STRUCTURAL BIOLOGY OF CITRUS CANKER, A BACTERIAL INFECTION DISEASE OF CITRUS PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1011447
• Project No.: CA-R-BCH-5051-H
• Project Start Date: Nov 22, 2016
• Project End Date: Sep 30, 2021

Project Director
Fan, L.

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
Biochemistry

Non Technical Summary
Citrus canker is a widespread bacterial disease in citrus-growing regions throughout the world. Infected trees exhibit gradual decline in health and fruit production until they eventually succumb to the disease. Currently there are no chemical or microbiological agents to control the disease. The disease is caused by Xanthomonas phytopathogens. The most widely spread and severe strain Xanthomonas citri subsp. citri (Xac) attacks all citrus varieties. However, the molecular mechanisms of Xac-citrus infection are not well understood. The Xac genome sequence has recently become available, opening the door for scientists worldwide to study Xac-citrus infection at the genomic level. Studies from functional genetic analysis and other methods have found that over 90 Xac genes are directly involved in citrus canker, providing a good model system for genome-wide studies of plant and pathogen interactions in general. These 90 genes are grouped into 15 categories: amino acid biosynthesis (3 proteins), biosynthesis of small molecules (6 proteins), cell structure (3 proteins), cellular process (1 protein), conserved hypothetical protein (19 proteins), DNA metabolism (5 proteins), energy metabolism (5 proteins), hypothetical protein (13 proteins), intermediary metabolism (3 proteins), macromolecule metabolism (5 proteins), mobile genetic element (3 proteins), pathogenicity, virulence, and adaptation (9 proteins), regulatory function (7 proteins), RNA metabolism (1 protein, 3 RNA molecules), and transport and binding proteins (8 proteins). We aim to determine the structures of these important Xac proteins by X-ray crystallography. This proposed project will lead to the determination of crystal structures of Xac proteins involved in multiple pathways important for Xac-citrus infection. The results will help understanding the biochemical functions of these Xac proteins, and therefore yield direct insights into the mechanism of Xac-citrus infection. The success of this project will provide many protein targets for the PI to follow up in order to completely understand the specific roles of these Xac proteins in citrus canker and the molecular mechanism of Xanthomonas-plant host interactions in general. Therefore, the proposed project will build a firm foundation for a lifetime long research integrated with education and training for undergraduate and graduate students. The ultimate goal of this research program is to develop structure-based chemical inhibitors which can effectively control the widespread citrus canker with acceptable effects on the environment.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0999	1000	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

citrus canker

xanthomonas

structural biology

Goals / Objectives
The ultimate goal of this research program is to understand the molecular mechanism of Xanthomonas-citrus infection and to develop structure-based chemical inhibitors which can effectively control the widespread citrus canker with acceptable effects on the environment. The specific objectives for this proposal are the following:1. To clone and express in E. coli Xac proteins encoded by the genes important for Xac-citrus infection as identified by functional genomic analysis and other methods including reported by literatures;2. To purify recombinant Xac proteins in large scale for crystallization;3. To identify crystallization conditions for targeted proteins by high throughput crystallization screenings;4. To determine the X-ray crystal structures of those proteins that will be crystallized well for high resolution data collection.

Project Methods
1. Clone and express in E. coli Xac proteins encoded by the genes important for Xac-citrus infection. Over 90 Xac protein-coding genes from different pathways are implicated in Xac-citrus infection. These genes will be cloned and tested for expression in E.coli. In order to facilitate purification, these Xac proteins will be expressed as His-tagged (or other tagged) recombinant proteins in E. coli.2. Purify recombinant Xac proteins in large scale for crystallization. The Xac proteins expressed in E. coli will be purified by affinity and conventional chromatographic methods using AKTA FPLC system. In order to ensure the success of the proposed studies, we will focus mainly on Xac proteins that are soluble after they are expressed in E. coli.3. Identify crystallization conditions for targeted proteins by high throughput crystallization screenings. State-of-the-art high throughput instruments and technology will be applied to prepare protein crystals using purified Xac recombinant proteins. Commercial screening kits will be used to screen several hundred or over thousand conditions for each target protein. The crystal initiation and growth will be monitored by computer-controlled imaging system 24/7. Initial crystals will be manually refined to get crystals for X-ray diffraction data collection.4. Determine the X-ray crystal structures of those proteins that will be crystallized well for high resolution data collection. Proteins crystallized in high quality will be used for data collection in house or at synchrotron beamlines to determine high resolution X-ray crystal structure. We expect to determine these crystal structures by standard Molecular Replacement method if a structural homolog is available in the protein data bank (PDB) or by MAD phasing using Se-Met substituted protein as we previously demonstrated in determining the structures of DNA repair helicases XPB and XPD.

