A high-throughput, near-saturating screen for type Ⅲ effector genes from Pseudomonas syringae

摘要

Pseudomonas syringae strains deliver variable numbers of type Ⅲ effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type Ⅲ effector genes. The screen relied on FACS and an arabinose-inducible hrpL σ factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type Ⅲ effector proteins by creating and testing full-length protein fusions to a reporter called Δ79AvrRpt2 that, when fused to known type Ⅲ effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Δ79AvrRpt2 is thus a marker for type Ⅲ secretion system-dependent translocation, the most critical criterion for defining type Ⅲ effector proteins. We describe our screen and the collection of type Ⅲ effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type Ⅲ proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL.-dependent type Ⅲ effector suites from two sequenced P. syringae pathovars and show that type Ⅲ effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.