PATHOGEN EFFECTORS AS MOLECULAR PROBES TO UNDERSTAND PLANT DISEASES AND DEVELOP MANAGEMENT STRATEGIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017747
• Project Start Date: Nov 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

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

Performing Department
Plant Pathology, Riverside

Non Technical Summary
World population has been quickly increased in the past century, and will reach 9.7 billion in 30 years. How to feed such a large population has become one of the most pressing challenges. As the major food source, crop yield has been greatly affected by microbial diseases, especially with the changing climate and environmental conditions. This threat of food security could be largely lessened by growing resistant plants. A fundamental understanding of how pathogens cause diseases is essential for the development of sustainable resistance in economically important crops.The main goal of this project is to facilitate the management of two important diseases - the citrus Huanglongbing (HLB, aka citrus greening disease) and Phytophthora diseases. HLB is currently considered the most destructive disease of citrus worldwide. All cultivars are affected by the HLB-associated bacterium Candidatus Liberibacter asiaticus (CLas) and there is no known resistance. Phytophthora are filamentous eukaryotic pathogens that exert major threats to food safety and human wellness. Hundreds of billions of dollars are lost each year due to crop diseases caused by Phytophthora species. A common theme in both pathogens is that they rely on effector proteins to manipulate plant cellular processes in order to cause disease. Using cutting-edge molecular biology, biochemistry and genetics approaches, this project will elucidate the mechanisms underlying the virulence activities of CLas and Phytophthora effectors. As such, plant genes that could confer sustainable disease resistance against these devastating pathogens will be identified. These resistance genes will be incorporated into elite varieties of citrus and other crops (such as potato) to enhance yield. As such, this project will directly benefit US agriculture. In addition, findings obtained from this project will have broad implications to fundamental principles of plant science, microbiology and plant pathology.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	4099	1040	50%
206	2499	1160	50%

Knowledge Area
206 - Basic Plant Biology; 204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
4099 - Microorganisms, general/other; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1160 - Pathology;

Keywords

effectoromics

plant immunity

citrus huanglongbing

liberibacter

oomycetes

rna silencing suppressors

small rnas

potato late blight

Goals / Objectives
Plant diseases cause enormous crop losses, thus threatening food security. The major goal of the research in my program is to understand the mechanisms underlying the arms race between plant hosts and microbial pathogens, and furthermore, to enhance disease management using this knowledge. In particular, we work on two important pathogens, the bacterial pathogen Candidatus Liberibacter asiaticus that causes the destructive citrus Huanglongbing (HLB) and the eukaryotic filamentous pathogens Phytophthora spp. that infects various crops and forest trees. Notorious Phytophthora diseases include potato late blight, sudden oak death, and soybean root rot. In the short-term, this project will advance our understanding of citrus HLB development and Phytophthora pathogenesis, identify plant defense-related genes, and develop biomarkers for disease detection. In the long-term, it will provide mechanistic insights into the governing principles of plant diseases and set the foundation for genetic engineering towards disease resistance.To achieve these goals, two objectives will be pursued in the next three years:Objective 1. Understanding the contribution of Sec-delivered Effectors (SDEs) of the HLB-associated pathogen to disease developmentObjective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectors

