Browsing by Author "Wang, Kunru"

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    Characterization of Novel Type VI Effectors of Acidovorax citrulli and Their Applicability to Biological Control of Plant Diseases
    Wang, Kunru (Virginia Tech, 2022-03-31)
    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.
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    Evaluate the guide RNA effectiveness via Agrobacterium-mediated transient assays in Nicotiana benthamiana
    Wang, Zhibo; Shea, Zachary; Li, Qi; Wang, Kunru; Mills, Kerri; Zhang, Bo; Zhao, Bingyu (Frontiers, 2023-02-20)
    CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the Agrobacterium-mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in Nicotiana benthamiana and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting Nicotiana benthamiana and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting NbEDS1, NbWRKY70, GmKTI1, and GmKTI3 had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting NbNDR1 was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.
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    Genomic features of bacterial adaptation to plants
    Levy, Asaf; Gonzalez, Isai Salas; Mittelviefhaus, Maximilian; Clingenpeel, Scott; Paredes, Sur Herrera; Miao, Jiamin; Wang, Kunru; Devescovi, Giulia; Stillman, Kyra; Monteiro, Freddy; Alvarez, Bryan Rangel; Lundberg, Alvarez Derek S.; Lu, Tse-Yuan; Lebeis, Sarah; Jin, Zhao; McDonald, Meredith; Klein, Andrew P.; Feltcher, Meghan E.; Rio, Tijana Glavina; Grant, Sarah R.; Doty, Sharon L.; Ley, Ruth E.; Zhao, Bingyu Y.; Venturi, Vittorio; Pelletier, Dale A.; Vorholt, Julia A.; Tringe, Susannah G.; Woyke, Tanja; Dangl, Jeffery L. (Nature Publishing Group, 2018-01-01)
    Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.
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    Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits
    Traore, Sy M.; Eckshtain-Levi, Noam; Miao, Jiamin; Sparks, Anita Castro; Wang, Zhibo; Wang, Kunru; Li, Qi; Burdman, Saul; Walcott, Ron; Welbaum, Gregory E.; Zhao, Bingyu (Wiley, 2019-06-01)
    Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.