1,721,056 research outputs found
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Understanding the Diversity of Plant Pattern Recognition Receptors to Engineer and Deploy Expanded Bacterial Recognition
No full textBacterial pathogens significantly threaten global agriculture production. Plants recognize these pathogens through cell-surface localized pattern recognition receptors (PRRs), which detect conserved microbial features and initiate immune responses. Many bacterial features perceived by PRRs are immunogenic epitopes derived from protein components such as flagellin and cold shock proteins. However, pathogens frequently evade immune detection by carrying modified epitopes that hinder PRR recognition. Although extensive research has examined the recognition specificity of PRRs, studies predominantly focus on the interaction between model pathogens and PRRs from model plants. Consequently, the diversity of PRR recognition capacities, particularly in non-cultivated species, remains poorly explored.In this thesis, I discussed the evolutionary path and the modes of functional diversification in plant PRRs. I surveyed the recognition of common microbial features in 97 Rutaceae genotypes and demonstrated the functionality of citrus chitin and flagellin receptors across responsive and non-responsive genotypes. I also examined six plant Flagellin-sensing 2 (FLS2) receptor homologs to characterize their recognition profiles against polymorphic flagellin epitope (flg22). I revealed that these receptors exhibit both expanded and distinct perception profile. Using a combination of diversity analyses, AlphaFold modeling, and amino acid property assessments, I identified key residues that contribute to expanded flagellin recognition, primarily located in the flg22 C‑terminus and co‑receptor binding regions. Through synthetic biology approaches, I successfully engineered expanded or altered recognition profile from Quercus variabilis and Vitis riparia FLS2 homologs against Ralstonia and Agrobacterium flagellin epitope. Finally, I introduced a dual PRR stack into potato cultivar ‘Atlantic’ to expand its recognition against bacterial 3-OH fatty acids and polymorphic flagellin. I demonstrated the transgenic potatoes are more resistant against soil-borne pathogen Ralstonia solanacearum, but not insect vectored Candidatus Liberibacter solanacearum.Collectively, this thesis provides insight into diversity of microbial feature recognition both within and across diverse plant families. This thesis also demonstrated that expanded bacterial flagellin recognition emerged independently and can be more common than previously appreciated. Finally, this work developed a general strategy to guide the rational engineering of PRRs and highlighted the efficacy of using PRR to control bacterial disease
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Role of post-translational modifications in regulating NADPH oxidase stability, reactive oxygen species production, and disease resistance in plants.
Get PDFAs the world’s population grows, we must seek to enhance food production. One hurdle in the way of achieving this goal is the devastating loss caused by plant pathogens and
pests. Plants are not completely defenseless against pathogenic attack and utilize a
multilayered immune system to prevent disease. Recognition of pathogens involves
dynamic signaling that activates multiple outputs with the overall goal of resistance. One
of these outputs is the generation of reactive oxygen species (ROS) during pathogen
recognition. In plants, production of ROS during infection is mediated by membrane
localized NADPH oxidases which are more commonly known as respiratory burst
oxidase homologs (RBOHs). Our understanding of activation of RBOHs is derived from
research on RBOHD from Arabidopsis thaliana. However, our knowledge of how
RBOHs are negatively regulated has been limited. In this dissertation, I studied a
pathway that negatively regulates stability of RBOH and ROS production in Arabidopsis
(Arabidopsis thaliana) and Tomato (Solanum lycopersicum). My results show that
RBOHD is negatively regulated through crosstalk between phosphorylation and
ubiquitination, which involves PIRE, an E3 ubiquitin ligase. Further analysis revealed
that PIRE is conserved in land plants as are the phosphorylation signaling residues in
RBOH homologs. Finally, utilizing gene editing tools, I show that this conserved
pathway can be exploited to enhance production of ROS and disease resistance in
tomato. Collectively, this dissertation work details the characterization of a novel
regulatory pathway for RBOHD in A. thaliana and highlights the potential to utilize this
knowledge as a foundation for translational work in tomato with the goal of enhancing
resistant to pathogens
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Characterizing the evolution and mechanisms of bacterial epitope perception and evasion of the plant immune systems
