Leaf Proteome Analysis of Transgenic Plants Expressing Antiviral Antibodies

Abstract

The expression of exogenous antibodies in plant is an effective strategy to confer protection against viral infection or to produce molecules with pharmaceutical interest. However, the acceptance of the transgenic technology to obtain self-protecting plants depends on the assessment of their substantial equivalence compared to non-modified crops with an established history of safe use. In fact, the possibility exists that the introduction of transgenes in plants may alter expression of endogenous genes and/or normal production of metabolites. In this study, we investigated whether the expression in plant of recombinant antibodies directed against viral proteins may influence the host leaf proteome. Two transgenic plant models, generated by Agrobacterium tumefaciens-mediated transformation, were analyzed for this purpose, namely, Lycopersicon esculentum cv. MicroTom and Nicotiana benthamiana, expressing recombinant antibodies against cucumber mosaic virus and tomato spotted wilt virus, respectively. To obtain a significant representation of plant proteomes, optimized extraction procedures have been devised for each plant species. The proteome repertoire of antibody-expressing and control plants was compared by 2-DE associated to DIGE technology. Among the 2000 spots detected within the gels, about 10 resulted differentially expressed in each transgenic model and were identified by MALDI-TOF PMF and muLC-ESI-IT-MS/MS procedures. Protein variations were restricted to a limited number of defined differences with an average ratio below 2.4. Most of the differentially expressed proteins were related to photosynthesis or defense function. The overall results suggest that the expression of recombinant antibodies in both systems does not significantly alter the leaf proteomic profile, contributing to assess the biosafety of resistant plants expressing antiviral antibodies.

Identification of Putative Stage-Specific Grapevine Berry Biomarkers and Omics Data Integration into Networks

“…Samples were prepared according to Tsugita and Kamo (1999) with some modifications (Di Carli et al, 2009). Extracted proteins were purified using the Clean-Up kit (GE Healthcare) and quantified using the DC Protein Assay (Bio-Rad).…”

“…Two-dimensional gel electrophoresis was carried out using the GE Healthcare DIGE system. Purified proteomes were covalently labeled using the CyDye DIGE Fluor (Cy2, Cy3, and Cy5) according to the manufacturer's instructions and analysis carried out as described previously (Di Carli et al, 2009). A random design with 12 gels and a dye-swap approach was used to avoid labeling artifacts and ensure statistical significance.…”

MS‐based analytical methodologies to characterize genetically modified crops

“…In another study, the expression of recombinant antibodies in two transgenic crops (tomato and tobacco)-as a strategy to confer self-protection against virus attack-did not significantly alter the leaf-proteome profile (Di Carli et al, 2009). However, Rocco et al (2008) observed that a tobacco transformed with the tomato prosystemin gene affected the expression of a number of proteins involved in FIGURE 1.…”

MS‐Based Methodologies for Transgenic Foods Development and Characterization

“…2-DGE provides the highest protein-resolution capacity with a low-instrumentation cost. This technique has been applied to compare protein profiles of GMOs, including wheat with improved functional properties (Di Luccia et al, 2005;Scossa et al, 2008), tomatoes with a genetically added resistance to virus and insect attacks (Corpillo et al, 2004;Di Carli et al, 2009), potatoes showing modified cell wall structure and a delayed sprouting process (Careri et al, 2003;Lehesrant et al, 2005) and insect-resistant GM maize (Albo et al, 2007;Zolla et al, 2008;Coll et al, 2011;Balsamo et al, 2011) versus their corresponding unmodified lines. The comparison of protein profiles of GMOs with those obtained from the unmodified lines often does not reveal more significant differences than those observed between different nonmodified cultivars/genotypes.…”