Basis of Xanthomonas Tissue-Specific Behavior During Plant Pathogenesis

BASIS OF XANTHOMONAS TISSUE-SPECIFIC BEHAVIOR DURING PLANT PATHOGENESIS

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1016673

Grant No.

2018-67013-28490

Cumulative Award Amt.

$700,000.00

Proposal No.

2018-05040

Multistate No.

(N/A)

Project Start Date

Sep 1, 2018

Project End Date

Aug 31, 2021

Grant Year

2018

Program Code

[A1171]- Plant Biotic Interactions

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Plant Pathology

Non Technical Summary
Pathogenic microbes such as bacteria cause vascular and non-vascular diseases of plants andlimit crop production globally. Vascular pathogenic bacteria are particularly destructive because they move long distances through the host veins often leading to widespread infection or even whole host death. Non-vascular pathogens are also agriculturally important but stay restricted to the site of infection, whichleadsto development of smallspots, specks or lesions. The basis for why some bacteria move in the vascular tissue while others remain localized to the non-vascular spaces remains unknown. This research aims to determine the host and pathogen factors that promote tissue-specific behavior in an important group of pathogenic bacteria called Xanthomonas. This project uses novel techniques in metabolomics, gene-expression and mutant analysis, microscopy to determine the basis for tissue-specificity.The outputs of this work will developnew insights into microbial pathogenesis, teaching techniques for undergraduate and graduate education and mentoring/professional development of young scientists focusing on agricultural research. This research topic focuses on crops and pathogens important to US agriculture and will fortify American agricultural science and scientific education.

Animal Health Component

(N/A)

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	1550	1040	50%
212	1550	1100	25%
212	1550	1080	25%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1550 - Barley;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1080 - Genetics;

Keywords

vascular pathogenesis

xanthomonas

xylem

Goals / Objectives
Plant pathogenic bacteria in the genus Xanthomonas cause vascular and non-vascular diseases of over 200 plant species. It is unclear what factors differentiate vascular from non-vascular Xanthomonas spp. This proposal aims to understand the biological basis of vascular pathogenesis in plant pathogenic bacteria.A single cell-wall degrading enzyme called CelA is shared among vascular bacteria in the Xanthomonadaceae family, but is absent in non-vascular Xanthomonas spp. Vascular pathogen, Xanthomonas translucens pv. translucens (Xtt), primarily colonizes the xylem and causes bacterial blight of barley. Non-vascular X. t. pv. undulosa (Xtu) does not colonize the xylem, but instead causes leaf streak by proliferating in the apoplast. The genome of Xtt but not Xtu encodes CelA. Notably, expressing CelA in Xtu permits vascular pathogenesis of barley. This proposal defines a strategy to test the hypothesis that CelA plays a role in bacterial pathogenesis in the xylem environment. The roles of CelA in affecting (1) host physiology by cell wall modification, (2) bacterial behavior in the xylem environment, and (3) plant responses and bacterial fitness during xylem colonization will be addressed. The specific objectivesare to:Objective 1: Define the activity and physiological impact of CelA on the xylem metabolome. Objective 2: Define the role of CelA in X. translucens vascular colonization, movement and virulence effector deliveryObjective 3: Determine plant responses and additional bacterial fitness factors impacted by CelA during vascular and non-vascular disease development in barley. Objective 4: Develop training based on visualization techniques created for this project.

Project Methods
This proposal aims to understand the biological basis of vascular pathogenesis in plant pathogenic bacteria. This project will explore the role of a cellulase (e.g. CelA) and other factors important tissu-especific behavior in Xanthomonas species. The objectives andmethods supporting the objectives include:Objective1: Define the activity and physiological impact of CelA on the xylem metabolome. This objective will use metabolomics to determine the molecular activities of cellulases on vascular, xylem pathogenesis.Objective 2: Define the role of CelA in X. translucens vascular colonization, movement and virulence effector delivery. This objective will use a combination of microscopy techniques and colonization assays to determine the impact of CelA on vascular movement. This objectivewill also employ a novel technique using Effector-Detector Plants to visualize how cellulase could alter bacterial virulence activities (e.g. effector protein injection).Objective 3: Determine plant responses and additional bacterial fitness factors impacted by CelA during vascular and non-vascular disease development in barley. This objective will use two novel techniques not widely employed in plant-microbe interactions: a) dual-RNA-seq and b)Tn-seq. Both techniques will provide a global characterization of a) gene expression of the host and pathogen simultaneously and b) global fitness factors for tissue-specific pathogenesis. Finally these genomic/transcriptomic experimentswill be combined for an exhaustive analysis including the transcriptomics to determine the basis for tissue-specific behavior.Objective 4: Develop training based on visualization techniques created for this project. This objective will use a technique from Objective 2 to teach plant and microbial molecular biology, agricultural science, plant-microbe interactions and genetic modification of crops. The PI and co-PIs already are developing the curriculumand will start presenting this workshop and coursé-based teaching tool at professional meetings at the end of summer 2018. They will strengthen this proposal with teaching evaluations and altering the curriculum to meet a specific audience's needs.

