Production of fluorescent antibody-labeling proteins in plants using a viral vector and the application in the detection of Acidovorax citrulli and Bamboo mosaic virus

Abstract

Serological methods are relatively convenient and simple for the detection of pathogens for front-line workers. On-site visualization of the test results plays a pivotal role in the process. However, an efficient, universal labeling agent for antibodies is needed for the development of efficient serological detection tools. In this study, a Bamboo mosaic virus (BaMV)-based viral vector was employed to express recombinant proteins, collectively designated GfED, consisting of Staphylococcus aureus Protein A domain ED (SpaED) fused to either the N- or C-terminal of an improved green florescent protein (GFP) with or without the coat protein (CP) of BaMV, efficiently in Chenopodium quinoa. The GfED in crude leaf extracts could specifically attach to IgG molecules of rabbits and mice, effectively labeling IgG with GFP, emitting green light at 506 nm when excited at 450 nm using simple, handheld equipment. To demonstrate the applicability of GfED in serological assays, we have developed a fluorescent dot blot assay for the rapid detection of Acidovorax citrulli (Ac), a bacterial pathogen of cucurbits, and BaMV, a viral pathogen of bamboos. By using the crude extracts of inoculated C. quinoa leaves expressing GfED as an IgG-labeling agent, the pathogens were easily and quickly detected through uncomplicated operations using simple equipment, with results observable by the naked eye. Examination using fluorescent microscopy and transmission electron microscopy revealed that the GfED subunits may assemble into virus-like particles, which were further involved in the formation of aggregates of GfED-antibody-antigen complexes with the potential for fluorescence signal enhancement. The results suggested that plant-expressed GfED may serve as a promising alternative of IgG-labeling agent for current serological assays.

Etiology, diagnostic approaches and management strategies of Acidovorax citrulli, a bacterial fruit blotch pathogen of cucurbits

“…Owing to the high potential risk of BFB disease development under transplant house and greenhouse conditions, it is crucial to develop high sensitivity, reliable, quick and precise tools for detecting A. citrulli strain from symptomatic fields (Tian et al 2016;Islam et al 2019). Several seed assay protocols are currently available, such as isolation on differential or semi-selective agar media (Schaad & Sechler 1999;Zhao et al 2009;Feng et al 2013) seedling grow-out assay (SGO), sweat box or "dome assay" (Venette et al 1987), carbon source utilization profiles, fatty acid methyl esters (FAME) and serological assays (Walcott et al 2004;Feng et al 2006;Matsuura et al 2008;Himananto et al 2011;Puttharungsa et al 2011;Horuz et al 2014;Melo et al 2014;Kuo et al 2018).…”

Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

“…The concept of a plant virus-based immunosorbent nanoparticle (VIN), a plant virus or virus-like particle displaying antibody-binding proteins, has been proposed to capture antibodies for biosensing (Kuo et al , 2018; Uhde-Holzem et al , 2016) and therapeutic antibody purification (Werner et al , 2006). This nascent technology is one approach to address the need to reduce capital intensity for equitable and accessible antibody-related healthcare solutions, which could be harnessed to treat more prevalent diseases with availability of inexpensive and adequate production and purification capacity (Buyel et al , 2017).…”

Functionalizing silica sol-gel with entrapped plant virus-based immunosorbent nanoparticles

“…The copyright holder for this preprint this version posted November 12, 2021. ; https://doi.org/10.1101/2021.11.12.468100 doi: bioRxiv preprint 4 In this study we present entrapment and utility of plant virus-based immunosorbent nanoparticles (VINs) in silica sol-gel matrices by pore confinement, representing a novel system configuration for VBNs in general. VINs display antibody-binding proteins on their external coat protein surface, and they have been used as simple and bioregenerable reagents for biosensing and therapeutic antibody purification 6,17,18 . This entrapment of VINs represents the first use of plant virus-based VBNs in a silica sol-gel matrix and the first application of VBN technology for utility in an intact silica sol-gel matrix.…”