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A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies.

ABSTRACT: Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner's guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.

SUBMITTER: Horstman A

PROVIDER: S-EPMC4100113 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies.

Horstman Anneke A Tonaco Isabella Antonia Nougalli IA Boutilier Kim K Immink Richard G H RG

International journal of molecular sciences 20140530 6

Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are pron ...[more]

PMID: 24886811

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Project description:BACKGROUND:Plasmodium vivax is the most widespread of the human malaria parasites in terms of geography, and is thought to present unique challenges to local efforts aimed at control and elimination. Parasite molecular markers can provide much needed data on P. vivax populations, but few such markers have been critically evaluated. One marker that has seen extensive use is the gene encoding merozoite surface protein 3-alpha (MSP-3?), a blood-stage antigen known to be highly variable among P. vivax isolates. Here, a sample of complete msp-3? gene sequences is analysed in order to assess its utility as a molecular marker for epidemiologic investigations. METHODS:Amplification, cloning and sequencing of additional P. vivax isolates from different geographic locations, including a set of Venezuelan field isolates (n?=?10), yielded a sample of 48 complete msp-3? coding sequences. Characterization of standard population genetic measures of diversity, phylogenetic analysis, and tests for recombination were performed. This allowed comparisons to patterns inferred from the in silico simulation of a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) protocol used widely. RESULTS:The larger sample of MSP-3? diversity revealed incongruence between the observed levels of nucleotide polymorphism, which were high in all populations, and the pattern of PCR-RFLP haplotype diversity. Indeed, PCR-RFLP haplotypes were not informative of a population's genetic diversity and identical haplotypes could be produced from analogous bands in the commonly used protocol. Evidence of frequent and variable insertion-deletion mutations and recurrent recombination between MSP-3? haplotypes complicated the inference of genetic diversity patterns and reduced the phylogenetic signal. CONCLUSIONS:The genetic diversity of P. vivax msp-3? involves intragenic recombination events. Whereas the high genetic diversity of msp-3? makes it a promising marker for some epidemiological applications, the ability of msp-3? PCR-RFLP analysis to accurately track parasites is limited. Local studies of the circulating alleles are needed before implementing PCR-RFLP approaches. Furthermore, evidence from the global sample analysed here suggests such msp-3? PCR-RFLP methods are not suitable for broad geographic studies or tracking parasite populations for an extended period of time.

Dataset Information

A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies.

Publications

A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies.

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