Bacterial secretion systems have been playing essential roles in modulating the microbiota of most ecological niches. Among a variety of secretion systems, the Type VI Secretion System (T6SS), a nanomachine widely distributed in Gram-negative bacteria, is gaining increasing attention due to its involvement in microbe-microbe and microbe-host interactions through secreting toxins into host cells, microbial competitors, and the extracellular milieu. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising biological control agents (BCAs). The main body of this dissertation will focus on the characterization of the T6SS of a phytopathogen, Acidovorax citrulli (strain AAC00-1), and the secreted T6 effectors, and will also discuss the possible application of AAC00-1 as a BCA.
The seed-borne, gram-negative A. citrulli is able to cause bacterial fruit blotch (BFB) disease and then result in devastating decrease in yields of important cucurbits including watermelon, melon, squash and cucumber. Our inter-microbial competition assays demonstrate that AAC00-1 contains an active T6SS and presents a dramatic antimicrobial activity against a variety of microbes, including Gram-negative bacteria, Gram-positive bacteria, and yeast, dependent upon its T6SS. A group of novel non-enzymatic effectors, Hyde1 proteins, delivered into prey cells through the T6SS, are responsible for this broad-spectrum antimicrobial activity. Expressing Hyde1 or its N-terminal transmembrane domain shows significant toxicity in both E. coli and AAC00-1, and the toxicity of Hyde1 can be counteracted by its immunity protein, Hyde2. A non-pathogenic AAC00-1 strain suppresses the growth of multiple deleterious phytopathogens in planta and protects plant host. Transgenic plants expressing either full-length Hyde1 or its transmembrane domain demonstrate improved resistance against both bacterial and oomycete pathogens. Altogether, we characterize the T6SS killing of AAC00-1, identify the determinant effectors and discuss the application of both AAC00-1 and its T6SS effector in plant disease management.
Additionally, in order to develop molecular tools better serving our T6SS-related studies, we successfully generate a series of salicylic acid (SA)-inducible vectors, functioning in A. citrulli, that can be used for inducible gene expression, protein purification and other applications. The core regulatory component that we employ, is a transcriptional regulator, Sal7AR-V295F, due to its responsiveness to salicylate. By cloning this fragment to a broad-host-range plasmid, in this study, we establish multiple SA-inducible vectors that may be used in most Gram-negative bacteria. When using the E. coli strain C41(DE3) as the expression host, protein purification can be conducted routinely, upon the addition of affinity tags to our vectors, such as the maltose-binding protein (MBP) tag. Combining the modified vectors with the robust NanoLuc binary Technology (NanoBiT), we are able to devise a novel bacteria two-hybrid system as an effective method to detect protein-protein interaction. Two complementary fragments of the NanoLuc protein, LgBiT and SmBiT, with extremely low affinity, are fused to potential interactors, and they will be brought into proximity and reconstitute NanoLuc bioluminescence upon the occurrence of interaction. This system is used in our T6SS study to validate the interaction between Hyde1 toxin and its cognate immunity protein. Another fragment, HiBiT, which automatically interacts with LgBiT and reconstitutes NanoLuc, is cloned to the SA-inducible vector as well, enabling us to generate a split-NanoLuc-based method, for the purpose of detecting secretion of tagged T6 toxins into the prey bacterial cells expressing LgBiT. Overall, our SA-inducible vectors and their further modifications enrich the molecular tool repertoire for T6SS-related studies. / Doctor of Philosophy / Effective crop disease management is critical for agricultural production. Chemical spray has been practiced as one major approach to control plant diseases for more than a decade. However, increase of pesticide application could threaten public health and the environment. Biological control has been considered as one of the effective and environmental-friendly alternative approaches for disease control. In this dissertation, we identify that, Acidovorax citrulli (strain AAC00-1), a Gram-negative pathogen causing bacterial fruit blotch (BFB) disease in Cucurbitaceae, could be a potential biological control agent (BCA), because it carries an active Type VI Secretion System (T6SS), and the T6SS has been shown to contribute to the protective effects of many plant-associated BCAs. T6S is believed to mediate inter-bacterial competition through secreting toxins into microbial competitors. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising BCAs. We demonstrate that AAC00-1 suppresses the growth of multiple phytopathogens, depending upon its T6SS. Expressing ten out of eleven microbial toxins, encoded by Hyde1 genes, in E. coli shows significant toxicity. The wild type AAC00-1 strain inhibits the growth of multiple Arabidopsis leaf bacterial isolates, while an AAC00-1 Hyde1 mutant loses this capacity. The antimicrobial activity of AAC00-1 is proven to be broad-spectrum since this strain also shows inhibitory effect on the growth of Gram-positive bacteria and yeast. In planta disease assay suggests that a non-pathogenic AAC00-1 mutant defective in Type III secretion system (T3SS) maintains its capacity to suppress disease symptoms and pathogen growth on plants infected with different phytopathogens. Our study demonstrates the viability of the employment of non-pathogenic A. citrulli as an effective BCA in plant disease management.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/109518 |
| Date | 31 March 2022 |
| Creators | Wang, Kunru |
| Contributors | Horticulture, Zhao, Bingyu, Vinatzer, Boris A., Li, Jianyong, Williams, Mark A. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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