- Research Article
- Open Access
Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants
© The Author(s) 2017
- Received: 26 April 2017
- Accepted: 18 July 2017
- Published: 20 September 2017
Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.
Influenza A viruses, negative-stranded enveloped orthomyxoviruses, are among the most serious respiratory pathogens. They cause severe and potentially fatal illnesses . Highly pathogenic avian flu influenza viruses are expected to cause the next global pandemic threat due to their relative easy spread by avian hosts and their ability to directly infect humans . During several H5N1 outbreaks, very large direct and indirect impacts on poultry and tourist industries in South-East Asia were observed . Recently, highly pathogenic avian influenza viruses (HPAI) A (H5N8) caused outbreaks in South Korea, China and Japan and were actually reported in many European countries as well as in the US and Canada (for an review, see ). Therefore, the development of an effective and cheap vaccination strategy to protect poultry is necessary. This should include high efficacy of the vaccine, easy and sure production strategies and an efficient way to handle distribution and application. Here, subunit vaccines from plants, which have the general advantages of low production cost, ease of scale up, low infrastructure cost, high stability and long shelf life are in the focus . Transient expression in tobacco plants has been developed as a very fast and efficient method to produce therapeutic proteins in plants (for a review, see ). A recently developed strategy is the production of virus-like particle (VLP)-based vaccines in the tobacco species N. benthamiana, by transient expression and downstream processing steps that include several filtrations, diafiltrations, continuous flow centrifugations and tangential flow filtration, or, alternatively, chromatographic methods [7, 8]. The production of VLP’s is accompanied by several constraints such as high down-stream cost and/or low expression levels. We produced trimeric H5 hemagglutinins in the endoplasmic reticulum of plant leaf cells. An artificially designed trimerization domain (GCN4-pII, ) was used to achieve stable trimers of H5 hemagglutinins from plants . The purified trimers were shown to be active in a hemagglutination assay and also induced neutralizing humoral immune responses as shown by mouse immunization and hemagglutination inhibition assays. We also developed a suitable cheap and efficient purification system from plant extracts by using ELPylation .
In the current article we wanted to check, if a further increase of the size of H5 multimers could improve the induction of immune responses. Because hemagglutinin forms trimers in its’ native structure on the surface of viruses we planned to keep the trimers as a basic structure of the oligomers. Further oligomerization should be caused by S·Tag–S·Protein interaction. Bovine pancreatic ribonuclease A, 124 amino acids in length, is cleaved by the protease subtilisin. The cleavage product consists of two tightly associated fragments: S-peptide (residues 1–20) and S·Protein (residues 21–124). Only residues 1–15 of S-peptide were found to be necessary to complex specifically with S·Protein. This shorter fragment is named as “S15” or the “S·Tag” sequence . High-affinity interaction between S·Protein and S·Tag of bovine pancreatic ribonuclease A was recently developed as target for protein purification  or for drug delivery . We applied this technology to generate H5 oligomers. To achieve this goal, a S·Tag was fused to H5 trimers and this construct was transiently co-expressed with different multimeric variants of S·Protein in planta.
Construction of plant expression vectors
Agrobacterium infiltration for expression of recombinant proteins was described in detail by Phan and Conrad, 2016 , and is briefly described here. Agrobacteria harboring the shuttle vectors for the expression of recombinant proteins (Figure 2) and the plant vector for expression of HcPro, which is a suppressor of gene silencing that has been found to enhance the expression levels of recombinant proteins in plant cells [17, 18] were pre-cultivated separately in LB medium with 50 µg/mL kanamycin, 50 µg/mL carbenicillin and 50 µg/mL rifampicin overnight at 28 °C and 140 rpm. The precultures were added to a new LB culture containing the appropriate antibiotics. After 24 h of cultivation, bacteria were harvested by centrifugation (4000g, 30 min, 4 °C) and resuspended in infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgSO4, pH 5.6). Agrobacteria harboring the shuttle vector for the expression of recombinant protein and the plant vector for the expression of HcPro were combined and diluted in infiltration buffer to a final OD600 of 1.0. N. benthamiana plants (6–8 weeks old) were infiltrated by completely submerging each plant in an Agrobacterium-containing cup standing inside a desiccator. Vacuum was applied for 2 min and then quickly released. The plants were then placed in the greenhouse at 21 °C, 16 h light per day. Five days after infiltration, leaf samples were harvested and stored at −80 °C. Two agrobacterial strains were mixed and combined with HcPro strain to co-express H5-S·Tag and S·Protein variants. Agrobacteria were then diluted in the infiltration buffer, and were used for vacuum infiltration described above.