Project Methods
Objective 1. Understanding the contribution of CLas SDEs to HLB developmentProfile SDE Expression during HLB development. We will determine the expression profiles of SDE genes using qRT-PCR. HLB symptom and CLas titer will be monitored so that potential correlations between SDE expression and HLB progression can be determined.Determine the virulence function of SDEs in citrus. We will generate transgenic citrusexpressing individual SDEs using Agrobacterium-mediated transformation. The transgenic plants will be examined for phenotypes reminiscent to HLB symptoms and disease susceptibility.Elucidate the molecular mechanisms by which SDEs promote CLas infection. We will identify the direct protein targets of SDEs in citrus using yeast two-hybrid screening. Specific SDE-target interactions will be confirmed using in vitro and in planta assays.Characterize the molecular details of SDE1/PLCP interaction. As a follow-up of our recently published work on SDE1, we will investigate the protein structure of SDE1 and map out the specific region/domain that is responsible for its protease inhibition activity. We will analyze citrus papain-like cysteine proteases for their potential contribution to HLB resistance using transcriptome, proteome and biochemical approaches.This work will be conducted on UC Riverside campus at the PI's laboratory and the newly established BSL3p facility off campus. The PI has access to all the equipment needed for the proposed experiments. Core facilities at the Center for Plant Cell Biology and the Institute for Integrative Genome Biology of UC Riverside will be utilized for this research.Objective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectorsCharacterize the protein structure of PSR2. We have obtained the crystal structure of a PSR2 homolog produced by the soybean pathogen Phytophthora sojae. Our results show that PSR2 forms a unique linear overall shape and is composed of seven tandem repeat units. Each unit forms a conserved α-helix-rich fold. We will use biochemical and genetic approaches to understand how each unit contributes to PSR2 function.Investigate the mechanisms underlying the virulence activity of PSR2. PSR2 promotes Phytophthora infection by suppressing host plant small RNA biogenesis. We will characterize the specific small RNA pathway that is affected by PSR2 and determine the function of the these specific small RNAs in plant immunity. We will also identify the host target(s) of PSR2 using yeast-two-hybrid screening and mass spectrometry. Potential function of PSR2-interacting protein(s) in plant immunity and/or small RNA biogenesis will be further examined using genetic approaches.Investigate the PSR2 family effectors in Phytophthora virulence. Sequence analysis revealed many effectors in Phytophthora that potentially contain the same repeat unit as PSR2. We will predict effectors with a PSR2-like fold from all the available Phytophthora genomes. We will then analyze their expression profiles and potential virulence activities. We will also establish the evolutionary relationship of PSR2-like effectors and investigate the role of this conserved repeat unit in the co-evolutionary arms race with plant hosts.This work will be conducted on UCR campus at the PI's laboratory. The PI has access to all the equipment needed for this research. Core facilities at the Center for Plant Cell Biology and the Institute for Integrative Genome Biology of UC Riverside will be employed for this research.

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

Outputs
Target Audience:Effectors are essential virulence factors produced by a large variety of parasites including bacteria, fungi, oomycetes, insects and nematodes. This project focuses on understanding the virulence functions of effectors produced by the bacterial pathogen Liberibacter asiaticus (causing the citrus Huanglongbing aka HLB) and the Phytophthora pathogens that causes potato late blight and soybean root and stem rot. Knowledge obtained from this research will provide broad implications on plant diseases, especially the mechanisms by which endemic and invasive pathogens infecting economically important crops. The crops studied in this project cause enormous damage to agriculture, therefore, this project directly benefits crop health in California and food safety worldwide. Theresearch supported by this projecthas reached out to a broad body of audience including graduate/undergraduate students, researchers, growers, and general public through classroom teaching and presentations at conferences, research institutes and universities, grower meetings and public events. The PI has participated in and led AES mission-oriented services by serving on committees that facilitate undergraduate and graduate education at UC Riverside. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided ample opportunities for student/postdoc training. During this review period, my group has trained fivepostdoctoral fellows, five Ph.D students, and three undergraduate students. Graduate students and postdocs were given opportunities to train undergraduate research assistants and write/review manuscripts and research proposals. Although they were unable to attend scientific conference due to the pandemic, all researchers were trained on the state-of-the art technologies in molecular biology, biochemistry, bioengineering, and cell biology. The PI served on advisory, guidance, qualifying exam, and dissertation committees for additional graduate students in the colleges of Natural and Life Science as well as Engineering. This project also supported her role as the continuing student advisor of the Genetics, Genomics and Bioinformatics graduate program. How have the results been disseminated to communities of interest?During the reportperiod, data generated from this project were reported in the form of journal publications (three research articles and two review papers),and invited virtual seminars given by the PD to several universities and conferences. This project involves extensive collaborations with UC Cooperative Extension specialists and USDA-ARS scientists. The research team works closely with stakeholders, especially citrus and potato growers, so that the research progress has been disseminated to end-users in a timely manner. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Understanding the contribution of Sec-delivered Effectors (SDEs) of the HLB-associated pathogen to disease development We will continue our investigations on the virulence contribution of individual SDEs to citrus HLB progression. We will also conduct in-depth expression analysis of SDEs in different citrus varieties and at different infection stages to further understand how they may affect plant physiology. SDEs withinteresting expression patterns will be further analyzed for target identification using proteomic approaches. Objective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectors Our recent exciting discoveries about PSR2 structure lead to many directions that warrant further investigation. Our on-going efforts include: 1) identify common target(s) of the Phytophthora effectors with PSR2-like repeat arrangements; 2) elucidate the mechanisms underlying the RNA silencing suppression activity of PSR2; 3) characterize how plant small RNAs enhance disease resistance.

