Plant Science

Protein–lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein–lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid–protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K⁺ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)

Nicotiana benthamiana (N. benthamiana) is a useful model system to transiently express protein at high level. This protocol describes in detail how to transiently express protein in N. benthamiana and how to carry out protein immunoprecipitation in this expression system. This protocol can be broadly used for investigation on protein-protein interaction, protein purification and other related protein assay.

Studying the biochemical interaction of ligands with their corresponding receptors requires highly sensitive detection and monitoring of the bound ligand. Classically, radioactively labelled ligands have been widely used as highly sensitive tools for such binding measurements. Disadvantages of radiolabelling include instability of products, high costs and risks of working with radioactivity. Thus, assays using chemiluminescent probes offer convenient, highly sensitive alternatives. Here we suggest acridinium esters as suitable conjugates to label ligands of interest. Chemical oxidation of acridinium esters triggers chemiluminescence, allowing quantitation of this compound down to amol concentrations in standard luminometers. The first report about acridinium esters in immunoassays date back to 1983 (Weeks et al., 1983) and demonstrated the ability to conjugate acridinium to peptides, followed by using such peptides to measure receptor – peptide ligand interactions (Joss and Towbin, 1994).

Recently, this binding assay was adapted for studying derivatives of the plant peptide IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and their interaction with the corresponding receptor HSL2 (HAESA-LIKE 2) was reported (Butenko et al., 2014). Here we describe how this sensitive, nonradioactive binding approach can be used to reveal receptor-ligand binding in plant material.

14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca²⁺-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of RSG. Our recent study revealed that NtCDPK1 forms a heterotrimer with RSG and 14-3-3 and that 14-3-3 was transferred from NtCDPK1 to phosphorylated RSG (Ito et al., 2014). In the course of the study, we found that 14-3-3 protects the phosphate group of RSG from λ protein phosphatase in vitro. Here, we describe a protocol for in vitro phosphatase protection assay. To detect the phosphorylation state of proteins, we used Phos-tag SDS-PAGE and autoradiography. This protocol can be adapted for the examinations whether the phosphoprotein-binding proteins protect the phosphate group of target proteins from phosphatases although protein kinases may be required for the phosphorylation of target proteins.

CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca²⁺/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms.

We developed an in vivo method to assay plant transcription factor (TF)–promoter interactions using the transient expression system in Nicotiana benthamiana (N. benthamiana) plants. The system uses the Arabidopsis stay green (SGR) gene as a reporter. Induction of SGR expression in N. benthamiana causes chlorophyll degradation and causes leaves to turn yellow.

Many cellular proteins interact with the cytoskeleton (both actin filaments and microtubules), either dynamically or permanently. This interaction is required during different aspects of the cell life, for example during the process of cell division. In addition, many enzymes interact transiently with actin filaments and microtubules in order to promote their cellular distribution. Several substances with inhibitory capacity can affect this binding and cause damages to cells. This protocol allows to analyze whether a protein interacts with either actin filaments or microtubules and, when applicable, the conditions controlling this interaction.

The test is based on the specific binding between the protein of interest and the cytoskeletal filaments. As shown schematically in the diagram of example (see below), the test starts from the cell lysate to which actin filaments (produced from monomeric actin) are added. The mixture (performed under different experimental conditions chosen by the operator) is then incubated so that the protein of interest (in the example, myosin) binds to actin filaments. The sample is then centrifuged in order to separate unbound or weakly-bound proteins from actin filaments to which both the protein of interest and, eventually, traces of less specific proteins are associated.

RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed ³²P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.

The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms.