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Inter-bacterial competition mediated by the type VI secretion system (T6SS): promising role in the biocontrol properties of Pseudomonas putida

Periodic Reporting for period 1 - BIOCONT6SS (Inter-bacterial competition mediated by the type VI secretion system (T6SS): promising role in the biocontrol properties of Pseudomonas putida)

Reporting period: 2016-09-01 to 2018-08-31

Plant pathogens (phytopathogens) are the causal agents of many severe crop diseases including bacterial wilt, crown gall and rot. These diseases are directly responsible for the decline in the yield and the quality of the crops and cause serious economic losses in the agricultural sector. Chemical pesticides has been successfully used for years to protect crop plants but has many downsides for the environment including soil erosion and the rise of resistance to these chemical products. The H2020 program has prioritised initiatives to promote environmentally sustainable agriculture. In this context, the biological control of plant diseases by beneficial microorganisms (biocontrol) is considered a promising alternative to pesticides to protect economically-crucial plant crops. Fully understanding the biocontrol mechanisms performed by well-established agents is key to progress in this field of research.
Pseudomonas putida is an important biocontrol agent, in this project, we aim to analyse the type VI secretion systems (T6SSs) to determine whether T6SSs play a role in the biocontrol properties of this strain. T6SSs are bacterial nanomachines used to inject toxic effectors into target cells. This secretion system plays an important role in inter-bacterial competition and thus it is susceptible to be an important mechanism of biocontrol used for beneficial bacteria to eradicate plant pathogens.
A preliminary in silico analysis revealed the existence of three T6SSs in P. putida KT2440 (K1, K2 and K3). The project was designed to advance in the study of P. putida T6SSs by addressing the following main issues: 1) performing a phylogenetic study of the T6SSs in P. putida strains; 2) broadening and completing the in silico study; 3) identifying P. putida competitors targeted by the system; 4) characterising the identified effectors and immunity proteins and 5) determine the role of Pseudomonas-T6SS in biocontrol.
The phylogenetic study of the T6SS in Pseudomonas putida strains has revealed that T6SSs is widely distributed in P. putida. In fact, 100% of the strains contain T6SS elements and 90% of them present at least one complete cluster. The T6S clusters found in P. putida strains are highly conserved and clad mainly in 3 groups: 1.2 2 and 4B respectively. The in silico analyses revealed that P. putida KT2440 contains three potential T6SSs that we have named K1- (group 4B), K2- and K3-T6SS (both from group 1.2). Each one of these systems contain the complete set of genes to encode the core components necessary to assemble a functional machinery (Tss components), accessory components (Tag proteins) and over eleven effectors including nucleases and pore-forming colicins together with their cognate immunity pairs (Tke1-Tki1, Tke2-Tki2, Tke3-Tki3, …). I have showed using techniques such as bacterial competition assays, secretion assays and heterologous expression of toxins in non-immune bacteria that the K1-T6SS is a potent antibacterial device which secretes toxic effectors including the putative nuclease Tke2. Remarkably, P. putida kills a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens such as Agrobacterium tumefaciens, Pseudomonas syringae, Pectobacterium caratovorum and Xanthomonas campestris. Furthermore, this study shows that the protection occurs in vitro but also in planta and that Nicotiana benthamiana leaves are protected from the attack of X. campestris in a T6SS-dependent manner. All these results show a new role for T6SS weapons as a novel mechanism of biological control.
In this project, the type VI secretion system has been described for the first time as an important mechanism of biocontrol. This potent antimicrobial weapon has turn out in an environmentally sustainable approach to eradicate plant pathogens, a promising alternative to the use of chemical pesticides. This new approach will lead to an efficient protection of crop plants from diseases caused for phytopathogens. This protection will translate in a boost in crop production without the downside effects of chemical pesticides such as soil erosion and contamination. The discovery of a new mechanism of biological control in this project offers promising strategies to exploit the biotechnological applications of the biocontrol agent P. putida. This strategy will most likely results in increased crop productivity and better crop quality with a clear impact in the agriculture and therefore the economy sector of the European Area