Impacts
What was accomplished under these goals? Objective 1. Understanding the contribution of Sec-delivered Effectors (SDEs) of the HLB-associated pathogen to disease development Citrus industry is under unprecedented threats from HLB, a bacterial disease associated with Candidatus Liberibacter asiaticus (Las). Since Las is uncultivable, mechanistic insights into its virulence activities remain largely unknown. We take an "effectoromics" approach to address this pressing question by functional characterization of Sec-delivered effectors (SDEs) encoded by Las. Previously, we found the effector SDE1 inhibits specific papain-like cysteine proteases in citrus (Clark et al., 2018),whichrepresents the first virulence mechanism described in Las, which is a milestone in HLB research. In the current report period, we published three research articles reporting new findings on the effector biology of Las. 1) we systematically analyzed the expression profiles of a set of SDEs that are conserved in all Las strains isolated from different geographic regions (Thapa et al., 2020, Molecular Plant Pathology). 2) we developed a nanosensor for HLB detection using SDE1 as the biomarker (Tran et al., 2020, Biosens Bioelectron). 3) we functionally characterized another Las effector SDE5 and found that it promotes HLB by manipulating programmed cell death process in citrus (Pang et al., 2020, Plant Physiology). Objective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectors Phytophthora species are important pathogens responsible for devastating diseases of major crops and forest trees. Each Phytophthora pathogen encodes a large number (hundreds to one thousand) of effectors, presumably for host manipulation. Understanding how these effectors contribute to virulence is essential to control Phytophthora diseases. My group is pioneered on the identification of Phytophthora effectors that promote infection by suppressing small RNA silencing in the hosts and discovered a specific class of plant small RNAs that can directly silence genes in the invading Phytophthora (Qiao et al, Nature Genetics, 2013; Qiao et al., PNAS, 2015; Hou et al., Cell Host & Microbe, 2019) . The most recent paper (Hou et al., 2019) was selected as one of the 10 "best of 2019" articles by the journal. In addition,we identified a conserved structural and functional module that is present in hundreds of Phytophthora effectors. This research provides novel insight into the mechanism by which new virulence activities may be evolved (He et al., PNAS,2019).In the current report period, wepublished two invited review articles summarizing these exciting new findings (Hudzik et al., 2020) and discussing how they can be used to enhance disease resistance (Hou and Ma, 2020). We have also made significant progress on understanding the molecular mechanisms of PSRvirulence activity. One paper was published in the journal New Phytologist (Zhang et al., 2019) describing the function of the conserved structural module in the RNA silencing suppression activity of PSR1.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Thapa SP, De Francesco A, Trinh J, Gurung FB, Pang Z, Vidalakis G, Wang N, Ancona V, Ma W, Coaker G. (2020) Genome-wide analyses of Liberibacter species provides insights into evolution, phylogenetic relationships, and virulence factors. Mol Plant Pathol. 21(5):716-731. doi: 10.1111/mpp.12925.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hudzik C, Hou Y, Ma W, Axtell MJ. (2020) Exchange of Small Regulatory RNAs between Plants and Their Pests. Plant Physiol. 182(1):51-62. doi: 10.1104/pp.19.00931.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hou Y, Ma W. (2020) Natural Host-Induced Gene Silencing Offers New Opportunities to Engineer Disease Resistance. Trends Microbiol. 28(2):109-117. doi: 10.1016/j.tim.2019.08.009.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang P, Jia Y, Shi J, Chen C, Ye W, Wang Y, Ma W, Qiao Y. (2019) The WY domain in the Phytophthora effector PSR1 is required for infection and RNA silencing suppression activity. New Phytol. 223(2):839-852. doi: 10.1111/nph.15836.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Tran TT, Clark K, Ma W, Mulchandani A. (2020) Detection of a secreted protein biomarker for citrus Huanglongbing using a single-walled carbon nanotubes-based chemiresistive biosensor. Biosens Bioelectron. 147:111766. doi: 10.1016/j.bios.2019.111766.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Pang Z, Zhang L, Coaker G, Ma W, He SY, Wang N. (2020) Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease. Plant Physiol. 184(2):792-805. doi: 10.1104/pp.20.00348.