Progress 11/22/16 to 09/30/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training for UCR undergraduate students and graduates. How have the results been disseminated to communities of interest?We have deposited protein structural data and coordinates to the Protein Data Bank (PDB) for free access by the public. In addition, we have published our results in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the past year, due to COVID-19, we had limited access to the campus for research activities. Fortunately, inthe previous years ahead of the final year, we have cloned and expressed more than a dozen of Xac proteins (aim1), most of these recombinant proteins were purified in large scales for crystallization trials (aim2), and we have sucessfully determined high resolution crystal structures of several Xac proteins (aim 3 and 4).

Publications

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research trainings for one UCR undergraduate student during this period. How have the results been disseminated to communities of interest?We published our results as research article in a peer-reviewed journal (Acta Crystal. D Struct. Biol.) What do you plan to do during the next reporting period to accomplish the goals?Genetic screenings performed by our collaborators in Brazil have shown that Xac3294 is important for citrus canker and is possibly involved in Xac biofilm formation. To understand the role of Xac3294 gene in citrus canker, we performed genetic analysis, which suggested Xac3294 likely interacts with the adjacent gene Xac3296. Bioinformatic analysis suggested that Xac3296 is a toll/interlecukin-1 receptor (TIR) domain-containing protein. We have cloned both Xac3294 and Xac3296 genesfrom Xac genomic DNA into expression vectors, and successfully prepared large quantities of the recombinant Xac3294 and Xac3296 protein from E. coli. Our plan for the coming year is 1. To continue screening crystallization conditions for Xac3294, Xac3296, and the complex. 2. To refine the crystallization conditions and obtain high quality crystals for X-ray crystallographic study.

Impacts
What was accomplished under these goals? This project characterized the structure and function of proteins encoded by Xanthomonas genes important to citrus canker. In this reporting period, 1) we published in a peer-reviewed journal (Acta Crystal. D.) a research article describing our results on biochemical and structural characterization of the protein encoded by the Xac3819 gene. 2) we expressed and purified Xac3294 gene, which is important to citrus canker phenotype, and its potential partner Xac3296 gene as recombinant protein in E. coli 3. we continued screeningconditions to crystallize Xac3294, xac3296 and together as a complex.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Structual and Biochemical characterization of a glutathione transferase from citrus canker pathogen Xanthomonas

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research trainings for one UCR undergraduate student during this period. How have the results been disseminated to communities of interest?We have deposited in the Protein Data Bank the structural coordinates of the protein encoded by Xac 3819 gene and its complex with glutathione. We also submitted the results as a journal article. What do you plan to do during the next reporting period to accomplish the goals?Genetic screenings performed by our collaborators in Brazil have shownthat Xac3294 is important for citrus canker and is possibly involved in Xac biofilm formation. To understand the role ofXac3294 gene in citrus canker, we performed genetic analysis, which suggested Xac3294 likely interacts with the adjacentgene Xac3296. Bioinformatic analysis suggested that Xac3296 is a toll/interlecukin-1 receptor (TIR) domain-containingprotein. We have cloned both Xac3294 and Xac3296 genesfrom Xac genomic DNA into expression vectors, and successfullyprepared large quantities of the recombinant Xac3294 andXac3296 protein from E. coli. Our plan for the coming year is 1. To continue screening crystallization conditions forXac3294,Xac3296, and the complex. 2. To refine the crystallization conditions and obtain high quality crystals for X-ray crystallographic study.

Impacts
What was accomplished under these goals? This project characterized the structure and function of proteins encoded by Xanthomonas genes important to citrus canker. In this reporting period, 1) we submitted a manuscript describing our results on biochemical and structural characterization of the protein encoded by theXac3819gene. 2) we expressed and purified Xac3294 gene, which is important to citrus canker phenotype, and its potential partner Xac3296 gene as recombinant protein in E. coli 3. we screened over a thousand conditions to crystallize Xac3294, xac3296 and together as a complex.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Structural and biochemical characterizations of a glutathione transferase from citrus canker pathogen Xanthomonas

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research trainings for one UCR undergraduate studentduring this period. How have the results been disseminated to communities of interest?We have deposited in the Protein Data Bank thestructural coordinates of the protein encoded by Xac 3819 gene and its complex withglutathione. What do you plan to do during the next reporting period to accomplish the goals?Last year, we made breakthrough on Xac3819. Because of limited funding from this award, we decided to foucs on Xac3819 instead of working on our originalplan with Xac3294.Genetic screenings performed by our collaborators in Brazil have shown that Xac3294 is important for citrus canker and ispossibly involved in Xac biofilm formation. To understand the role of Xac3294 gene in citrus canker, we performed geneticanalysis, which suggested Xac3294 likely interacts with the adjacent gene Xac3296. Bioinformatic analysis suggested thatXac3296 is a toll/interlecukin-1 receptor (TIR) domain-containing protein. We have cloned both Xac3294 and Xac3296 genesfrom Xac genomic DNA into expression vectors. Our plan for the coming year is 1. To prepare large quantities of the recombinant proten complex of Xac3294/Xac3296 from E. coli. 2. To screen crystallization conditions for this complex 3. To refine the crystallization conditions and obtain high quality crystals for X-ray crystallographic study.

