Use of Dominant-negative HrpA Mutants to Dissect Hrp Pilus Assembly and Type III Secretion in Pseudomonas syringae pv. tomato

Abstract

The Hrp pilus plays an essential role in the long-distance type III translocation of effector proteins from bacteria into plant cells. HrpA is the structural subunit of the Hrp pilus in Pseudomonas syringae pv. tomato (Pst) DC3000. Little is known about the molecular features in the HrpA protein for pilus assembly or for transporting effector proteins. From previous collections of nonfunctional HrpA derivatives that carry random pentapeptide insertions or single amino acid mutations, we identified several dominant-negative mutants that blocked the ability of wild-type Pst DC3000 to elicit host responses. The dominant-negative phenotype was correlated with the disappearance of the Hrp pilus in culture and inhibition of wild-type HrpA protein self-assembly in vitro. Dominant-negative HrpA mutants can be grouped into two functional classes: one class exerted a strong dominant-negative effect on the secretion of effector proteins AvrPto and HopPtoM in culture, and the other did not. The two classes of mutant HrpA proteins carry pentapeptide insertions in discrete regions, which are interrupted by insertions without a dominant-negative effect. These results enable prediction of possible subunit-subunit interaction sites in the assembly of the Hrp pilus and suggest the usefulness of dominant-negative mutants in dissection of the role of the wild-type HrpA protein in various stages of type III translocation: protein exit across the bacterial cell wall, the assembly and/or stabilization of the Hrp pilus in the extracellular space, and Hrp pilus-mediated long-distance transport beyond the bacterial cell wall. The bacterial type III secretion system (TTSS)1 is a longdistance protein transport system, moving bacterial effector proteins from the bacterial cytoplasm into a eukaryotic cell (1-4). During this long-distance transport, several physical barriers must be traversed: the bacterial cell wall, the host extracellular matrix layer (e.g. plant cell wall or animal mucous layer/glycocalyx), and the host plasma membrane. The TTSS has adapted to such long-distance transport by assembling extracellular needle/pilus-like appendages of various lengths (3, 4). For example, the TTSS of mammalian pathogenic bacteria assembles a needle complex, which consists of a base structure embedded in the bacterial cell wall and a primarily extracellular hollow needle of 6 -8 nm in diameter and 50 -80 nm in length (5-10). In enteropathogenic Escherichia coli, the needle is connected with another extracellular filament called the EspA filament (11,12). The EspA filament is 12 nm in diameter and can be several micrometers long (13,14). The TTSS of plant pathogenic bacteria assembles an extracellular appendage called the Hrp pilus, which is 6 -8 nm wide and several micrometers long (15-20). The needle, EspA filament, and Hrp pilus are believed to be tunnels linking the type III "secreton" embedded in the bacterial cell wall and a type III "translocon" in the host plasma membrane. The secreton allows the exit of effector proteins across the bacte...

The Needle Component of the Type III Secreton of Shigella Regulates the Activity of the Secretion Apparatus

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Type III Secretion and Effectors Shape the Survival and Growth Pattern of Pseudomonas syringae on Leaf Surfaces

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Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria

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