Preparation of Antibody Against Immunodominant Membrane Protein (IMP) of Candidatus Phytoplasma aurantifolia

Document Type: Research Paper


1 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, IR Iran

2 Department of Microbial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran, Karaj, IR Iran Department of Plant Viruses, Iranian Research Institute of Plant Protection, Tehran, IR Iran

3 Razi Vaccine and Serum Research Institute, Tehran, IR Iran


Background: The witches’ broom disease of lime caused by Candidatus Phytoplasma aurantifolia, is the most devastating disease of acidian lime in the southern parts of Iran.
Objectives: At present, no effient method has been developed for controlling the disease, therefore quarantine approaches such as early detection and subsequent eradication of infected trees is very important. Toward this aim, developing a reliable and sensitive detection method would be the fist step to prevent transportation of infected plant materials to other places.
Background: The witches’ broom disease of lime caused by Candidatus Phytoplasma au‌rantifolia, is the most devastating disease of acidian lime in the southern parts of Iran.
Objectives: At present, no efficient method has been developed for controlling the dis‌ease, therefore quarantine approaches such as early detection and subsequent eradica‌tion of infected trees is very important. Toward this aim, developing a reliable and sensi‌tive detection method would be the first step to prevent transportation of infected plant materials to other places.
Materials and Methods: In this study, Immunodominant membrane protein (IMP) of the pathogen was selected as a target for detection and preparation of polyclonal anti‌body. The IMP is the major protein present on the surface of phytoplasma cells. For this purpose, the DNA region encoding IMP gene was isolated and cloned into pET28a bacte‌rial expression vector. The recombinant protein was expressed in a large scale in Escheri‌chia coli. Purification was performed under native conditions and the purity and integ‌rity of produced recombinant protein were confirmed by western immuno blot analysis using anti His-tag and anti-IMP polyclonal antibodies. The purified recombinant IMP was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with the alkaline phosphatase enzyme.
Results: The purified antibodies and conjugates were applied for efficient detection of infected plants in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and dot immunosorbent assay (DIBA).
Conclusions: These antibodies were proven to be very powerful tools to detect the Can‌didatus Phytoplasma aurantifolia in plants.


1. Background

The witches’ broom disease of lime (WBDL) is a destruc­tive disease caused by CandidatusPhytoplasma auran­tifolia (1). It was first observed in Oman (2) then it was reported in the United Arab Emirates (3) and Iran (4). It is estimated that over 98% of lime trees in Oman are in­fected with WBDL (5). The lime trees infected with WBDL show different symptoms such as compactness and their very small, pale green leaves, stunting, yellowing and shortening of internodes. As the disease progresses, the leaves dry, many witches’ brooms appear, and in four or five years the infected tree collapses. No flowers or fruits are produced in case of witches’ brooms and the ones produced on normal shoots are reduced in size (5). Phy­toplasmas are wall-less prokaryotes which belong to Mol­licutes, and are known with their specific characteristics such as small genomic size from 530 to 1350 kb (6), low G + C percent (7), nonculturable in cell free media (8), transmission and spread by insect vectors mainly leaf hoppers (Cicadellidae) and plant hoppers (Delphacid­ae), and multiplication in the phloem sieve tubes. They are phloem-limited bacterial pathogens which colonize their host persistently and cause great losses in economi­cally important plants like ornamentals, vegetables and fruit trees (9). Phytoplasmas are surrounded by a single cell membrane. The membrane proteins of phytoplas­mas appear to do their function directly in the cytoplasm of the host plant and transmitting insect cells (10). A sub­set of membrane proteins are in the phytoplasma cells, of which the immunodominant membrane proteins (IMPs) are the major part (11). The IMPs are located on the external surface of the cell membrane (12), and probably play important roles in the attachment to their host cell surface (13). Considering the rapid spread of the disease, a correct, precise and highly sensitive detection method is necessary for quick identification of infected samples and prevention of infected plant materials translocation. Several diagnostic techniques have been developed to detect phytoplasmas. Molecular methods such as poly­merase chain reaction (PCR) (14), DNA hybridization (15) and electron microscopy (16) have been described. Each of these methods has inherent disadvantages. PCR and DNA hybridization require specialized equipment. In con­trast, serological detection is convenient and economical method which allows examination of many samples in a short time. Up to now, several polyclonal and monoclonal antibodies have been produced against numerous phyto­plasmas including clover phyllody (CP) (17), aster yellows (AY) (18), ash yellows (19), faba bean phyllody (20) and lime witches’ broom (21). Considering the fact that phyto­plasmas have not been cultured in vitro, such antibodies have been produced by purifying or partially purifying phytoplasma cells from infected plants. These antibod­ies have some disadvantages such as low titers, cross re­activity with plant components and weak specificity to the target phytoplasma. To overcome these difficulties, recombinant IMP has been successfully used to produce antibody against several phytoplasmas causing apple proliferation (AP) (22), Western X-disease (WX) (23) and onion yellows (OY) (13). Moreover, traditional approach to prepare antibody against phytoplasmas is based on immunizing animal with antigen preparations from in­fected plants. This approach results in antisera with rela­tively low titer, contamination with plant-derived immu­nogens and occurrence of cross reactions with healthy crude extracts (13). As it has been applied for several plant viruses (24, 25), the cloning and expression of the phyto­plasma gene fragment in E. coli and purification of the protein are proposed as a means to advance our ability to overcome these limitations.

2. Objectives

This work aimed to describe the application of recombi­nant IMP to develop specific antibody against Candidatus Phytoplasma aurantifolia and produce a DAS-ELISA sero­logical kit for efficient detection of infected plants.

3. Materials and Methods

3.1. Materials

All chemicals were supplied from the Fermentas (Vil­nius, Lithuania), Qiagen (Hilden, Germany), Roche (Mannheim, Germany), Sigma (Deisenhofen, Germany) and Fluka (Neu-Ulm, Germany) Companies. The QIA ex­pressionist and Ni-NTA agarose matrix (Qiagen, Hilden, Germany) were used for expression and purification of recombinant protein. Immobilon-P transfer membrane (PVDF) (0.45 μm) was from Sigma (Deisenhofen, Germa­ny). DNA Extraction Kit (Roche, Mannheim, Germany) and restriction enzymes, PCR materials and InsTAclo­neTM PCR Cloning Kit were prepared from Fermentas (Vilnius, Lithuania). Synthetic oligonucleotides were ob­tained from MWG-Biotech (Ebersberg, Germany).

3.2. Phytoplasma and DNA Extraction From the Infected Plants

Phytoplasma was maintained by serial transmission from diseased to healthy plants using graft transmission in Citrus aurantifolia from a lime plant infected by WBDL (Kindly provided by Dr. Mohsen Mardi, ABRII, Karaj, Iran). These plants were used as the main source to isolate IMP and subsequent analysis. All healthy and diseased plants were grown in an insect-proof greenhouse. Stems and leaves of the WBDL infected lime plant were frozen with liquid nitrogen and ground to a fine powder. Total DNA was isolated and purified using Cetyltrimethylammoni­um bromide (CTAB) method (26).

3.3. Isolating, Cloning and Sequencing of the IMP Gene

The primer set IMP-pET28-For (5´-CAACGTCGACAAAAT­CACAAAGAAAATTTTTTAC-3´), and IMP-pET-28-Rev (5´-CAACGCGGCCGCTTATGATAATTTTAAATCTG-3´) con­taining the restriction sites of the SalI and NotI enzymes. They were designed according to the IMP sequence (Ac­cession No. GU339497.1) and were used to isolate the gene encoding IMP from infected plant. The PCR amplification was performed in a 50 μL reaction volume containing 1 ng of extracted template DNA, 5 μL PCR buffer (10×), 50 mM MgCl2 2.5 μL, 2.5 mM deoxynucleotide triphosphates 1 μL, 10 pmol of each primers (IMP-pET28-For and IMP-pET-28-Rev) and 5U/μL Taq DNA polymerase 0.5 μL (Fermentas, Vilnius, Lithuania). PCR was performed under the follow­ing conditions: initial denaturation at 94°C for 5 min, fol­lowed by 35 cycles of denaturation at 94°C for 1 min, an­nealing at 55°C for 1 min and extension at 72°C for 1.5 min and a final extension at 72°C for 12 min. Amplification products were analyzed by electrophoresis on agarose gel (1 %), in 1× TAE buffer and stained with ethidium bro­mide (0.5 μg.mL-1).  Amplified fragments were recovered from gel using DNA Extraction Kit (Roche, Mannheim, Germany) and ligated into the plasmid vector pTZ57R/T (InsTAcloneTM PCR Cloning Kit, Fermentas, Vilnius, Lithuania). The recombinant plasmid was used for trans­formation of Escherichia coli strain DH5α by heat-shock protocol (27). Intact clones containing right sequences were initially selected after digestion with SalI and NotI enzymes. To make sure that the amplification and con­struction processes have not affected the base sequence of IMP gene, the cloned gene was sequenced by Macrogen (Korea) using universal primers for pTZ57R/T.

