A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice.

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

Glutamate-GABA imbalance mediated by miR-8-5p and its STTM regulates phase-related behavior of locusts.

The aggregation of locusts from solitary to gregarious phases is crucial for the formation of devastating locust plagues. Locust management requires research on the prevention of aggregation or alternative and greener solutions to replace insecticide use, and insect-derived microRNAs (miRNAs) show the potential for application in pest control. Here, we performed a genome-wide screen of the differential expression of miRNAs between solitary and gregarious locusts and showed that miR-8-5p controls the γ-aminobutyric acid (GABA)/glutamate functional balance by directly targeting glutamate decarboxylase (Gad). Blocking glutamate-GABA neurotransmission by miR-8-5p overexpression or Gad RNAi in solitary locusts decreased GABA production, resulting in locust aggregation behavior. Conversely, activating this pathway by miR-8-5p knockdown in gregarious locusts induced GABA production to eliminate aggregation behavior. Further results demonstrated that ionotropic glutamate/GABA receptors tuned glutamate/GABA to trigger/hamper the aggregation behavior of locusts. Finally, we successfully established a transgenic rice line expressing the miR-8-5p inhibitor by short tandem target mimic (STTM). When locusts fed on transgenic rice plants, Gad transcript levels in the brain increased greatly, and aggregation behavior was lost. This study provided insights into different regulatory pathways in the phase change of locusts and a potential control approach through behavioral regulation in insect pests.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

Stress responsive ZmWRKY53 gene increases cold tolerance in rice.

Plant WRKY transcription factors are responsible for biotic and abiotic stresses and play an important role in enhancing their adaptability. The AtWRKY33 is a gene that functions in response to abiotic stresses such as low temperature, drought, salinity, etc. In this study, a recombinant vector YG8198-ZmWRKY53 carrying the ZmWRKY53, an interspecific homolog of the dicotyledonous AtWRKY33, was transferred to rice plants by Agrobacterium mediated transformation. The ectopic expression of the ZmWRKY53 in transgenic rice plants conferred cold tolerance with a higher accumulation of free proline and water-soluble sugars, an increase in chlorophyll content, a decrease in electrolyte leakage rate and MDA levels compared to control plants. This result suggests that ZmWRKY53 may confer cold tolerance in rice.

Field performance and nitrous oxide emissions of transgenic nitrogen use efficient rice lines cultivated in tropical paddy fields.

Nitrogen (N) fertilizers make up the majority of the input used in rice production, and their excess application leads to significant environmental pollution. Developing rice varieties with improved nitrogen use efficiency (NUE) is essential to maintain the sustainability of rice production. This study aims to evaluate the performance of transgenic Oryza sativa japonica cv. Kitaake expressing the barley (Hordeum vulgare) alanine aminotransferase (HvAlaAT) gene in response to different levels of N fertilizer application under tropical paddy field conditions. Results from this study demonstrate that transgenic nitrogen use efficient Kitaake rice (Kitaake NUE) displays a grain yield increase of up to 41% compared to Kitaake null. Transgenic Kitaake NUE expressing the HvAlaAT gene displays a higher N uptake and achieves a higher nitrogen use efficiency compared to control plants while maintaining lower nitrous oxide (N2O) fluxes. The reduction in N2O emissions in Kitaake NUE compared to Kitaake null ranges from 37.5 to 96.3%. The transgenic Kitaake NUE used in this study has potential as a donor to improve the nitrogen use efficiency of indica rice for better adaptability to tropical conditions.

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

Phenotypic and microarray analysis reveals salinity stress-induced oxidative tolerance in transgenic rice expressing a DEAD-box RNA helicase, OsDB10.

Helicases are the motor proteins not only involved in the process of mRNA metabolism but also played a significant role in providing abiotic stresses tolerance. In this study, a DEAD-box RNA helicase OsDB10 was cloned and functionally characterized. The transcript levels of OsDB10 were increased both in shoot and root upon salt, heat, cold, and ABA application and was more prominent in shoot compared to root. Genomic integration of OsDB10 in transgenic rice was confirmed by PCR, Southern blot and qRT-PCR analysis. The transgenic plants showed quicker seed germination, reduced necrosis, higher chlorophyll, more survival rate, better seedling growth, and produced more grain yield under salinity stress. Furthermore, transgenic lines also accumulated less Na+ and high K+ ions and salinity tolerance of the transgenic were also assayed by measuring different bio-physiological indices. Moreover, the OsDB10 transgenic plants showed enhanced tolerance to salinity-induced oxidative stress by scavenging ROS and increased activity of antioxidants enzymes. Microarray analysis showed upregulation of transcriptional regulations and metabolic reprogramming as OsDB10 overexpression modulates the expression of many other genes. Altogether, our results confirmed that OsDB10 is a functional DEAD-box RNA helicase and played vital roles in plant defence response against salinity stress.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Co-expression of four penaeidins in transgenic rice seeds: an alternative strategy for substitute antibiotic agricultural products.

