DWARF AND LOW-TILLERING 2 functions in brassinosteroid signaling and controls plant architecture and grain size in rice.

Brassinosteroids (BRs) are a class of steroid phytohormones that control various aspects of plant growth and development. Several transcriptional factors (TFs) have been suggested to play roles in BR signaling. However, their possible relationship remains largely unknown. Here, we identified a rice mutant dwarf and low-tillering 2 (dlt2) with altered plant architecture, increased grain width, and reduced BR sensitivity. DLT2 encodes a GIBBERELLIN INSENSITIVE (GAI)-REPRESSOR OF GAI (RGA)-SCARECROW (GRAS) TF that is mainly localized in the nucleus and has weak transcriptional activity. Our further genetic and biochemical analyses indicate that DLT2 interacts with two BR-signaling-related TFs, DLT and BRASSINAZOLE-RESISTANT 1, and probably modulates their transcriptional activity. These findings imply that DLT2 is implicated in a potentially transcriptional complex that mediates BR signaling and rice development and suggests that DLT2 could be a potential target for improving rice architecture and grain morphology. This work also sheds light on the role of rice GRAS members in regulating numerous developmental processes.

An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation.

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we have isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity, likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

DGW1, encoding an hnRNP-like RNA binding protein, positively regulates grain size and weight by interacting with GW6 mRNA.

Grain size and weight determine rice yield. Although numerous genes and pathways involved in regulating grain size have been identified, our knowledge of post-transcriptional control of grain size remains elusive. In this study, we characterize a rice mutant, decreased grain width and weight 1 (dgw1), which produces small grains. We show that DGW1 encodes a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family protein and preferentially expresses in developing panicles, positively regulating grain size by promoting cell expansion in spikelet hulls. Overexpression of DGW1 increases grain weight and grain numbers, leading to a significant rise in rice grain yield. We further demonstrate that DGW1 functions in grain size regulation by directly binding to the mRNA of Grain Width 6 (GW6), a critical grain size regulator in rice. Overexpression of GW6 restored the grain size phenotype of DGW1-knockout plants. DGW1 interacts with two oligouridylate binding proteins (OsUBP1a and OsUBP1b), which also bind the GW6 mRNA. In addition, the second RRM domain of DGW1 is indispensable for its mediated protein-RNA and protein-protein interactions. In summary, our findings identify a new regulatory module of DGW1-GW6 that regulates rice grain size and weight, providing important insights into the function of hnRNP-like proteins in the regulation of grain size.

Grass-specific ABERRANT MICROSPORE DEVELOPMENT 1 is required for maintaining pollen fertility in rice.

Pollen development includes a series of biological events that require precise gene regulation. Although several transcription factors (TFs) have been shown to play roles in maintaining pollen fertility, the major regulatory networks underlying tapetum development and pollen wall formation are largely unknown. Herein, we report that ABERRANT MICROSPORE DEVELOPMENT1 (AMD1), a protein annotated previously as unknown protein, is required for tapetum development and pollen exine patterning in rice (Oryza sativa L.). AMD1 encodes a grass-specific protein exhibiting transactivation activity in the nucleus and is spatiotemporally expressed in the tapetum and microspores during pollen development. Further biochemical assays indicate that AMD1 directly activates the transcription of DEFECTIVE POLLEN WALL (DPW) and POLYKETIDE SYNTHASE2 (OsPKS2), which are both implicated in sporopollenin biosynthesis during exine formation. Additionally, AMD1 directly interacts with TAPETUM DEGENERATION RETARDATION (TDR), a key TF involved in the regulation of tapetum degradation and exine formation. Taken together, we demonstrate that AMD1 is an important regulatory component involved in the TDR-mediated regulatory pathway to regulate sporopollenin biosynthesis, tapetum degradation, and exine formation for pollen development. Our work provides insights into the regulatory network of rice sexual reproduction and a useful target for genetic engineering of new male-sterile lines for hybrid rice breeding.

SWOLLEN TAPETUM AND STERILITY 1 is required for tapetum degeneration and pollen wall formation in rice.

