DISSECTING DEFENSE SIGNALING PATHWAYS IN SOYBEAN (GLYCINE MAX) AND ARABIDOPSIS THALIANA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014539
• Project Start Date: Oct 13, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Plant Pathology

Non Technical Summary
Plant diseases have major negative impacts on crop plants produced for food, feed, and fiber. Disease-related croploss estimates exceed $100 billion worldwide. Current plant protection strategies involve the use of genetic resistance,chemical treatments, and farming practices. These methods generally offer partial protection often only againstspecific pathogen strains. Strategies involving the induction of intrinsic defense responses, or recognition of pathogenspecificelicitors, offer viable alternatives and have the potential to protect against broad spectrum of pathogens.Developing sustainable crop protection approaches such as these requires a fair knowledge of the signalingmechanisms involved in defense. Thus, the elucidation of signal transduction pathway(s) following pathogenrecognition could eventually aid the targeted manipulation of defense responses without affecting crop yield. Theproposed research aims to understand a novel pathway the regulates systemic immunity against a broad spectrum of pathogens.The long-term goal is to identify factors that can induce broad-spectrum resistance, withoutsignificantly affecting plant growth, development, and ultimately yields. In addition this work will examine the overlap between signaling induced in response to infection by beneficial versus pathogenic microorganisms.This work will ultimately benefit the UnitedStates agricultural economy because it is directly relevant to the second largest crop produced in the United States,soybean, and could be applicable to other crops as well. Understanding how plants respond to microbes could also beused for promoting interactions with beneficial organisms.

Animal Health Component

5%

Research Effort Categories

Basic

95%

Applied

5%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1410	1040	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1410 - Beans (dry);

Field Of Science
1040 - Molecular biology;

Keywords

systemic immunity

rhizobacteria

soybean

bacterial blight

soybean mosaic virus

Goals / Objectives
Characterize defense signaling mechanisms in soybeanExamine mechanisms of pathogen effector recognitionStudy the signaling overlap in plant interactions with pathogenic and beneficial microbesCharacterize the roles of primary metabolic components in plant defenseIdentify the role of glycerolipid signals during systemic immunityStudy functional overlap of SAR signaling components in soybean

Project Methods
1a. Examine mechanisms of pathogen effector recognition This aim focuses on identifying and functionally characterizing soybean components that mediate pathogen recognition in soybean. Using various approaches, we identified soybean proteins that interact with P. syringae pv. glycinea (Psg) and SMV effector proteins.The functions of the identified effector-interacting proteins in plant defense signaling will be studied by examining the effects of overexpression and silencing the encoding genes in soybean. Plants overexpressing/silenced for the target genes will be evaluated for their responses to specific pathogens.1b. Study the signaling overlap in plant interactions with pathogenic and beneficial microbes Legume plants like soybean develop symbiotic relationships with diazotrophic bacteria called rhizobia. During this symbiosis, plants receive essential reduced nitrogen from the bacteria in return for carbon provided to the bacteria. Symbiosis involves the formation of the root nodule, a specialized plant organ containing the optimal environment for rhizobia to convert nitrogen into ammonia. Entry into compatible plant roots is initiated by the exchange of signals between the bacteria and plant root hair cells. During nodule development, bacterial numbers in the infected cells increase dramatically, requiring membrane and lipid biosynthesis, in the plant and bacteria. Notably, plant-symbiotic interactions share many similarities with plant-pathogen interactions (Deakin and Broughton, 2009). We have generated several soybean lines that are knocked-down for the expression of conserved components involved in plant defense (freeze-dried tissue from confirmed knock-down plants can be stored long term at -80oC). In this objective, we will examine the potential involvement of these components in promoting/inhibiting soybean interactions with root-nodulating bacteria.We will assess the expression of known defense-related genes in soybean root tissue at various time points post rhizobium inoculation.Next, we determine the effects of silencing the defense-related genes on root nodulation by rhizobia.2a. Identify the role of glycerolipid signals during systemic immunitySAR involves the systemic movement of signals from the site of primary infection to non-infected portions of the plant. 14C-G3P transport assay shows that G3P is derivatized in the infected leaf and transported to the systemic tissue (Chanda et al., 2012). We will use four complementary approaches to identify the bioactive derivative(s) because that will enable us to predict their biosynthesis route (enzymatic and/or chemical), determine underlying signaling, and position it in the signaling pathway with respect to other identified components.i. Determine integrity of the 3 carbon G3P backboneii. Ultra-performance LC (UPLC)-MS analyses of the bioactive fraction:iii. NMR analyses of the bioactive fractioniv. Test bioactivity of the identified G3P-derivative(s)2b. Study functional overlap of SAR signaling components in soybeanOur studies show that like A. thaliana, G3P and SA can induce SAR in soybean and that G3P-deficient soybean plants are unable to induce SAR (Chanda et al., 2012 and unpublished data). Furthermore, petiole exudate from pathogen-infected soybean plants can induce SAR in healthy A. thaliana plants (unpublished data). Together, this suggests significant overlap in the SAR signaling pathways of soybean and A. thaliana. Here, we will test the SAR related functions of soybean orthologs of known SAR signaling components from A. thaliana. Based on sequence analysis, we have identified soybean orthologs for many of the key regulators of the A. thaliana SAR signaling pathway. Full-length cDNAs for the identified sequences will be amplified from the G. max genome using sequence-specific primers and functionally characterized for their roles in defense to various pathogens.

