311. This insertion occurred predominantly in temperate japonica rice. Comparisons on the near-isogenic lines showed that the qPH8.2 allele from NIP exhibits pleiotropic effects on plant growth, including reduced plant height, leaf length, photosynthetic capacity, delayed heading date, decreased yield, and increased tiller angle. These results indicate that qPH8.2 from temperate japonica triggers adverse effects on plant growth and yield when introduced into the indica rice, highlighting the importance of the inter-subspecies crossing breeding programs.

ITRAQ-based protein profiling and functional identification of four genes involved in rice basal resistance against Magnaporthe oryzae in two contrasting rice genotypes

Rice blast, caused by Magnaporthe oryzae, is one of the most destructive rice diseases. Developing blast-resistant rice cultivars represents the most economical and environmentally friend strategy for managing the disease. In our previous study, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative protein quantification was carried out to investigate the resistance gene Piz-t gene-mediated resistance response to infection in two contrasting rice genotypes of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and its wild-type Nipponbare (NPB). Here, from the comparisons of differentially expressed proteins (DEPs) of NPB-Piz-t to the avirulent isolate KJ201 (KJ201-Piz-t)and the virulent isolate RB22 (RB22-Piz-t) with mock-treated NPB-Piz-t (Mock-Piz-t), NPB to the virulent isolate KJ201(KJ201-NPB) and RB22 (RB22-NPB) with mock-treated NPB (Mock-NPB), 1, 1, and 6 common DEPs were, respectively, identified at 24, 48 and 72 h post-inoculation (hpi) in the susceptible comparisons of RB22-Pizt/Mock-Piz-t, KJ201-NPB/Mock-NPB, and RB22-NPB/Mock-NPB, involving in gi|54,290,836 and gi|59,800,021 were identified in the resistance comparison KJ201-Piz-t/Mock-Piz-t at 48 and 72 hpi respectively. Moreover, four genes of Os01g0138900 (gi|54,290,836), Os04g0659300 (gi|59,800,021), Os09g0315700 (gi|125,563,186) or Os04g0394200 (gi|21,740,743) were knocked out or overexpressed in NPB using gene over-expression and CRISPR/Cas9 technology, and results verified that the Os01g0138900 obviously affected the rice blast resistance. Further, expression and targeted metabolomics analysis illuminated the resistance response of cysteine-containing substances as gi|59,800,021 under blast infection. These results provide new targets for basal resistance gene identification and open avenues for developing novel rice blast resistant materials.

Genomic variations combined with epigenetic modifications rewire open chromatin in rice.

Cis-regulatory elements (CREs) fine-tune gene transcription in eukaryotes. CREs with sequence variations play vital roles in driving plant or crop domestication. However, how global sequence and structural variations are responsible for multi-level changes between indica and japonica rice (Oryza sativa) is still not fully elucidated. To address this, we conducted multi-omics studies using MH-seq (MNase hypersensitivity sequencing) in combination with RNA-seq, ChIP-seq and BS-seq between the japonica rice variety Nipponbare (NIP) and indica rice variety 93-11. We found that differential MHSs exhibited some distinct intrinsic genomic sequence features between NIP and 93-11. Notably, through MHS-GWAS integration, we found that key sequence variations may be associated with differences of agronomic traits between NIP and 93-11, which is partly achieved by MHSs harboring CREs. In addition, SV-derived differential MHSs caused by TE insertion, especially by non-common TEs among rice varieties, were associated with genes with distinct functions, indicating that TE driven gene neo- or subfunctionalization is mediated by changes of chromatin openness. This study thus provides insights into how sequence and genomic structural variations control agronomic traits of NIP and 93-11; it also provides genome editing targets for molecular breeding aiming at improving favorable agronomic properties.

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.

Rice lipid transfer protein, OsLTPL23, controls seed germination by regulating starch-sugar conversion and ABA homeostasis.

