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

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.

Involvement of hrpX and hrpG in the Virulence of Acidovorax citrulli Strain Aac5, Causal Agent of Bacterial Fruit Blotch in Cucurbits

“…As hrpG and hrpX were found to be essential for HR induction by A. citrulli , we investigated whether PTI-associated ROS was altered during the A. citrulli –host interaction. Thus, we examined the ROS levels induced by the A. citrulli strains in N. benthamiana leaves using chemiluminescence (Liu et al, 2015 ). ΔhrpG and ΔhrpX elicited significantly higher levels of ROS (~12-fold and ~8-fold, respectively) than the WT strain, which induced ROS to a level similar to that of DC3000 (Figure 5 ).…”

“…ROS were measured by chemiluminescence as described previously (Liu et al, 2015 ), with slight modifications. N. benthamiana leaves were inoculated with cell suspensions of A. citrulli Aac5, ΔhrpG, and ΔhrpX, and respective complementation strains (~10 8 CFU/mL).…”

“…Subsequently, 100 μL of 0.5 mM L-012 (Wako, Guangzhou, China) in 10 mM MOPS-KOH buffer (pH 7.4) were added to each well. The intensity of ROS generation was determined as described by Liu et al ( 2015 ). N. benthamiana leaf disks infiltrated with P. syringae pv.…”

Genomic insights into a plant growth‐promoting Pseudomonas koreensis strain with cyclic lipopeptide‐mediated antifungal activity

“…The final construct was designated as pK18-LokA, which was then introduced into strain S150 to make a lokA deletion mutant, and this was followed by two recombination steps (Liu, Zhang, Zhang, Liu, & Wei, 2015). The 7.0 kb fragment that was digested by Hind III and Xba I was inserted at the same sites on the shuttle vector pB-BR1MCS-2 to generate the complementation plasmid pBBR-LokA.…”

Identification and Functional Analysis of AopN, an Acidovorax Citrulli Effector that Induces Programmed Cell Death in Plants

“…The cotyledons were photographed at 4 days after inoculation using an embedded operation system (EOS) 70D camera (Canon, Beijing, China). Quantitative analysis was then performed with inoculated watermelon cotyledons collected at 2 and 4 days, and bacterial populations were evaluated as described previously [ 42 ]. Each experiment was independently repeated three times.…”