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

Outputs
Target Audience:Plant science research community Program leaders at national funding agencies including National Science Foundation and USDA State and national soybean growers State corn growers Undergraduate students-Unversity of Kentucky High school students-multiple counties, KY Middle school students-multiple counties, KY Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: One postdoctoral scholar, three graduate students, two undergraduate students and one high school studentreceived training in plant molecular pathology based scientific research via engaging in daily laboratory research, one-on-one meetings with the PI and weekly group meetings. How have the results been disseminated to communities of interest?1. Publications in peer reviewed journals: Total 7 2. Invited talks at Plant & Animal Genome XXVIII conference 3. Local talks: Student presentations at the Univeristy of KY undergraduate student research showcase (April 2020); Guest lecture in undergraduate class ABT101 (October 2020) 4. Presentations to stakeholder groups: Annual meeting with the Kentucky Soybean Promotion Board (March 2020) What do you plan to do during the next reporting period to accomplish the goals? Continue research related to all major objectives listed above and disseminate research widely through various venuesas before. Continue postdoctoral and graduate student training. Continue mentoring undergraduate student interns. Continue to train high school research interns and conduct plant biology workshops for area middle/high school studentand their families.

Impacts
What was accomplished under these goals? Characterize defense signaling mechanisms in soybean 1) Examined thenovel systemic signaling pathway that we recently discovered in soybean. This pathway enables the plant to simultaneously restrict root nodulation by poor nitorgen fixersi while prevent pathogen infection in the leaf. This is highly attractive mechanism with tremendous potential for use in agricutlure. We are currently dissecting the ptathway to identify nodes that could be manipulated to promote nitrogen fixing ability and pathogen resistance in crop plants like soybean. 2) Studied the involvement of systemic acquired resistance (form of long lasting, braod spectrum plant systemic immunity) signaling molecules indentified from studies in Arabidopsis. We find most signals are highly conserved and are generated via identical biosynthetic pathways although the enzymes contributing to biosynthesis can be differentially regulated and the existence of multiple isoforms can result in redundancy effects. Characterize the roles of primary metabolic components in plant defense 1) Showed that salicylic acid (SA) is indeed a transported systemic signal during systemic acquired resistance (SAR)-this is major breakthrough because it discounts a long accepted notion in the field that SA transport is inconsequential to SAR. 2) Demonstrated that the plant cuticle is important for systemic transport of the plant hormone salicylic acid-this is an important fiding as it identifies a key signaling role for thecuticle in plant immunity and has implications for application using cuticle properties to develop biomimics. 3) Identified the signaling mechanism via which the amino acid breakdown product piecolic acid regulates systemic immunity in plants. Thsi knowledge opens up avenues for practical applications of pipecolic acid as a disease protecting chemical for use in agriculture.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kachroo P, Kachroo A (2019) Plant Immunity: a bird's-eye view. Plant Science 279:1- 2
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang C, Liu R, Lim GH, de Lorenzo L, Yu K, Zhang K, Hunt AG, Kachroo A, Kachroo P (2019) Pipecolic acid confers systemic immunity by regulating free radicals. Science Advance 4:eaar4509
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Klessig DF, Manohar M, Shine MB, Koch A, Danquah WB, Luna E, Park H-J, Kolkman JM, Turgeon G, Nelson R, Leach JE, Williamson VM, Kogel K-H, Kachroo A, Schroeder FC (2019) Nematode ascaroside enhances resistance in a broad spectrum of plantpathogen systems. Journal of Phytopathology 167: 265-272
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Paul NC, Nam SS, Park W, Yang J-W, Kachroo A (2019) First report of storage tuber rot in sweet potato (Ipomoea batatas) caused by Plenodomus destruens in Korea. Plant Disease 103: 1020
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kachroo P, Kachroo A (2020) Lipid-modulated trafficking in plants. Molecular Plant 13:351-353
• Type: Journal Articles Status: Published Year Published: 2020 Citation: DeMers LC, Redekar NR, Kachroo A, Tolin SA, Li S, Saghai Maroof MA (2020) A transcriptional regulatory network of Rsv3-mediated extreme resistance against Soybean mosaic virus. PLoS One 15:e0231658
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lim GH, Liu H, Yu K, Liu R, Shine MB, Fernandez J, Burch-Smith T, Mobley JK, McLetchie N, Kachroo A, Kachroo P (2020) The plant cuticle regulates apoplastic transport of salicylic acid during systemic acquired resistance. Science Advance 6:eaaz0478
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kachroo P, Liu H, Kachroo A (2020) Salicylic acid: transport and long-distance immune signaling. Current Opinion in Virology 42:53-57
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kachroo A, Kachroo P. (2020) Mobile signals in systemic acquired resistance. Current Opinion in Plant Biology 58:41-47