Progress 11/01/18 to 09/30/19

Outputs
Target Audience:Effectors are essential virulence factors produced by a large variety of parasites including bacteria, fungi, oomycetes, insects and nematodes. My group has been focusing our research on understanding the virulence functions of bacterial and oomycete effetors. Knowledge obtained from this research will provide broad implications on plant diseases, especially the mechanisms by which endemic and invasive pathogens infecting economically important crops. This project investigates diseases of citrus and potato that cause enormous damage to agriculture, thereby directly benefit crop health in Californa and food safety worldwide. This researchhas reached out to a broad body of audience including graduate/undergraduate students, researchers, growers, and general public through classroom teaching and presentations at conferences, research institutes and universities, grower meetings and public events. The PIhas participated in and led AES mission-oriented services by serving on committees thatfacilitate undergraduate and graduate education at UC Riverside. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided ample opportunities for student/postdoc training. During this review period, my group has trained five postdoctoral fellows, sixPh.D students, and sixundergraduate students. Graduate students and postdocswere given opportunities to train undergraduate research assistants andwrite/review manuscripts and research proposals.Theywere also given the opportunites toattend scientific conferences and grower meetings, including the APS annual conference (Cleveland, August 2019) and the International Research Congress of HLB(Riverside, March 2019).Postdoc trainees had the opportunities to attend workshops designed to facilitate career development. All researchers were trainedon the state-of-the-art technologies in molecular biology, biochemistry, bioengineering, and cell biology. The PIserved on advisory, guidance, qualifying exam, and dissertation committees for additionalgraduate students in the colleges of Natural and Life Science as well as Engineering. This project also supported herrole asthe continuing student advisor of the Genetics, Genomics and Bioinformatics graduate program. How have the results been disseminated to communities of interest?Findings obtained from this project were reported to related research community and commodities (citrus and potato).During the review period, data generated from this project have been reported in the form of journal publications (two papers published) and conference presentations (eight oral presentations by the PI, two oral presentations by postdoc/graduate students, and numerous poster presentations by all the participants of the project).This project involves extensive collaborations with UC Cooperative Extension specialists and USDA-ARS scientists. The research team works closely with stakeholders, especially citrus and potato growers, so that the research progress has beendisseminated to end-users in a timely manner. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Understanding the contribution of Sec-delivered Effectors (SDEs) of the HLB-associated pathogen to disease development We will continue our investigations on the virulence contribution of SDEs to citrus HLB progression. In particular, we have generated transgenic citrus plants expressing individual SDEs and/or their potential target proteins. These plants are currently characterized for altered response to Las infection. We will also conduct in-depth expression analysis of SDEs in different citrus varieties and at different infection stages to further understand how they may affect plant physiology. SDEs with interesting expression patterns will be further analyzed for target identification using proteomic approaches. Objective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectors Our recent exciting discoveries about PSR2 structure lead to many directions that warrant further investigation. Our on-going efforts include: 1) identify common target(s) of the Phytophthora effectors with PSR2-like repeat arrangements; 2) determine the virulence activities of these PSR2-like effectors; 3) elucidate the mechanisms underlying the RNA silencing suppression activity of PSR2; 4) understand the mechanisms driving the evolution of these PSR2-like effectors in Phytophthora with a focus on recombination-based domain shuffling.

