Browsing by Author "Zhao, Bingyu"

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    Characterization of Effector Genes in Acidovorax citrulli the Causing Agent of Bacteria Fruit Blotch Disease of Cucurbits
    Traore, Sy M. (Virginia Tech, 2014-08-08)
    Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit.
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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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    Characterization of signaling pathways underlying key growth and development processes in Populus trichocarpa
    Rigoulot, Stephen Bradley (Virginia Tech, 2018-09-05)
    The project goals for this dissertation were to manipulate Populus trichocarpa source-sink relationships to optimize this woody crop species for specific agricultural traits such as increased growth rate, stress tolerance and/or improvements in overall biomass accumulation. We targeted specific tissues such as xylem, where alterations in the relationship of source and sink tissues can lead to the control of xylem cell deposition or of various wood properties. This led to the characterization of 165 protein-protein interactions and 20 protein-DNA interaction which constitute numerous woody tissue related subnetworks. One such network, centered on the DIVARACATA and RADIALIS INTERACTING FACTOR (PtrDRIF), identified PtrWOX13c as an interacting protein. Characterization of PtrWOX13c shows that it displays the ability to control promoters related to lignin biosynthesis genes and overexpression phenotypes show alterations in axillary branch activity. Genes which control the differentiation and specialization of cells such as members of the WOX family are also highly responsive to abiotic stress which can force major changes in plant metabolism and nutrient mobilization. ABA, a prominent plant phytohormone with known roles in the adaptation to stress has shown novel connections in the regulation of growth promoting complexes such as TOR through antagonistic regulatory actions of the SnRK2 protein kinase in Arabidopsis. Characterization of the core ABA signaling in P. trichocarpa has identified a regulatory clade A protein phosphatase which interacts with numerous PtrSnRK2 proteins and when overexpressed in hybrid poplar results in increased height and node production potentially by indirect control of growth promoting complexes like TOR through SnRK2 inhibition. This work has also demonstrated that in addition to the involvement of phytohormones in the regulation of plant development, sugar phosphates such as T6P can exert significant control of plant architecture. Together, these studies comprise the discovery and subsequent characterization of novel wood associated networks, hormone pathways and sugar signaling in the manipulation of P. trichocarpa source-sink relationships for the promotion of biomass accumulation.
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    Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L.
    Ruiz-Rojas, Juan Jairo (Virginia Tech, 2010-11-01)
    Cultivated strawberry (Fragaria × ananassa) is an octoploid (2n=8x=56) species that belongs to the Rosaceae family and the high ploidy level makes genetic and molecular studies difficult. However, its commercial success because of its unique flavor and nutritious qualities has increased interest in the development of genomic resources. Fragaria vesca L. is a diploid (2n=2x=14) species with a small genome size (206 Mbp), short reproductive cycle, and facile vegetative and seed propagation that make it an attractive model for genomic studies. The availability of an efficient transformation methodology for Fragaria vesca has facilitated the use of a T-DNA mutagenesis system to develop a collection of several hundred insertional T-DNA mutants at Virginia Tech, using either of two commercially available vectors, pCAMBIA 1302 and 1304. In this study, we have used expression of the green fluorescent protein (GFP) as a tool to identify homozygous mutant lines. Three different approaches were conducted, first we identified 11 homozygous lines by PCR, then another 55 homozygous lines by absence of segregation of GFP expression in T2 seedlings, and finally we attempted to distinguish homozygous from hemizygous lines by relative GFP expression measured using a commercially available GFP meter. The latter methodology was unsuccessful due to uncontrolled variability in the readings. Continuing the characterization of our mutant population, we used thermal asymmetric interlaced PCR (TAIL-PCR) to obtain the nucleotide sequence of the genomic DNA regions that flank the T-DNA insertion sites in independent transgenic strawberry lines. Primers were designed that would amplify the derived strawberry flanking sequences in the two parents of an interspecific mapping population between the two diploid species, F. vesca x F. bucharica. The amplified products were sequenced and examined for the occurrence of SNPs (single nucleotide polymorphisms). The same primers were then used on the F2 mapping population. Segregation of SNP markers with previously mapped genetic markers allowed us to position 74 SNP markers, and hence their corresponding insertional mutants, on a well-populated genetic linkage map for the diploid strawberry. Finally, we analyzed the insertion site from more than 190 mutants looking at both the right and left borders of the T-DNA where microsimilarities of a few base pairs between ends of T-DNA and genomic DNA were observed, indicating that T-DNA integration had not occurred randomly in strawberry. We have also characterized the insertion sites through gene annotation found in the strawberry genome database.
