Performing Department
College of Nat & Agr Sciences
Non Technical Summary
Traditionally, it was thought that the bacteria must rapidly reprogram themselves for the plant lifestyle, but we will test the hypothesis that bacteria also sense the fluid dynamics that occur during the transmission process within the insect to not only initiate gene expression cascades that enable detachment from the foregut, but also turn on genes that are important for initial plant colonization prior to deposition in the plant. Thus, the bacterial sensing of fluid hydrodynamic forces experienced in the insect prime the bacteria so they are pre-programmed for a plant lifestyle as they are leaving the insect head. Our goal is to identify components of the X. fastidiosa/insect/plant interface that can be used as targets to disrupt vector transmission and readiness for the transition to the plant environment. Simultaneously, we will learn a vast amount about the biology of the X. fastidiosa/insect/plant interface. In the next phase of this work, we will focus on the insect component of this tri-trophic interaction and test select X. fastidiosa mutants for their acquisition and transmission phenotypes using the blue-green sharpshooter and V. vinifera as our model systems. In the Americas, X. fastidiosa has caused significant damage and remains a major threat to agricultural crops and ecological and ornamental landscapes. In terms of agricultural crops this includes not only grapes but also citrus (Citrus Variegated Chlorosis), coffee (Coffee leaf scorch), stone fruits (Phony peach) among many others. In addition, Europe was historically considered to be free of X. fastidiosa, but the bacterium was recently detected in Italy. In 2013, olive trees in the Apulia region of Southern Italy began exhibiting leaf scorch symptoms that were later confirmed to be caused by X. fastidiosa. Since then, thousands of olive trees have died from Olive Quick Decline, and X. fastidiosa has been detected in various plants species in France, Spain, and Portugal. Thus, the findings of this work may have direct implications in identifying vulnerabilities in the tri-trophic pathogen/insect/plant interface that can be leveraged for disease control for other very important diseases caused by X. fastidiosa, such as Olive Quick Decline and Citrus Variegated Chlorosis.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Goals / Objectives
The overall goal of this project is to determine if plant pathogenic bacteria sense environmental mechanics (fluid flow) to drive adaptive behaviors that mediate the transition from their insect vector to the plant environment. Historically, research on how bacteria sense their environment has primarily focused on the role of chemical signals in bacterial behavior. However, recent advances in bacterial sensing have incorporated mechanistic studies on the biophysical parameters that govern bacterial behaviors. These studies have demonstrated that hydrodynamic fluid flow and rheology (elasticity and viscosity of the bacterial extracellular matrix) interfaces with the bacterial cell surface to affect changes in bacterial behaviors. Insect-vectored, bacterial plant pathogens are uniquely poised as an informative system to investigate the effect fluid dynamics has on bacterial gene expression and subsequent bacterial behaviors. This is due to the exposure of insect-transmitted bacteria to high and sometimes extremely high fluid shear stress during the ingestion and egestion process that occurs in the tube-like mouthparts of insects during the feeding process. We have created fluid dynamic simulations that model these flow rates within the 3-dimensional space of an insect foregut, thus, we can approximate similar flow rates in vitro. Additionally, once inside the plant and, particularly the xylem, vascular-dwelling bacteria are exposed to high shear stress in the xylem sap stream during transpiration. In this project, we will focus on Xylella fastidiosa and the forces it is exposed to in the foregut of its sharpshooter vectors. For the purposes of the proposed project, we will focus on flow rates and drag forces that occur in the precibarium because X. fastidiosa is exposed to a range of drag forces within this region that include some of the highest drag forces within the foregut, particularly in the epipharnyx portion of the precibarium. Moreover, the precibarium is a critical platform to test our hypotheses outlined in this proposal because the precibarium is a key exit point for the bacterium during egestion as it is deposited into the plant tissue from the insect's mouthparts. Overall goals of the project: The short-term goals of this project are to assess the transcriptional changes that occur in response to hydrodynamic flow. The medium-term goals are to mechanistically test genes and their gene products as important contributors to adaptation to the plant environment prior to deposition in the plant by the insect. The long-term future goals are to identify vulnerabilities at the interface of this multi-trophic system that can be targeted for disrupting pathogen transmission and, ultimately, disease control.
