Seeing red: a modified RUBY reporter assay for visualizing in planta protein–protein interactions

Proteins rarely act alone. Up to 80% of all proteins exist in protein complexes (Berggård et al., 2007) composed of multiple proteins working in unison to perform a variety of cellular processes. ATP synthase, for example, is made up of 17 protein subunits (Song et al., 2018) that interact with each other in specific ways in order to make ATP. In addition to the formation of these large complexes, protein–protein interactions also dictate ligand recognition, signal transduction, protein maturation, organelle targeting, cell recognition, and many other integral cellular processes. Determining how proteins interact is therefore critically important. Several assays exist for assessing the ability of proteins to interact with one another, such as yeast two-hybrid screening, co-immunoprecipitation, and fluorescence resonance energy transfer. However, none of these techniques can be performed in planta without destructive sampling or fluorescent microscopes. During his PhD with Peter Dodds at the Commonwealth Scientific and Industrial Research Organization (CSIRO) and the Australian National University, Jian Chen studied interactions between pathogen effector molecules and plant resistance proteins. This is one of the foundations of plant pathogen research, as the specific interactions between effector proteins and resistance proteins determine the virulence of plant pathogens. While doing this research, Chen realized there were no simple, in planta protein–protein interaction assays with visual outputs. This is when he came across a new gene expression assay called RUBY that fit what he was looking for. The RUBY reporter system is beautiful in its simplicity: it involves the conversion of a ubiquitous small molecule (the amino acid tyrosine) into the bright red pigment betalain, which gives beets (Beta vulgaris) their signature color (He et al., 2020). Although the RUBY reporter was initially used to monitor gene expression, Chen realized that the same technique could be modified to assess protein–protein interactions. The first step was to replace the existing RUBY promoter with the GAL4 transcription factor upstream activating sequence (UAS) next to a minimal CaMV35S promoter. This would allow assessment of protein interactions by fusing one protein of interest with the GAL4 DNA binding domain and another protein of interest with the herpes simplex virus VP16 transcriptional activation domain. If the two proteins interacted in planta, the DNA binding and activation domains would come into proximity, driving expression of the RUBY reporter gene and leading to the production of betalain, which is observable with the naked eye. Chen proved the efficacy of this new system in Nicotiana benthamiana and began using it to assess protein–protein interactions between plant resistance proteins and pathogen effectors (Chen et al., 2023). Specifically, he was interested in the control of stem rust in wheat (Triticum aestivum), which is caused by Puccinia graminis f. sp. tritici. This pathogen was largely under control in the latter half of the 20th century, but is receiving renewed attention since new more virulent strains have arisen (Upadhyaya et al., 2021). Because identifying resistance to these new virulent rust isolates is necessary, Chen chose to examine the Sr27 resistance protein, a potentially useful source of resistance that recognizes a corresponding AvrSr27 effector in the pathogen. Although Chen could not confirm this interaction using yeast two-hybrid screens, with the new RUBY assay he was able to show that Sr27 does directly interact with AvrSr27 (Figure 1a). The AvrSr27 effector family directly interacts with the Sr27 resistance protein. (a) Sites 1–4 on the leaf shown were infiltrated Agrobacterium cultures containing a UAS-RUBY construct in addition to: (1) GAL/Sr27 + VP16/AvrSr27-1; (2) GAL/Sr27 + VP16/AvrSr27-2; (3) GAL/Sr27 + VP16/avrSr27-3; or (4) GAL/Sr27 + VP16/AvrP (negative control). (b) Betalain accumulation as shown by absorbance at 538 nm. Modified from Chen et al. (2023). To further characterize this relationship, Chen developed a method to quantify the binding affinity between two proteins using the RUBY reporter system by spectrophotometrically assessing absorbance at 538 nm, which is the absorbance spectrum of betalain. He then compared the binding affinity of Sr27 with three different allelic variants of AvrSr27, and showed that Sr27 binds much more strongly with AvrSr27-1 than with AvrSr27-2 or avrSr27-3 (Figure 1b). The observation that Sr27 does bind to avrSr27-3 is important as it is the virulence allele of this effector, i.e. it does not activate a resistance response in wheat plants. This virulence allele is expressed at a lower level in the fungus during infection, which, combined with its reduced binding affinity with Sr27, likely enables the pathogen to escape detection (Upadhyaya et al., 2021). Developing this RUBY system made his research into pathogen effectors and resistance proteins easier, but this technique will surely find adopters in fields outside plant pathology. Chen mentioned that another team recently modified the RUBY system to visualize DNA–protein interactions (Sun et al., 2023). After finishing his PhD, Chen decided to stay at CSIRO as a postdoc in the plant disease resistance group. His focus remains on determining the molecular basis of recognition specificity by resistance proteins against cereal rust diseases. He has also started to use his RUBY protein–protein detection system to assess downstream signaling components in the defense response initiated by TIR-NLR proteins, a group of immune receptors that recognize pathogen effectors and activate effector-triggered immunity.