Impacts
What was accomplished under these goals? Objective 1. Understanding the contribution of Sec-delivered Effectors (SDEs) of the HLB-associated pathogen to disease development Citrus industry is under unprecedented threats from HLB, a bacterial disease associated with Candidatus Liberibacter asiaticus (Las). Since Las is uncultivable, mechanistic insights into its virulence activities remain largely unknown. We take an "effectoromics" approach to address this pressing question by functional characterization of Sec-delivered effectors (SDEs) encoded by Las. We found the effector SDE1 inhibits specific papain-like cysteine proteases in citrus and promotes bacterial infection. This work was published in the journal Nature Communications (Clark et al., 2018) and represents the first virulence mechanism described in Las, which is a milestone in HLB research. A patent has been filed to employ genetic engineering technologies to modify selected cysteine proteases in order to enhance HLB resistance. We have also systematically analyzed the expression profiles of a set of SDEs that are conserved in all Las strains isolated from different geographic regions. This work has been submitted to the journal Molecular Plant Pathology for publications. Objective 2. Understanding the function and evolution of the PSR2 family of Phytophthora effectors Phytophthora species are important pathogens responsible for devastating diseases of major crops and forest trees. Each Phytophthora pathogen encodes a large number (hundreds to one thousand) of effectors, presumably for host manipulation. Understanding how these effectors contribute to virulence is essential to control Phytophthora diseases. My group is pioneered on the identification of Phytophthora effectors that promote infection by suppressing small RNA silencing in the hosts. In particular, our research has focused an effector called PSR2. During this review period, we continued to make groundbreaking discoveries on this line of research. Using PSR2 as a molecular probe, we discovered a specific class of plant small RNAs that can directly silence genes in the invading Phytophthora. As a pool, these sRNAs potentially silence multiple gene targets in the pathogen through a "shotgun" mechanism. These findings fill important conceptual gaps in host-induced gene silencing as a natural defense mechanism by defining the major sRNA executors and the evolutionary dynamics of the sRNA-producing sequences. This work was published in the journal Cell Host & Microbe (Hou et al., 2019) as a cover story with a commentary. In addition to functional analysis, we have also invesitaged effector evolution in the context of co-evolutionary arms race with plant hosts. By analyzing the protein structure of PSR2, we identified a conserved structural and functional module that is present in hundreds of Phytophthora effectors. This module is arranged as tandem repeats via a unique concatenation mechanism, which is also highly conserved. This research provides novel insight into the mechanism by which new virulence activities may be evolved. This work was published in the journal PNAS (He et al., 2019).

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. He, J., Ye, W., Choi, DS, Wu, B., Zhai, Y., Guo, B., Duan, S., Wang, Y., Gan, J., Ma, W.*, Ma, J.* (2019) Structural analysis of Phytophthora suppressor of RNA silencing 2 (PSR2) reveals a conserved modular fold contributing to virulence. Proc Natl Acad Sci USA 116: 8054-8059.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 2. Hou, Y., Zhai, Y., Feng, L., Karimi, H.Z., Rutter, B.D., Zeng, L., Choi, D.S., Zhang, B., Gu, W., Chen, X., Ye, W., Innes, R.W., Zhai, J., Ma, W.* (2019) A Phytophthora effector suppresses trans-kingdom RNAi to promote disease susceptibility. Cell Host & Microbe. 25: 153-165.