Get PDFBoth plants and animals are impacted by diverse biotic threats. To limit disease, plants use protein receptors to recognize and respond to pathogen protein epitopes or effectors. Pathogens have evolved strategies to circumvent recognition to proliferate and cause disease. Pathogens can also persist on non-hosts, leading to reservoir populations and subsequent costly outbreaks. Despite considerable resources focused on understanding the interactions between pathogens and model organisms, we lack considerable knowledge in how the natural diversity of bacterial pathogens, particularly Gram-positive actinobacteria, impact plant immune perception, colonization, and disease susceptibility. Using a combination of comparative genomics, genetics, and biochemistry, I leveraged natural genetic variation to understand the evolution of pathogen epitopes and elucidate a driver of pathogen evasion in a Gram-positive actinobacteria. Pathogen recognition and receptor signaling is crucial in host-pathogen interactions, but most studies use a single pathogen epitope and thus, the impact of multi-copy epitopes on pathogen outcomes is unknown. Through comparative genomics of thousands of plant-associated bacterial genomes, I characterized the naturally-evolved bacterial epitope landscape and their impact on pathogen outcomes. I revealed that natural variation was constrained yet experimentally testable and both epitope sequence and copy number variation altered pathogen-immune outcomes. Through genetic and biochemical analyses, I uncovered a mechanism for pathogen immune evasion, intrabacterial antagonism, where a non-immunogenic epitope blocks perception of immunogenic forms encoded in a single genome. One such intrabacterial antagonist, cold shock protein CspB, was conserved in actinobacteria including Clavibacter, a genus comprised of several crop pathogens including tomato, potato, wheat, and corn. As a non-model system, I developed a genetic toolkit to manipulate Clavibacter and test the role of CspB in blocking immune perception of one host species, tomato. While I was able to build and validate the genetic tools through deletion of several critical virulence genes, I was unable to generate a null mutant of the cspB gene in C. michiganensis, likely due to its high GC-content between 73-78%. Instead, I validated our intrabacterial antagonism model though a combination of biochemical assays and genetic transfer of cspB to another foliar pathogen of tomato, Pseudomonas syringae pathovar tomato DC3000. I show via bacterial titers that expression of antagonist cspB blocked perception of other native encoded immunogenic cold shock proteins in a receptor-dependent manner.
Collectively, I revealed a mechanism for immune evasion and showcased the importance of analyzing all epitope copies within a genome. I also provided evidence that Gram-positive actinobacteria interface with the plant immune system, a paradigm previously put into question due to insufficient evidence. Finally, I developed a genetic toolkit which may aid in characterizing other genotypic-phenotypic outcomes in the non-model bacterium. While my research has shown that we can leverage natural genetic variation to generate hypotheses and understand their impact on phenotypic outcomes, major questions remain in the evolution, functional biology, and signaling in plant-microbe interactions, which is addressed in the final chapter. Findings from the research questions posed may provide critical insights for subsequent advancements in bioengineering for disease resistance
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Investigating Candidatus Liberibacter solanacearum effectors role in solanaceous hosts
Get PDFEmerging vector borne diseases have significant ecological and economic impacts throughout the world. Because emergence of a disease is unpredictable, understanding of the pathogen manipulation of host and vector is critical to manage the disease. Successful bacterial pathogens modify host physiology, including defences, by secreting small proteins called effectors. Candidatus Liberibacter solanacearum (CLso) is an emerging, unculturable, slow growing, phloem-limited bacterium associated with multiple economically important diseases in Solanaceous crops. CLso is transmitted by the potato and tomato psyllid Bactericera cockerelli. Currently there is no genetic resistance to CLso in cultivated plant germplasm and increasing resistance to insecticides pose a high risk for disease epidemics. In this dissertation, I studied Lso effectors to gain insight into disease development. SEC secreted effectors were identified from four different CLso haplotypes. CLso effector subcellular localization, cell-to-cell movement and expression patterns were determined. Our results demonstrate that CLso differentially deploys suites effectors capable of targeting diverse eukaryotic subcellular compartments to modify its insect vector and tomato host. We also evaluated the ability of CLso effectors to suppress early markers of plant defense in response to perception of pathogen and vector features. Despite diverse subcellular localizations, the majority of tested effectors were unable to suppress host immune responses, indicating CLso relies on only a few effectors to suppress plant defense, unlike other well characterized plant pathogenic bacteria. Plants infected with bacterial vector-borne pathogens exhibit altered plant morphology and color. These alterations increase attractiveness of insect vectors and facilitate disease spread. To understand the role of CLso effectors in plant metabolism and architecture we deployed a PVX-mediated screening. We identified a suite of effectors that induce chlorosis and enhance viral symptoms in Nicotiana benthamiana. We also identify one effector able to induce systemic necrosis in tomato cv Micro-tom. Understanding the mechanisms that underlies CLso effectors induced necrosis will be an important step to generate resistant cultivars. Collectively, this study represents the first characterization of CLso effector repertoire and highlights key effectors that may shed light onto phloem disease biology
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The perception and evolution of microbial features from vector-borne plant pathogenic bacteria
