The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

Expression of OsBiP4 and OsBiP5 is highly correlated with the endoplasmic reticulum stress response in rice

Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under ER stress conditions induced by DTT treatment, OsBiP1 knockdown, OsBiP1 overexpression, OsIRE1 overexpression, or various exogenous recombinant proteins in transgenic rice. In contrast, OsBiP4&5 did not accumulate in OsIRE1 knockdown transgenic rice even after DTT treatment. When the subcellular localization of OsBiP4&5 was investigated in seed endosperm cells under the ER stress condition, OsBiP4&5 were localized to the ER, but did not participate in ER-derived protein body (PB-I) formation in a different manner to OsBiP1. These results indicate that OsBiP4&5 levels were positively correlated with stress levels in the ER. Taken together, these results suggest that OsBiP4&5 are ER stress-related BiP proteins that are regulated by OsIRE1/OsbZIP50 pathway and that they may have a distinct function from that of OsBiP1 in rice.     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

Increased lysine content in rice grains by over-accumulation of BiP in the endosperm

Over-accumulation of lysine-rich binding protein (BiP) in the rice endosperm caused strong endoplasmic reticulum (ER) stress and reduced seed storage proteins, resulting in a relative increase in nutritionally balanced non-seed storage proteins. We show that transgenic rice with over-accumulated BiP was a high-lysine rice germplasm and that the over-accumulation of BiP in the endosperm offered a unique strategy to improve the lysine content of cereal grains.     

The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60?C80?% of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.     

Change in subcellular localization of overexpressed vaccine peptide in rice endosperm cell that is caused by suppression of endogenous seed storage proteins

Rice-based peptide vaccine based on T cell epitopes acts as an ideal oral tolerogen for the treatment of type 1 allergic diseases. To improve production yields of oral tolerogen against Japanese cedar pollen allergy, hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, Cry j 1 and Cry j 2, was produced in transgenic rice seed by expression of its codon optimized gene under the control of the endosperm-specific 26 kD globulin (Glb-1) promoter containing its signal peptide and the simultaneous suppression of endogenous seed storage proteins (SSPs) by RNA interference. Accumulation level of 7Crp peptide produced as a secretory protein was remarkably enhanced by suppression of both the 13–14 kDa prolamins and GluA and GluB glutelins as compared to those under suppression of either of them or in wild type rice. When these SSPs were down-regulated, the 7Crp peptide was observed to be localized in ER lumen as well as ER derived PBs (PB-Is). Especially, accumulation as self-aggregates in ER lumen increased by reduction of the endogenous 13–14 kDa prolamins. It is interesting to note that the absence of C terminal KDEL ER retention signal from the 7Crp peptide resulted in higher level accumulation (116 µg/grain) than that containing the KDEL.     

OsFes1C,a potential nucleotide exchange factor for OsBiP1, is involved in the ER and salt stress responses

The endoplasmic reticulum (ER) quality control system monitors protein homeostasis and relies on the activity of many molecular chaperones. Binding immunoglobulin protein (BiP) is a major ER luminal chaperone that is involved in most functions of the organelle. BiP activity is tightly regulated by nucleotide exchange factors (NEFs). However, information about NEFs in plants is limited. We obtained a Fes1-like protein (OsFes1C) through isobaric tags for relative and absolute quantitation-based proteomics analysis of ER-stressed rice (Oryza sativa) seeds. Unlike its homologs in yeast and mammals, which are located in the cytosol and respond to heat stress, OsFes1C is an ER membrane protein and responds to ER and salt stresses. OsFes1C interacts directly with OsBiP1 and the interaction is inhibited by ATP but promoted by ADP, suggesting that OsFes1C acts as a potential NEF of OsBiP1 in vivo. Overexpression or suppression of OsFes1C led to hypersensitivity to ER stress and affected the growth of rice. Furthermore, we established that OsFes1C directly interacts with a putative salt response protein and is involved in the salt response. Taken together, our study marks an important step toward elucidating the functional mechanisms of an identified ER stress response factor in rice.     

Rice BiP3 regulates immunity mediated by the PRRs XA3 and XA21 but not immunity mediated by the NB-LRR protein,Pi5

Plant innate immunity is mediated by pattern recognition receptors (PRRs) and intracellular NB-LRR (nucleotide-binding domain and leucine-rich repeat) proteins. Overexpression of the endoplasmic reticulum (ER) chaperone, luminal-binding protein 3 (BiP3) compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) mediated by the rice PRR XA21 [12]. Here we show that BiP3 overexpression also compromises resistance mediated by rice XA3, a PRR that provides broad-spectrum resistance to Xoo. In contrast, BiP3 overexpression has no effect on resistance mediated by rice Pi5, an NB-LRR protein that confers resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae). Our results suggest that rice BiP3 regulates membrane-resident PRR-mediated immunity.     

Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site

Human IL‐10 (hIL‐10) is a therapeutic treatment candidate for inflammatory allergy and autoimmune diseases. Rice seed‐produced IL‐10 can be effectively delivered directly to gut‐associated lymphoreticular tissue (GALT) via bio‐encapsulation. Previously, the codon‐optimized hIL‐10 gene was expressed in transgenic rice with the signal peptide and endoplasmic reticulum (ER) retention signal (KDEL) at its 5′ and 3′ ends, respectively, under the control of the endosperm‐specific glutelin GluB‐1 promoter. The resulting purified hIL‐10 was biologically active. In this study, the yield of hIL‐10 in transgenic rice seed was improved. This protein accumulated at the intended deposition sites, which had been made vacant through the selective reduction, via RNA interference, of the endogenous seed storage proteins prolamins or glutelins. Upon suppression of prolamins that were sequestered into ER‐derived protein bodies (PB‐I), hIL‐10 accumulation increased approximately 3‐fold as compared to rice seed with no such suppression and reached 219 μg/grain. In contrast, reducing the majority of the glutelins stored in protein‐storage vacuoles (PB‐II) did not significantly affect the accumulation of hIL‐10. Considering that hIL‐10 is synthesized in the ER lumen and subsequently buds off in ER‐derived granules called IL‐10 granules in a manner similar to PB‐Is, these results indicate that increases in the available deposition space for the desired recombinant proteins may be crucial for improvements in yield. Furthermore, efficient dimeric intermolecular formation of hIL‐10 by inhibiting interaction with Cys‐rich prolamins also contributed to the enhanced formation of IL‐10 bodies. Higher yield of hIL‐10 produced in rice seeds is expected to have broad application in the future.     

Expression of hypoallergenic Der f 2 derivatives with altered intramolecular disulphide bonds induces the formation of novel ER-derived protein bodies in transgenic rice seeds

House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy.     

Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens

The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% µg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.     

Analysis of ER stress in developing rice endosperm accumulating β‐amyloid peptide

The common neurodegenerative disorder known as Alzheimer’s disease is characterized by cerebral neuritic plaques of amyloid β (Aβ) peptide. Plaque formation is related to the highly aggregative property of this peptide, because it polymerizes to form insoluble plaques or fibrils causing neurotoxicity. Here, we expressed Aβ peptide as a new causing agent to endoplasmic reticulum (ER) stress to study ER stress occurred in plant. When the dimer of Aβ_1–42 peptide was expressed in maturing seed under the control of the 2.3‐kb glutelin GluB‐1 promoter containing its signal peptide, a maximum of about 8 μg peptide per grain accumulated and was deposited at the periphery of distorted ER‐derived PB‐I protein bodies. Synthesis of Aβ peptide in the ER lumen severely inhibited the synthesis and deposition of seed storage proteins, resulting in the generation of many small and abnormally appearing PB bodies. This ultrastructural change was accounted for by ER stress leading to the accumulation of aggregated Aβ peptide in the ER lumen and a coordinated increase in ER‐resident molecular chaperones such as BiPs and PDIs in Aβ‐expressing plants. Microarray analysis also confirmed that expression of several BiPs, PDIs and OsbZIP60 containing putative transmembrane domains was affected by the ER stress response. Aβ‐expressing transgenic rice kernels exhibited an opaque and shrunken phenotype. When grain phenotype and expression levels were compared among transgenic rice grains expressing several different recombinant peptides, such detrimental effects on grain phenotype were correlated with the expressed peptide causing ER stress rather than expression levels.     

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.     

Generation of a transgenic rice seed-based edible vaccine against house dust mite allergy

As an alternative approach to conventional allergen-specific immunotherapy, transgenic rice seed expressing a major house dust mite (HDM) allergen, Der p 1, was developed as an edible vaccine. The C-terminal KDEL-tagged Der p 1 allergen specifically accumulated in seed endosperm tissue under the control of the endosperm-specific GluB1 promoter. Der p 1 reached a maximum concentration of 58 μg/grain and was deposited in the endoplasmic reticulum (ER)-derived protein body I (PB-I). Plant-derived Der p 1 was posttranslationally modified with high-mannose-type glycan structures. Glycosylated Der p 1 displayed reduced IgE binding capacity in comparison with its unglycosylated counterpart in vitro. Our results indicate that transgenic Der p 1 rice seeds are a safe, potential oral delivery vaccine for the treatment of HDM allergy.     

过量表达内质网小分子热激蛋白增强番茄的衣霉素抗性

真核细胞内质网腔内未折叠蛋白的过度积累会引起内质网胁迫（ER胁迫）,继而激活未折叠蛋白应答（UPR）信号途径,诱导内质网定位的分子伴侣的大量表达（如BiP和calnexin等）。本工作将CaMV35S启动子驱动的内质网小分子热激蛋白基因（ER-sHSP）导入番茄,发现ER-sHSP的过量表达提高了转基因番茄整株对衣霉素的抗性。衣霉素处理使未转基因番茄中BiP和calnexin基因的表达迅速升高,转基因番茄中这两个基因的表达也有增加,但表达强度明显低于未转基因番茄。说明ER-sHSP能够减轻ER胁迫,并可能参与UPR信号转导途径。     

Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed

A 7Crp peptide composed of seven major human T cell epitopes derived from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 is an ideal tolerogen for peptide immunotherapy against Japanese cedar pollinosis. To maximize the accumulation level of the 7Crp peptide in transgenic rice seed, we tested endosperm specific promoters and intracellular localizations suitable for stable accumulation. A 7Crp peptide carrying the KDEL ER retention signal directed by the 2.3-kb promoter of the glutelin GluB-1, which contains a signal peptide, accumulated at the highest level of about 60 μg/grain. Notably, the 7Crp peptide predominantly accumulated in ER-derived protein bodies irrespective of the presence of various sorting signals or expression as a fusion protein with glutelin. We attribute this abnormal pattern of accumulation to the formation of disulfide bonds between the 7Crp peptide and cysteine-rich (Cys-rich) prolamin storage proteins. Furthermore, the formation of these aggregates induced the chaperone proteins BiP and PDI as an ER stress response.