2 in 1 Vectors Improve in Planta BiFC and FRET Analysis.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes, and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2-in-1 cloning approach, and their application in plant cell biology: ratiometric bimolecular fluorescence complementation (rBiFC), FRET acceptor photobleaching (FRET-AB), and fluorescent lifetime imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Rice OsHsp16.9A interacts with OsHsp101 to confer thermotolerance.

Class I small heat shock proteins (CI sHSPs), OsHsp16.9A and OsHsp18.0, share 74% identity in amino acid sequences and accumulate in response to heat shock treatments. Individual rice transformants overexpressing OsHsp16.9A and OsHsp18.0 exhibit distinct thermoprotection/thermotolerance modes. Under high temperature stress, OsHsp16.9A-overexpressing lines showed higher seed germination rate, seedling survival, and pollen germination than wild-type controls, while OsHsp18.0 overexpression provided higher thermoprotection/thermotolerance for seedling survival. To elucidate the functional roles of OsHsp16.9A, mass spectrometry was used to identify OsHsp16.9A-interacting proteins. OsHsp101 was consistently identified in the OsHsp16.9A protein complex in several mass spectrometry analyses of seed proteins from OsHsp16.9A-overexpressing lines. Both OsHsp16.9A and OsHsp101 proteins accumulated during similar developmental stages of rice seeds and formed a heat-stable complex under high temperature treatments in in vitro assays. Co-localization of OsHsp16.9A and OsHsp101 was observed via ratiometric bimolecular fluorescence complementation analyses. Amino acid mutation studies revealed that OsHsp16.9A glutamate residue 74 and amino acid residues 23-36 were essential for OsHsp16.9A-OsHsp101 interaction. Moreover, overexpressing OsHsp16.9A in OsHsp101 knockdown mutants did not increase the seed germination rate under heat stress, which further confirmed the functional roles of OsHsp16.9A-OsHsp101 interaction in conferring thermotolerance to rice plants.

Variation in Membrane Trafficking Linked to SNARE AtSYP51 Interaction With Aquaporin NIP1;1.

SYP51 and 52 are the two members of the SYP5 Qc-SNARE gene family in Arabidopsis thaliana. These two proteins, besides their high level of sequence identity (85%), have shown to have differential functional specificity and possess a different interactome. Here we describe a unique and specific interaction of SYP51 with an ER aquaporin, AtNIP1;1 (also known as NLM1) indicated to be able to transport arsenite [As(III)] and previously localized on PM. In the present work we investigate in detail such localization in vivo and characterize the interaction with SYP51. We suggest that this interaction may reveal a new mechanism regulating tonoplast invagination and recycling. We propose this interaction to be part of a regulatory mechanism associated with direct membrane transport from ER to tonoplast and Golgi mediated vesicle trafficking. We also demonstrate that NIP1;1 is important for plant tolerance to arsenite but does not alter its uptake or translocation. To explain such phenomenon the hypothesis that SYP51/NIP1;1 interaction modifies ER and vacuole ability to accumulate arsenite is discussed.

2in1 Vectors Improve In Planta BiFC and FRET Analyses.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.