Preparation, Selection And "Internal Image" Characterization Of Monoclonal Antibodies Against Harpin_Xoo From Xanthomonas Oryzae PV. Oryzae

The hrp (hypersensitive response and pathogenicity) gene clusters in Gram-negative phytopathogenic bacteria determine hypersensitive response (HR) in nonhost plants and pathogenicity in host plants of the bacteria. A hrp gene cluster usually contains genes coding for the components of the typeⅢsecretion pathway, effectors and the proteins that regulate the productions and transportations of effectors. Harpins are non-specific elicitors produced from Xanthomonas oryzae pv. oryzae and regulated by hrp genes. They are exported and delivered from the bacteria into the cytosol of host plant cells and have common characters:glycine-rich, heat stable and protease K-sensitive. They function at the bacterial-plant cell surface interface and have the ability to induce pathogenicity in susceptible plants and to elicit a hypersensitive reaction (HR) in nonhost plants or resistant cultivars of host plants. The application of harpins in many plants is to induce disease and insect resistance, trigger many plant-reaction phenotypes, promote plant growth, and enhance yield and quality by activating distinct signaling pathways. HarpinXoo, identified recently, is encoded by hrfl gene from Xanthomonas oryzae pv. oryzae and contains 139 amino acids (15.6kDa). They have similar biological activities with other harpins, while show some unque natures, such as containing cysteine.Plants possess an innate immune system enabling them to defend themselves against attacking pathogens. Using a large set of receptors, the immune system has developed different strategies to realize disease resistance. The HR of higher plants, characterized by rapid, localized necrosis of plant cells at the sites of pathogen infection, is an active defense of plants against many pathogens. The dead cells limit pathogen around the sites of infection, and prevent the further expansion of pathogen realizing local disease-resistance. The signaling pathway of pant disease-resistance was further activated, making plants acquire system resistance to many types of pathogens invaded later. The most prominent feature is the event of eliciting a hypersensitive response, when harpin was infiltrated into nonhost plants. To respond to harpin, the presence of harpin must be recognized by the plant cell. How and where harpin’s active site is recognized has not been clear completely. Here, we introduced a new strategy to study the problem of plant-pathogen, empolying an anti-idiotypic antibody based on Jerne’s immune network to probe harpin receptor(s) in nonhost plant.1 Extraction and purification of HarpinXoo proteinHrf1 gene amplified from genomic DNA of Xanthomonas oryzae pv. oryzae strain PXO99 was digested with NdeⅠand HindⅢ, subsequently purified, inserted between the NdeⅠand HindⅢsites of the pET30(a+) vector containing an IPTG promoter. The resulting plasmids, named as pET30a(+)-hrf1, were analyzed by restriction enzyme digestion and DNA sequencing, and transformed into E. coli BL21 (DE3) for expression of the fusion proteins. The recombinant E. coli BL21/pET-hrf1 was incubated at 37℃for 3h at 225 rpm under inducing withlmM IPTG, and then the E. coli cells were harvested by centrifugation at 4℃. HarpinXoo fusion protein was extracted from the harvested cells by boiling them for 10 min in 100℃, purified through Ni affinity chromatography and analyzed on SDS-PAGE. The molecular massed of Harpinxoo is 15.6kDa, in consistence with the prediction by a pertinent software program. This result is an essential basis for further studies on functions of the gene and protein in plants.2 Preparation and purification of rabbit polyclonal anti-HarpinXoo antibodies (Ab1)New Zealand white rabbits were immunized subcutaneously at multiple sites and intramuscularly with 500μg of purified HarpinXoo emulsified with an equal volume of Freund complete adjuvant and boosted at bi-weekly intervals with 200μg of antigen emulsified with Freund incomplete adjuvant. Six weeks later, the rabbits were bled and the anti-HarpinXoo serum titer was determined by the ELISA method with pre-immune sera analyzed in parallel. Protein A/G affinity chromatography was performed on a 2-mL settled gel pre-packed column to purify Ab1. HR induced by Harpinxoo can be inhibited and even completely blocked by Ab1, which further demonstrates that Ab1 binds to one or more harpin’s active sites, which are likely to be combining sites of receptor. This makes it an interesting candidate for generating anti-idotypes.3 Preparation of monoclonal anti-HarpinXoo antibody with inhibitory activities in vitro and in vivoFemale Balb/c mice received four times of immunization with purified harpinXoo. The mice with the highest serum antibody titer were selected to produce hybridomas. Six monoclonal harpinXoo hybridomas were selected, subcloned and identified. ELISA additivity tests revealed that four epitopes in harpinXoo are defined by six monoclonal antibodies, of which one epitope recognized by the mAb designated 6G4 was found to part of the functional sites of harpinXoo, depending upon their immunoinactivation properties. In vitro studies showed that 6G4 was able to bind to harpinXoo competing with total protein of tobacco leaves. Most importantly, in vivo,6G4 blocked partially tobacco HR elicited by HaprinXoo.6G4 appear to be a suitable antigen to produce aniti-iditypic antibodies against harpinXoo, which have the properties of the internal images of harpinXoo actives sites. Elucidating the effect of 6G4 in vitro and in vivo will provide more insight into identifying key functional regions in Harpins that induce HR in nonhost plant.4 Preparation and characterization of monoclonal Ab2 having the properties of an internal image of HarpinXooF(ab’)2 fragments retaining antigen binding activity, which were generated by digesting the purified rabbit polyclonal anti-harpin antibodies (Ab1) using immobilized pepsin, were used to produce mouse anti-idiotypic antibodies having the properties of "internal image" of HarpinXoo- For the preparation of mAb Ab2, mice were immunized with different doses of F(ab’)2 fragments in the presence of adjuvant. The concentration of Ab2 in mouse sera increased with an increase in the number of immunizations. When the inhibition rate reached 50%, the order of the three mouse sera dilutions were:mouse 1 (917)< mouse 2 (1094)< mouse 3 (2540). So we selected mouse 3 having the best inhibitory effect on the binding of Ab1 to HarpinXoo to generate hybridomas.The anti-idiotypic antibody that has the properties of an internal image of the protein HarpinXoo was screened by using standard ELISA and competitive inhibition ELISA. Clones were screened by indirect ELISA first,224 were positive against Ab1 polyclonal antibody, but only 23 were able to block more than 50% of the binding of Ab1 to Harpinxoo. After a first selection round,6B2 was selected to be fully characterized and to be propagated as a stable cell line. The results from the competition ELISA demonstrate that the binding of 6B2 mAb to the fragments of Ab1 was inhibited by HarpinXoo but not by other proteins, such as BSA, indicating that the binding probably occurred close to or on the combining site of the idiotype. In addition, we showed that 6B2 competes with Harpinxoo for binding to the harpin putative receptor in nonhost plants, which was the crucial point of this study. This fact provides strong evidence that 6B2 binds to the putative receptor at the harpin binding site. The binding of 6B2 to the harpin putative receptor could be the result of cross-linking to the receptor or of the induction of a conformational change in the receptor. However, it is also possible that both mechanisms are active together. Additional evidence for the anti-idiotype nature of 6B2 was provided by the induction of an Ab3 response.6B2 mAb induced a strong response against harpin in rabbits and elicited Ab3 antibodies with the same specificity as the rabbit polyclonal antibodies Ab1. These results implied that 6B2 mAb has ligand-like properties, i.e. that it behaves as the internal image of harpin protein and therefore 6B2 can be classified as Ab2βaccording to Jerne’s nomenclature.In conclusion, Antigen mimicry by anti-idiotypic antibodies is employed as a reliable strategy to probe receptors that are present in very low concentrations in the organism. This approach calls on knowledge at the crossroads of various fields such as general protein chemistry, enzymology, and immunology, as well as protein-protein interactions, for which the scope of applications is wide, for example:functional mimicry, using as a probe to identify cell surface receptors and immunohistochemical localization.