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    A Comprehensive Analysis of Rust Disease Resistance in the Bioenergy Plant Switchgrass (Panicum virgatum L.)
    Frazier, Taylor Price (Virginia Tech, 2016-01-14)
    Switchgrass is a C4 perennial grass that is currently being developed for use as a second generation lignocellulosic biofuel crop. For switchgrass to be fully utilized as a bioenergy crop, large-scale plantings of elite switchgrass germplasm, possibly in monoculture, are likely to occur. This practice may increase the selection pressure on plant pathogens, such as switchgrass rust, which could result in devastating disease epidemics. The identification and deployment of quantitative trait loci (QTLs) and major plant disease resistance genes (R) in switchgrass breeding programs could offer broad spectrum and durable disease resistance in commercial switchgrass cultivars. 'Alamo', a lowland cultivar, is generally resistant to switchgrass rust whereas 'Dacotah', an upland cultivar, is highly susceptible. I hypothesized that major R genes and/or QTLs were contributing to the differences in disease phenotypes of these two cultivars. In this dissertation, bioinformatics and molecular biology approaches were employed to dissect the genetic mechanisms underlying switchgrass rust disease resistance. Novel pseudo-F2 mapping populations were created from a cross derived from 'Alamo' and 'Dacotah'. RNA-sequencing of the pseudo-F2 progenies of 'Alamo' x 'Dacotah' was used to construct a genetic linkage map and to identify potential QTLs correlating with disease resistance. In addition, a homology-based computational method was used to identify 1,011 potential NB-LRR R genes in the switchgrass genome (v 1.1). These potential R genes were characterized for polymorphism and expression differences between 'Alamo' and 'Dacotah'. Moreover, I found that some NB-LRR genes are developmentally regulated in switchgrass. One of the major objectives of switchgrass breeding programs is to develop cultivars with improved feedstock quality; however, changes in the components of the plant cell wall may affect disease resistance. I hypothesized that genetically modified switchgrass plants with altered cell wall components will respond differently than the wild-type to switchgrass rust. Transgenic switchgrass plants overexpressing AtSHN3, a transcription factor with known functions in epicuticular wax accumulation and cell wall deposition, were created. I found that AtSHN3-overexpressing transgenic switchgrass lines were more susceptible than wild-type plants in their response to switchgrass rust. Overall, the results of this dissertation provide a platform for elucidating the molecular mechanisms underlying resistance of switchgrass to switchgrass rust. These findings will help breeders create switchgrass cultivars with improved disease resistance, and will ultimately allow switchgrass to be used for sustainable biomass production.
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    Detection of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch Disease of Cucurbits, Prevention via Seed Treatments and Disease Resistance Genes
    Kiremit, Merve (Virginia Tech, 2021-04-02)
    Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background.
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    Developing Novel Management Options for Pear Fire Blight and Apple Bitter Rot with Characterization of Apple European Canker in Virginia
    Correa Borba, Matheus (Virginia Tech, 2024-06-27)
    In the realm of tree fruit cultivation, the management of various diseases affecting pome fruits like apples and pears is crucial for sustaining production. This study amalgamates findings from three distinct disease investigations to propose an integrated approach to their management. Firstly, in pursuit of mitigating shoot blight severity caused by Erwinia amylovora and preventing fire blight cankers on pear trees, a two-year evaluation was conducted. The study assessed the efficacy of preventive treatments, including foliar spray and trunk injection applications of Giant Knotweed Extract (RSE) alongside antibiotics. Results highlighted the effectiveness of RSE in controlling both shoot blight severity and canker incidence, offering a sustainable alternative to antibiotics. Secondly, in addressing the bitter rot of apples caused by Colletotrichum spp., eighteen fungicide treatments were evaluated over two years, focusing on newer fungicide options to mitigate fungicide resistance development. Fungicides such as Omega, Aprovia, Ferbam, Captan, Ziram, and Cabrio were proven reliable management tools, complementing the existing effective fungicides that growers heavily depend on. Lastly, the emergence of European canker (Neonectria ditissima) on cider apple cultivars was investigated. Molecular analysis confirmed the presence of N. ditissima as the causal agent, posing a significant threat to cider apple production. Koch's postulates were fulfilled through various tests, proving the pathogenicity of N. ditissima. Further research avenues, including genome sequencing were conducted to enhance understanding and control of a devastating pathogen like N. ditissima. Integrating findings from these studies proposes a comprehensive management strategy incorporating preventive spray programs, alternative fungicides, and pathogen identification to combat these diseases effectively, ensuring sustainable production of apples and pears in orchards. This holistic approach offers growers a multifaceted toolkit to manage diseases effectively, safeguarding apple and pear orchards' productivity and economic viability.
