Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

KEY MESSAGE: We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

Clinical trials of Cry j 1 and Cry j 2 T-cell epitope peptide-expressing rice in patients with Japanese cedar pollinosis.

BACKGROUND: In allergic models, administration of rice that expresses a hybrid peptide consisting of 7 major T cell epitopes of Cry j 1 and Cry j 2 (7Crp), suppressed allergic symptoms, IgE elevation and specific T cell response to Japanese cedar pollen. OBJECTIVE: To evaluate the efficacy and safety of 7Crp-expressing rice in patients with Japanese cedar pollinosis. METHODS: A 24-week randomized, double-blind, placebo-controlled study was performed to see the efficacy of 7Crp on allergic symptoms using scoring systems, in which 45 patients were assigned to take either 5 g, 20 g test rice, or placebo daily. A 96-week open study was also conducted to determine its inhibitory effect on serum IgE and T cell proliferative response for Japanese cedar pollen, in which 10 patients consumed 5 g test rice daily. RESULTS: No adverse events associated with the test rice occurred, and the intake rate was more than 96%. The test rice did not show suppression of symptoms related to Japanese cedar pollinosis within 24 weeks. However, intake of 5 g test rice led to a significant decrease in T cell response to Japanese cedar pollen during and after the second disperse season in a 96-week open trial, whereas the specific IgE titer remained unchanged. CONCLUSIONS: Tolerability and safety of 7Crp-expressing rice was accepted. Daily intake of up to 20 g transgenic rice did not provide beneficial effects on Japanese cedar pollinosis within 24 weeks, however, continuous intake of 5 g rice might reduce allergen specific T cell response.

Long-term oral administration of transgenic rice containing cedar pollen T-cell epitopes potentially improves medication- and allergy-related quality-of-life scores.

Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their clinical symptoms did not improve. Objective: We examined the clinical efficacy of low-dose (5 g and 20 g) intake of the transgenic rice administered for two successive seasons. Methods: In this randomized, double-blind, placebo controlled study, transgenic rice seeds (5 g or 20 g) were orally administered to the participants for 24 weeks in each of two successive JC pollen seasons. We analyzed T-cell proliferation and cytokine expression, and monitored symptom and medication scores during the pollen season. Quality of life (QOL) was evaluated by using the Japanese Allergic Rhinitis Quality of Life Standard Questionnaire (JRQLQ). Results: Specific T-cell proliferation after stimulation with 7Crp, Cry j 1, and Cry j 2 was significantly suppressed in the second JC pollen season. No significant differences were found among the three groups (5 g, 20 g, and placebo) with regard to clinical symptoms or medication scores in the first season. However, the medication scores and face scale for overall condition of JRQLQ improved in the 5-g transgenic rice group in the second season, although careful re-examination with a large sample size is necessary to confirm the results. Conclusion: Low-dose oral administration of transgenic rice that contains 7Crp significantly reduced allergen-specific T-cell responses and improved medication scores during the second season of administration. Thus, oral intake of the transgenic rice has the potential to induce immune tolerance to JC pollen allergens when administered for at least two successive seasons.

Immunological and Symptomatic Effects of Oral Intake of Transgenic Rice Containing 7 Linked Major T-Cell Epitopes from Japanese Cedar Pollen Allergens.

BACKGROUND: A rice-based peptide vaccine containing 7 linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens, Cry j 1 and Cry j 2, was developed. Here, we examined the efficacy and safety of this transgenic rice in JC pollinosis patients. METHODS: Transgenic rice (5, 20, and 80 g) was administered orally. We measured the T-cell proliferative activity against 7Crp, Cry j 1, and Cry j 2; the cytokine expression levels; and specific IgE and IgG4 production levels. In addition, the symptom and medication scores were monitored during the pollen season, and quality of life (QOL) was evaluated. RESULTS: T-cell proliferative activities to Cry j 1, Cry j 2, and 7Crp were significantly depressed in a dose-dependent manner. Oral intake of 80 g transgenic rice for 20 weeks resulted in significant suppression of allergen-specific T-cell proliferation with downregulation of IL-13 and upregulation of IL-10 levels but no changes to specific IgE and IgG4 levels. The QOL symptom scores for allergic rhinitis were not significantly improved. CONCLUSIONS: Allergen-specific T-cell responses were significantly reduced by oral intake of transgenic rice in a dose-dependent manner. However, neither medication score nor QOL symptom scores could be improved during the JC pollen season with oral intake of transgenic rice for 20 weeks.

