Bioengineering secreted proteases convert divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases Rcr3 (Required for Cladosporium resistance-3) and Pip1 (Phytophthora- inhibited protease-1) of tomato (Solanum lycopersicum) are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signalling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues, and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Evolution of a guarded decoy protease and its receptor in solanaceous plants.

Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.

Assessing the Physiological Cost of Active Videogames (Xbox Kinect) Versus Sedentary Videogames in Young Healthy Males.

OBJECTIVES: The aims of this study were twofold: (1) to compare the physiological costs of active videogames (AVGs) and sedentary videogames (SVGs) and (2) to compare the exercise intensities attained during AVGs with the exercise intensity criteria for moderate and vigorous physical activity, as stated in current physical activity recommendations for improving public health. MATERIALS AND METHODS: Nineteen young males participated in the study (age, 23 ± 3 years; height, 178 ± 6 cm; weight, 78 ± 15 kg). Participants completed a maximum oxygen uptake ([Formula: see text]) test and a gaming session, including AVGs ("Reflex Ridge," "River Rush," and "Boxing" for the Microsoft [Redmond, WA] Kinect™) and SVGs ("FIFA 14" [Electronic Arts, Burnaby, BC, Canada] and "Call of Duty" [Activision, Santa Monica, CA]). Heart rate (HR) and oxygen uptake [Formula: see text]) were recorded continuously during all videogames. Rating of perceived exertion (RPE) was taken every 3 minutes during AVGs and SVGs. Energy expenditure (EE), expressed as metabolic equivalents (METs), was calculated. One MET was defined as the volume of oxygen consumed at rest in a seated position and is equal to 3.5 mL of O2/kg of body mass/minute. The exercise intensity for each game was expressed as a percentage of [Formula: see text] and percentage of age-predicted maximum HR (HRmax). RESULTS: Exercise intensity (percentage HRmax, percentage [Formula: see text], and RPE) and EE (METs) were significantly higher during active gaming compared with sedentary gameplay (P < 0.01). AVGs elicited moderate levels of exercise intensity (64-72 percent HRmax) in line with current recommended physical activity guidelines. CONCLUSIONS: Our results indicate AVGs provoke physiological responses equivalent to a moderate-intensity physical activity.