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    Development of a Transposon Based Activation Tagged Mutant Population in Tomato for Functional Genomic Analysis
    Carter, Jared Daniel (Virginia Tech, 2012-04-17)
    Tomato serves as an important model organism for Solanaceae in both molecular and agronomic research. With whole genome sequencing in progress, there is a need to study functional genetics through mutant lines that exceed the practical limitations imposed by the popular research cultivar, Micro-Tom. This project utilized Agrobacterium transformation and the transposon tagging construct, Ac-DsATag-Bar_gosGFP, to produce activation tagged and knockout mutants in the processing tomato variety, M82. The construct contained hygromycin resistance (hyg), green fluorescent protein (GFP), and maize transposase (TPase) on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 10,568 selfed and M82 backcross progeny was produced from the functional T0 line. This population was screened by spraying with 0.05% Liberty® herbicide, followed by a 100 mg/L hygromycin leaf painting procedure to select for Ds only (herbicide tolerant and hygromycin sensitive) individuals. The T-DNA genotype of Ds only plants was confirmed through multiplex PCR and the location of insertions within the genome was determined through TAIL-PCR. Resulting product sequences were blasted against the pre-publication tomato genome browser to determine insertion sites. A population of 309 independent transposants dispersed to all twelve chromosomes from the original insertion site on chromosome five has been developed. The transposon tagged lines are currently being immortalized in seed stocks.
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    Development of tools to study the association of transposons to agronomic traits
    Yan, Haidong (Virginia Tech, 2020-05-21)
    Transposable elements (Transposons; TEs) constitute the majority of DNA in genomes and are a major source of genetic polymorphisms. TEs act as potential regulators of gene expression and lead to phenotypic plasticity in plants and animals. In crops, several TEs were identified to influence alleles associated with important agronomic traits, such as apical dominance in maize and seed number in rice. Crops may harbor more TE-mediated genetic regulations than expected in view of multifunctional TEs in genomes. However, tools that accurately annotate TEs and clarify their associations with agronomic traits are still lacking, which largely limits applications of TEs in crop breeding. Here we 1) evaluate performances of popular tools and strategies to identify TEs in genomes, 2) develop a tool 'DeepTE' to annotate TEs based on deep learning models, and 3) develop a tool 'TE-marker' to identify potential TE-regulated alleles associated with agronomic traits. As a result, we propose a series of recommendations and a guideline to develop a comprehensive library to precisely identify TEs in genomes. Secondly, 'DeepTE' classifies TEs into 15-24 super families according to sequences from plants, metazoans, and fungi. For unknown sequences, this tool can distinguish non-TEs and TEs in plant species. Finally, the 'TE-marker' tool builds a TE-based marker system that is able to cluster rice populations similar to a classical SNP marker approach. This system can also detect association peaks that are equivalent to the ones produced by SNP markers. 'TE-marker' is a novel complementary approach to the classical SNP markers that it assists in revealing population structures and in identifying alleles associated with agronomic traits.
