Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death

Many bacterial avirulence (Avr) proteins, including the Pseudomonas syringae proteins, AvrRpt2 and AvrB, appear to be recognized inside the host plant cell by resistance mechanisms mediated by the cognate resistance (R) genes. It is thought that Avr proteins are either delivered directly into the host cell via the bacterial type III secretion system (TTSS) or taken up by the plant cell following secretion into the apoplast through the TTSS. Recently, it was shown that the Xanthomonas campestris AvrBs2 protein can be delivered directly into the host plant cell by the TTSS. However, it is not known whether other type III effectors of phytopathogens behave similarly. Here, using a novel protein transfection method, we demonstrate that AvrRpt2 and AvrB must enter the plant cell to be recognized by R gene-mediated mechanisms. First, we established a hypersensitive cell death assay for protoplasts using the membrane-impermeable, nuclear-staining dye, YO-PRO-1, and transgenic Arabidopsis plants that carry an inducible avrRpt2 gene. Second, we transfected E. coli-produced AvrRpt2 or AvrB proteins into Arabidopsis protoplasts using a protein transfection kit based on the carrier peptide Pep-1, and demonstrated that hypersensitive cell death occurs in a gene-for-gene-specific manner. In contrast, these Avr proteins failed to elicit hypersensitive cell death when they were applied to protoplasts without the carrier peptide. We conclude that our preparations of E. coli-produced AvrRpt2 and AvrB are active, that AvrRpt2 and AvrB must be delivered into the plant cell to be recognized, and that a method based on a carrier peptide can be used to introduce proteins into plant cells.