VIRULENCE PROTEINS ON THE XYLELLA FASTIDIOSA CELL SURFACE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1009165
• Project Start Date: Feb 3, 2016
• Project End Date: Jun 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Xylella fastidiosa is the causative agent of numerous plant diseases relevant to the California agricultural economy, including Pierce's Disease of grapevines. Successful colonization of the host plant requires the presence of specific proteins on the bacterial cell surface. One important group is the autotransporter (AT?1) proteins. AT-1 proteins are dedicated to the secretion of a single specific polypeptide (the passenger domain) across the outer membrane and frequently contribute to the virulence of Gram-negative pathogens. Previously, we generated knock-out mutations in two AT-1 proteins XatA and XatB and showed that their absence impacts X. fastidiosa virulence, making these proteins attractive targets for control strategies. Completion of the objectives outlined in this proposal will provide important insights into XatA and XatB regulation and function under laboratory conditions. The results from this analysis will then be used to determine which mutations will be subjected to further analysis in the greenhouse. As soon as we have identified promising mutations, we will consult with our collaborators at UC Davis and UC Riverside and discuss how these results might be used to develop an effective control method for Pierce's Disease.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1131	1100	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1100 - Bacteriology;

Keywords

xylella fastidiosa

pierce's disease

temecula grapevine diseases

autotransporter proteins

adhesins

type v secretion

Goals / Objectives
In many Gram-negative bacteria, the synthesis and function of afimbrial adhesins is tightly regulated and we predict that the afimbrial adhesins of X. fastidiosa will be subjected to a similar type of regulation. Our strategy will be to determine the level of regulation (transcriptional, translational, localization, extracellular processing), identify the cellular factors responsible for this regulation, and generate mutations in xatA and xatB that disrupt their regulation and function. The long-term goal will be to introduce these mutants into plants to determine their impact on adherence, colonization and migration within the xylem, and on symptom production in susceptible host plants.Objectives of the Proposed Research:(1) Identify the molecular mechanisms responsible for the regulation and function of the X. fastidiosa afimbrial adhesins, XatA and XatB.(2) Generate mutations that impact the structure and function of XatA and XatB.(3) Examine how mutations generated in Objective 2 impact the ability of X. fastidiosa to colonization both susceptible (grapevines, Vitus vinifera) and tolerant (Arabidopsis thaliana) hosts.