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    Dissection of Innate Immunity in Tomato and Tolerance to Bacterial Wilt in Solanaceae species
    Naumenko, Anastasia Nikolayevna (Virginia Tech, 2013-04-05)
    Unlike mammals, plants do not have specific immune cells. However, plants can still recognize pathogens and defend themselves. They do that by recognizing microbial-associated molecular patterns (MAMPs) and secreted pathogen proteins, called effectors. MAMP-triggered immunity (MTI) relies on recognition of MAMPs by leucine-rich repeats (LRRs) pattern-recognition receptors (PRRs). The best-studied LRR PRR is Flagellin-Sensitive 2 (Fls2), the receptor of a 22-amino acid long epitope of bacterial flagellin, called flg22. In this project, alleles of FLS2 of different tomato cultivars were sequenced and compared to each other to get insight into natural selection acting on FLS2 and to identify residues important for ligand binding. This information may be used in the future to engineer Fls2 for improved ability to recognize flagellin. MTI can be suppressed by effectors secreted by bacteria into plant cells through the type III secretion system. On the other hand, plants are equipped with repertoires of resistance proteins, which can recognize some pathogen effectors. If a pathogen carries an effector that is recognized, effector-triggered immunity (ETI) is activated and the plant is resistant. Here, eggplant breeding lines were screened for their ability to activate ETI upon recognition of effectors of the soil borne pathogen Ralstonia solanacearum, a causative agent of bacterial wilt. Four effectors were found to trigger plant defenses in some of the lines. This is the first step in cloning the genes coding for the responsible resistance proteins. These genes may be used in the future for engineering tomato and potato for resistance to bacterial wilt.
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    Elucidating essential roles of oomycee effector proteins in immune suppression and in targeting hormonal pathways in the host plant
    Deb, Devdutta (Virginia Tech, 2013-09-25)
    Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms.
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    Elucidating three novel mechanisms of Pseudomonas syringae pathogenicity
    Clarke, Christopher R. (Virginia Tech, 2012-02-10)
    Pseudomonas syringae is an important bacterial plant pathogen that, as a species, is known to cause disease on hundreds of different plant species. However, any individual pathovar of P. syringae typically only causes disease on one or a few plant species, which constitute the host range of the pathovar. Plants are generally resistant to most pathogens primarily because the plant innate immune system is capable of recognizing conserved microbial-associated molecular patterns (MAMPs). Adapted pathovars of P. syringae secrete effector proteins through a Type Three Secretion System (T3SS) to suppress the immune response elicited by their MAMPs. However, secretion of effectors can also trigger a strong plant immune response if the plant harbors resistance proteins capable of recognizing the secreted effectors. Successful pathovars, therefore, must secrete a combination of effectors capable of suppressing MAMP/Pattern-Triggered Immunity (PTI) without eliciting Effector-Triggered Immunity. Here we identify several novel strategies employed by P. syringae to overcome the plant immune system and cause disease. First, we demonstrate that, in place of the canonical T3SS used by all known pathogens of P. syringae, several apparently nonpathogenic isolates of P. syringae employ a novel T3SS that is functional but not necessary for colonization of plants. Despite being closely related to pathogenic isolates of P. syringae, the isolates employing the noncanonical T3SS do not cause disease on any tested plants and instead appear to act more as commensal organisms. Second, we advance the understanding of PTI by identifying a second region of bacterial flagellin that triggers PTI in addition to the archetypical MAMP flg22, which is recognized by the archetypical plant receptor FLS2. This new elicitor, termed flgII-28, is also detected by FLS2 and appears to be under selection in very closely related lineages of P. syringae. Alleles of flagellin present in one recently expanded and agriculturally problematic lineage of P. syringae appear to trigger less PTI on their host plant, tomato, than the ancestral allele suggesting that avoidance of PTI through allelic diversity in MAMPs is an effective alternative strategy to suppression of PTI through delivery of effectors. Finally, we start to elucidate a role for chemotaxis (chemical-directed movement) in P. syringae pathogenicity. Not only is chemotaxis required for pathogenicity of P. syringae on plants, but it also appears to contribute to delimiting the host range of several P. syringae pathovars. These results highlight that additional aspects of P. syringae pathogenicity, such as chemotaxis, can directly contribute to defining the host range of individual P. syringae pathovars. The current paradigm of P. syringae pathogenicity posits that MAMPS and the repertoire of effector proteins are the primary determinant of the host range of any P. syringae pathovar; in contrast these results inspire a more nuanced view of pathogenicity that considers multiple aspects of the infection process.