The co-expression of multiple antimicrobial peptides (AMPs) in genetically modified (GM) crops can give plants a broader antibacterial spectrum and lower the pathogen risk of drug resistance. Therefore, four penaeidins (shrimp-derived AMPs) were fused and encoded in an artificial gene (PEN1234), driven by the seed-specific promoter Pzein, with the aim of co-expression in seeds of transgenic rice. The resistant rice plants, acquired via Agrobacterium-mediated transformation and glufosinate screening, were identified by PCR and the modified disk-diffusion method, and eight GM lines with high AMP content in the seeds were obtained. Among them, the PenOs017 line had the largest penaeidin content, at approximately 251-300 µg/g in seeds and 15-47 µg/g in roots and leaves. The AMPs in the seeds kept their antibacterial properties even after the seed had been boiled in hot water and could significantly inhibit the growth of methicillin-resistant Staphylococcus aureus, and AMPs in the leaves could effectively inhibit Xanthomonas oryzae pv. Oryzae. The results indicate that PenOs017 seeds containing AMPs are an ideal raw-material candidate for antibiotic-free food and feed, and may require fewer petrochemical fungicides or bactericides for disease control during cultivation than conventional rice.

The ppk-expressing transgenic rice floating bed improves P removal in slightly polluted water.

With significant economic advantages, the plant floating bed has been widely utilized in the ecological remediation of eutrophic water because of the excessive phosphorus (P) and nitrogen discharge in China. Previous research has demonstrated that polyphosphate kinase (ppk)-expressing transgenic rice (Oryza sativa L. ssp. japonica) (ETR) can increase the P absorption capacity to support rice growth and boost rice yield. In this study, the floating beds of ETR with single copy line (ETRS) and double copy line (ETRD) are built to investigate their capacity to remove aqueous P in slightly polluted water. Compared with the wild type Nipponbare (WT) floating bed, the ETR floating beds greatly reduce the total P concentration in slightly polluted water though the ETR floating beds have the same removal rates of chlorophyll-a, NO3--N, and total nitrogen in slightly polluted water. The P uptake rate of ETRD on the floating bed is 72.37% in slightly polluted water, which is higher than that of ETRS and WT on the floating beds. Polyphosphate (polyP) synthesis is a critical factor for the excessive phosphate uptake of ETR on the floating beds. The synthesis of polyP decreases the level of free intracellular phosphate (Pi) in ETR on the floating beds, simulating the phosphate starvation signaling. The OsPHR2 expression in the shoot and root of ETR on the floating bed increased, and the corresponding P metabolism gene expression in ETR was changed, which promoted Pi uptake by ETR in slightly polluted water. The Pi accumulation further promoted the growth of ETR on the floating beds. These findings highlight that the ETR floating beds, especially ETRD floating bed, have significant potential for P removal and can be exploited as a novel method for phytoremediation in slightly polluted water.

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Use of Germination to Enhance Resveratrol Content and Its Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is triggered by a variety of danger signals and is now a worldwide concern. Resveratrol, a natural nonflavonoid polyphenol found in naturally consumed plants and foods, has a wide spectrum of bioactive potency. We successfully generated resveratrol-enriched rice by introducing the resveratrol biosynthesis gene into Dongjin rice. In this study, resveratrol- and piceid-enriched rice (DJ526) was investigated for its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells compared to normal rice (DJ). In addition, the 5-day-old germinated DJ526 (DJ526_5) was tested for its anti-inflammatory effects. The piceid and resveratrol amounts increased in DJ526_5 by germination. Treatment of LPS-stimulated RAW264.7 cells with resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) significantly decreased the production of nitric oxide (NO) and the inflammatory mediator prostaglandin E2 (PGE2), downregulated proinflammatory gene expression, and inhibited nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. These findings demonstrated the anti-inflammatory mechanisms of resveratrol-enriched rice in LPS-stimulated RAW264.7 cells. Furthermore, resveratrol-enriched rice could be a potential source of anti-inflammatory agents.

iTRAQ based proteomic analysis of rice lines having single or stacked blast resistance genes: Pi54/Pi54rh during incompatible interaction with Magnaporthe oryzae.

