New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

Background  

The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells

Changes in vacuolar structure and the expression at the RNA level of a tonoplast aquaporin (BobTIP26-1) were examined in cauliflower (Brassicaoleracea L. var. botrytis) under water-stress conditions. Gradual drying out of slices of cauliflower floret tissue caused its collapse, with a shrinkage in tissue and cell volumes and an apparent vesiculation of the central vacuole, whereas osmotic stress resulted in plasmolysis with a collapse of the cytoplasm and the central vacuole within. Osmotic stress caused a rapid and substantial increase in BobTIP26 mRNA in slices of floret tissue. Exposure of tissue slices to a regime of desiccation showed a slower but equally large rise in BobTIP26 mRNA followed by a rapid decline upon rehydration. In situ hybridization showed that BobTIP26-2 mRNA is expressed most highly in meristematic and expanding cells of the cauliflower florets and that desiccation strongly increased the expression in those cells and in differentiated cells near the xylem vessels. These data indicate that under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells. Received: 21 October 1998 / Accepted: 10 February 1999     

Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles

The Arabidopsis thaliana Tonoplast Intrinsic Protein 1;1 (AtTIP1;1) is a member of the tonoplast aquaporin family. The tissue-specific expression pattern and intracellular localization of AtTIP1;1 were characterized using GUS and GFP fusion genes. Results indicate that AtTIP1;1 is expressed in almost all cell types with the notable exception of meristematic cells. The highest level of AtTIP1;1 expression was detected in vessel-flanking cells in vascular bundles. AtTIP1;1-GFP fusion protein labelled the tonoplast of the central vacuole and other smaller peripheral vacuoles. The fusion protein was not found evenly distributed along the tonoplast continuum but concentrated in contact zones of tonoplasts from adjacent vacuoles and in invaginations of the central vacuole. Such invaginations may result from partially engulfed small vacuoles. A knockout mutant was isolated and characterized to gain insight into AtTIP1;1 function. No phenotypic alteration was found under optimal growth conditions indicating that AtTIP1;1 function is not essential to the plant and that some members of the TIP family may act redundantly to facilitate water flow across the tonoplast. However, a conditional root phenotype was observed when mutant plants were grown on a glycerol-containing medium.     

The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group.

Aquaporins, proteins that enhance the permeability of biological membranes to water, are widely distributed in living organisms. They are 26- to 29-kD proteins that belong to the major intrinsic protein (MIP) family of channels. By searching the Arabidopsis thaliana expressed sequence tag database and by using the polymerase chain reaction with oligonucleotides to conserved plant aquaporin domains, we identified 23 expressed Arabidopsis MIP genes. Eight of these had been previously identified as active aquaporins, and two additional ones are now reported to have water-transport activity in Xenopus laevis oocytes. One of these is highly expressed in suspension-cultured cells. On a dendrogram these 23 MIP sequences cluster into three groups: the first group has 11 members and contains the plasma membrane aquaporins, the second group also has 11 members and contains the tonoplast aquaporins, and the third group has only a single member. This MIP protein, provisionally called At-NLM1, is most closely related to the Gm-NOD26 protein that is found in the bacteroid membranes of soybean (Glycine max L.) nodules; At-NLM1 is an active aquaporin when expressed in oocytes. With a semiquantitative slot-blot analysis technique, we determined the expression levels of 22 MIP genes in the various organs. The quantitative polymerase chain reaction was used to determine the effects of various stress treatments on the expression of NLM1.     

TAT-Mediated Protein Transduction of Nogo Extracellular Peptide 1-40 and its Biological Activity

Aim Nogo extracellular peptide 1-40 (NEP1-40), a Nogo-66 antagonistic peptide, is one of the potential candidates for therapeutic intervention after central nervous system injury. This study is focused on the generation of TAT-NEP1-40 fusion protein and its transducible effects and biological activity. Methods TAT-NEP1-40 fusion protein was expressed in vitro. Transducible effects of TAT-NEP1-40 were analyzed by using immunofluorescence staining or Western blot in vitro and in vivo. The biological activity of TAT-NEP1-40 was assessed by its effects against oxygen and glucose deprivation (OGD)-induced PC12 cell damages. Results Our results showed that the TAT-NEP1-40 fusion protein was successfully expressed, purified, and refolded. Western blot analysis and immunofluorescence staining confirmed the delivery of TAT-NEP1-40 protein into PC12 cells and rat brains. OGD caused cell apoptosis or death, decreased cell viability, increased lactate dehydrogenase release in medium and the Bax/Bcl-2 ratio, all of which were prevented by the TAT-NEP1-40 fusion proteins when added exogenously to culture medium. In addition, TAT-NEP1-40 promoted neurite outgrowth of PC12 cells exposed to OGD. Conclusion These results demonstrate that the TAT-NEP1-40 can be successfully generated and efficiently transduced into PC12 cells and rat brains. The TAT-NEP1-40 can protect PC12 cells against OGD and promote neurite outgrowth. This finding suggests that the transducible TAT-NEP1-40 fusion protein offers a possibility of the development of novel therapy for cerebral injuries via delivery of the biologically active TAT-NEP1-40 fusion protein into injured sites. Qiang Wang and Xingchun Gou contributed equally to this article.     

