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 Pseudomonas syringae pv. actinidiae (Psa), 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 Psa M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of Psa 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 tssM and tssJ 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 tssM and tssJ, for further analyses. Western blot results revealed that tssM and tssJ 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 Psa 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 hrpR, structural genes hrpZ, hrcC, hopP1, and effector genes hopH1 and hopM1 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 Psa, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.
Highlights
The bacterial canker of kiwifruit is caused by the virulent form of Pseudomonas syringae pv. actinidiae (Psa) and is the most prevalent disease in the kiwifruit industry (Pinheiro et al, 2020)
We found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of Psa M228 based on a genome-wide analysis
The results showed that T6SS is an important pathogenic determinant in Psa M228 and plays a role in bacterial competition, biofilm formation, hydrogen peroxide tolerance, proteolytic ability, and T3SS function
Summary
The bacterial canker of kiwifruit is caused by the virulent form of Pseudomonas syringae pv. actinidiae (Psa) and is the most prevalent disease in the kiwifruit industry (Pinheiro et al, 2020). Pathogenic bacteria can infect kiwifruits repeatedly, breed in cortex, expand up and down, and even move to the xylem and central column of the plant, causing severe pathogenicity under appropriate conditions (Gao et al, 2016). Due to the latent and short-term outbreak characteristics of Psa, bacterial canker has been identified as a destructive disease of kiwifruit, which results in production losses worldwide. This disease has received more attention in the main kiwifruit planting areas owing to the huge economic losses (Vanneste, 2017; Hang et al, 2018; Kim et al, 2019; Williams et al, 2020). Identifying the pathogenic mechanism will play a vital role in effective prevention and control of the disease
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