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    Enhancing Seed Quality and Application of Soybean through High Test Weigh and Low Trypsin Inhibitors
    Shea, Zachary (Virginia Tech, 2023-02-09)
    Soybean is an integral oilseed crop that is used all over the world. Despite this, soybean has been experiencing a decrease in seed quality seen by the decreasing trend in test weight, and is limited in its uses due to the antinutritional factor, trypsin inhibitor (TI). This project first worked to identify single nucleotide polymorphisms (SNPs) associated with high test weight through a genome-wide association study (GWAS) as well as determine the relationship between important seed composition traits and test weight. Additionally, it focused on identifying and knocking out KTI genes specifically expressed in soybean seed tissue through CRISPR/Cas9. Lastly, this project investigated further use of soybean in aquaculture by evaluating the performance of Rainbow trout fed diets with low-TI, 'VT Barrack' soy meal. Nine SNPs on chromosome 15 were found to be significantly associated with high test weight as and eight potential gene candidates were identified. Test weight was found to be significantly, and negatively related to seed oil content and had inconsistent correlations with protein and sugar content. The KTI genes KTI1, Glyma01g095000, and KTI3, Glyma08g341500, were found to be only expressed in seed tissues and multiple KTI knock-out soybean plants with decreased seed TI content and activity were developed. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health.
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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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    Exploring candidate genes and rhizosphere microbiome in relation to iron cycling in Andean potatoes
    Xiao, Hua (Virginia Tech, 2017-06-05)
    Fe biofortification of potato is a promising strategy to prevent Fe deficiency worldwide either through traditional breeding or biotechnological approaches. These approaches require the identification of candidate genes to uptake, transport and store Fe in potato tubers. We employed multiple approaches including SNP genotyping, QTL analysis, identifying genes orthologous to Arabidopsis ferrome, yeast complementation assay and genetic transformation to avoid the limitation from a single approach. We revealed several candidate genes potentially associated with potato plant Fe acquisition, PGSC0003DMG400024976 (metal transporter), PGSC0003DMG400013297 (oligopeptide transporter), PGSC0003DMG400021155 (IRT1) and IRTunannotated (an ortholog to the IRT gene that is not annotated in the potato genome). The microorganisms in the rhizosphere react intensely with the various metabolites released by plant roots in a variety of ways such as positive, negative, and neutral. These interactions can influence the uptake and transport of micronutrients in the plant roots. Therefore, the contribution of soil microorganisms in the rhizosphere to improve Fe supply of plants may play a key role in Fe biofortification, especially under real world field-based soil scenarios. We thus investigated rhizosphere microbial community diversity in Andean potato landraces to understand the role of plant-microbial interaction in potato Fe nutrient cycling. From the analysis of the high-throughput Illumina sequences of 16S and ITS region of ribosomal RNA gene, we found that both potato landraces with low and high Fe content in tubers and a landrace on which low or high Fe content fertilizer was applied to the leaf surface had large impacts on the rhizosphere fungal community composition. Indicator species analysis (ISA) indicated that Operational Taxonomic Units (OTUs) contributing most to these impacts were closely related to Eurotiomycetes and Leotiomycetes in the phylum Ascomycota, Glomeromycetes in the phylum Glomeromycota and Microbotryomycetes in the phylum Basidiomycota. Lots of species from these groups have been shown to regulate plant mineral nutrient cycling. Our research revealed potential candidate genes and fungal taxa involved in the potato plant Fe nutrient dynamics, which provides new insights into crop management and breeding strategies for sustainable Fe fortification in agricultural production.
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    Flowering Gene Homologs Regulate Seasonal Growth Changes in Poplar
    Sheng, Xiaoyan (Virginia Tech, 2018-10-19)
    The adaptation of trees to temperate and boreal climates depends on their ability to respond to environmental signals that are markers of seasonal changes in order to survive winter and maximize growth. The genus Populus (poplars) is a model system for identifying the genes and molecular mechanisms that regulate growth and dormancy transitions. Photoperiod and temperature regulate both vegetative and floral phenology. FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FD) are key regulators of flowering time in Arabidopsis and other plants. The distinct functions of three poplar FD-LIKE (FDL) genes and two FTs were explored through gain-of-function, dominant repression, and CRISPR/CAS9-mediated gene editing. We studied trees in controlled environments, including manipulation of daylength and temperature to mimic an annual seasonal growth and dormancy cycle. Our studies showed that the FDL proteins share less than complete functional equivalency. Among the three paralogs, only FDL2.2 promoted precocious flowering, whereas FDL1 and FDL3 appear to have distinct roles in vegetative growth and phenology. Whereas overexpression of any FDL gene delays short day-induced growth cessation and bud set, only FDL3 coordinately altered leaf development and the transition to secondary growth in a photoperiod-dependent manner. For the first time, we demonstrate distinct functions of the two FT paralogs in vegetative phenology. Study of ft1ft2 double mutants and ft1-specific mutants showed that FT1 promotes dormancy release, whereas FT2 is necessary to sustain growth. Collectively, our results reveal that poplar FTs and FDLs have distinct roles in controlling different aspects of vegetative phenology and woody shoot development.