The pollen wall is important for protecting the male gametophyte and for fertilization. The lipid components of the pollen wall are mainly synthesized and transported from the sporophytic tapetum. Although several factors related to lipid biosynthesis have been characterized, the molecular mechanisms underlying lipid biosynthesis during pollen development in rice (Oryza sativa L.) remain elusive. Here, we showed that mutation in the SWOLLEN TAPETUM AND STERILITY 1 (STS1) gene causes delayed tapetum degradation and aborted pollen wall formation in rice. STS1 encodes an endoplasmic reticulum (ER)-localized protein that contains domain of unknown function (DUF) 726 and exhibits lipase activity. Lipidomic and transcriptomic analyses showed that STS1 is involved in anther lipid homeostasis. Moreover, STS1 interacts with Polyketide Synthase 2 (OsPKS2) and Acyl-CoA Synthetase 12 (OsACOS12), two enzymes crucial in lipidic sporopollenin biosynthesis in pollen wall formation, suggesting a potentially lipidic metabolon for sporopollenin biosynthesis in rice. Collectively, our results indicate that STS1 is an important factor for lipid biosynthesis in reproduction, providing a target for the artificial control of male fertility in hybrid rice breeding and insight into the function of DUF726-containing protein in plants.

A ubiquitin-specific protease functions in regulating cell death and immune responses in rice.

Ubiquitin-specific proteases (UBPs) process deubiquitination in eukaryotic organisms and are widely involved in plant development and responses to environmental stress. However, their role in cell death and plant immunity remains largely unknown. Here, we identified a rice lesion mimic mutant (LMM) and cloned its causative gene, LMM22. Both dysfunction and overexpression of LMM22 gave rise to the hypersensitive response-like cell death, reactive oxygen species bursts, and activated defence responses. LMM22 encodes an active UBP that is localised to the endoplasmic reticulum (ER) and displays a constitutive expression pattern in rice. LMM22 interacts with SPOTTED LEAF 35 (SPL35), a coupling of ubiquitin conjugation to ER degradation domain-containing protein that is known to participate in ubiquitination and the regulation of cell death and disease response in rice. Additional analyses suggest that LMM22 can positively regulate and stabilise the abundance of SPL35 protein likely through its deubiquitination activity. These data therefore improve our understanding of the function of UBP in rice innate immune responses by demonstrating that LMM22 functions as a critical regulator of SPL35 in cell death and disease resistance.

Ectopic Expression of a Maize Gene ZmDUF1645 in Rice Increases Grain Length and Yield, but Reduces Drought Stress Tolerance.

As the human population grows rapidly, food shortages will become an even greater problem; therefore, increasing crop yield has become a focus of rice breeding programs. The maize gene, ZmDUF1645, encoding a putative member of the DUF1645 protein family with an unknown function, was transformed into rice. Phenotypic analysis showed that enhanced ZmDUF1645 expression significantly altered various traits in transgenic rice plants, including increased grain length, width, weight, and number per panicle, resulting in a significant increase in yield, but a decrease in rice tolerance to drought stress. qRT-PCR results showed that the expression of the related genes regulating meristem activity, such as MPKA, CDKA, a novel crop grain filling gene (GIF1), and GS3, was significantly changed in the ZmDUF1645-overexpression lines. Subcellular colocalization showed that ZmDUF1645 was primarily localized on cell membrane systems. Based on these findings, we speculate that ZmDUF1645, like the OsSGL gene in the same protein family, may regulate grain size and affect yield through the cytokinin signaling pathway. This research provides further knowledge and understanding of the unknown functions of the DUF1645 protein family and may serve as a reference for biological breeding engineering to increase maize crop yield.

Secretory lipid transfer protein OsLTPL94 acts as a target of EAT1 and is required for rice pollen wall development.

The plant pollen wall protects the male gametophyte from various biotic and abiotic stresses. The formation of a unique pollen wall structure and elaborate exine pattern is a well-organized process, which needs coordination between reproductive cells and the neighboring somatic cells. However, molecular mechanisms underlying this process remain largely unknown. Here, we report a rice male-sterile mutant (l94) that exhibits defective pollen exine patterning and abnormal tapetal cell development. MutMap and knockout analyses demonstrated that the causal gene encodes a type-G non-specific lipid transfer protein (OsLTPL94). Histological and cellular analyses established that OsLTPL94 is strongly expressed in the developing microspores and tapetal cells, and its protein is secreted to the plasma membrane. The l94 mutation impeded the secretory ability of OsLTPL94 protein. Further in vivo and in vitro investigations supported the hypothesis that ETERNAL TAPETUM 1 (EAT1), a basic helix-loop-helix transcription factor (bHLH TF), activated OsLTPL94 expression through direct binding to the E-box motif of the OsLTPL94 promoter, which was supported by the positive correlation between the expression of EAT1 and OsLTPL94 in two independent eat1 mutants. Our findings suggest that the secretory OsLTPL94 plays a key role in the coordinated development of tapetum and microspores with the regulation of EAT1.

Genome-wide association study-based identification genes influencing agronomic traits in rice (Oryza sativa L.).