Project Methods
Objective 1: Identify the molecular mechanisms responsible for the regulation and function of the X. fastidiosa afimbrial adhesins, XatA and XatB.(1a) Are the genes encoding XatA and XatB regulated at the transcriptional level? To address this question, we will conduct qRT-PCR analysis on RNA isolated from X. fastidiosa cells grown under conditions that favor planktonic growth (PIM6D-P) versus biofilm formation (PIM6D-B). We will also examine RNA isolated from two X fastidiosa mutants: the RpfF mutant, which is defective in quorum sensing molecule DSF and the CgsA mutant, which is defective in intracellular signaling by cyclic di-GMP. Both RpfF and CgsA mutants have a profound impact of X. fastidiosa pathogenicity (2). If transcriptional regulation is observed, the next step will be to identify the regulatory element upstream of the xatA (or xatB) promoter responsible for this regulation. Our strategy will be to generate a set of stacked deletions in the xatA regulatory region and then place the resulting DNA fragments upstream of a promoterless lucerifase gene. [We have previously shown that lucerifase can be used as a reporter gene in X. fastidiosa (4).] The results from these experiments should establish whether xatA and/or xatB are regulated at the transcriptional level under a specific set of growth conditions or by the two signaling pathways.(1b) Is the release of the passenger domain regulated? For this analysis, wild-type X. fastidiosa cells will be harvested from PIM6D-P and PIM6D-B. We will also prepare cultures of the RpfF and CsgA mutants. The cultures will then be subjected to protocols designed to generate two fractions (5, 7). The crude membrane fraction will contain both inner and outer membrane proteins, whereas the supernatant fraction will include both outer membrane vesicles and extracellular proteins. Western analysis will then be performed using antibodies previously generated against the passenger domains of XatA and XatB to determine if the release of the passenger domain is regulated under laboratory conditions. If these preliminary experiments are successful, we plan to use the antibodies to determine if the XatA and XatB passenger domains are present in the xylem fluid of infected grapevines using published protocols (1), and if so, does the amount change as the infection progresses. By the conclusion of this series of experiments, we should have a better understanding of how growth and xylem environment influence the maturation of these two adhesins. Objective 2: Generate mutations that impact the structure and function of XatA and XatB.At first glance, it would seem that release of the passenger domain from an adhesin autotransporter would be counterproductive since it would result in less attachment. However, if properly regulated, low-level cleavage could promote bacterial migration and therefore allow the bacterium to colonize new surfaces in the host. Moreover, some adhesins such as the E. coli Ag43 and AIDA autotransporters are multifunctional proteins and the release of their passenger domains increases their alternative functions, which include autoprotease activity, autoaggregation, and biofilm formation (3, 6). Therefore, to increase our understanding of XatA and XatB function, we plan to map the functional regions of the proteins. The first step will be to generate mutations in xatA that interfere with the release of the XatA passenger domain from the cell surface. The preliminary work will be carried out by two undergraduate researchers and will focus on development of the methods necessary to generate and screen for the desired mutations. Once the mutant strains have been generated, their phenotypic properties will be examined under laboratory conditions using previously published protocols (5). Objective 3: Examine how structure/function mutations in xatA and xatB impact the ability of X. fastidiosa to colonization both susceptible (grapevines, Vitus vinifera) and tolerant (Arabidopsis thaliana) hosts.We will then examine how the mutations impact the interactions with a susceptible and tolerant host plant. The key will be to take full advantage of the expertise of other members of the PD research community. To successfully complete this objective, we will consult with Dr. Kirkpatrick (Department of Plant Pathology at UC Davis) and Dr. Caroline Roper, who have extensive experience in studying the interactions between plant pathogens and their hosts. Our previous collaborations with these laboratories has been productive and the expertise provided by these PD researchers will be critical to the successful completion of Objectives 3. Completion of these experiments should identify the regions of XatA and XatB that are important for their processing and localization and which regions within these proteins are important for their colonization of the plant xylem.LITERATURE CITED:1. Aguero, C., S. Uratsu, C. Greve, A. Powell, J. Labavitch, C. Meredith, and A. Dandekar. 2005. Evaluation of tolerance to Pierce's disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene. MOL PLANT PATHOL 6:43-51.2. Chatterjee, S., N. Killiny, R. P. Almeida, and S. E. Lindow. 2010. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission. Mol Plant Microbe Interact 23:1356-1363.3. Girard, V., and M. Mourez. 2006. Adhesion mediated by autotransporters of Gram-negative bacteria: Structural and functional features. Research in Microbiology 157:407-416.4. Igo, M. M. 2007. Presented at the Pierce's Disease Research Symposium San Diego, CA.5. Matsumoto, A., S. L. Huston, N. Killiny, and M. M. Igo. 2012. XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temecula1. MicrobiologyOpen.6. van der Woude, M. W., and I. R. Henderson. 2008. Regulation and function of Ag43 (flu). Annu Rev Microbiol 62:153-169.7. Voegel, T. M., J. G. Warren, A. Matsumoto, M. M. Igo, and B. C. Kirkpatrick. 2010. Localisation and Characterization of Xylella fastidiosa Hemagglutinin Adhesins. Microbiology.