Progress 09/01/18 to 08/31/21

Outputs
Target Audience:Our target audience includes basic and applied reseachers and stakeholders in industry. In particular this research is important to thosefocused on the basis of bacterial plant pathogenesis. The research targets crops such as cereals critical to the US economy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We developed a workshop based in visualization of bacterial virulence that has been presented at the American Phytopathological Society's Annual meeting (Objective 4). We trained postdoctoral researchers and graduate and undergraduate research assistants as part of this project. This will train the next workforce motivated to solve agricultural problems faced by American farmers especially cereal producers. How have the results been disseminated to communities of interest?We published multiple articles on the topic of tissue-specificity. We also project additional publications to result from data gathered from this research around tissue-specificity. These include: Gluck-Thaler E*, Cerutti A*, Perez-Quintero A*, Butchacas J, Roman-Reyna V, Madhaven VN, Shantharaj D, Merfa MV, Pesce C, Jauneau A, Vancheva T, Lang JM, Allen C, Verdier V, Gagnevin L, Szurek B, Cunnac S, Beckham G, De La Fuente L, Patel HK, Sonti RV, Bragard C, Leach JE, Noe?l LD, Slot JC, Koebnik R**, Jacobs JM**. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles. Science Advances. 2020. doi: 10.1126/sciadv.abc4516 *co-first authors, **co-corresponding authors Nordstedt, N*,Roman-Reyna, V*,Jacobs, J, Jones, M (2021) Comparative Genomic Understanding of Gram-Positive Plant Growth PromotingLeifsonia. Phytobiomes, 10.1094/PBIOMES-12-20-0092-SC Dia NC, Morinière L, Cottyn B,Bernal E,Jacobs JM, Koebnik R, Osdaghi E, Potnis N, Pothier JF.Xanthomonas hortorum- beyond gardens: current taxonomy, genomics, and virulence repertoiresMolecular Plant Pathology in press doi: 10.1111/mpp.13185 Cohen SP, Luna EK, Lang JM, Ziegle J, Chang C, Leach JE, Le-Saux MF, Portier P, Koebnik R,Jacobs JM†.High-Quality Genome Resource ofXanthomonas hyacinthiGenerated via Long-Read Sequencing. Plant Dis. 2020 Apr;104(4):1011-1012. doi: 10.1094/PDIS-11-19-2393-A. Epub 2020 Feb 17. Lang JM, Pérez-Quintero AL, Koebnik R, DuCharme E, Sarra S, Doucoure H, Keita I, Ziegle J,Jacobs JM, Oliva R, Koita O, Szurek B, Verdier V, Leach JE. A Pathovar ofXanthomonasoryzaeInfecting Wild Grasses Provides Insight into the Evolution of Pathogenicity in Rice Agroecosystems. Front Plant Sci. 2019 doi:10.3389/fpls.2019.00507 Roman-Reyna V, Pesce C, Luna E, Vancheva T, Chang C, Ziegle J, Bragard C, Koebnik R, Lang JM, Leach JE,Jacobs JM†. Genome resource of barley bacterial blight and leaf streak pathogenXanthomonastransluscenspv. translucens strain UPB886. 12 Aug 2019 https://doi.org/10.1094/PDIS-05-19-1103-A A video presentation from the American Society for Plant Biologists Plantae Presents is publicly available: https://plantae.org/plantae-presents-sheng-yang-he-and-jonathan-jacobs/ https://www.youtube.com/watch?v=QgQzXBe7sg4&feature=emb_logo What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) We have discovered with our team and collaborators that CelA (herein called CbsA) was required for successful colonization of the plant xylem. We defined the specific activities of CbsA for successful xylem colonization and are awaiting to prepare this research for publication. We have determined that CbsA enzymatic bioproducts are required for xylem-colonization. 2) We demonstrated that CbsA is required for succesful xylem colonization by typically non-vascular subgroup X. translucens pv. undulosa. Heterlogous experession of CbsA allows for host vascular colonization. Loss of CbsA function in vascular X. translucens pv. translucens enhances the ability of this pathogen to colonize the non-vascular tissue. We developed preliminary data thatCbsA plays a role in hydathode entry.3) We are currently defining the plant responses to CbsA activity for xylem colonization. We have also determined that CbsA is a dominant factor during compeitive fitness between wild-type X. translucens pv. translucens compared to a cbsA mutant. 4) We developed educational workshops around research in this grant. We have presented these workshops at the APS Plant Health Meetings.