Seed germination is vital for ensuring the continuity of life in spermatophyte. High-quality seed germination usually represents good seedling establishment and plant production. Here, we identified OsLTPL23, a putative rice non-specific lipid transport protein, as an important regulator responsible for seed germination. Subcellular localization analysis confirmed that OsLTPL23 is present in the plasma membrane and nucleus. The knockout mutants of OsLTPL23 were generated by CRISPR/Cas9-mediated genome editing, and osltpl23 lines significantly germinated slower and lower than the Nipponbare (NIP). Starch and soluble sugar contents measurement showed that OsLTPL23 may have alpha-amylase inhibitor activity, and high soluble sugar content may be a causal agent for the delayed seed germination of osltpl23 mutants. Transcript profiles in the germinating seeds exhibited that the abscisic acid (ABA)-responsive genes, OsABI3 and OsABI5, and biosynthesis genes, OsNCED1, OsNCED2, OsNCED3 and OsNCED4, are obviously upregulated in the osltpl23 mutants compared to NIP plants, conversely, ABA metabolism genes OsABA8ox1, OsABA8ox2 and OsABA8ox3 are stepwise decreased. Further investigations found that osltpl23 mutants displays weakened early seedling growth, with elevated gene expresssion of ABA catabolism genes and repressive transcription response of defence-related genes OsWRKY45, OsEiN3, OsPR1a, OsPR1b and OsNPR1. Integrated analysis indicated that OsLTPL23 may exert an favorable effect on rice seed germination and early seedling growth via modulating endogenous ABA homeostasis. Collectively, our study provides important insights into the roles of OsLTPL23-mediated carbohydrate conversion and endogenous ABA pathway on seed germination and early seedling growth, which contributes to high-vigor seed production in rice breeding.

Global whole-genome comparison and analysis to classify subpopulations and identify resistance genes in weedy rice relevant for improving crops.

Common weedy rice plants are important genetic resources for modern breeding programs because they are the closest relatives to rice cultivars and their genomes contain elite genes. Determining the utility and copy numbers of WRKY and nucleotide-binding site (NBS) resistance-related genes may help to clarify their variation patterns and lead to crop improvements. In this study, the weedy rice line LM8 was examined at the whole-genome level. To identify the Oryza sativa japonica subpopulation that LM8 belongs to, the single nucleotide polymorphisms (SNPs) of 180 cultivated and 23 weedy rice varieties were used to construct a phylogenetic tree and a principal component analysis and STRUCTURE analysis were performed. The results indicated that LM8 with admixture components from japonica (GJ) and indica (XI) belonged to GJ-admixture (GJ-adm), with more than 60% of its genetic background derived from XI-2 (22.98%), GJ-tropical (22.86%), and GJ-subtropical (17.76%). Less than 9% of its genetic background was introgressed from weedy rice. Our results also suggested LM8 may have originated in a subtropical or tropical geographic region. Moreover, the comparisons with Nipponbare (NIP) and Shuhui498 (R498) revealed many specific structure variations (SVs) in the LM8 genome and fewer SVs between LM8 and NIP than between LM8 and R498. Next, 96 WRKY and 464 NBS genes were identified and mapped on LM8 chromosomes to eliminate redundancies. Three WRKY genes (ORUFILM02g002693, ORUFILM05g002725, and ORUFILM05g001757) in group III and one RNL [including the resistance to powdery mildew 8 (RPW8) domain, NBS, and leucine rich repeats (LRRs)] type NBS gene (ORUFILM12g000772) were detected in LM8. Among the NBS genes, the RPW8 domain was detected only in ORUFILM12g000772. This gene may improve plant resistance to pathogens as previously reported. Its classification and potential utility imply LM8 should be considered as a germplasm resource relevant for rice breeding programs.

Fine Mapping of qTGW7b, a Minor Effect QTL for Grain Weight in Rice (Oryza sativa L.).