Progress 02/03/16 to 06/30/19

Outputs
Target Audience:The target audience of this project is scientific researchers interested in diseases caused by the xylem-limited, plant pathogen Xylella fastidiosa. X. fastidiosa is the causative agent of numerous plant diseases relevant to the California and global agricultural economy, including diseases of grape, almond, citrus, olive, alfalfa, peach, and coffee. X. fastidiosa has a wide plant host range and can be introduced into susceptible plants by insects that feed on the plant vascular system. Historically, X. fastidiosa has been geographically limited to the Americas. However, the findings of X. fastidiosa in Italian olive groves in 2013 and on ornamental plants in France underscore its importance as a re-emerging global plant pathogen. My laboratory has been examining the role of specific proteins associated with the X. fastidiosa cell surface in its virulence. We have identified two cell surface proteins that help X. fastidiosa adhere to solid surfaces and influences its ability to colonize and to cause disease in grapevines. We also identified two secreted proteins that may affect different stages of the infection cycle. Finally, we examined the role of these proteins in the tolerance of X. fastidiosa to oxidative stress. Many of the greenhouse experiments were carried out by our collaborator Dr. Caroline Roper and her colleagues at the University of California, Riverside. The ultimate goal of this project is to develop ways to interfere with the function of these cell surface proteins and thereby prevent X. fastidiosa from colonizing the host xylem, a critical step in the initiation of the disease state. The results of oxidative stress experiments were published in the journal Molecular Plant Pathology (Wang et al., 2017). We also described the importance of X. fastidiosa cell surface proteins in virulence in a review article (Zaini et al., 2015). Finally, my laboratory has presented on these results in seminars on campus and Dr. Roper's group has presented these findings at UC Riverside. These formats have allowed us to disseminate our latest findings to scientists, cooperative extension agents, and other researchers interested in discovering a possible treatment for this fatal disease of grapevines. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Sherry Huston was a Staff Research Associate in the laboratory and was responsible for generating the mutant strains used in this study. Dr. Huston joined our laboratory in Fall 2009 and was familiar with the methods used for studying X. fastidiosa both under laboratory conditions and in grapevines conducted most of the experiments described in this report. Dr. Huston also supervised the work of Corinn Small, a Microbiology undergraduate who helped characterize the mutants under laboratory conditions at UC Davis. The grapevine experiments were performed by Claudia Castro, a plant pathology graduate student in the laboratory of Dr. Caroline Roper at UC Riverside. How have the results been disseminated to communities of interest?The results of oxidative stress experiments were published in the journal Molecular Plant Pathology (Wang et al., 2017). We also described the importance of X. fastidiosa cell surface proteins in virulence in a review article (Zaini et al., 2015). Finally, my laboratory has presented on these results in seminars on campus and Dr. Roper's group has presented these findings at UC Riverside. These formats have allowed us to disseminate our latest findings to scientists, cooperative extension agents, and other researchers interested in discovering a possible treatment for this fatal disease of grapevines. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Xylella fastidiosa has a wide host range and is found in the xylem of many plants. In some plants, X. fastidiosa is able to grow without causing serious damage to the host. However, in susceptible plants, its growth within the xylem can clog the xylem vesicles and prevent the movement of water within the plant. This results in scorched leaves, shriveled fruit and the eventual death of the plant. X. fastidiosa is spread from infected plants to uninfected plants by xylem-feeding insects, such as sharpshooters and spittlebugs. Therefore, in order to disrupt the X. fastidiosa infectious cycle, it is important to understand how this pathogen is able to colonize and interact with the xylem tissue of susceptible plants and with the foregut of insect vectors. This project resulted in the identification of proteins that allow X. fastidiosa to adhere to the host cell surfaces and to escape any host defense mechanisms. As our experimental system, we have chosen to study strains of X. fastidiosa that cause Pierce's Disease of grapevines (PD). Our previous work had established the importance of a group of cell surface proteins known as the AT-1 autotransporters in this process. AT-1 systems are dedicated to the secretion of a single specific polypeptide, the passenger domain, across the outer membrane. Because they are not present on the surface of animal and plant cells, these proteins have been identified as rational targets for the design of novel vaccines and control strategies. Activities: The passenger domains of X. fastidiosa AT-1 autotransporters can be divided into two categories: adhesin proteins and enzymes. During the previous review period, we characterized two genes (xatA and xatB) that were predicted to be involved in adhesion. Our examination of the properties of strains carrying mutations in these genes revealed that xatA and xatB mutants were unable to adhere to glass and to form a mature biofilm under laboratory conditions. Moreover, the strain carrying the mutation in xatA did not produce Pierce's Disease symptoms when infected into grapevines. The properties of the xatA mutant suggest that the XatA protein plays an important role in the initial colonization of the host plant by the bacteria. A complete description of this study can be found in our manuscript entitled "XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temecula1" (Matsumoto et al. 2012). During the current review period, we characterized mutations in three genes predicted to encode AT-1 extracellular proteases. The production of extracellular proteases is thought to be important for allowing the bacterium to colonize and move between vessels in the grapevine xylem. Interestingly, we discovered that deletion of a single protease does not always result in the same phenotype. For example, one of the protease mutants exhibited a hyper-virulent phenotype in grapevines (PD symptoms appeared earlier than observed in a wild type infection). In contrast, the other protease mutant caused very few symptoms and the onset of symptoms was delayed when compared to wild type. Based on the properties of the mutant strains, it seems likely that these two AT-1 extracellular proteases are important for different stages in the infection process. Finally, we generated a strain containing mutations in all three proteases. This mutant strain is defective in biofilm formation and autoaggregation under laboratory conditions. Studies in grapevines revealed that although the strain missing of all three proteases could be found in the xylem, the infected grapevines do not exhibit scorched leaves or other symptoms of Pierce's Disease. This result supports our hypothesis that the AT-1 proteases could serve as rational targets for the design of novel vaccines and control strategies.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The focus of this project is the bacterial pathogen Xylella fastidiosa, which is the causative agent responsible for a number of plant diseases important to the California agricultural economy. The target audience is scientists and cooperative extension agents increased in controlling the spread of these diseases and undergraduate researchers interested in understanding the role of basic research in fields important to the Agricultural Experimental Station. During the period under review, I focused on the aspects of the project that could be conducted by undergraduate researchers. One mechanism that is particularly promising is the Course-based Undergraduate Research Experiences (CUREs) associated with the freshman seminar series. Changes/Problems:Due to changes in the level of external funding for this project, the experiments are much simpler than originally anticipated. Therefore, although the goals are the same, the experiments have been redesigned to lower the cost and to make it easier for them to be carried out by undergraduates. What opportunities for training and professional development has the project provided?My laboratory is very small and the work is primarily carried out by myself and undergraduates. The students are trained in a variety of laboratory techniques. They are also expected to read scientific manuscripts related to their project and to analyze data. Finally, they are encouraged to present their preliminary results at informal meetings in order to become comfortable answering questions about their work. How have the results been disseminated to communities of interest?The main way the results of our experiments are disseminated is through informal seminars at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?The major focus of the next review period will be to finish up the experiments necessary to publish our study of Xf cell surface and secreted proteins.