Rice is one of the most important cereal crops, providing the daily dietary intake for approximately 50% of the global human population. Here, we re-sequenced 259 rice accessions, generating 1371.65 Gb of raw data. Furthermore, we performed genome-wide association studies (GWAS) on 13 agronomic traits using 2.8 million single nucleotide polymorphisms (SNPs) characterized in 259 rice accessions. Phenotypic data and best linear unbiased prediction (BLUP) values of each of the 13 traits over two years of each trait were used for the GWAS. The results showed that 816 SNP signals were significantly associated with the 13 agronomic traits. Then we detected candidate genes related to target traits within 200 kb upstream and downstream of the associated SNP loci, based on linkage disequilibrium (LD) blocks in the whole rice genome. These candidate genes were further identified through haplotype block constructions. This comprehensive study provides a timely and important genomic resource for breeding high yielding rice cultivars.

DEAP1 encodes a fasciclin-like arabinogalactan protein required for male fertility in rice.

Arabinogalactan proteins (AGPs) are widely distributed in plant cells. Fasciclin-like AGPs (FLAs) belong to a subclass of AGPs that play important roles in plant growth and development. However, little is known about the biological functions of rice FLA. Herein, we report the identification of a male-sterile mutant of DEFECTIVE EXINE AND APERTURE PATTERNING1 (DEAP1) in rice. The deap1 mutant anthers produced aberrant pollen grains with defective exine formation and a flattened aperture annulus and exhibited slightly delayed tapetum degradation. DEAP1 encodes a plasma membrane-associated member of group III plant FLAs and is specifically and temporally expressed in reproductive cells and the tapetum layer during male development. Gene expression studies revealed reduced transcript accumulation of genes related to exine formation, aperture patterning, and tapetum development in deap1 mutants. Moreover, DEAP1 may interact with two rice D6 PROTEIN KINASE-LIKE3s (OsD6PKL3s), homologs of a known Arabidopsis aperture protein, to affect rice pollen aperture development. Our findings suggested that DEAP1 is involved in male reproductive development and may affect exine formation and aperture patterning, thereby providing new insights into the molecular functions of plant FLAs in male fertility.

Genome-wide association study and transcriptome analysis discover new genes for bacterial leaf blight resistance in rice (Oryza sativa L.).

BACKGROUND: Rice (Oryza sativa) bacterial leaf blight (BLB), caused by the hemibiotrophic Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases affecting the production of rice worldwide. The development and use of resistant rice varieties or genes is currently the most effective strategy to control BLB. RESULTS: Here, we used 259 rice accessions, which are genotyped with 2 888 332 high-confidence single nucleotide polymorphisms (SNPs). Combining resistance variation data of 259 rice lines for two Xoo races observed in 2 years, we conducted a genome-wide association study (GWAS) to identify quantitative trait loci (QTL) conferring plant resistance against BLB. The expression levels of genes, which contains in GWAS results were also identified between the resistant and susceptible rice lines by transcriptome analysis at four time points after pathogen inoculation. From that 109 candidate resistance genes showing significant differential expression between resistant and susceptible rice lines were uncovered. Furthermore, the haplotype block structure analysis predicted 58 candidate genes for BLB resistance based on Chr. 7_707158 with a minimum P-value (-log 10 P = 9.72). Among them, two NLR protein-encoding genes, LOC_Os07g02560 and LOC_Os07g02570, exhibited significantly high expression in the resistant line, but had low expression in the susceptible line of rice. CONCLUSIONS: Together, our results reveal novel BLB resistance gene resources, and provide important genetic basis for BLB resistance breeding of rice crops.

Identification of rice (Oryza sativa L.) genes involved in sheath blight resistance via a genome-wide association study.

Rice sheath blight (RSB) is an economically significant disease affecting rice yield worldwide. Genetic resistance to RSB is associated with multiple minor genes, with each providing a minor phenotypic effect, but the underlying dominant resistance genes remain unknown. A genome-wide association study (GWAS) of 259 diverse rice varieties, with genotypes based on a single nucleotide polymorphism (SNP) and haplotype, was conducted to assess their sheath blight reactions at three developmental stages (seedlings, tillering and booting). A total of 653 genes were correlated with sheath blight resistance, of which the disease resistance protein RPM1 (OsRSR1) and protein kinase domain-containing protein (OsRLCK5) were validated by overexpression and knockdown assays. We further found that the coiled-coil (CC) domain of OsRSR1 (OsRSR1-CC) and full-length OsRLCK5 interacted with serine hydroxymethyltransferase 1 (OsSHM1) and glutaredoxin (OsGRX20), respectively. It was found that OsSHM1, which has a role in the reactive oxygen species (ROS) burst, and OsGRX20 enhanced the antioxidation ability of plants. A regulation model of the new RSB resistance though the glutathione (GSH)-ascorbic acid (AsA) antioxidant system was therefore revealed. These results enhance our understanding of RSB resistance mechanisms and provide better gene resources for the breeding of disease resistance in rice.