3.4. Heterologous Expression of the IMP Encoding Gene

The clone containing the intact sequence of IMP was selected and the gene encoding IMP was subcloned into the SalI and NotI sites of pET28a bacterial expression vec­tor and the new construct was designated pET28-IMP and transformed to E. coli strain BL21 (DE3). Expression of the IMP was induced under native conditions following the manufacturer’s instructions (The QIA expressionist TM, Hilden, Germany) and purification was performed through Immobilized metal ion affinity chromatogra­phy (IMAC) in column containing Ni-NTA agarose beads (Qiagen, Hilden, Germany) following the manufacturer’s instructions. For extracting soluble protein, cell disrup­tion was performed by vortexing with glass beads and sonication (Misonix, USA). Successful expression and pu­rification steps were confirmed by SDS-polyacrylamide gels (SDS-PAGE) (stacking gel 4%, pH 6.8; separating gel 12%, pH 8.8) (28).

3.5. Antibody Preparation

Two white inbred rabbits were used for immunization. Five intramuscular injections in the hind legs were per­formed at intervals of two weeks. Each injection con­tained about 100 µg of IMP recombinant protein and equal volumes of complete Freund’s adjuvant (Sigma, Deisenhofen, Germany) for the first injection and incom­plete Freund’s adjuvant for the subsequent injections. Animals were bled 4 to 5 times from the marginal ear vein at 14 day intervals to estimate antibody titer by ELI­SA. Finally, blood was collected from rabbits’ heart 14 days after the fifth immunization. The serum fraction was col­lected and stored at -20°C.

3.6. Purification of IgG and Conjugation With Alkaline Phosphatase

Antibody purification from serum was performed using Protein A spin column according to the manufacturers’ manual (AbDSerotec, UK). The concentration and the pu­rity of antibody were determined by SDS-PAGE. Purified antibody was conjugated to alkaline phosphatase using LYNX Rapid alkaline phosphatase antibody conjugation kit based on the manufacturer’s manual (AbDSerotec kit, UK).

3.7. Western-Blot Analysis

Purified IMP protein and the proteins extracted from the WBDL infected and healthy plants were separated by SDS-PAGE. Proteins were transferred to Millipore poly­vinylidene difluoride (PVDF) membrane (Immobilon-P transfer membrane; Sigma Deisenhofen, Germany) ac­cording to the instructions of the manufacturer.  The membrane was blocked with PBS buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 X 2H2O, 1.5 mM KH2PO4; pH 7.5) containing 2% (w/v) skim milk powder and blotted pro­teins probed with a primary antibody (anti-His tag at a dilution of 1/1000 and anti-IMP polyclonal antibody at a dilution of 1/500). The alkaline phosphatase-conjugated secondary antibody was used at a dilution of 1/3000 (Ab­cam, USA). The target proteins were finally revealed by adding substrate 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) (Sigma, Deisen­hofen, Germany).