Impacts
What was accomplished under these goals? The goal of this project is to identify virulence factors that allow Xylella fastidiosa to adhere to the host cell surfaces and to escape any host defense mechanisms. As our experimental system, we have chosen to study strains of X. fastidiosa that cause Pierce's Disease of grapevines (PD). One of the major goals of this project is to understand how a category of cell surface proteins known as afimbrial adhesins influence of the ability of X. fastidiosa to colonize the plant xylem. In previous work, we established that X. fastidiosa missing the afimbrial adhesin XatA or XatB are not able to colonize the host plant. During the period under review, we primarily focused on Objective 2: Generate mutations that impact the structure and function of XatA and XatB. Our strategy has been to mutagenize plasmids carrying the genes encoding these proteins. The initial mutagenesis protocols were relative simple and were easily carried out by an undergraduate. Unfortunately, after some preliminary screening, none of the mutated plasmids carried mutations of interest. In Spring 2017, our collaborator at UC Riverside initiated a series of greenhouse experiments to examine the impact of selected mutations on X. fastidiosa virulence. These experiments were continued in Spring 2018 to generate a more useful dataset. We are still collecting and analyzing the data generated from these experiments. Our goal is to prepare a manuscript describing the results from this analysis and submit it for publication during the next review period.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience of this project is scientists interested in diseases caused by the bacterial plant pathogen Xylella fastidiosa and undergraduate researchers interested in understanding the role of basic research in fields important to the Agricultural Experimental Station. Due to changes in the funding of this project, I focused on the low cost aspects of the project and sent materials to my collaborator at UC Riverside for the greenhouse studies. I also explored different strategies for introducing undergraduates to research. One mechanism that is particularly promising is the Course-based Undergraduate Research Experiences (CUREs) associated with the freshman seminar series. Although outside the review period, I worked with other members of my department to develop a CURE focusing on basic microbiology, which was offered in Winter 2018. Changes/Problems:Due to changes in the level of external funding for this project, the experiments are much simpler than originally anticipated. Therefore, although the goals are the same, the experiments have been redesigned to lower the cost and to make it easier for them to be carried out by undergraduates. What opportunities for training and professional development has the project provided?My laboratory is very small and the work is primarily carried out by myself and undergraduates. The students are trained in a variety of laboratory techniques. They are also expected to read scientific manuscripts related to their project and to analyze data. Finally, they are encouraged to present their preliminary results at informal meetings in order to become comfortable answering questions about their work. How have the results been disseminated to communities of interest?The main way the results of our experiments are disseminated is through informal seminars at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?The major focus of the next review period will be to finish up the experiments necessary to publish our study of Xf cell surface and secreted proteins. This may involve additional greenhouse experiments.