OsSHOC1 and OsPTD1 are essential for crossover formation during rice meiosis.

Meiosis is essential for eukaryotic sexual reproduction and plant fertility, and crossovers (COs) are essential for meiosis and the formation of new allelic combinations in gametes. In this study, we report the isolation of a meiotic gene, OsSHOC1, and the identification of its partner, OsPTD1. Osshoc1 was sterile both in male and female gametophytes, and it showed a striking reduction in the number of meiotic COs, indicating that OsSHOC1 was required for normal CO formation. Further investigations showed that OsSHOC1 physically interacted with OsPTD1 and that the latter was also required for normal CO formation and plant fertility. Additionally, the expression profiles of both genes were consistent with their functions. Our results suggest that OsSHOC1 and OsPTD1 are essential for rice fertility and CO formation, possibly by stabilizing the recombinant intermediates during meiosis.

A Silent Exonic Mutation in a Rice Integrin-α FG-GAP Repeat-Containing Gene Causes Male-Sterility by Affecting mRNA Splicing.

Pollen development plays crucial roles in the life cycle of higher plants. Here we characterized a rice mutant with complete male-sterile phenotype, pollen-less 1 (pl1). pl1 exhibited smaller anthers with arrested pollen development, absent Ubisch bodies, necrosis-like tapetal hypertrophy, and smooth anther cuticular surface. Molecular mapping revealed a synonymous mutation in the fourth exon of PL1 co-segregated with the mutant phenotype. This mutation disrupts the exon-intron splice junction in PL1, generating aberrant mRNA species and truncated proteins. PL1 is highly expressed in the tapetal cells of developing anther, and its protein is co-localized with plasma membrane (PM) and endoplasmic reticulum (ER) signal. PL1 encodes an integrin-α FG-GAP repeat-containing protein, which has seven ß-sheets and putative Ca2+-binding motifs and is broadly conserved in terrestrial plants. Our findings therefore provide insights into both the role of integrin-α FG-GAP repeat-containing protein in rice male fertility and the influence of exonic mutation on intronic splice donor site selection.

GRAIN LENGTH AND AWN 1 negatively regulates grain size in rice.

Grain size is an important factor determining yield in rice. Here, we identified a recessive mutant gene, grain length and awn 1 (gla1), which caused a significant increase in grain length and weight, and was associated with long awns. The gla1 mutation was mapped to a single-nucleotide polymorphism in a gene encoding a cytoplasmically-localized mitogen-activated protein kinase (MAPK) phosphatase. Overexpression of GLA1 caused a decrease in grain length, and the GLA1 protein interacted with OsMAPK6. These results suggest that GLA1 may serve as a negative regulator of the OsMAPKK4-OsMAPK6 cascade, controlling grain size via the dephosphorylation of OsMAPK6.

Comparison of gene co-networks reveals the molecular mechanisms of the rice (Oryza sativa L.) response to Rhizoctonia solani AG1 IA infection.

Rhizoctonia solani causes rice sheath blight, an important disease affecting the growth of rice (Oryza sativa L.). Attempts to control the disease have met with little success. Based on transcriptional profiling, we previously identified more than 11,947 common differentially expressed genes (TPM > 10) between the rice genotypes TeQing and Lemont. In the current study, we extended these findings by focusing on an analysis of gene co-expression in response to R. solani AG1 IA and identified gene modules within the networks through weighted gene co-expression network analysis (WGCNA). We compared the different genes assigned to each module and the biological interpretations of gene co-expression networks at early and later modules in the two rice genotypes to reveal differential responses to AG1 IA. Our results show that different changes occurred in the two rice genotypes and that the modules in the two groups contain a number of candidate genes possibly involved in pathogenesis, such as the VQ protein. Furthermore, these gene co-expression networks provide comprehensive transcriptional information regarding gene expression in rice in response to AG1 IA. The co-expression networks derived from our data offer ideas for follow-up experimentation that will help advance our understanding of the translational regulation of rice gene expression changes in response to AG1 IA.

The OsmiR396c-OsGRF4-OsGIF1 regulatory module determines grain size and yield in rice.

Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth-regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF-interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c-OsGRF4-OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.

Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis from Sichuan Basin in China.

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.

Characterization of cry2-type genes of bacillus thuringiensis strains from soilisolated of sichuan basin, china.

Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%), followed by cry2Aa (6.8%) and cry2Ab alone (2.5%), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.