Total soluble protein extraction
Five days after vacuum infiltration of Agrobacterium, 20 g of leaf samples were harvested, ground in liquid nitrogen and homogenized in 60 mL of PBS buffer (pH 7.4) using a commercial blender. The extracts were clarified by centrifugation (16 200 g, 30 min, 4 °C). Total soluble protein contents of clear plant extracts were determined by Bradford assay .
Protein concentrations of all plant extracts were diluted to 3 µg/µL. A H5-S·Tag plant extract was then combined with a single S·Protein variant plant extract in equal volume. The mixtures were rotated at 4 °C for 1 h and used for hemagglutination assay. H5 oligomer or H5-S·Tag in crude plant extracts was semi-quantified by Western blotting. A series of known concentrations of the anti TNFα-nanobody-ELP standard protein  was used to construct blot signal intensities. Hemagglutinin contents in the plant crude extracts were determined by comparing their blot signal intensities and blot signal intensities of the standard protein.
SDS-PAGE and Western blotting
Extracted plant proteins, purified proteins, or an anti TNFα-nanobody-ELP standard protein  were separated by reducing SDS-PAGE (10% polyacrylamide) and then electrotransferred to nitrocellulose membranes. The Western blotting procedure was carried out using monoclonal anti-c-myc antibodies following the protocol described by Gahrtz and Conrad . Sheep anti-mouse IgG, horseradish peroxidase-linked whole antibody was used as the secondary antibody (secondary antibodies, GE Healthcare UK limited, Little Chalfont Buckinghamshire, UK) followed by enhanced chemiluminescence-based detection (ECL). A total of 10 ng of the IMAC and SEC purified hemagglutinin was separated by reducing SDS-PAGE (10% polyacrylamide) and electrotransferred to nitrocellulose membranes. To detect H5-specific mouse antibodies, ten mouse sera from each group were mixed, and the membranes were incubated with the respective mixtures (200 times dilution). Specific signals were detected as described above.
Protein purification by IMAC
Five days after vacuum infiltration of Agrobacterium, leaf samples were harvested, frozen in liquid nitrogen and homogenized using a commercial blender. Total proteins were extracted in 50 mM Tris buffer (pH 8.0). The extracts were clarified by centrifugation (75 600 g, 30 min, 4 °C) and then filtrated through paper filters. The clarified extracts were mixed with Ni–NTA agarose resin that had been washed twice with water beforehand. After mixing for 30 min at 4 °C, the mixture was added to a chromatography column. Thereafter, the column was extensively washed (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, pH 8.0). Recombinant proteins were then eluted from the column with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 125 mM Imidazole, pH 8.0), put into dialysis bags, concentrated in PEG 6000 and dialyzed against PBS.
Purification of H5 oligomer using Galanthus nivalis (GLN)-linked agarose
Frozen leaf samples (40 g) were homogenized in liquid nitrogen. Total soluble proteins were extracted in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.5). The extract was centrifuged twice (75 600 g, 30 min, 4 °C) and mixed with 10 mL of GLN resin that had been washed twice with water and once with PBS. After mixing at 4 °C for 30 min, the mixture was applied to a chromatography column. Thereafter, the column was washed two times with 30 mL PBS. The recombinant protein was then eluted from the column with 10 mL elution buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 200 mM α-methyl mannoside, pH 7.4). The protein solution was dialyzed against PBS at 4 °C overnight and concentrated using PEG 6000. The protein products were loaded on Superose™ 6 increase 10/300GL.