Grain weight is a key trait that determines rice quality and yield, and it is primarily controlled by quantitative trait loci (QTL). Recently, attention has been paid to minor QTLs. A minor effect QTL qTGW7 that controls grain weight was previously identified in a set of chromosomal fragment substitution lines (CSSLs) derived from Nipponbare (NPB)/93-11. Compared to NPB, the single segment substitution line (SSSL) N83 carrying the qTGW7 introgression exhibited an increase in grain length and width and a 4.5% increase in grain weight. Meanwhile, N83 was backcrossed to NPB to create a separating population, qTGW7b, a QTL distinct from qTGW7, which was detected between markers G31 and G32. Twelve near-isogenic lines (NILs) from the BC9F3 population and progeny of five NILs from the BC9F3:4 population were genotyped and phenotyped, resulting in the fine mapping of the minor effect QTL qTGW7b to the approximately 86.2-kb region between markers G72 and G32. Further sequence comparisons and expression analysis confirmed that five genes, including Os07g39370, Os07g39430, Os07g39440, Os07g39450, and Os07g39480, were considered as the candidate genes underlying qTGW7b. These results provide a crucial foundation for further cloning of qTGW7b and molecular breeding design in rice.

The telomere-to-telomere gap-free genome of four rice parents reveals SV and PAV patterns in hybrid rice breeding.

The telomere-to-telomere gap-free genome of four rice parents reveals SV and PAV patterns in hybrid rice breeding.

Exogenous Melatonin Improves the Quality Performance of Rice under High Temperature during Grain Filling

With the increasing greenhouse effect, high temperature has become the most unfavorable environmental factor for the rice grain filling process, affecting rice yield and quality mainly through changing the composition and structure of starch in rice grains. Research has focused on the rational management of water and fertilizer, and spraying of exogenous chemicals, which have become important measures to alleviate high temperature stress of rice. As a multifunctional molecule, melatonin has the potential to improve plant stress resistance by enhancing the scavenging efficiency of reactive oxygen species (ROS), thus protecting plants from the adverse effects of abiotic stress. The present study used a typical japonica rice variety Nipponbare (NPB) as the experimental material, which was treated with high temperature and melatonin during grain-filling stages. The effects of exogenous melatonin on the rice growth and quality traits, as well as starch synthesis, in response to high temperature were analyzed systematically. Exogenous melatonin significantly increased the rice leaf photosynthetic and heat-resistance properties. Melatonin could alleviate the effects of high temperature on the key physicochemical properties related to rice quality. Furthermore, milled rice from NPB plants treated with melatonin had better endosperm appearance under high temperature. Further study found that exogenous melatonin could stabilize the chain length distribution of starch in NPB (especially amylopectin), which implied that melatonin could be used in rice cultivation to alleviate the effect of high temperature on quality, optimization of amylopectin synthesis can also improve rice quality. The results of the present study provide a new idea and research direction to alleviate high temperature stress of rice in the context of global warming.

Fine Mapping of Two Interacting Loci for Transmission Ratio Distortion in Rice (Oryza sativa L.)

Transmission ratio distortion (TRD) denotes the observed allelic or genotypic frequency deviation from the expected Mendelian segregation ratios in the offspring of a heterozygote. TRD can severely hamper gene flow between and within rice species. Here, we report the fine mapping and characterization of two loci (TRD4.1 and TRD4.2) for TRD using large F2 segregating populations, which are derived from rice chromosome segment substitution lines, each containing a particular genomic segment introduced from the japonica cultivar Nipponbare (NIP) into the indica cultivar Zhenshan (ZS97). The two loci exhibited a preferential transmission of ZS97 alleles in the derived progeny. Reciprocal crossing experiments using near-isogenic lines harboring three different alleles at TRD4.1 suggest that the gene causes male gametic selection. Moreover, the transmission bias of TRD4.2 was diminished in heterozygotes when they carried homozygous TRD4.1ZS97. This indicates an epistatic interaction between these two loci. TRD4.2 was mapped into a 35-kb region encompassing one candidate gene that is specifically expressed in the reproductive organs in rice. These findings broaden the understanding of the genetic mechanisms of TRD and offer an approach to overcome the barrier of gene flow between the subspecies in rice, thus facilitating rice improvement by introgression breeding.