Project Methods
Objective 1. Functional characterization of X. fastidiosa's response to hydrodynamic shear force. Our preliminary work identified several classes of putative virulence factors that were induced under one flow condition. The purpose of this objective will be to expand on this work to test a set of flow conditions that fall within the spectrum of what our fluid simulation model predicted to occur within the precibarium of the insect. We will also functionally test if the set of flow-induced genes we have generated thus far are linked to in planta virulence by generating deletion mutants in individual flow-induced genes and testing them in disease and plant colonization bioassays using V. vinifera grapevines. We will continue to add to this list of testable genes as we progress through the project and perform additional global transcriptomic experiments. Using the initial flow rate parameters we have used in the past as a foundation, we will integrate additional flow rates that impose drag forces relevant to those predicted in our hydrodynamic fluid model. To prepare seed inoculum for the flow cells, X. fastidiosa will be propagated in a defined medium (XFM) amended with plant carbohydrates (pectin and carboxy-methyl cellulose) to approximate the xylem sap of V. vinifera. The flow cells will subsequently be seeded with 103 cfu/ml of bacteria. Cells will be propagated in the chamber initially under stagnant conditions (0 ml/min) to allow for cell attachment and biofilm establishment. The bacteria will be imaged using a camera connected to an inverted microscope (Axio Observer 5, Zeiss) housed in the Roper laboratory. Matlab (Mathworks, Natick, MA) will be used to analyze the images. We will then collect bacterial cells and analyze X. fastidiosa gene expression under the different flow rates tested. Specifically, the transcriptional profiles will be analyzed for significant differential gene expression patterns across all treatments (no flow vs. the different rates of flow outlined above) for the attached cells. We will also compare the transcriptomes of attached vs. detached cells for each flow condition using these same bioinformatic tools. Each flow condition will be repeated three times for all strains being tested in this objective and in objectives 2 and 3.In this objective we will also functionally test the genes that were significantly up-regulated by flow by constructing deletion mutants in the respective genes and performing in planta disease bioassays and spatial colonization studies in V. vinifera grapevines. This will test our hypothesis that genes that are induced by flow are important factors for life inside the plant host. Certified pathogen-tested V. vinifera grapevines cv. Cabernet sauvignon will be reared from cuttings obtained from Foundation Plant Services (Univ of CA, Davis). To assess the impacts of the resulting mutants in in planta virulence and systemic plant colonization, we will mechanically inoculate plants using the needle inoculation method routinely used in the Roper laboratory to inoculate grapevines. Each vine plant will be inoculated twice with a 20 μl drop of a 108 cfu/ml suspension of either wild type X. fastidiosa or an individual deletion mutant. We will inoculate 15 plants/strain and repeat each experiment 3 times. Plants inoculated with 1X phosphate buffered saline (PBS) will be used as negative controls. All plants will be rated on an established PD severity scale of 0-5 with 0=healthy up to 5=dead. Data will be analyzed by quantifying the area under the disease progress curve, survival index and a generalized estimated equations (GEE) model. We will also assess the X. fastidiosa titer in the plants by isolating X. fastidiosa from the petioles at the point of inoculation (POI, local infection) and 20 nodes above the point of inoculation (systemic infection) to determine the ability of the deletion mutants to colonize and move systemically within the xylem as compared to wild type X. fastidiosa that readily achieves systemic colonization of the grapevine xylem. All plants will be inoculated as described for the disease assessment bioassays described above. Local colonization will be determined by harvesting the petiole closest to the POI. Systemic colonization will be determined by harvesting the petiole 20 nodes above the POI. Plants inoculated with 1X PBS will be used as negative controls.Objective 2. Examine the role of Type IV pili as mechanosensitive proteins that control flow-induced gene expression. Here we will test our hypothesis that X. fastidiosa senses changes in fluid dynamics via its Type IV pili. Type IV pili are dynamic surface appendages that rapidly polymerize and depolymerize to enable movement along a surface via successive pilus extension, tip attachment, and retraction. This surface-based motility is referred to as twitching- based motility. Phenotypically, Type IV pili initiate up-stream migration in response to fluid flow in X. fastidiosa. Our preliminary data indicates that major transcriptional reprogramming occurs under flow conditions as compared to no flow conditions. To test our hypothesis that mechanosensing of flow is hard wired into the Type IV pili system, we will profile the transcriptomes of three Type IV pili mutants (and their cognate complemented strains) and compare them to the wild type parental strain under the different flow regimes in both flow cells (flat and 3D printed) we outlined in Obj. 1All mutants have already been constructed as part of the SEED project in the Roper lab. In the proposed project, these will be complemented with their cognate wild type gene as described in Obj. 1. The mutants and their complements will be compared to the wild type parent strain in flow cells using the same flow parameters we utilize in Obj. 1. Each flow treatment will have a paired no-flow treatment for RNASeq analysisObjective 3. Examine regulatory responses linked to mechanosensing of flow. Our preliminary data indicate that two response regulators were up-regulated under flow suggesting that these are cogs in the network that controls mechanosensing for X. fastidiosa. In this objective, we will test the hypothesis that these transcriptional regulators are a key mechanistic part of how X. fastidiosa responds to changes in its hydrodynamic environment to proactively prepare itself for deposition into the plant. Initially we will generate mutants by site directed mutagenesis as described in Obj. 1. and phenotype the mutants in planta to determine if these deletions have an impact on virulence by testing it in plant disease and systemic colonization bioassays in grapevines as described in Obj.1. We will explore the regulons of these mutants under flow and no flow conditions by integrating them into the flow cell studies outlined in Obj. 1 using the RNASeq methods also described in Obj. 1. All mutants will be complemented and the complemented strains will be used as comparative treatments in all assays described here.