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Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of cross-species networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

Proper copper (Cu) homeostasis is required by living organisms to maintain essential cellular functions. In the model plant Arabidopsis (Arabidopsis thaliana), the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor participates in reprogramming global gene expression during Cu insufficiency in order to improve the metal uptake and prioritize its distribution to Cu proteins of major importance. As a consequence, spl7 null mutants show morphological and physiological disorders during Cu-limited growth, resulting in lower fresh weight, reduced root elongation, and chlorosis. On the other hand, the Arabidopsis KIN17 homolog belongs to a well-conserved family of essential eukaryotic nuclear proteins known to be stress activated and involved in DNA and possibly RNA metabolism in mammals. In the study presented here, we uncovered that Arabidopsis KIN17 participates in promoting the Cu deficiency response by means of a direct interaction with SPL7. Moreover, the double mutant kin17-1 spl7-2 displays an enhanced Cu-dependent phenotype involving growth arrest, oxidative stress, floral bud abortion, and pollen inviability. Taken together, the data presented here provide evidence for SPL7 and KIN17 protein interaction as a point of convergence in response to both Cu deficiency and oxidative stress.

To study gene function, generation of loss-of-function mutants by RNA interference (RNAi) approaches and T-DNA insertional mutagenesis are widely used in plant science and have proven to be very successful in determining gene functions. AGAMOUS-LIKE18 ( AGL18 ) is a MADS-box gene that is expressed