Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean

Abstract

The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5 and the NIa protease. To test this, we re-localized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in N. benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV would enhance RPS5 activation by TuMV. Significantly, over-expressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either Agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.