Size exclusion chromatography
A total of 34 µg protein/0.5 mL of purified H5 oligomers and H5-S·Tag, respectively, were loaded onto a Superose™ 6 increase 10/300GL column (GE Healthcare). The high molecular weight kit contains standard proteins with molecular weights in the range of 44–2000 kDa, which were loaded onto the column to estimate the molecular weight of the proteins of interest. Five hundred microliters per fraction were collected for hemagglutination test and Western blot analysis.
For ELISA, affinity-purified trimeric hemagglutinin was used as an antigen for the plate coating and was further purified via the Superose™ 6 increase 10/300GL column with starting concentrations of 1.25 mg in 0.5 mL.
The hemagglutinin contents (H5 oligomer and H5-S·Tag) in plant extracts were semi-quantified by Western blotting. Plant extracts containing 100 ng of either H5 oligomers or H5-S·Tag were selected for mouse immunization. In the control groups, a plant extract containing S·Protein-TP and a non-transformed plant extract that had the same amount of total soluble protein as plant extracts containing H5 oligomers and H5-S·Tag were used. All plant extracts were formulated with the Emulsigen®-D adjuvant (MVP Technologies, 4805 G Street, Omaha, NE 68117, USA) at 20% final concentration. Seven–nine-week-old male C57/Bl6/J mice (Charles River Laboratories, Research Models and Services, Germany GmbH; twelve per group) were subcutaneously immunized with Emulsigen®-D adjuvant-formulated plant extracts at days 0, 14 and 28. One week after the 2nd and 3rd immunizations, mice were bled via the retro-orbital sinus. Mouse sera were collected individually for hemagglutination inhibition and ELISA tests.
Hemagglutination test and hemagglutination inhibition assay
The hemagglutination test was based on a standard protocol . The dilution that induced complete hemagglutination was defined as one hemagglutination unit (HAU). The HI assay was performed similarly based on a standard procedure . Because of unavailability of the A/duck/Viet Nam/TG24-01/2005 (H5N1) virus in an inactivated form, the heterologous inactivated virus strain rg A/swan/Germany/R65/2006(H5N1) was used for HI assay. The deduced hemagglutinin amino acid sequence similarity of both strains is 96%. A 25 µL aliquot of serum from a single mouse was placed in the first well of a microtiter plate containing 25 µL PBS, and two-fold serial dilutions were made across the row of 8 wells. A 25 µL volume containing 4 HAU of the inactivated rg A/swan/Germany/R65/2006(H5N1) virus was added to the reaction and incubated at 25 °C for 30 min. Then, 25 µL of 1% chicken red blood cells was added, and the plates were incubated at 25 °C for 30 min. The HI titer is presented as the reciprocal of the highest dilution of serum that could completely inhibit hemagglutination.
Microtiter plates (ImmunoPlateMaxisorp, Nalgen Nunc International, Roskilde, Denmark) were coated with 100 µL of 0.5 µg/mL of IMAC- and SEC-purified hemagglutinin trimers in phage PBS (100 mM NaCl, 32 mM Na2HPO4, 17 mM Na2HPO4, pH 7.2) and incubated overnight at room temperature. After blocking with 3% (w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween20 in PBS (PBST) for 2 h, 100 µL of the specific dilution (6 × 10−4) was applied and incubated at 25 °C for 1 h. Plates were washed 5 times with PBST, incubated with alkaline phosphatase-conjugated rabbit anti-mouse IgG diluted (2000 times) in 1% (w/v) BSA and washed again. The enzymatic substrate, p-nitrophenyl phosphate (pNPP) in 0.1 M diethanolamine-HCl (pH 9.8) was added, and the absorbance signal was measured at 405 nm after a 1 h incubation at 37 °C.
Statistical analyses of HI data and ELISA results were performed using Mann–Whitney Rank-Sum test and T test (ELISA) in Sigma Plot software. p-values < 0.05 were defined as significant.