Deployment of single or multiple blast resistance (R) genes in rice plant is considered to be the most promising approach to enhance resistance against blast disease caused by fungus Magnaporthe oryzae. At the proteome level, relatively little information about R gene mediated defence mechanisms for single and stacking resistance characteristics is available. The overall objective of this study is to look at the proteomics of rice plants that have R genes; Pi54, Pi54rh and stacked Pi54 + Pi54rh in response to rice blast infection. In this study 'isobaric tag for relative and absolute quantification' (iTRAQ)-based proteomics analysis was performed in rice plants at 72-h post inoculation with Magnaporthe oryzae and various differentially expressed proteins were identified in these three transgenic lines in comparison to wild type during resistance response to blast pathogen. Through STRING analysis, the observed proteins were further examined to anticipate their linked partners, and it was shown that several defense-related proteins were co-expressed. These proteins can be employed as targets in future rice resistance breeding against Magnaporthe oryzae. The current study is the first to report a proteomics investigation of rice lines that express single blast R gene Pi54, Pi54rh and stacked (Pi54 + Pi54rh) during incompatible interaction with Magnaporthe oryzae. The differentially expressed proteins indicated that secondary metabolites, reactive oxygen species-related proteins, phenylpropanoid, phytohormones and pathogenesis-related proteins have a substantial relationship with the defense response against Magnaporthe oryzae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01327-3.

MoSDT1 triggers defense response through modulating phosphorylated proteins in rice.

KEY MESSAGE: MoSDT1, a rice blast fungus transcription factor, is as an inducer to activate defense response through mainly mediating phosphorylated proteins in rice. Pathogen effector proteins play a dual role in infecting the host or triggering a defense response. Our previous research found a Magnaporthe oryzae effector, MoSDT1, which could activate the rice defense response when it was overexpressed in rice. However, we still know little about the mechanisms on how MoSDT1 in vivo or in vitro influences the resistance ability of rice. Our results showed that decreased ROS and increased lignin contents appeared along with significant upregulation of defense-related genes, raffinose synthesis gene, and phenylalanine ammonialyase gene. Moreover, we revealed that the contents of lignin were increased, which was in accordance with the upregulation of its precursor phenylalanine gene despite the fact that the glutamate-/thiamine-responsive genes were inhibited in MoSDT1 transgenic rice, and these indicated that MoSDT1 triggered the defense system of rice in vivo. Interestingly, in vitro studies, we further found that MoSDT1 induced the defense system by ROS synthesis, callose deposition, PR gene expression and SA/JA synthesis/signal genes using the purified prokaryotic expression system in rice plants. In addition, this defense response was confirmed to be activated by the zinc finger domain of MoSDT1 via prokaryotic expression of MoSDT1 truncated mutants in rice plants. To elucidate the regulative effects of MoSDT1 on protein phosphorylation in rice, phosphoproteome analysis was performed in both MoSDT1-transgenic and wild type  rice. We found that MoSDT1 specifically up-regulated the expression levels of a few phosphorylated proteins, which were involved in multiple functions, such as biotic/abiotic stress and growth. In addition, the motifs in these specific proteins ranked the top among the top-five conserved motifs in the MoSDT1-transgenic rice. MoSDT1 played a crucial role in enhancing rice resistance by modulating several genes and signaling pathways.

Conversion of sheath blight susceptible indica and japonica rice cultivars into moderately resistant through expression of antifungal ß-1,3-glucanase transgene from Trichoderma spp.