Analyses on the Infection Process of Rice Virus and the Spatiotemporal Expression Pattern of Host Defense Genes Based on a Determined-Part Inoculation Approach.

Rice viral diseases adversely affect crop yield and quality. Most rice viruses are transmitted through insect vectors. However, the traditional whole-plant inoculation method cannot control the initial inoculation site in rice plants because the insect feeding sites in plants are random. To solve this problem, we established a determined-part inoculation approach in this study that restricted the insect feeding sites to specific parts of the rice plant. Rice stripe virus (RSV) was used as the model virus and was inoculated at the bottom of the stem using our method. Quantitative real-time PCR and Western blot analyses detected RSV only present at the bottom of the Nipponbare (NPB) stem at 1 day post-inoculation (dpi), indicating that our method successfully controlled the inoculation site. With time, RSV gradually moved from the bottom of the stem to the leaf in NPB rice plants, indicating that systemic viral spread can also be monitored using this method. In addition, a cultivar resistant to RSV, Zhendao 88 (ZD88), was inoculated using this method. We found that RSV accumulation in ZD88 was significantly lower than in NPB. Additionally, the expression level of the resistant gene STV11 in ZD88 was highly induced at the initial invasion stage of RSV (1 dpi) at the inoculation site, whereas it remained relatively stable at non-inoculated sites. This finding indicated that STV11 directly responded to RSV invasion to inhibit virus accumulation at the invasion site. We also proved that this approach is suitable for other rice viruses, such as Rice black-streaked dwarf virus (RBSDV). Interestingly, we determined that systemic infection with RSV was faster than that with RBSDV in NPB, which was consistent with findings in field trails. In summary, this approach is suitable for characterizing the viral infection process in rice plants, comparing the local viral accumulation and spread among different cultivars, analyzing the spatiotemporal expression pattern of resistance-associated genes, and monitoring the infection rate for different viruses.

Coordination of m6A mRNA methylation and gene transcriptome in rice response to cadmium stress

N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNAs of all higher eukaryotes. However, the role of the m6A methylomes in rice is still poorly understood. With the development of the MeRIP-seq technique, the in-depth identification of mRNAs with m6A modification has become feasible. A study suggested that m6A modification is crucial for posttranscriptional regulation related to Cd2+-induced malignant transformation, but the association between m6A modification in plants and Cd tolerance has not been reported. We investigated the m6A methylomes in the roots of a cadmium (Cd)-treated group and compared them with the roots in the control (CK) group by m6A sequencing of cv. 9311 and cv. Nipponbare (NIP) plants. The results indicated that Cd leads to an altered modification profile in 3,406 differential m6A peaks in cv. 9311 and 2,065 differential m6A peaks in cv. NIP. KEGG pathway analysis of the genes with differentially modified m6A peaks indicated that the “phenylalanine”, “tyrosine and tryptophan biosynthesis”, “glycine”, “adherens junctions”, “glycerophospholipid metabolism” and “threonine metabolism” signalling pathways may be associated with the abnormal root development of cv. 9311 rice due to exposure to Cd. The “arginine”, “proline metabolism”, “glycerolipid”, and “protein processing in endoplasmic reticulum” metabolism pathways were significantly enriched in genes with differentially modified m6A peaks in cv. NIP. Unlike that in Arabidopsis, the m6A-modified nucleotide position on mRNAs (m6A peak) distribution in rice exhibited a preference towards both the stop codon and 3′ untranslated regions (3′ UTRs). These findings provide a resource for plant RNA epitranscriptomic studies and further increase our knowledge on the function of m6A modification in RNA in plants.