Recombinant hemagglutinin-S·Tag fusion proteins and S·Protein variants are produced in planta
Trimeric hemagglutinin containing S·Tag and S·Protein variants were designed and expressed in planta, to generate influenza hemagglutinin oligomers based on the specific interaction of bovine S·Protein and the S·Tag (Figure 1). We fused the S·Tag (15 amino acid in length) c-terminally to the artificial trimerization domain (GCN4-pII) of the H5 hemagglutinin. This trimerization domain was proven to stabilize influenza hemagglutinin as trimers in planta . An ubiquitous plant promoter (CaMV35S), a signal peptide and an endoplasmic reticulum (ER) retention signal (KDEL) allow for the production and accumulation of trimers in the ER of leaf cells after transient expression (Figure 2). The accumulation of such trimers has been analyzed by Western blot via the c-myc tag after separation of plant crude extracts at denaturing conditions in a SDS gel.
We constructed an expression vector, that allows for the production of potentially multimeric wild-type S·Protein  in the plant ER (Figure 2) and performed transient expression experiments in N. benthamiana leaves. The analysis of crude extracts of these leaves by Western blot after separation at denaturating conditions revealed a major band that corresponds to a S·Protein monomer (Figure 3B, S·Protein, lane 7).
H5-S·Tag and S·Protein constructs were co-expressed in the ER of leaf cells. After co-infiltration of N. benthamiana with the appropriate Agrobacterium strains, leaf crude extracts were analyzed by Western blot after separation at denaturating conditions. Two major bands, reflecting H5-S·Tag and S·Protein, each corresponding in size to the molecular weights of the single expressed proteins were detected (Figure 3B, S·Protein::S·Tag, lane 9).
Expression and functionality profiles of recombinant influenza hemagglutinin and S·Protein variants
Protein expression confirmed by Western blot
Hemagglutination unit (HAU)
Plant extracts containing each single recombinant protein
Combination with H5-S·Tag in vitro
Expression and functionality profiles of recombinant influenza hemagglutinin and S·Protein variants
Protein expression confirmed by Western blot
Hemagglutination unit (HAU)
Co-expression with H5-S·Tag in plants in vivo
Screening for an optimal hemagglutinin oligomerization tool
The hemagglutination assay is based on the ability of influenza hemagglutinin to bind sialic acid receptors presented on the surface of chicken red blood cells. Cross-linkages between influenza hemagglutinin and red blood cell cause the formation of a lattice called hemagglutination. In the hemagglutinin oligomers (Figure 1), cross-linkages between hemagglutinin trimers were already built via S·Protein and S·Tag interaction, while influenza trimers lack these linkages. Therefore, when hemagglutinin oligomers were mixed with given amount of red blood cells, high hemagglutination titers will be expected compared with those of trimeric hemagglutinin. This assay was used to screen for formation of hemagglutinin oligomers in plant crude extracts. The hemagglutination titers caused by the plant crude extracts containing single H5-S·Tag or S·Protein, as well as co-expressed proteins (H5-S·Tag and S·Protein) were all very low (Figure 3A; Table 2). We hypothesized that the wild-type S·Protein was not being sufficiently multimerized. Therefore, we fused different oligomerization motifs, such as the trimerization motif GCN4-pII, the dimerization motif GCN4 wild-type  and a tailpiece (TP) element of mouse IgM to the c-terminal end of the S·Protein sequence to multimerize the S·Protein. TP elements are responsible for the interchain connections between constant parts of single IgM chains to penta- or hexamers via disulfide bridges  (Figures 1 and 2). The principal plant expression constructs coding for S·Protein-pII, S·Protein-GCN4 and S·Protein-TP are shown in Figure 2. The plant expression has been tested by a transient assay in N. benthamiana. Each single crude extract was analyzed by a hemagglutination assay but no hemagglutination activity could be measured (Table 1). These S·Protein variants (S·Protein-pII, S·Protein-GCN4, and S·Protein-TP, respectively), were co-expressed with H5-S·Tag. In parallel, extracts containing the single S·Protein variants, were each mixed in vitro with extracts containing H5-S·Tag. These extracts respectively mixtures were tested in hemagglutination assays. Only the variant co-expression of S·Protein-TP with H5-S·Tag (named as H5 oligomers) was found to cause a very high hemagglutination titer of 256 (Figure 3A; Table 2). All other variants, including the S·Protein-TP with H5-S·Tag mixtures conducted in vitro, did not cause increased hemagglutination titers (Tables 1 and 2). The analysis of H5 oligomer extracts, H5-S·Tag extracts and S·Protein-TP extracts by Western blot revealed major bands of expected sizes (Figure 3B, lanes 5, 6 and 8). Notably, the co-expression of H5-S·Tag and S·Protein-TP had no significant effect on the accumulation of hemagglutinin (Figure 3B). Hemagglutinin was accumulated in plants with around 0.2% of total soluble proteins estimated by Western blot (data not shown).