Publications

Type: Journal Articles Status: Published Year Published: 2021 Citation: Nordstedt, N, Roman-Reyna, V, Jacobs, J, Jones, M (2021) Comparative Genomic Understanding of Gram-Positive Plant Growth Promoting Leifsonia. Phytobiomes, doe: 10.1094/PBIOMES-12-20-0092-SC
Type: Journal Articles Status: Published Year Published: 2022 Citation: Dia NC, Morini�re L, Cottyn B, Bernal E, Jacobs JM, Koebnik R, Osdaghi E, Potnis N, Pothier JF. Xanthomonas hortorum beyond gardens: current taxonomy, genomics, and virulence repertoires Molecular Plant Pathology in press doi: 10.1111/mpp.13185
Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: ASPB Plantae Presents "Defining tissue-specificity of plant colonization by bacteria" Jonathan M. Jacobs https://plantae.org/plantae-presents-sheng-yang-he-and-jonathan-jacobs/ https://www.youtube.com/watch?v=QgQzXBe7sg4&feature=emb_logo
Type: Journal Articles Status: Published Year Published: 2020 Citation: Gluck-Thaler E, Cerutti A, Perez-Quintero A, Butchacas J, Roman-Reyna V, Madhaven VN, Shantharaj D, Merfa MV, Pesce C, Jauneau A, Vancheva T, Lang JM, Allen C, Verdier V, Gagnevin L, Szurek B, Cunnac S, Beckham G, De La Fuente L, Patel HK, Sonti RV, Bragard C, Leach JE, No�l LD, Slot JC, Koebnik R, Jacobs JM. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles. Science Advances. 2020. doi: 10.1126/sciadv.abc4516
Type: Journal Articles Status: Published Year Published: 2020 Citation: Roman-Reyna V, Luna EK, Pesce C, Vancheva T, Chang C, Ziegle J, Bragard C, Koebnik R, Lang JM, Leach JE, Jacobs JM. 2020. Genome Resource of Barley Bacterial Blight and Leaf Streak Pathogen Xanthomonas translucens pv. translucens strain UPB886. Plant Dis. doi: 10.1094/PDIS-05-19-1103-A.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Lang JM, P�rez-Quintero AL, Koebnik R, DuCharme E, Sarra S, Doucoure H, Keita I, Ziegle J, Jacobs JM, Oliva R, Koita O, Szurek B, Verdier V, Leach JE. 2019 A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems. Front Plant Sci. 2019 Apr 30;10:507. doi: 10.3389/fpls.2019.00507.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience: Our target audience includes basic and applied reseachers interested in plant bacteriology. We focus our research on crops important to the US Economy. Changes/Problems:The COVID19 pandemic slowed our progress, but we still met our objectives. What opportunities for training and professional development has the project provided?As part of objective 4, we have developed training for a postdoctoral researche,a graduate student and undergraduates as part of this project. We developed an educationalworkshop that trains students in understanding molecular aspects of virulence through visualization tools like microscopy. This workshop is called "Effector Detector Plants" has been presented at the American Phytopathological Society's annual Plant Health meetings. It was delayed to February 2022 online from COVID19 delays. How have the results been disseminated to communities of interest?This research has been presented at the American Phytopathological Society's annual Plant Health meeting. We regularly engage with stakeholders including cereal growers to highlight our findings and their impacts. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) We have discovered with our team and collaborators that CelA (herein called CbsA) is conserved across Xanthomonas species. We determined itwas required for successful colonization of the plant xylem. We are currently defining the specific activities of CbsA for successful xylem colonization.2) We demonstrated that CbsA is required for succesful xylem colonization by typically non-vascular subgroup X. translucens pv. undulosa. Heterlogous experssion of CbsA allows for host vascular colonization. 3) We are defining the plant responses to CbsA activity for xylem colonization.

