Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus

Brief info about the research group

• Martin Drucker research group focuses on the transmission of plant viruses using insect vectors. Focusing on the interactions of the virus with the host and vectors, and all the complexity these events present.
• Currently working on the GreenPeas project, focused on studying the Pea Enation Mosaic Virus (PEMV) transmision and infection mechanisms and dynamics to pea (Pisum sativum), as well as the identification of genes that provide resistance to the plants.

• https://orcid.org/0000-0002-9765-1189
• https://www.researchgate.net/profile/Martin-Drucker-2
• https://www.inrae.fr/centres/occitanie-montpellier

Mahin Pouresmaeil, Mattia Dall’Ara, Maria Salvato, Valentina Turri, Claudio Ratti,Cauliflower mosaic virus: Virus-host interactions and its uses in biotechnology and medicine,Virology,Volume580,2023,Pages112-119,ISSN0042-6822,

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Brief overview of the background info for the paper

• Cauliflower mosaic virus (CaMV) was the first discovered plant virus with genomic DNA that uses reverse transcriptase for replication. The P6 protein acts as the matrix protein of the cytoplasmic virus factories (VFs), by serving as a transactivator of viral protein translation, by interacting with host defenses and being the main determinant of disease symptoms.
• Arabidospsis thaliana and Brassica rapa used as experimental models.
• Split Green Fluorescent Protein (GFP)

https://doi.org/10.1016/j.virol.2023.02.008.(https://www.sciencedirect.com/science/article/pii/S0042682223000314)

DOI: 10.13140/RG.2.2.32720.25609

doi: 10.1016/j.virol.2024.110240

Summary

• In this paper the objective is to insert the GFP11 tag with site directed mutagenesis in the CaMV genome to tag the P6 protein and localize it upon infection in GFP1-10 Arabidopsis mutants and in wild type turnip. The complementation of the GFP halves will be used to characterize infection, elucidate early infection events, localize the P6 protein and quantify its accumulation.

Summary

• Novelty: Use split GFP system to overcome size limitations of using other fluorescent proteins and use the reconstitution of gfp to localize viral proteins and sites of accumulation.
• Nanobodies can be used to immunoprecipitate reconstituted GFP, as an more specific alternative to antibodies
• Significance: Tagging with split GFP is functional and reliable to use as alternative to other protein tags. New possibilities for small genomes. Assessing the effects of the tag on protein dynamics, expression and infection.
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Results

• Fig 2. Symptom development in CaMV-infected plants and stability of the insertion.

Results

Results

Results

Results

Results

Most interesting points from the discussion

• GFP11 tag can be maintained through generations and multiple mechanical insertions.
• The stability of the viral genome was not compromised, neither the P6 sequence.
• Symptoms were milder and delayed than in CaMVwt.
• P6 could be directly related to lower translation levels by affecting transactivation or being a physical impediment
• Inclusions were similar between WT and 11P6 CaMV infections. However, they were rounder/ovoid in mutant; possible additional data on N-terminus of P6 being key to accumulation or stability of virus?

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Most interesting points from the discussion

• Contrasting results on early infection.
• Comparable assay in protoplasts
• No nuclear P6 observed; accumulation below detection limits
• Reconstituted splitGFP stable enough for immunoprecipitation.

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What are the strengths and weaknesses of the paper

Strenghts

• In vivo data; easily observable
• Highly reproducible
• Innovative and specific
• Possibilities for tagging in very small genomes
• Localization of proteins in vivo and immunoprep

Weaknesses

• Only used one split GFP variant
• Requires a really high accumulation for it to be detectable (lowers protein production and infection rate) Thus, may not be suitable for proteins with low expression profiles
• Not overexpression of GFP1-10 in A.thaliana nor inducible mutants to tackle early infection dynamics.
• No C-terminal tagging possibilities offered.
• ORF2 could have been deleted and increased the size of insert
• No translation nor transcription experiments to asses P6 role on it.