Formation of H5 oligomers in planta but not in vitro
The protein expression of each S-tagged H5 trimers and S·Protein-TP in different plants and the in vitro combination of the extracts caused hemagglutination titers of 0 (Table 1), although production of the different components at the expected molecular weights was shown by Western blot (Figure 5B, summarized in Tables 1 and 2). The low hemagglutination titers reflect that hemagglutinin oligomers were not formed in vitro. As shown in previous studies, functionally active S·Protein was only yielded in the presence of the S·Tag. Obviously, the S·Tag serves as a template for proper folding of S·Protein [12, 13, 26]. We suppose, that S·Protein-TP is expressed in an inactive form in the absence of H5-S·Tag and therefore it does not interact with H5-S·Tag in vitro. Exclusively, the co-expression of H5-S·Tag and S·Protein-TP caused high hemagglutination titers as well as very high molecular weights by production in planta in the ER, thus allowing folding and oligomerization by closure of disulfide bridges. This variant presents a new and innovative way to generate functional H5 oligomers in planta.
H5 oligomers are highly immunogenic
We conclude that plant crude extracts containing H5 oligomers are more immunogenic than the trimeric H5-S·Tag containing extracts as measured by an indirect ELISA. H5 oligomers induce neutralizing antibodies to a significantly higher extend compared to trimeric H5-S·Tag vaccines. The stability of H5 derivatives in crude extracts is important for the feasibility of the concept. The immunogenic extracts were stored at 4 °C for 1 week without loss of antigen content, as revealed by Western blot and hemagglutination titer (see Additional file 2).
The transient expression system has emerged as an alternative platform to produce influenza subunit vaccine candidates because of its capacity to be easily scaled up and because of the rapid production process [7, 29]. The method of infiltration of plant leaves with Agrobacteria harboring the transgene is a robust and technologically simple method . Two major approaches have been developed to efficiently express influenza vaccine candidates: expression of hemagglutinin ectodomains from swine flu H1N1 strains  or from avian flu H5N1 strains  and production of enveloped hemagglutinins as VLPs [7, 8, 29]. All plant-made hemagglutinin ectodomains tested to date were expressed as soluble monomeric [10, 32] as well as trimeric proteins stabilized by trimeric motifs [10, 31]. Artificially trimerized hemagglutinin ectodomains without S·Tag fusion, mimicking hemagglutinin homotrimers on the viral surface, enhanced immunogenicity, induced neutralizing antibodies  and reduced the necessary vaccine doses . In the actual study, trimeric hemagglutinin fused with S·Tag (H5-S·Tag) in crude extracts was confirmed again to induce neutralizing antibodies (Figures 8B and D). Here, we present a strategy to produce hemagglutinin trimer-based antigen oligomers. This concept is based on the specific interaction of bovine S·Protein and the S·Tag, both of them are cleavage products of ribonuclease A by the proteinase subtilisin . The trimerization by a specific domain (GCN4-pII motif [9, 10]) served as a founding structure of putative oligomers to design basic structures resembling native hemagglutinin homotrimers (see above). These hemagglutinin trimers should be further multimerized by interaction of an S·Tag fused to hemagglutinin with S·Proteins via co-expression of both proteins in the ER of plant cells. S·Proteins themselves are expected to consist of a mixture of monomeric, dimeric, trimeric and tetrameric proteins (or even more), as could be concluded from a previous study using ribonuclease A  (reviewed by ). Obviously, this was not sufficient to produce oligomeric H5 in the ER of plant cells (Tables 1 and 2). The fusion of dimerization and trimerization domains to the C-terminus of the S·Proteins and the subsequent co-expression in the plant cell ER (Figures 3A and B) also did not induce oligomers in high concentrations as could be concluded