3.8. Enzyme linked Immunosorbent Assay (ELISA)

Indirect ELISA was performed to determine the IMP polyclonal antibodies titer.  A Nunc-Immuno™ Maxi­Sorb™ 96-wells microtiter plate (Thermo Fisher Scientific Inc.) was initially coated with 10 μg.mL-1 of purified re­combinant IMP using a carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3 pH 9.6) and plate was left over­night at 4°C. The plate was blocked with 2% (w/v) skim milk (Fluka, Neu-Ulm, Germany) in 1×PBS. Serial dilutions of serum (1/512-1/262144) in 1×PBS was added to the coated plate and incubated at 37°C for 2 hours.  Bound antibod­ies were detected by adding 1/3000 diluted alkaline phos­phatase-conjugated goat anti-rabbit IgG (Abcam, UK) for 2 hours at 37°C. Finally, p-nitrophenyl phosphate (pNPP) as substrate (Sigma, Deisenhofen, Germany) was added and incubated at room temperature for 20 - 60 min fol­lowed by measuring the absorbance values at 405 nm us­ing TECAN Microplate reader (Switzerland). To determine specificity of the prepared antibody against phytoplas­mas, double antibody sandwich-ELISA (DAS-ELISA) was performed as described previously by Clark and Adams (29). Wells of the plate were coated with anti-IMP poly­clonal antibody diluted to 1:500 in 1×PBS and incubated at 37°C for 2 hours. Extraction of plant material from healthy and infected lime were performed with mortar and pestle or in a plastic bag with a roller. Plant protein were extracted 1:3 (w/v) in extraction buffer (1×PBS pH 7.5, 5 mM EDTA, 5 mM β-mercaptoethanol or 2% (v/v) polyvi­nylpyrrolidone, molecular weight 10000 (PVP-10) in PBS). The plant extracts and purified protein control (IMP, posi­tive control) were added to the plate and incubated over­night at 4°C. Next, the alkaline-phosphatase–conjugated anti-IMP polyclonal antibody was added at a dilution of 1:500 and incubated at 37°C for 2 hours. Finally, the sub­strate (pNPP) was added and after 30 min absorbance values were read at 405 nm. The sample was identified as positive if the mean DAS-ELISA (A405 nm) value of sample exceeded at least twice that of the healthy control(s).

3.9. Dot Immunobinding Assay (DIBA)

Protein extracts of healthy and infected lime plants were diluted 1:10, 1:20 and 1:50 in 1×PBS buffer and 4 µL of each were disposed on nitrocellulose membrane. Sat­uration of the free binding sites was performed with 2% (w/v) skim milk in 1×PBS. The target protein was detected by 1:500 diluted AP-labeled anti-IMP IgG. The bound anti­body was revealed by adding substrate NBT/BCIP.

3.10. Specificity of the Anti-IMP Polyclonal Antibody

Specificity of the polyclonal antibody prepared against recombinant IMP of lime witches’ broom was evaluated with several phytoplasma-infected plant samples includ­ing almond infected with Candidatus Phytoplasma phoe­nicium (30), alfalfa infected with alfalfa witches, broom and sesame infected with a phytoplasma associated with sesame phyllody (31) using DAS-ELISA.

4. Results

4.1. Detection and Cloning of the IMP Gene

The gene encoding IMP was amplified using specific primers from total DNA extracted from the infected plant (Figure 1).

SalI/NotI Restriction sites were introduced into the for­ward and reverse primers for subcloning in to desired vectors. The PCR product was directly cloned into the pTZ57R/T vector and integrity of IMP gene in obtained clones was evaluated by restriction analysis, PCR ampli­fication and sequencing. The clone harboring the right sequence was selected for subcloning in pET28a bacte­rial expression vector. Sequencing analysis revealed that the cloned DNA into pTZ57R/T vector consists of 519 bp, starting from the ATG initiation codon and stopping at translation termination codon of TAA and 100% identity with IMP gene of Candidatus Phytoplasma aurantifolia (Table 1).

The multiple sequence alignment of the IMP with the sequences of the NCBI databases and ClustalW program indicated that this gene encodes a protein of 172aa which has the most similarity with membrane proteins of the members of Peanut WB group; faba bean phyllody, al­falfa witches’ broom, peanut witches’ broom (PNWB) and sweet potato witches’ broom (SPWB) (Figure 2). Like the WX, AP, CP and SPWB IMPs, this gene contains no cysteine. All of the IMP genes isolated from phytoplasmas have a high lysine content ranging from 11.0 mol% for CP to 14.6 mol% in the AY IMP, and tryptophan (W) are encoded by TGG (23). This antigenic gene has twenty-seven AAA lysine codons (15.7 mol%), which make it the most abundant amino acid and also two tryptophan residues are encod­ed by TGG.

4.2. Expression of Recombinant IMP Protein

To produce sufficient protein for immunization of rab­bit and further characterization of prepared antibodies, the IMP gene was inserted into the pET28a bacterial ex­pression vector downstream of a 6×His-tag and expressed in E. coli BL21 (DE3) under native conditions. The ex­pressed IMP in bacteria cells was purified by purifica­tion of recombinant protein on Ni-NTA-agarose column. Disruption of bacterial cells using ultrasonic waves was more efficient than other approaches such as glass beads. Expression and purification resulted in the production of varying amounts of fusion protein after induction with 1 mM IPTG. SDS-PAGE analysis confirmed the high purity and integrity of IMP and showed a protein with expected size of about 19 kDa (Figure 3). Generally, the total yield of purified protein in the culture medium varied from 6 to 18 mg.L-1.

4.3. Immunization, Determination of Antibody Titer and Antibody Purification

Recombinant IMP was used to immunize two rabbits. The antibody titer was determined by indirect ELISA and after the fifth boosting, the final polyclonal antibody ti­ter was about 1:131072. The IgG was purified from serum and monitored for purity by SDS-PAGE which appeared as approximately 25 kDa and 50 kDa bands. Also the IgG concentration was quantified with comparison to known amount of a standard protein, BSA, which was calculated to be about 1 mg.mL-1.

4.4. Western Immunoblot Analysis

Western blot analysis using anti 6×His tag monoclonal antibodies proved successful expression of IMP in E. coli cells (data not shown). Specificity of produced polyclonal antibodies against recombinant IMP was confirmed by western blotting analysis. A distinct band around 19 kDa was detected in the purified protein (Figure 4).

Subsequent western blotting analysis proved specific­ity of polyclonal antibodies against IMP presented in infected plants (data not shown). Complementary DIBA analysis was performed to further evaluate the specificity of the prepared antibody against recombinant and na­tive IMP. The results proved binding ability of antibody against IMP present in infected sap as well as against re­combinant IMP (Figure 5).

4.5. DAS-ELISA Technique

To establish an effective method to detect the infected plant, DAS-ELISA analysis was performed. This technique required preparation of a scaffold for quantifying patho­gen and making direct comparison between infected plants. Applying DAS-ELISA proved the ability of prepared antibodies for successful detection and differentiation of infected samples from the healthy ones at a dilution of 1:500 (Figure 6). Serial dilutions of prepared polyclonal antibody proved that the dilution of 1:2000 could be ap­plied for further diagnostic purposes.

Each value represents the mean of 3 replicates. Absor­bance values were read at 405 nm after 30 min of incu­bation. The differences among the groups and between each two groups were statistically significant P < 0.05 except for alfalfa plants infected by witches’ broom. To determine binding activity of the prepared antibody against other phytoplasma agents, the DAS-ELISA analy­sis was performed using plants infected with witches’ broom diseases of almond, alfalfa and sesame phyllody. The results revealed cross reactivity of prepared antibody with phytoplasmas present in almond and sesame, but no reaction was observed with infected alfalfa plants by witches’ broom disease (Figure 6).