AtPTR4 and AtPTR6 are differentially expressed,tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.     

The yeast osmosensitive mutant fps1Delta transformed by the cauliflower BobTIP1;1 aquaporin withstand a hypo-osmotic shock

Osmoregulation plays an important role in cellular responses to osmotic stress in plants and in yeast. Aquaporins contribute to osmotic adjustment by facilitating transport of water or solutes across membranes. The tonoplastic water channel BobTIP1;1 (original name BobTIP26-1) genes are upregulated during dessication stress in cauliflower meristematic tissue. To investigate the physiological importance of BobTIP1;1, we expressed it in a Saccharomyces cerevisiae osmosensitive mutant fps1Delta. We showed that the defect in the yeast glycerol plasma membrane transporter is complemented by a plant cDNA encoding the aquaporin BobTIP1;1 which is localized in the vacuolar membrane of the complemented yeast cells. To our knowledge, this is the first example of a plant aquaporin for which localization in the vacuolar membrane of yeast cells is related to an osmoresistant phenotype under hypo-osmotic shock.     

Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.     

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

 Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity. Received: 2 July 1998 / Accepted: 14 September 1998     

Identification with a photoaffinity reagent of a tonoplast protein involved in vacuolar malate transport of Catharanthus roseus

The effect of N-(4-azido-salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a K_ti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of ¹²⁵I-labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate-binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.     

Generation of B6‐Ddx4em1(CreERT2)Utr,a novel CreERT2 knock‐in line,for germ cell lineage by CRISPR/Cas9

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock‐out mouse studies. In this study, we established a novel knock‐in mouse line, B6‐Ddx4 ^{em1(CreERT2)Utr}, which expresses CreERT2 recombinase under the control of the endogenous DEAD‐box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen‐treated B6‐Ddx4 ^{em1(CreERT2)Utr}::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6‐Ddx4 ^{em1(CreERT2)Utr} is beneficial for studying female and male germ cell development.     

Isolation and functional characterization of PgTIP1, a hormone-autotrophic cells-specific tonoplast aquaporin in ginseng

The suppression subtractive hybridization technique was used to identify differentially expressed genes between hormone-autotrophic and hormone-dependent Panax ginseng callus lines. A tonoplast intrinsic protein cDNA (PgTIP1) was found to be highly and specifically expressed in hormone-autotrophic ginseng cells, which was slightly up-regulated by cytokinin while significantly down-regulated when treated with auxin. PgTIP1 encodes a polypeptide of 250 amino acids which shows sequence and structure similarity with tonoplast aquaporins in plants. The water channel activity of PgTIP1 was demonstrated by its expression in Xenopus laevis oocytes. When over-expressed in Arabidopsis thaliana, PgTIP1 substantially altered the plant's vegetative and reproductive growth and development. Arabidopsis plants over-expressing PgTIP1 showed significantly enhanced seed size and seed mass plus greatly increased growth rate compared with those of the wild type. Moreover, the seeds from PgTIP1 over-expressing Arabidopsis had 1.85-fold higher fatty acid content than the wild-type control. These results demonstrate a significant function of PgTIP1 in the growth and development of plant cells.     

Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.     

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_n (n?=?2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96?hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.     

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l⁻¹ 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.     

Quantitative Stress Responses of the V0V1-ATPase of Higher Plants Detected by Immuno-electron Microscopy

Immuno-electron microscopy of negatively stained isolated tonoplast vesicles was used to quantify stress responses of the H⁺-transporting tonoplast ATPase (V₀V₁-ATPase; EC 3.6.1.1) of the C₃/CAM intermediate Mesembryanthemum crystallinum L. and the C₃ plant Hordeum vulgare L. This approach has the advantage that it relates quantitative adaptations at the level of membrane enzymes directly to membrane area and thus is independent of concomitant changes of relative amounts of other membrane proteins which may perturb conclusions when data are expressed on a tonoplast protein basis. It was shown that in M. crystallinum the amount of V₀V₁-ATPase per unit membrane area increased slightly with ageing and pronouncedly with salinity stress. In H. vulgare under salt stress there was an increase in V₀V₁-ATPase amount only in the highly salt tolerant cv. California Mariout and not in the moderately tolerant cv. Carina. This corroborates conclusions from earlier work, where results were expressed on a protein basis, although this was not to be expected a priori. In all comparative ecophysiological studies using tonoplast vesicles at least some key-point tests with the immunonegative staining technique should be included for the sake of prudence. The data obtained here via immunonegative staining of isolated tonoplast vesicles are in very close agreement with much earlier assessments of area and whole cell-related activities given by measurements of entire isolated vacuoles and morphometric analysis, which further corroborates the suitability of the approaches. The data presented here for the first time allow calculations of the coverage of the tonoplast surface of M. crystallinum with V₀V₁-ATPase holoenzyme protein and with total tonoplast protein, i.e. 1.5 to 2.3 . 10^?15 g V₀V₁-ATPase per μm² and 7.4 to 8.8 . 10^?15 g protein per μm², respectively.     

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.