Testing the independent action hypothesis of plant pathogen mode of action: a simple and powerful new approach

The independent action hypothesis is a simple model of pathogen infection that can make many useful predictions on infection kinetics and, therefore, a number of different tests of independent action have been developed. However, some of these analyses are rather sophisticated, limiting their appeal to experimentalists, and it is also unclear how well the different tests perform. Here, we developed and evaluated a simple and robust new test of independent action. Our new test is based on using a constant inoculum dose of one pathogen variant, varying the dose of a second variant, and then quantifying the infection response for the first variant. We simulated infection data in which we introduced deviations from independent action, experimental variation, or both. Simulations showed that our new procedure has many advantages over the existing tests of independent action, especially if only systemic-infection data are available. We also performed experimental tests of our new procedure using two marked Tobacco etch virus (TEV) variants. We found minor deviations from the independent action model, which were not detected by previous tests using existing methods, exemplifying the utility of this approach. We discuss the implications for TEV infection kinetics and consider how to reconcile different dose-dependent effects.