from low hemagglutination titers (Figure 3A; Tables 1 and 2). However, the plant extracts achieved after co-expression of H5-S·Tag and S·Protein-TP (containing the TP sequence at the c terminus) showed significantly increased hemagglutination titers compared to hemaglutination titers induced by H5-S·Tag or S·Protein-TP, respectively (Figures 3A and B; Table 2). This could be explained by the different structures of dimerization or, trimerization motives and disulfide bonds. The dimerization and trimerization domains are parallel, coiled coil structures  which can force their fusion partners to dimerize or trimerize in their fixed angles. However, the disulfide bonds formed by cysteine residues in the TP elements are flexible joins allowing S·Protein-TPs to fold correctly into an active form and allowing S·Protein-TPs to bend, twist, and flex into an optimal position to bind H5-S·Tag to finally form oligomers.
We conclude, that large oligomers were built. When a tail piece sequence was fused to wild-type S·Protein, monomeric, dimeric, trimeric or tetrameric S·Protein-TP joined with themselves or with others to generate multimerized S·Protein-TP via disulfide bonds. The plant ER, containing protein disulphide isomerases, is the perfect compartment for these processes. The resulting multimeric S·Protein-TPs have multiple valences to bind trimeric H5-S·Tag proteins to form H5 oligomers. This process is partially presented in Figure 1. In fact, H5 oligomers are a mixture of different numbers of H5-S·Tag and S·Protein-TP incorporated into the complexes (Figures 1, 4 and 5). Larger complexes cause higher hemagglutination titers (Figure 5). These large complexes cause improved neutralizing immune responses as shown by higher hemagglutination inhibition titers (Figure 8B and D). The low p values (p ≤ 0.001) comparing the HI titers after immunizations with either H5 oligomer or H5-S·Tag after 2 and 3 immunizations document the significance of these differences. Other crucial prerequisites for the successful development of a veterinary vaccine against avian flu are speed and practicability. Agrobacterium infiltration systems allow for the production of large amounts of proteins only a few days after finishing the cloning procedure, and thus, this concept generally shares speed and convenience . The lack of down-stream processing effort also fits into the general timeline demands for flu vaccines . Downstream cost for recombinant expression systems can represent up to 80% of the overall processing cost . These high downstream costs are a major bottleneck limiting the commercial production of plant-based pharmaceuticals . Thus, the successful use of crude extracts for immunization as performed in the actual study can significantly lower down-stream cost. This is essentially important for veterinary vaccines, where cost have to be low to fit into economical parameters of animal-based production . The principles shown and discussed here will generally allow for the development of low cost vaccines with unlimited scalability as precautions for veterinary immunotherapies . In the actual paper, we use a synthetic biology approach to combine different principles of protein–protein interaction from different organisms to design an innovative vaccine concept in plants .
The authors declare that they have no competing interests.
HTP, HHC, THV and UC conceived the study and designed the experiments. TTH, UG and HTP performed experiments. UC, HTP and TTH wrote the manuscript with the input of all other authors. All authors read and approved the final manuscript.
We thank J. Veits from FLI Riems for providing inactivated influenza viruses and U. Apel and H. Ziebell, JKI Quedlinburg for help with the animal experiments. The help of E. Stöger, BOKU Vienna and E. Sorge, IPK, in critical reading the manuscript is also gratefully acknowledged. This work was supported by the Federal Ministry of Education and Research-BMBF, Germany, 031A283 and by the Ministry of Science and Technology-MOST, Vietnam, NĐT.07.GER.15.
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