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

Outputs
Target Audience:Plant science research community Program leaders at national funding agencies including National Science Foundation and USDA US Congressional staff and state representatives State and national soybean growers State corn growers Undergraduate students-Unversity of Kentucky High school students-multiple counties, KY Middle school students-multiple counties, KY Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities One postdoctoral scholar, three graduate students, two undergraduate students and four high school students received training in plant molecular pathology based scientific research via daily laboratory research andone-on-one meetings with the PI. Community workshops provided training in basic plant biology techniques to area middle and high school students and their families. Professional development Postdoctoral scholar and graduate students received professional development training via participation in undergraduate and high school student mentoring, participation in hands-on activities/workshops for middle/high school students in KY, oral/poster research presentations at national conferences. Students and postdocs also hadnetworking opportunities withother scientists via attendance at national research conferences and hosting invited speakers in the Department of Plant Pathology. How have the results been disseminated to communities of interest? Publications in peer reviewed journals-total 4 Agricultural Research Congressional Exhibition at the Coalition for National Science Foundation (CNSF) exhibition for congress, US Capitol, DC (April 2019) Invited talks at annual society meetings: Plant Health 2019, annual conference of the American Phytopathological Society, Cleveland, OH(August 2019) Invited talk at the Corteva Plant Sciences Symposium 2019, University of Illinois, Urbana-Champaign, IL (October 2019) Local talks:Student presentation at the Univeristy of KY undergraduate student research showcase (April 2019);Student presentation at the Kentucky Science Pathways program for high school students (August 2019);Guest lecture inundergraduate class ABT101 (November 2019) Presentations to stakeholder groups: Annual meeting with the Kentucky Soybean Promotion Board (March and December 2019, Princeton and Lexignton, KY); annual meeting with Ky Corn growers association (November 2019, Elizabethtown, KY) Reserach highlights in local news media outlet: UKNow(https://news.ca.uky.edu/article/uk-researchers-find-new-soybean-pathway-responds-pathogenic-and-beneficial-microbes) What do you plan to do during the next reporting period to accomplish the goals? Continue research related to all major objectives listed above and disseminate research widely through various venues as before Continue postdoctoral and graduate student training, andcomplete the planned graduation of one graduate student Continue to train high school research interns and conduct plant biology workshops for area middle/high school student and their families