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    Functional analysis of novel protein-protein interactions involving ROP GTPases in Arabidopsis thaliana and Populus trichocarpa
    Jia, Xiaoyan (Virginia Tech, 2013-09-02)
    We are using the yeast two-hybrid (Y2H) system to identify novel protein-protein interactions (PPI) relevant to wood formation. Bait proteins for Y2H binary assays and screening against a xylem cDNA prey library were selected from approximately 400 Populus trichocarpa genes that are at least 8-fold more highly expressed in differentiating secondary xylem versus phloem-cambium, and designated here as poplar biomass (PB) genes. Here we report some of the interactions involving selected PB proteins and efforts to characterize their functions in Populus and Arabidopsis. Members of the ROP GTPase family, PB15 in poplar and ROP11 in Arabidopsis, interact with the domain of unknown function (DUF) 620 (DUF620) proteins (e.g., PB129 in poplar). Ectopic co-expression of PB15 and PB129 in Arabidopsis caused outgrowths at the base of flower pedicels and altered leaf morphology. Interestingly, the co-expression phenotype could not be observed in transgenic plants that are only expressing either one of the interacting partners separately. Transgenics altered in expression of PB15 and/or PB129 were prepared in Populus and characterization of transgenic trees will be performed in greenhouse and field. In addition to DUF620 family proteins, ROP11 also interacts with the COP9 subunit CSN5A in Arabidopsis. We confirmed the interaction of ROP11 and CSN5A in Y2H and employed available mutants for ROP11 and CSN5A in Arabidopsis to genetically characterize this interaction. Surprisingly, loss of ROP11 was found to rescue the csn5a-2 pleiotropic phenotype. Ectopic expression of a ROP11 dominant negative mutant in the csn5a-2 background also complemented the stunted growth phenotype. Transcript analysis and gel blot assays showed that CSN5A transcript levels remained unchanged in all rescue lines, whereas CSN5A protein levels increased relative to WT. Taken together, we concluded that ROP11 negatively regulate CSN5A protein level in plant by some as yet unknown mechanism.
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    Functional Characterization of Four Xanthomonas euvesicatoria Type III Effectors
    Wang, Zhibo (Virginia Tech, 2020-03-19)
    Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells.
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    Genetic Improvement of Switchgrass Cell Wall Content, Leaf Angle and Flowering Time
    Xu, Bin (Virginia Tech, 2011-06-10)
    Switchgrass (Panicum virgatum L.) is a candidate bioenergy crop. Somatic embryogenic (SE) calli are used for genetic transformation in switchgrass. A superior switchgrass line, HR8, was developed using recurrent tissue culture selection from cv. Alamo. HR8 SE calli were genetically transformable using Agrobacterium at an efficiency of ~12%. We used HR8 somatic embryogenic calli for genetic improvement of switchgrass. The lignin content of feedstock has been proposed as one key trait impacting biofuel production. 4-Coumarate: Coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthetic pathway. Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down-regulated. RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. The transgenic plants had uncompromised biomass yield. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency for biofuel production. Erect leaf is a desirable trait to adjust the overall plant architecture to perceive more solar energy and thereby to increase the plant biomass production in a field population. We overexpressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1), in switchgrass. Surprisingly, AtLOV1 induced smaller leaf angle by changing morphologies of epidermal cells in the leaf collar region, affecting lignin content and monolignol composition, and also causing delayed flowering time in switchgrass. Global gene-expression analysis of AtLOV1 transgenic plants demonstrated an array of genes has altered expressions. Potential downstream genes involved in the pleiotropic phenotypic traits of the transgenic plants are discussed.