Improvement of production yield and extraction efficacy of recombinant protein by high endosperm-specific expression along with simultaneous suppression of major seed storage proteins.

Human transforming growth factor-ß1 (hTGF-ß1)　was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins　(SSPs)　through RNA interference (RNAi). When driven by the 26 kDa α-globulin endosperm-specific promoter, it accumulated up to the markedly high level of 452 µg/grain. However, exchange with other seed-specific promoters such as 18 kDa oleosin and AGPase promoters resulted in remarkable reduction to the levels of 62 and 48 µg/grain, respectively, even though endogenous SSPs were reduced to the similar level. These production levels were almost similar to those (42 and 108 µg/grain) produced by the glutelin GluB-1 endosperm-specific promoter and the maize ubiquitin constitutive promoter without reduction of SSPs, respectively. When extracted from these transgenic rice seeds with reduced SSPs with various buffers, it could be solubilized with denaturant solution, which was in remarkable contrast with those without depressed SSPs which required further supplementation of reducing agent for extraction. This difference was associated with the fact that it was mainly deposited to ER-derived structures though self-aggregation or interaction with remaining prolamin via intermolecular disulfide bonds.

Safety and efficacy of rice seed-based oral allergy vaccine for Japanese cedar pollinosis in Japanese monkeys.

Japanese cedar (Cryptomeria japonica) pollinosis　(JCP) is one of the major seasonal IgE-mediated type I allergies from February to April each year. Not only human patients but also Japanese monkeys (Macaca fuscata) are afflicted with this pollinosis in Japan, which exhibit similar clinical allergic symptoms such as allergenic rhinitis and conjunctivitis. Therefore, monkeys naturally sensitized to JC pollen allergens are expected to serve as a suitable animal model for exploiting the allergy vaccine for JCP, since allergen-specific immunotherapy is the only curative treatment for allergy diseases. We generated transgenic rice containing the hypoallergenic JC pollen Cry j 1 and Cry j 2 allergen derivatives as tolerogen. In this study, safety and efficacy of transgenic rice seed were evaluated by oral administration to Japanese monkeys. Healthy monkeys were not sensitized to Cry j 1 and Cry j 2 allergens, even　when administered for one to ten months. By contrast, peripheral blood mononuclear cell　(PBMC) proliferation and IgE antibody specific　to these allergens were reduced in Japanese monkeys with JCP. Especially, suppression of allergen-specific PBMC proliferation was observed within only two months after administration. These findings indicate that this transgenic rice acts as effective tolerogen to induce oral immune tolerance against JC allergens.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

Transgenic rice seeds expressing altered peptide ligands against the M3 muscarinic acetylcholine receptor suppress experimental sialadenitis-like Sjögren's syndrome.