Impacts
What was accomplished under these goals? The Xac3819 gene (XacGST) from the phytopathogen Xanthomonas axonopodis pv. citri strain 306 (Xac) was reported to be important for citrus canker. In this period, 1) we have determined thefirst crystal structures of XacGST apo-form (PDB ID: 6NXV) and GSH-bound complex (PDB ID: 6NV6). Both structures show the canonical GST-like fold and contain a conserved theta class serine residue (Ser12), in which the serine hydroxyl group is used to activate the GSH thiol for nucleophilic attack on electrophilic substrates. Although we observed XacGST as a dimer in crystals,gel filtration chromatography suggests that XacGST is a monomer in solution. 2) we characterized the biochemical properties of XacGST showingXacGST is able to usebromoacetate as a substrate with a Km value of 6.10 mM, a kcat value of 41.56 s-1, and a kcat/Km value of 6.80 mM-1s-1, under saturating glutathione concentration. After structural and sequence analysis, we havedefinedXacGST as a dehalogenase theta class GST.We believe that XacGST is an essential GST to protectXanthomonas axonopodis citriagainst major mechanisms of cell detoxification and defense against cellular oxidative stress during plant-pathogen interaction at the infection interface. This study provides new insights in understanding the roles of XacGST in citrus plant-phytopathogen interaction as a potential target protein to control ofXacin citrus crops. 3) based on amino acid sequence conservation and structural comparisons with GSH bound XacGST structure, we have classified other 9 unclassified and uncharacterized GSTs fromXacgenome.While Xac0894, Xac1299, Xac1461, Xac1474, Xac2394, Xac2460, Xac3819, Xac4352 are theta class members, Xac1007 and Xac2230 are likely beta class GSTs.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: PDB deposit: 6NXV Crystal structure of the theta class glutathione S-transferase from the citrus canker pathogen Xanthomonas axonopodis pv. citri, apo form
• Type: Other Status: Submitted Year Published: 2019 Citation: PDB deposit: 6NV6 Crystal structure of the theta class glutathione S-transferase from the citrus canker pathogen Xanthomonas axonopodis pv. citri with glutathione bound.

Progress 11/22/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research trainings for two UCR undergraduate students during this period. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Our plan is 1. To prepare large quantities of the recombinant proten complex of Xac3294/Xac3296 from E. coli. 2. To screen crystallization conditions for this complex 3. To refine the crystallization conditions and obtain high quality crystals for X-ray crystallographic study.

Impacts
What was accomplished under these goals? Citrus canker is a widespread bacterial disease in citrus-growing regions throughout the world. Infected trees exhibit gradual decline in health and fruit production until they eventually succumb to the disease. Currently there are no chemical or microbiological agents to control the disease. The disease is caused by Xanthomonas phytopathogens. The most widely spread and severe strain Xanthomonas citri subsp. citri (Xac) attacks all citrus varieties. However, the molecular mechanisms of Xaccitrus infection are not well understood. Genetic screenings performed by our collaborators in Brazil have shown that Xac3294 is important for citrus canker and is possibly involved in Xac biofilm formation. To understand the role of Xac3294 gene in citrus canker, we performed genetic analysis, which suggested Xac3294 likely interacts with the adjacent gene Xac3296. Bioinformatic analysis suggested that Xac3296 is a toll/interlecukin-1 receptor (TIR) domain-containing protein. We have cloned both Xac3294 and Xac3296 genes from Xac genomic DNA into expression vectors. Single expression of Xac3294 as a recombinant protein in E. coli produced mostly insoluble protein, supporting its possible role in biofilm formation. However, when we co-expressed Xac3294 and Xac3296 together in E. coli, the solubility of Xac3294 protein increased significantly. We further observed that Xac3294 and Xac3296 recombinant proteins form a heterodimeric complex, confirming our prediction from bioinformatic analysis. We have established an experimental protocol to express and purifythe Xac3294/Xac3296 complex from E. coli, suitablefor structural study by X-ray crystallography.

Publications