Publications

Type: Journal Articles Status: Published Year Published: 2021 Citation: Nordstedt, N, Roman-Reyna, V, Jacobs, J, Jones, M (2021) Comparative Genomic Understanding of Gram-Positive Plant Growth Promoting Leifsonia. Phytobiomes, doe: 10.1094/PBIOMES-12-20-0092-SC
Type: Journal Articles Status: Published Year Published: 2022 Citation: Dia NC, Morini�re L, Cottyn B, Bernal E, Jacobs JM, Koebnik R, Osdaghi E, Potnis N, Pothier JF. Xanthomonas hortorum beyond gardens: current taxonomy, genomics, and virulence repertoires Molecular Plant Pathology in press doi: 10.1111/mpp.13185
Type: Journal Articles Status: Published Year Published: 2021 Citation: ASPB Plantae Presents "Defining tissue-specificity of plant colonization by bacteria" Jonathan M. Jacobs https://plantae.org/plantae-presents-sheng-yang-he-and-jonathan-jacobs/ https://www.youtube.com/watch?v=QgQzXBe7sg4&feature=emb_logo

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:Our target audience includes basic and applied reseachers focused on the basis of bacterial plant pathogenesis. We are particularly focused on cereals, which has target audiences in government, industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have developed a workshop based in visualization of bacterial virulence that has been presented at the American Phytopathological Society's Annual meeting. We have trained postdoctoral researchers and graduate research assistants as part of this project. This will train the next workforce motivated to solve agricultural problems faced by American farmers. How have the results been disseminated to communities of interest?We have published multiple articles associated with this research. In particular the foundational findings that CbsA plays a critical role in the evolution of Xanthomonas species. Gluck-Thaler E*, Cerutti A*, Perez-Quintero A*, Butchacas J, Roman-Reyna V, Madhaven VN, Shantharaj D, Merfa MV, Pesce C, Jauneau A, Vancheva T, Lang JM, Allen C, Verdier V, Gagnevin L, Szurek B, Cunnac S, Beckham G, De La Fuente L, Patel HK, Sonti RV, Bragard C, Leach JE, Noël LD, Slot JC, Koebnik R**, Jacobs JM**. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles. Science Advances. 2020. doi: 10.1126/sciadv.abc4516 *co-first authors, **co-corresponding authors What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1)We have discovered with our team and collaborators that CelA (herein called CbsA) wasrequired for successful colonization of the plant xylem. We are currently defining the specific activities of CbsA for successful xylemcolonization. We have determined that cell wall bioproducts of CbsA are required for xylem-colonizationactivity.2) We demonstrated that CbsA is required for succesful xylem colonization by typically non-vascular subgroup X. translucens pv. undulosa. Heterlogous experssion of CbsA allows for host vascular colonization.Loss of CbsA function in vascularX. translucens pv. translucens enhances the ability of this pathogen to colonize the non-vascular tissue. 3) We are currently defining the plant responses to CbsA activity for xylem colonization. We have also determined that CbsA is a dominant factor during compeitive fitness between wild-type X. translucens pv. translucens compared to a cbsA mutant.

Publications

Type: Journal Articles Status: Published Year Published: 2020 Citation: Gluck-Thaler E*, Cerutti A*, Perez-Quintero A*, Butchacas J, Roman-Reyna V, Madhaven VN, Shantharaj D, Merfa MV, Pesce C, Jauneau A, Vancheva T, Lang JM, Allen C, Verdier V, Gagnevin L, Szurek B, Cunnac S, Beckham G, De La Fuente L, Patel HK, Sonti RV, Bragard C, Leach JE, No�l LD, Slot JC, Koebnik R**, Jacobs JM**. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles. Science Advances. 2020. doi: 10.1126/sciadv.abc4516 *co-first authors, **co-corresponding authors
Type: Journal Articles Status: Published Year Published: 2019 Citation: Lang JM, P�rez-Quintero AL, Koebnik R, DuCharme E, Sarra S, Doucoure H, Keita I, Ziegle J, Jacobs JM, Oliva R, Koita O, Szurek B, Verdier V, Leach JE. 2019 A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems. Front Plant Sci. 2019 Apr 30;10:507. doi: 10.3389/fpls.2019.00507.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Roman-Reyna V, Luna EK, Pesce C, Vancheva T, Chang C, Ziegle J, Bragard C, Koebnik R, Lang JM, Leach JE, Jacobs JM. 2020. Genome Resource of Barley Bacterial Blight and Leaf Streak Pathogen Xanthomonas translucens pv. translucens strain UPB886. Plant Dis. doi: 10.1094/PDIS-05-19-1103-A.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Cohen SP, Luna E, Lang JM, Ziegle J, Chang C, Leach JE, Le-Saux M, Portier P, Koebnik R, and Jacobs JM. High-quality complete genome resource of Xanthomonas hyacinthi generated via long-read sequencing. 2020. Plant Disease. https://doi.org/10.1094/PDIS-11-19-2393-A Epub 12 Dec. 2019