Supramolecular Structure and Functional Analysis of the Type III Secretion System in Pseudomonas fluorescens 2P24

Ping Liu,Wei Zhang,Li-Qun Zhang,Xingzhong Liu,Hai-Lei Wei

doi:10.3389/fpls.2015.01190

Abstract

The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in non-pathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus, and a membrane-embedded base. We show that strain 2P24 deploys a harpin homolog protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS.

Highlights

In natural systems, plants are attacked continuously by a broad spectrum of pathogens and, at the same time, they are protected by a large number of beneficial microorganisms
P. fluorescens Type III Secretion System is referred to as a needle complex, is comprised of three functional modules: (i) a cylindrical basal body that spans the bacterial inner and outer membranes with a presumed central rod to build a channel, (ii) a needle/pilus structure that extrudes from the bacterial outer membrane and functions as a conduit for effector transfer, and (iii) a translocon complex that produces a pore in the host plasma membrane to inject the effectors (Galán and Collmer, 1999; Wei and Collmer, 2012)
It was reported that the hrp genes encoding the TTSS machinery in P. syringae are the conserved center region (CCR) of a tripartite pathogenicity island that includes exchangeable (EEL) and conserved (CEL) effector loci (Alfano et al, 2000)

Summary

Introduction

Plants are attacked continuously by a broad spectrum of pathogens and, at the same time, they are protected by a large number of beneficial microorganisms. A large variety of structural studies on animal and human bacterial T3SS was conducted. Tomato DC3000, a pilus is comprised of a major subunit of the T3SS, the HrpA1 protein, which extends to the plant cell (Roine et al, 1997). This pilus is flexible and its length of ∼2 μm is much greater than that of the needles found in animal pathogens (Roine et al, 1997). An in situ immunogold labeling was used to visualize the extrusion of the effector and harpin proteins from the tip of the Hrp pilus, which demonstrates that the bacterial pilus can function as a conduit for protein delivery (Jin et al, 2001; Li et al, 2002)

Methods

Results

Conclusion