Objective: We previously reported that Rag1-/- mice inoculated with splenocytes from M3 muscarinic acetylcholine receptor (M3R) knockout mice immunized with an M3R peptide mixture developed sialadenitis-like Sjögren's syndrome (M3R-induced sialadenitis [MIS]). We also found that intravenous administration of altered peptide ligand (APL) of N-terminal 1 (N1), which is one of the T-cell epitopes of M3R, suppressed MIS. In this study, we aimed to evaluate the suppressive ability and its mechanisms of rice seeds expressing N1-APL7 against MIS.Methods: Rice seeds expressing N1 and N1-APL7 were orally administered to MIS mice for 2 weeks. The changes in saliva flow and sialadenitis (salivary gland inflammation) were analyzed. The M3R-specific T-cell response in the spleen and the expression of regulatory molecules in the cervical lymph nodes and mesenteric lymph nodes were also analyzed.Results: Oral administration of N1-APL7-expressing rice seeds significantly recovered reduction in saliva flow and suppressed sialadenitis when compared with treatment with nontransgenic rice seeds and N1 rice seeds. IFNγ production from M3R-reactive T cells tended to decline in the N1-APL7 rice-treated group as compared with those in the other groups. In the N1-APL7 rice-treated group, the mRNA expression levels of Foxp3 in the cervical-lymph-node CD4+ T cells were higher than those in the other groups.Conclusion: Oral administration of N1-APL7-expressing rice suppressed MIS via suppression of M3R-specific IFNγ and IL-17 production and via enhancement of regulatory molecule expression.Key messagesWe generated N1-peptide- or N1-APL7-expressing rice seeds. Oral administration of N1-APL7-expressing rice seeds significantly recovered the reduction of saliva flow and suppressed sialadenitis via the suppression of M3R specific IFNγ and IL-17 production and via enhancement of regulatory T (Treg) cells.

Development of Rice-Seed-Based Oral Allergy Vaccines Containing Hypoallergenic Japanese Cedar Pollen Allergen Derivatives for Immunotherapy.

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.

T-cell activation by transgenic rice seeds expressing the genetically modified Japanese cedar pollen allergens.

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.

Specific region affects the difference in accumulation levels between apple food allergen Mal d 1 and birch pollen allergen Bet v 1 which are expressed in vegetative tissues of transgenic rice.

KEY MESSAGE: Specific domain of the Mal d 1 was identified to be mainly involved in higher accumulation level in vegetative tissues of transgenic rice than the Bet v 1. Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When green fluorescent protein (GFP) fused to either of these allergens was expressed as a secretory protein in transgenic rice by ligating an N terminal signal peptide and a C terminal KDEL ER retention signal under the control of the maize ubiquitin constitutive promoter, the GFP:Mald1 highly accumulated in various tissues, whereas accumulation level of the GFP:Betv1 was remarkably reduced in vegetative tissues except for seed. Analysis by RT-PCR exhibited that there was little difference in their transcript levels, indicating the involvement of post-transcriptional regulation. To investigate the cause of such difference in accumulation levels, deletion analysis of the Mal d 1 and domain swapping between them were carried out in transgenic rice. The results showed that the region between positions 41-90 in the Mal d 1 is predominantly implicated in higher level accumulation in vegetative tissues as well as seed as compared with the Bet v 1. The GFP:Mald1 was localized in oligomeric form within ER lumen or ER-derived particles in vegetative tissues, whereas in seed mainly deposited into novel huge ER-derived protein bodies with the size of 5-10 µm in aleurone cells.

Oral Immunotherapy for Allergic Conjunctivitis Using Transgenic Rice Expressing Hypoallergenic Antigens.

Pollinosis, or allergic conjunctivitis and rhinitis induced by pollen, is one of the most common diseases worldwide. In Japan, Japanese cedar (Cryptomeria japonica) pollinosis is a predominant allergic condition that affects more than one-third of all Japanese individuals. Pharmacological treatments of allergic conjunctivitis include administration of antiallergic eye drops containing an antihistamine or mast cell stabilizer. However, these topical treatments provide transient relief from symptoms. The only available curative treatment for allergic diseases is allergen-specific immunotherapy. Sublingual immunotherapy for pollinosis has been found to be effective for suppression of ocular and nasal symptoms, but patient compliance is low. Oral administration of staple foods engineered to express allergens is a possible means of delivering antigens for immunotherapy, and its convenience would be expected to improve compliance. With the aim of developing more convenient, effective, and safe immunotherapies for allergic diseases, we have generated rice-based edible vaccines expressing antigens derived from dust mites or pollen from Japanese cedar, birch, or ragweed. In this study, we summarize the results of our immunotherapy studies using transgenic rice. Oral immunotherapy with transgenic rice seeds containing hypoallergenic modified forms of Japanese cedar pollen antigens was effective for both preventing allergic conjunctivitis and suppressing established disease in mice. Oral administration of transgenic rice seeds is thus a promising approach to immunotherapy for conjunctivitis and rhinitis induced by Japanese cedar pollen.