Get PDFVector-borne bacterial plant diseases have a tremendous impact on agriculture around the world. These pathogens infect a myriad of crops, from solanaceous crops such as potato and tomato to tree crops such as citrus. These pathogens are unique because they are heavily reliant on their plant and insect hosts and as a result, have lost many essential biosynthetic pathways that free-living bacteria require. Because of the loss of these pathways, these bacteria are incredibly fastidious and many are still unculturable, making them difficult organisms to study in the laboratory. Our approach to studying these organisms is by examining the perception of microbial features by plants, which is one of the key mechanisms of plant immunity. In this dissertation, I examine two different sides of microbial feature perception in vector-borne bacterial plant pathosystems: a) the ability of plants to perceive a variety of microbial features from pathogens, and b) the perception of microbial features from vector-borne pathogens versus their free-living relatives. My results show that in Rutaceae, a large family of plants containing citrus Huanglongbing-susceptible citrus and their relatives, variation in the perception of microbial features occurs both within and between Rutaceae sub-tribes. Additionally, we were able to discover ten Rutaceae genotypes that could perceive a microbial feature from Candidatus Liberibacter asiaticus, the causative agent of citrus Huanglongbing. These ten genotypes may be useful to study further for resistance mechanisms that could be transferred to susceptible citrus. In another study, I also demonstrate that vector-borne bacterial plant pathogens tend to carry less copies of immunogenic microbial features in their genomes. The microbial features from vector-borne bacterial plant pathogens also tend to be non-immunogenic in plants. Interestingly, a microbial feature present in a citrus-Huanglongbing pathogen is perceived in solanaceous plants, while a feature present in a solanaceous plant-infecting pathogen is not perceived. I have also co-authored research on two different studies of Candidatus Liberibacter pathogens, which are vector-borne bacteria that cause devastating diseases on several plant hosts. One study is dedicated to understanding the evolution of bacteria in the Liberibacter genus, and another is dedicated to understanding the mechanisms behind SDE1, an effector from Candidatus Liberibacter asiaticus. Collectively, this dissertation work highlights a landscape of immune responses across vector-susceptible plants as well as characterizes microbial features from vector-borne pathogens to gain a broader understanding of how plants may or may not be able to defend themselves from these pathogens
Woody Host-Specific Type III Effector HopBL2 Is Essential for Pseudomonas savastanoi Virulence and Associates With Plasmodesmata
No full textThe type III secretion system in Pseudomonas syringae complex pathogens delivers type III effectors (T3Es) into plant cells to manipulate host processes, enhance survival, and promote disease. While substantial research has focused on herbaceous pathogens, T3Es in strains infecting woody hosts are less understood. This study investigates the HopBL family of effectors in Pseudomonas savastanoi, a pathogen of woody plants. HopBL1 and HopBL2, core effectors in P. savastanoi, are restricted to phylogroup 3 strains of the P. syringae complex, all isolated from woody hosts. Phylogenetic analysis suggests recent horizontal acquisition of these effectors across multiple P. syringae pathovars, integrated into genomic islands flanked by mobile genetic elements. Structural analysis shows that both HopBL effectors contain SUMO protease and DNA-binding domains, with HopBL1 also possessing an ethylene-responsive motif, all characteristic of XopD from Xanthomonas spp. Despite low sequence identity, HopBL effectors exhibit structural similarity to XopD, with HopBL1 showing greater resemblance, particularly in the arrangement of these domains. Functional assays in olive and oleander revealed strain-specific contributions of HopBL1 and HopBL2 to virulence. In oleander, the natural host of P. savastanoi pv. nerii, mutation of either effector gene resulted in reduced symptom development. We show that HopBL2 localised predominantly to subnuclear foci and associated with plasmodesmata, with partial overlap observed along microtubules, suggesting a potential role in cytoskeleton manipulation. These findings underscore the importance of T3Es unique to P. syringae strains infecting woody hosts and their adaptation to modulate host cellular structures to promote disease.This research was supported by project grant PID2020-115177RB-C21 from the Spanish Ministry of Science and Innovation (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033/, the European Regional Development Fund (ERDF)–‘A way to make Europe’, and the National Institutes of Health (NIH) under (Grant 2R35GM136402)Peer reviewe
Pathogen Specialization
Get PDFThe ability to infect new hosts can drive the evolution and specialization of secreted pathogen proteins
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Purification and Detection of Ubiquitinated Plant Proteins Using Tandem Ubiquitin Binding Entities
Get PDFThe timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasome or removal of modifications by deubiquitination enzymes (DUBs). Tandem ubiquitin binding entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBs. Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEs after transient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitination of a target protein. This methodology provides an effective method for identification of ubiquitin ligase substrates and can be coupled with TUBEs targeting specific ubiquitination linkages
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