Impacts
What was accomplished under these goals? Characterize defense signaling mechanisms in soybean: Identified and characterized a novel systemic signaling pathway invovling root-shoot-root signaling in soybean plants. Examine mechanisms of pathogen effector recognition: Identified a ubiquitin ligase protein involved in recognition of the soybean mosaic virus effector based on protein-protein interaction studies. Study the signaling overlap in plant interactions with pathogenic and beneficial microbes: Discovered that molcular components of resistance protein-mediated signaling pathways also function in genetic exclusion of non-desirable root nodulating bacteria in soybena roots. Demonstrated that some chemical signals required for activating systemic acquired resistance against pathogenic microbes alos function in systemic immune responses to root-nodulating bacteria and thereby likely influence nitrogen fixation efficiency in soybean. Identify the role of glycerolipid signals during systemic immunity: Discovered that glycerol-3-phosphate, the precursor of plant lipids, is a key systemic signaling molecule that enables genetic control of nodulation by nitrogen-fixing bacteria in the root. Study functional overlap of SAR signaling components in soybean: Showed that the overall SAR signaling pathway identified in Arabidopsis is conserved in soybean and that the SAR inducing mobile signal is conserved between Arabidopsis and soybean.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Plant Immunity: a bird's-eye view. 2019 Kachroo P, Kachroo A. Plant Sci. 279:1-2
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Signaling mechanisms underlying systemic acquired resistance to microbial pathogens. 2019 Shine MB, Xiao X, Kachroo P, Kachroo A. Plant Sci. 279:81-86
• Type: Journal Articles Status: Published Year Published: 2019 Citation: The analogous and opposing roles of double-stranded RNA-binding proteins in bacterial resistance. 2019 Lim GH, Zhu S, Zhang K, Hoey T, Deragon JM, Kachroo A, Kachroo P. J. Exp. Bot.70(5):1627-1638.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Glycerol-3-phosphate mediates rhizobia-induced systemic signaling in soybean. 2019 Shine MB, Gao QM, Chowda-Reddy RV, Singh AK, Kachroo P, Kachroo A. Nat. Commun. 10(1):5303.

Progress 10/13/17 to 09/30/18

Outputs
Target Audience:Plant science research community State and national soybean growers Undergraduate students K-12 students Middle and HIgh school teachers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided professional training for one postdoctoral researcher, one visiting scientist, two graduate students, twoundergraduate students, and threehigh school students. How have the results been disseminated to communities of interest?1) Publications in peer reviewed journals 2) Invited talks at annual society meetings:17th Biennial Conference on the Molecular and Cellular Biology of the Soybean, Athens, GA (August 2018) 3) International Invited talks:National Institue for Crop Science, S. Korea (September2018), Yonsei University S. Korea (September2018),Chonnam National University (September2018) 4) Local Research Presentations: Seminar series at the University of Kentucky, Lexington, KY; (March 2018); Guest lecture in undergraduate class ABT101 (October 2018) 5) Presentation to stakeholder groups: annual meeting with the Kentucky Soybean Promotion Board (December 2018) 6) Reserachhighlights in local news media outlets: Mid America Farmer Grower Spetmeber 2018 vol 38, no.37; UKNow (http://uknow.uky.edu/research/uk-research-finds-connection-between-plant-defense-development); College of Agriculture Food & Environment news (https://news.ca.uky.edu/article/uk-researchers-make-important-finding-plant-disease-resistance) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Study the signaling overlap in plant interactions with pathogenic and beneficial microbes:Identified metabolites that simultaeneously regulate the root exclusion of undesirable microbes and foliar resistance to pathogenic microbes. Identified genes that encode the biosynthetic activities that contribute to production of those metabolites. Functionally characterized those genes in the defense signaling response of soybean plants. A manuscript is in preparation. Study functional overlap of SAR signaling components in soybean: Studied the effect of knockdown of activities contributing to the accumulation of various SAR related metabolites. This work is on-going. Characterize defense signaling mechanisms in soybean: Continued functional characterization of soybena proteins interacting with viral and bacterial effectors using biochemical, metablomic and genetic analyses.This work is on-going. Identify the role of glycerolipid signals during systemic immunity: Characterized pathogen response and other SAR related functions in mutants defective for various aspects of glycerolipid biosynthesis in Arabidopsis.This work is on-going.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Lim GH, Hoey T, Zhu S, Clavel M, Yu K, Navarre D, Kachroo A, Deragon JM, Kachroo P. 2018 COP1, a negative regulator of photomorphogenesis, positively regulates plant disease resistance via double-stranded RNA binding proteins. PLoS Pathogens 14(3):e1006894
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kachroo P, Kachroo A. 2018 Plants Pack a Quiver Full of Arrows. Cell Host Microbe. 23(5):573-575
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang C, Liu R, Lim GH, de Lorenzo L, Yu K, Zhang K, Hunt AG, Kachroo A, Kachroo P. 2018 Pipecolic acid confers systemic immunity by regulating free radicals. Science Advances 4(5):eaar4509.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Paul NC, Nam SS, Yang J-W, Kachroo A (2018) First report of blue mold caused by Penicillium oxalicum in sweet potato (Ipomoea batatas) in Korea. Plant Disease 102:6
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Paul NC, Nam SS, Kachroo A, Kim YH, Yang J-W. (2018) Characterization and pathogenicity of sweet potato (Ipomoea batatas) black rot caused by Ceratocystis fimbriata in Korea. European Journal of Plant Pathology. DOI:10.1007/s10658-018-1522-8