Transgene-independent heredity of RdDM-mediated transcriptional gene silencing of endogenous genes in rice.

To induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double-strand RNA of each promoter region and thus induced RNA-directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulphide isomerase (PDIL1-1) and luminal binding protein (BiP1); an endoplasmic reticulum stress-inducible gene (OsbZIP50); and genes with seed-specific expression encoding α-globulin (Glb-1) and glutelin-B4 (GluB4). TGS of four genes was obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50, 63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1-1 (33.3%) and Glb-1 TGS lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T-DNA) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the -1 to -250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post-transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of RdDM to new plant breeding techniques in rice are discussed.

Compensatory rebalancing of rice prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies.

KEY MESSAGE: Bioactive peptide was produced by fusion to rice prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type prolamin/APL12 led to reduction of other type(s) prolamin/APL12 to maintain the limited amounts of prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of prolamins deposited within PB-Is.

Efficacy of oral immunotherapy with a rice-based edible vaccine containing hypoallergenic Japanese cedar pollen allergens for treatment of established allergic conjunctivitis in mice.

BACKGROUND: We have previously shown that prophylactic oral administration of transgenic rice seeds expressing hypoallergenic modified antigens suppressed the development of allergic conjunctivitis induced by Japanese cedar pollen. We have now investigated the efficacy of oral immunotherapy with such transgenic rice for established allergic conjunctivitis in mice. METHODS: BALB/c mice were sensitized with two intraperitoneal injections of Japanese cedar pollen in alum, challenged with pollen in eyedrops, and then fed for 16 days with transgenic rice seeds expressing modified Japanese cedar pollen allergens Cry j 1 and Cry j 2 or with nontransgenic rice seeds as a control. They were then challenged twice with pollen in eyedrops, with clinical signs being evaluated at 15 min after the first challenge and the eyes, blood, spleen, and lymph nodes being isolated at 24 h after the second challenge. RESULTS: The number of eosinophils in the conjunctiva and the clinical score for conjunctivitis were both significantly lower in mice fed the transgenic rice than in those fed nontransgenic rice. Oral vaccination with transgenic rice seeds also resulted in a significant increase in the production of IFN-γ by splenocytes, whereas it had no effect on the number of CD4+CD25+Foxp3+ regulatory T cells in the spleen or submandibular or mesenteric lymph nodes. CONCLUSIONS: Oral administration of transgenic rice seeds expressing hypoallergenic allergens ameliorated allergic conjunctivitis in the established setting. Such a rice-based edible vaccine is potentially both safe and effective for oral immunotherapy in individuals with allergic conjunctivitis.

An overview on the strategies to exploit rice endosperm as production platform for biopharmaceuticals.

Cereal seed has been utilized as production platform for high-value biopharmaceutical proteins. Especially, protein bodies (PBs) in seeds are not only natural specialized storage organs of seed storage proteins (SSPs), but also suitable intracellular deposition compartment for recombinant proteins. When various recombinant proteins were produced as secretory proteins by attaching N terminal ER signal peptide and C terminal KDEL endoplasmic reticulum (ER) retention signal or as fusion proteins with SSPs, high amounts of recombinant proteins can be predominantly accumulated in the PBs. Recombinant proteins bioencapsulated in PBs exhibit high resistance to digestive enzymes in gastrointestinal tract than other intracellular compartments and are highly stable at ambient temperature, thus allowing oral administration of PBs containing recombinant proteins as oral drugs or functional nutrients in cost-effective minimum processed formulation. In this review, we would like to address key factors determining accumulation levels of recombinant proteins in PBs. Understanding of bottle neck parts and improvement of specific deposition to PBs result in much higher levels of production of high quality recombinant proteins.

