Identification of new type III effectors and analysis of the plant response by competitive index

In recent years, many efforts have been directed towards the identification of new type III-secreted effectors, and the completion of the secretomes of several Pseudomonas syringae pathovars. Several functional and bioinformatic screenings have been used to search for candidates, which have been tested for translocation into the plant cell, an essential criterion for the identification of new type III effector proteins. The most common translocation assay is based on the use of Delta AvrRpt2 as a reporter. When fused to a type III effector protein, Delta AvrRpt2 is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 resistance protein. This approach has been used widely and has allowed the identification of a considerable number of new effectors in a fast and convenient manner. However, as the hypersensitive response is a semi-quantitative assay, and the conditions do not resemble those occurring in nature, effectors with low expression or translocation efficiency could fail to translocate sufficient Delta AvrRpt2 to trigger a clear hypersensitive response. In keeping with these limitations, this test has failed to detect some true effectors that have been confirmed as such by other means. In order to increase the sensitivity of this method, we have developed a modification of the Delta AvrRpt2-based translocation assay using a competitive index in mixed infection to monitor the limitation of growth associated with the induction of the hypersensitive response. We have tested several effector candidates from P. syringae pv. phaseolicola and other P. syringae pathovars, and have compared the results obtained by our competitive index translocation assay with those obtained by standard hypersensitive response assays. We have identified six type III secretion system-translocated proteins using this approach, five of which failed to be identified by hypersensitive response assays. In addition, we have analysed the defence response triggered by one of these effectors using competitive index assays.