Rice is an important food crop for three billion people worldwide. The crop is vulnerable to several diseases. Sheath blight caused by fungal pathogen Rhizoctonia solani is a significant threat to rice cultivation accounting for up to 50% yield losses. The pathogen penetrates leaf blades and sheaths, leading to plant necrosis; and major disease resistance gene against the pathogen is not available. This study describes development of sheath blight resistant transgenic indica and japonica rice cultivars through introduction of antifungal ß-1,3-glucanase transgene cloned from Trichoderma. The transgene integration and expression in transformed T0 rice plants was examined by PCR, RT-PCR, qRT-PCR demonstrating up to 5-fold higher expression as compared to non-transgenic plants. The bioassay of T0, T1 and homozygous T2 progeny plants with virulent R. solani isolate revealed that plants carrying high level of ß-1,3-glucanase expression displayed moderately resistant reaction to the pathogen. The optical micrographs of leaf sheath cells from moderately resistant plant after pathogen inoculation displayed presence of a few hyphae with sparse branching; on the contrary, pathogen hyphae in susceptible non-transgenic plant cells were present in abundance with profuse hyphal branching and forming prominent infection cushions. The disease severity in T2 progeny plants was significantly less as compared to non-transgenic plants confirming role of ß-1,3-glucanase in imparting resistance.

Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine.

BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.

Development of marker-free rice with stable and high resistance to rice black-streaked dwarf virus disease through RNA interference.

The rice black-streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Asia. RNA interference (RNAi) has been widely applied to develop antiviral varieties in plants. So far, only a few studies reported the application of RNAi in rice against RBSDV and most of them are lack of enough data to support its breeding potential, which limited the progress on developing RBSDV-resistant variety. In this study, we generated three RNAi constructs to specifically target three RBSDV genes (S1, S2 and S6), respectively. We confirmed that RNAi targeting RBSDV S6 conferred rice with almost full immunity to RBSDV through phenotyping test in eight consecutive years in both artificial inoculation and field trials, while RNAi of S1 or S2 only leads to partially increased resistance. The S6RNAi was also found conferring strong resistance to southern rice black-streaked dwarf virus (SRBSDV), a novel species closely related to RBSDV that outbroke recently in Southern China. In particular, no adverse effects on agronomical and developmental traits were found in S6RNAi transgenic lines. The marker-free transgenic lines with S6RNAi, driven by either maize ubiquitin-1 promoter or rice rbcS green tissue expression promoter, in elite rice background should have great potential in breeding of resistant varieties to both RBSDV and SRBSDV and provide a basis for further safety evaluation and commercial application.

Overexpression of the homoterpene synthase gene, OsCYP92C21, increases emissions of volatiles mediating tritrophic interactions in rice.

Plant defence homoterpenes can be used to attract pest natural enemies. However, the biosynthetic pathway of homoterpenes is still unknown in rice, and the practical application of such indirect defence systems suffers from inherent limitations due to their low emissions from plants. Here, we demonstrated that the protein OsCYP92C21 is responsible for homoterpene biosynthesis in rice. We also revealed that the ability of rice to produce homoterpenes is dependent on the subcellular precursor pools. By increasing the precursor pools through specifically subcellular targeting expression, genetic transformation and genetic introgression, we significantly enhanced homoterpene biosynthesis in rice. The final introgressed GM rice plants exhibited higher homoterpene emissions than the wild type rice and the highest homoterpene emission reported so far for such GM plants even without the induction of herbivore attack. As a result, these GM rice plants demonstrated strong attractiveness to the parasitic wasp Cotesia chilonis. This study discovered the homoterpene biosynthesis pathway in rice, and lays the foundation for the utilisation of plant indirect defence mechanism in the "push-pull" strategy of integrated pest management through increasing precursor pools in the subcellular compartments and overexpressing homoterpene synthase by genetic transformation.

Functional rice with tandemly repeated Cbl-b ubiquitin ligase inhibitory pentapeptide prevents denervation-induced muscle atrophy in vivo.

Ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) play a critical role in nonloading-mediated skeletal muscle atrophy: Cbl-b ubiquitinates insulin receptor substrate-1 (IRS-1), leading to its degradation and a resulting loss in muscle mass. We reported that intramuscular injection of a pentapeptide, DGpYMP, which acts as a mimic of the phosphorylation site in IRS-1, significantly inhibited denervation-induced skeletal muscle loss. In order to explore the possibility of the prevention of muscle atrophy by diet therapy, we examined the effects of oral administration of transgenic rice containing Cblin (Cbl-b inhibitor) peptide (DGYMP) on denervation-induced muscle mass loss in frogs. We generated transgenic rice seeds in which 15 repeats of Cblin peptides with a WQ spacer were inserted into the rice storage protein glutelin. A diet of the transgenic rice seeds had significant inhibitory effects on denervation-induced atrophy of the leg skeletal muscles in frogs, compared with those receiving a diet of wild-type rice.