Role of the Type VI Secretion System in the Pathogenicity of Pseudomonas syringae pv. actinidiae, the Causative Agent of Kiwifruit Bacterial Canker

Abstract

The type VI secretion system (T6SS), a macromolecular machine, plays an important role in the pathogenicity of many Gram-negative bacteria. However, the role of T6SS in the pathogenicity of <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> (<i>Psa</i>), the pathogen of kiwifruit bacterial canker, is yet to be studied. Here, we found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of <i>Psa</i> M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of <i>Psa</i> M228, T6SS and its 13 core gene deletion mutants were constructed and their pathogenicity was determined. The deletion mutants showed different degrees of reduction in pathogenicity compared with the wild-type strain M228; in <i>tssM</i> and <i>tssJ</i> mutants, pathogenicity was significantly reduced by 78.7 and 71.3%, respectively. The pathogenicity results were also confirmed by electron microscopy. To further confirm that the reduction in pathogenicity is related to the function of T6SS, we selected the T6SS gene cluster, comprising <i>tssM</i> and <i>tssJ</i>, for further analyses. Western blot results revealed that <i>tssM</i> and <i>tssJ</i> were necessary for hemolytic co-regulatory protein secretion, indicating that they encode a functional T6SS. Further, we explored the mechanism by which T6SS affects the pathogenicity of <i>Psa</i> M228. The ability of bacterial competition, biofilm formation, hydrogen peroxide tolerance, and proteolytic activity were all weakened in the deletion mutants M228ΔT6SS, M228ΔtssM, and M228ΔtssJ. All these properties of the two gene complementation mutants were restored to the same levels as those of the wild-type strain, M228. Quantitative real-time results showed that during the interaction between the deletion mutant M228ΔT6SS and the host, expression levels of T3SS transcriptional regulatory gene <i>hrpR</i>, structural genes <i>hrpZ</i>, <i>hrcC</i>, <i>hopP1</i>, and effector genes <i>hopH1</i> and <i>hopM1</i> were down-regulated at different levels. Taken together, our data provide evidence for the first time that the T6SS plays an important role in the pathogenicity of <i>Psa</i>, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.