Suppression of GPI-induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands.

OBJECTIVE: To investigate the effects and mechanisms of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI325-339) in mice model of GPI induced arthritis (GIA). METHODS: We generated transgenic rice expressing APL12 which was analog peptide of hGPI325-339. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined IL-17 production from splenocytes and inguinal lymph node (iLN) and mesenteric lymph nodes (mLN) cells and analyzed the expression levels of functional molecules from splenocytes and iLN cells. RESULTS: Prophylactic treatment of GIA mice with APL12 transgenic rice seeds (APL12-TG) significantly improved the severity of arthritis, histopathological arthritis scores, and decreased titers of serum anti-GPI antibodies, BAFF mRNA in iLN cells, IL-17 production in splenocytes and iLN cells compared with non-transgenic rice-treated mice. APL12-TG-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4+CD25+ cells in the spleen. CONCLUSION: APL12-TG improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on regulatory T cells in spleen.

Suppression of glucose-6-phosphate-isomerase induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of glucose-6-phosphate-isomerase.

OBJECTIVE: To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI325-339) in mice model of GPI-induced arthritis (GIA). METHODS: We generated transgenic rice expressing T-cell epitope of hGPI325-339 and APL12 contained in the seed endosperm. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined for IL-17 production in splenocytes and inguinal lymph node (iLN) cells, and analyzed the expression levels of functional molecules in splenocytes. RESULTS: Prophylactic treatment of GIA mice with APL12 transgenic (APL12-TG) rice seeds significantly reduced the severity of arthritis and titers of serum anti-GPI antibodies compared with non-transgenic (Non-TG) rice-treated mice. APL12-TG and hGPI325-339 transgenic (hGPI325-339-TG) rice seeds improved the histopathological arthritis scores and decreased IL-17 production compared with non-TG rice-treated mice. APL12-TG rice-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4 + CD25 + Foxp3+ cells in the spleen compared with non-TG rice- and hGPI325-339-TG rice-treated mice. CONCLUSION: APL12-TG rice seeds improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on Treg cells in spleen.

Deposition mode of transforming growth factor-ß expressed in transgenic rice seed.

KEY MESSAGE: Mouse TGF-ß highly accumulated by expressing as a secretory homodimeric protein in transgenic rice endosperm. It was tightly deposited in ER-derived PBs by interaction with cysteine-rich prolamins. TGF-ß is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the induction of regulatory T cells. In order to utilize rice-based TGF-ß as a tool to promote oral immune tolerance induction, high production of TGF-ß is essentially required. When the codon-optimized mTGF-ß was expressed as a secretory protein by ligating an N-terminal signal peptide and C-terminal KDEL ER retention signal under the control of the endosperm-specific rice storage protein glutelin GluB-1 promoter, accumulation level was low in stable transgenic rice seeds. Then, to increase the accumulation level of mTGF-ß, it was expressed as fusion proteins by inserting into the C terminus of acidic subunit of glutelin GluA and the variable region of 26 kDa globulin. When fused with the glutelin, it could accumulate well as visible bands by CBB staining gel, but not for the 26 kDa globulin. Unexpectedly, expression of homodimeric mTGF-ß linked by a 6×Gly1×Ser linker as secretory protein resulted in higher level of accumulation. This expression level was further enhanced by reduction of some endogenous prolamins by RNA interference. The monomeric and dimeric mTGF-ßs were deposited in ER-derived PBs containing prolamins. When highly produced in rice seed, it is notable that most of ER-derived PBs were distorted and granulated. Step-wise extraction of storage proteins from rice seeds suggested that the mTGF-ß strongly interacted with cysteine-rich prolamins via disulfide bonds. This result was also supported by the finding that reducing agent was absolutely required for mTGF-ß extraction.

Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts.

In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants.