lmmunomodulazione dell'infezione virale da CMV in piante transgeniche di pomodoro
Author(s)
Villani, Maria Elena
Date Issued
March 21, 2006
Type
Doctoral Thesis
Abstract
Immunomodulation by the ectopic expression of intracellular antibodies ('intrabodies') has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to property fold and function in the reducing environment of the cytoplasm, which prevents the formation ofessential disulfide bonds. In our laboratory an antibody phage display library has been built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. This antibody library (named 'F8 library') allowed the isolation of new intrinsically stable antibodies expressed as soluble molecules in the bacterial cytoplasm and with new specificity (Desiderio et al., 2001), even though none were to be used as intracellular antibodies.
This library has been the starting point for my Ph.D. thesis. We wished to assess the intracellular performance of antibodies derived from the 'F8 library'. To this purpose we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro 'biopanning' on immobilized virions, two scFvs were isolated and biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of N-ic.otiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tornato plants
(cv.Micro-Tom, Meissner et al., 1997). Challenge with high viraI dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection, showing that both scFvs when expressed in transgenic plants behave as intrabodies, accumulating as soluble proteins in the plant cytoplasm and have the ability to affect the plant phenotype upon virus challenge. Co-immunoprecipitation of the scFv-CMV complex from inoculated leaves of transgenic plants proves the occurrence of an in vivo interaction. This result suggests that, most likely, the scFv exert its in vivo modulation by restraining the infection into the inoculated leaves and impairing some other fundamental functions of CP, preventing CMV from moving long distance. This is further confirmed by a recovery test that demonstrates the complete absence of the virus in the apical leaves. In conclusion, we have shown that from our single-framework antibody repertoire, after classical in vitro screening, we isolated active intrabodies against a plant virus that conferred a new agronomic trait. Our library, containing stable scFvs able to modulate specific function in vivo, should automatically ensure that the isolated antibodies do bind their cognate antigens in the reducing environment of the cytoplasm, without having to recourse to further protein engineering andl or complicated selection procedures. F8 library-derived antibodies can also find application in functional genomics studies and, in the long run, might even be used in human therapy applications.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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