Impacts
What was accomplished under these goals? The goal of this project is to identify virulence factors that allow Xylella fastidiosa to adhere to the host cell surfaces and to escape any host defense mechanisms. As our experimental system, we have chosen to study strains of X. fastidiosa that cause Pierce's Disease of grapevines (PD). One of the major goals of this project is to understand how a category of cell surface proteins known as afimbrial adhesins influence of the ability of X. fastidiosa to colonize the plant xylem. In previous work, we established that X. fastidiosa missing the afimbrial adhesin XatA or XatB are not able to colonize the host plant. During the period under review, we primarily focused on Objective 2: Generate mutations that impact the structure and function of XatA and XatB. Our strategy was to mutagenize plasmids carrying the genes encoding these proteins. The initial mutagenesis protocols were relative simple and could be easily carried out by an undergraduate. As the undergraduates have gained more experience, they are beginning to use more sophisticated molecular methods. After some preliminary screening, the mutated plasmids will be sent out for sequencing. In Spring 2017, our collaborator at UC Riverside initiated a series of greenhouse experiments to examine the impact of selected mutations on X. fastidiosa virulence. We are still collecting and analyzing the data generated from these experiments. A major focus of the next review period will be to prepare a manuscript describing the results from this analysis.

Publications

Progress 02/03/16 to 09/30/16

Outputs
Target Audience:The target audience of this project is scientists interested in diseases caused by the bacterial plant pathogen Xf. Our basic research on this bacterium provides important information about the physiology of this microorganism. Understanding how the proteins required for Xf to colonize and thrive in the xylem of infected plants has important implications for scientists, cooperative extension agents, and other researchers interested in discovering a possible treatment for this fatal disease of grapevines. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The work in my laboratory is primarily carried out by myself and undergraduates. The students are trained in a variety of laboratory techniques. They are also expected to read scientific manuscripts related to their project and to analyze data. Finally, they are encouraged to present their preliminary results at group meetings in order to become comfortable answering questions about their work. Once they have made significant progress on their project, they are encouraged to present their work at the Undergraduate Research Conference on campus. How have the results been disseminated to communities of interest?During the period under review, we published the results from the oxidative stress study in Molecular Plant Pathology, a high visibility journal in this field. Due to the time required for the greenhouse experiments, we did not have sufficient data to present our work at the annual Pierce's Disease Conference. However, the work has been presented at UC Riverside by my collaborator Dr. Caroline Roper and her graduate student Claudia Castro and by myself at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?The major focus of the next review period will be to finish up the experiments necessary to publish our study of Xf cell surface and secreted proteins. This may involve additional greenhouse experiments.

Impacts
What was accomplished under these goals? The bacterium X. fastidiosa (Xf) is responsible for diseases in a number of economically important plants, including citrus, maple, alfalfa, peach, plum, sycamore, elm, almond, coffee and oleander. Historically, Xf has been geographically limited to the Americas. However, the recent findings of Xf in olive groves in Italy and on ornamental plants in France underscore its importance as a re-emerging global plant pathogen. Our research has focused on identifying genes that impact Xf virulence. We have focused on two categories of genes. The first category consists of genes involved in the oxidative stress response. During the period under review, we completed this analysis in collaboration with Dr. Caroline Roper (UC Riverside). The manuscript describing this work was published in August 2016 (Wang et al., 2016. Mol. Plant Pathol. doi:10.1111/mpp.12456). The second category consists of genes that encode cell surface proteins and secreted proteins. During the period under review, we continued our laboratory analysis of mutations in these genes and selected the most promising mutations for further analysis. In Spring 2016, we initiated a series of greenhouse experiments to examine the impact of selected mutations on Xf virulence. We are still collecting and analyzing the data generated from these experiments. A major focus of the next review period will be to prepare a manuscript describing the results from this analysis.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, P., Lee, Y., Igo, M. M. and Roper, M. C. (2016), Tolerance to oxidative stress is required for maximal xylem colonization by the xylem-limited bacterial phytopathogen, Xylella fastidiosa. Molecular Plant Pathology. doi:10.1111/mpp.12456