5. Discussion

The generation of specific antibodies against obligate parasites is greatly complicated due to the problems as­sociated with obtaining pure material for immunization. Toward this aim and for efficient and simple detection of infected plants, present study described both produc­tion of specific antibodies against WBDL using recombi­nant IMP and development of serological methods such as DAS-ELISA and DIBA. A single major antigenic protein with a molecular mass ranging from 15 to 32 kDa has been identified in several phytoplasmas (3, 13, 22, 23, 32-36). Proteins from different strains usually have great amino acid and antigenic variation. All of the proteins have a central hydrophilic region, which may be on the outside of the phytoplasma cell, and one or two transmembrane domains. In this study, a protein with a molecular mass of about 19 kDa and a pI value of 9.29 was detected. This protein showed similarities to an antigenic membrane protein of the sweet potato witches’ broom (SPWB) agent described by Yu et al. (37). The multiple sequence align­ment showed that the NH2-terminal amino acids of 1 to 20 are fully conserved between the N-termini of FBP, Alf WB-Y, PNWB, SPWB, and WBDL. In previous studies two distinct regions, a strongly hydrophobic NH2-terminus (amino acids 10 - 50) and a highly hydrophilic C terminus (amino acids 50 - 172), have been identified in the hydro­phobicity profile of the deduced amino acid sequence of the major antigenic protein of SPWB phytoplasma (37). Regarding 70% amino acid sequence identity between the major antigenic protein of SPWB phytoplasma and WBDL, a strongly hydrophobic NH2-terminus within the cell membrane (aa 1 - 14), a highly hydrophilic C-terminus ex­posed at the cell surface (aa 38 - 172) and also a transmem­brane protein (aa 15 - 37) were predicted for IMP of WBDL. The obtained purified protein was used for the immuni­zation of rabbit and preparation of a polyclonal antibody against the lime witches’ broom disease. The alkaline phosphatase labeled antibody was used in DAS-ELISA test and it detected the pathogen and exhibited a high speci­ficity. Although DIBA test revealed a lesser sensitivity in comparison with DAS-ELISA, yet a smaller amount of anti­gen is required and detection of a large number of plants under field conditions would be rapid and easy. Consid­ering the fact that phytoplasma infected sesame and almond plants react positively with anti-IMP polyclonal antibody, it can be concluded that there are serological associations between these phytoplasmas and phyto­plasma associated with the LWB disease. Both polyclonal and monoclonal antibodies have been generated against phytoplasmas for their detection and differentiation. The relative sensitivities of polyclonal antibodies produced against several phytoplasmas such as: aster yellows, pea­nut witches’ broom, phytoplasma associated with faba bean, sesamum phyllody, apple proliferation and sandal spike phytoplasma were determined using indirect ELISA procedure (20, 22, 38, 39). Polyclonal antibodies can be provided rapidly, at less expense, with less technical skill than required to produce monoclonal antibodies but it has mainly the disadvantage of cross reactivity with re­lated antigens and limited products obtained in this way. Recombinant DNA and molecular display technologies have provided new opportunities to create recombinant antibodies. Phage display, involves the introduction of peptide sequences (such as the antigen-binding domains of recombinant antibodies) into the coat protein gene of a bacteriophage displayed on the virion surface. Phage antibody display libraries can be screened and the corre­sponding antibody gene can then be recovered from the phage genome (40). This technique is simple, cheap, and rapid and requires no special equipment. “The ethical and financial burdens of animal use are avoided because of the exclusion of immunization” (41). It has been shown that immunoassays are more reliable and sensitive in de­tecting the phytoplasmas in their host and for establish­ing relationships among phytoplasmas (33). The purity of the immunogen has significant effect on the sensitivity and specificity of immunoassays. As phytoplasmas can­not be cultured on cell free media, therefore, obtaining recombinant proteins which are very highly purified immunogen and produce good quality antisera when in­jected into rabbits is recommended. The antibody gener­ated against IMP can be used for detection of procedures and also for other purposes like the characterization of secretion pathways or the study of host-pathogen inter­action. In conclusion, witches’ broom disease of lime has become the most important disease of lime in south of Iran during the last two decades, and there was no effi­cient strategy to control the disease. To restrict the spread of the infected plant materials, for quarantine purposes, eradication programs and resistance breeding trials, sen­sitive and specific detection tools were needed. Antibod­ies against IMP of Candidatus Phytoplasma aurantifolia have been proven to be very powerful tools to detect the pathogen.


We would like to thank Dr. M. Ghaeb-Zamharir and SA Esmailzadeh-Hosseini for providing infected samples and Dr. G. Hosseini Salekdeh and Mr. H. Rassol for their valuable cooperation in conducting the protein purifica­tion experiment.

Authors’ Contribution

None declared.

Financial Disclosure

None declared.


Financial support of the national project of WBDL and Agriculture Biotechnology Research Institute of Iran (AB­RII) is acknowledged.

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