A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice

Grain size is one of the most important factors that control rice yield, as it is associated with grain weight (GW). To date, dozens of rice genes that regulate grain size have been isolated; however, the regulatory mechanism underlying GW control is not fully understood. Here, the quantitative trait locus qGL5 for grain length (GL) and GW was identified in recombinant inbred lines of 9311 and Nipponbare (NPB) and fine mapped to a candidate gene, OsAUX3. Sequence variations between 9311 and NPB in the OsAUX3 promoter and loss of function of OsAUX3 led to higher GL and GW. RNA sequencing, gene expression quantification, dual-luciferase reporter assays, chromatin immunoprecipitation-quantitative PCR, and yeast one-hybrid assays demonstrated that OsARF6 is an upstream transcription factor regulating the expression of OsAUX3. OsARF6 binds directly to the auxin response elements of the OsAUX3 promoter, covering a single-nucleotide polymorphism site between 9311 and NPB/Dongjin/Hwayoung, and thereby controls GL by altering longitudinal expansion and auxin distribution/content in glume cells. Furthermore, we showed that miR167a positively regulate GL and GW by directing OsARF6 mRNA silencing. Taken together, our study reveals that a novel miR167a-OsARF6-OsAUX3 module regulates GL and GW in rice, providing a potential target for the improvement of rice yield.

The Interaction between Rice Genotype and Magnaporthe oryzae Regulates the Assembly of Rice Root-Associated Microbiota

BackgroundUtilizating the plant microbiome to enhance pathogen resistance in crop production is an emerging alternative to the use of chemical pesticides. However, the diversity and structure of the microbiota, and the assembly mechanisms of root-associated microbial communities of plants are still poorly understood.ResultsWe invstigated the microbiota of the root endosphere and rhizosphere soils of the rice cultivar Nipponbare (NPB) and its Piz-t-transgenic line (NPB-Piz-t) when infected with the filamentous fungus Magnaporthe oryzae (M. oryzae) isolate KJ201, using 16S rRNA and internal transcribed spacer 1 (ITS1) amplicon sequencing. The rhizosphere soils showed higher bacterial and fungal richness and diversity than the endosphere except for fungal richness in the rhizosphere soils of the mock treatment. Bacteria richness and diversity increased in the endospheric communities of NPB and Piz-t under inoculation with KJ201 (referred to as ‘NPB-KJ201’ and ‘Piz-t-KJ201’, respectively) compared with the corresponding mock treatments, with the NPB-KJ201 showing the highest diversity in the four bacterial endocompartments. In contrast, fungal richness and diversity decreased in the endospheric communities of NPB-KJ201 and Piz-t-KJ201, relative to the corresponding mock treatments, with NPB-KJ201 and Piz-t-KJ201 having the lowest richness and diversity, respectively, across the four fungal endocompartments. Principal component analysis (PCA) indicated that the microbiota of Piz-t-KJ201 of root endophytes were mostly remarkablely distinct from that of NPB-KJ201. Co-occurrence network analysis revealed that the phyla Proteobacteria and Ascomycota were the key contributors to the bacterial and fungal communities, respectively. Furthermore, a comparative metabolic analysis showed that the contents of tryptophan metabolism and indole alkaloid biosynthesis were significantly lower in the Piz-t-KJ201 plants.ConclusionsIn this study, we compared the diversity, composition, and assembly of microbial communities associated with the rhizosphere soils and endosphere of Piz-t-KJ201 and NPB-KJ201. On the basis of the different compositions, diversities, and assemblies of the microbial communities among different compartments, we propose that the host genotype and inoculation pattern of M. oryzae played dominant roles in determining the microbial community assemblage. Further metabolomics analysis revealed that some metabolites may influence changes in bacterial communities. This study improves our understanding of the complex interactions between rice and M. oryzae, which could be useful in developing new strategies to improve rice resistance through the manipulation of soil microorganisms.