MOLECULAR CHARACTERIZATION OF PLANT-MICROBE INTERACTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017662
• Project Start Date: Oct 9, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Biochemistry & Molecular Biology

Non Technical Summary
Plant diseases cause crop loss, reduce food production and threaten global food security(Savary et al., 2012). Focusing on two distinct pathosystems that cause Fusarium vascular wilts and the Basil downy mildew (BDM), respectively, we proposal to establish apipeline to dissect host-pathogen interactions and provide novel means to develop disease resistant cultivars in order to manage plant diseases that threaten food security.Fusarium oxysporumspecies complex can cause vascular wilt on over 100 cultivated plant species (Beckman 1987, Moore et al. 2001), including the notorious Panama disease that nearly eliminated banana production during a 1950s outbreak(Koenig et al., 1997; Stover, 1972; Waite and Stover, 1960). Today, Panama disease remains one of the major diseases of banana worldwide(Fourie et al., 2011; Ploetz, 2006a, b). Because of its significance,F. oxysporumis listed among the top 10 fungal pathogens bythe journal "Molecular Plant Pathology"(Dean et al., 2012). In Massachusetts and New England area, wilt diseases caused byFusarium oxysporumare common agricultural problems that affect the production of vegetables, including asparagus, tomatoes, eggplant, ornamentals, and variety of flowers (Elmer WH and Ferandino FJ, 2009). Widespread resistance to existing fungicides and the persistence of thick-walled fungal clamidospores in the soil compound the difficulty of controlling these diseases and emphasizing the importance of appropriate control measures.Similarly, the Basil downy mildew (BDM), caused by the obligate oomycetePeronospora belbahrii,has devastated US production of sweet basil (Ocimum basilicum),one of the most important culinary herbs grown in the US and worldwide. The economic value of sweet basil as a potted herb was estimated to be approximately 15 million dollars annually(Wyenandt et al. 2015).In Massachusetts, basil is grown in fields, high-tunnels, and greenhouses for cut herb and potted plant sales. Fusarium wilt of basil caused byFusarium oxysporumf. sp.basilicumfirst emerged in Massachusetts in 1992.Basil downy mildew was first reported in the United States in Florida in the fall of 2007, and spread north to Massachusetts and the east coast in 2008, resulting in devastating crop losses.Since that time breeding programs have produced resistant cultivars, and seed testing has helped to reduce the introduction of inoculum.However, 100% of the US production acreage (~15,000) remains at-risk to BDM and Fusarium wilts, two economically important diseases. To control BDM, chemical treatments show promise under low disease pressure, but only offer partial relief to growers under high inoculum levels. Furthermore, no organic controls were found to be effective leaving all those involved in certified organic production without options (Wyenandt et al 2015).The development of resistant plants remains one of the most practical and cost-efficient strategies for managing such diseases, but this strategy is hampered by our limited understanding of the molecular mechanisms involved in host-pathogen interactions. Focusing on two important plant pathogens,F. oxysporumandP. belbahrii, we aim to dissect host-pathogen interactionscombining state of the art of genetic, biochemical and biophysical techniques. In addition, these two selected systems represent two distinct modes of action at host-pathogen interfaces, as Fusarium wilts attack plant roots and BDM infects plant leave tissues. BDM is an obligated biotrophic pathogen and Fusarium wilts have a biotrophic to necrotrophic transition. This comparative study will not only advance scientific knowledge, but also provide new avenues for disease management and the development of wilt resistant plants.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1499	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1040 - Molecular biology;

Keywords

plant-microbe interactions

fusarium oxysporum

Goals / Objectives
The goals of this proposal are to use cutting edge genetic, biochemical and biophysical techniques to understand molecular mechanisms that underpin the disease development of plant pathogens; to use these results to develop molecular markers for the functional dissection of fungal-plant interactions; and to characterize the molecular mechanisms of the identified fungal effectors and plant resistance proteins. These studies will also establish a pipeline for evaluating the molecular mechanisms of effectors, provide new insight into how plant pathogens infect host plants and have the potential to reveal new avenues for plant disease control. If accomplished, this project will not only advance scientific knowledge; the knowledge gained through such studies would also provide new avenues for disease management and the development of wilt resistance plants.

Project Methods
1:Reverse genetics to confirm the functional importance of key effectors:The expression of all selected candidate effectors will be confirmed using qRT-PCR. The split-marker approach(Catlett et al., 2003)will be the used to generate targeted mutants for confirmed candidate effectors in theF. oxysporumgenomes.At least three independenttransformantsof each mutant will bescreened for the presence of the marker and the absence of the target gene. All selected mutants will be assayed using the establishedF. oxysporum-Arabidopsispathosystem.They will also be tested fordefects in viability, mycelial growth, sporulation, germination and response to stress using establishedin vitroassays. For added assurance, we will follow up with a complementation assay in which a functional copy of the gene will be expressed in the mutant background. Since the transformation system is not established in thePeronospora belbahriisystem, only qRT-PCR will be performed for effectors.2:biochemical and biophysical characterization of effector proteinsin vitro: 2.1The effector of interest will be amplified using cDNA through RT-PCR to remove intronic sequences. The effector will be expressed under a constitutive promoter, tagged with a fluorescent protein (FP), e.g. monomeric enhanced green fluorescent protein (meGFP). The FP tag will be added at the C-terminus of the effector. Two strategies will be used to express the fluorescent protein tagged effectors.2.2 Gel-based biochemistry to characterize post-translational modifications:The focus of this set of experiments is to understand the mature and active form of a given effector and they are designed to detect disulfide bonds, glycosylation, phosphorylation, and protein-protein interactions.2.3 Biophysical characterizationof important effectors in vitro.This goal investigates targets of a candidate effector.Two environments, with (secretion of fungal expression system) and without (secretion of bacterial expression system) other factors from fungal secretions, will be used to study the interaction of the effector with plant cell membranes (protoplasts and membrane vesicles) using biophysical measurements.This set of experiments will address the binding properties with plant plasma membrane, specific lipids or protein binding partners.

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

Outputs
Target Audience: Please note that research and outreach were seriously affected by the pandemic. In particular, due to pandemic restrictions we were unable to work with high school students and research was delayed due both to lab closures from March to June, 2020 and the time required to restart research. In 2020 this project continued the efforts to target three types of audiences. Increase the awareness of wilt diseases in academia: PI Ma presented knowledge gained through this project at Clemson University and at SC20, the international conference for high performance computing, networking, storage and analysis held virtually Nov. 9-19, 2020. Disseminate genomics to vegetable growers, seed companies and other stakeholders: UMass served as host for the USDA/NIFA 2020 Basil Workshop: Managing Basil Under Increasingly Challenging Conditions. This workshop was originally conceived as a 30-40 in-person meeting focusing on basil research being performed at UMass, Rutgers University, Cornell University and the University of Florida. However, due to the pandemic, the meeting morphed into a virtual 2-day meeting held on Zoom December 10-11, 2020. This free workshop had over 200 registrants from across the US including home gardeners, commercial basil growers, extension agents, seed and chemical company representatives, and basil researchers. The meeting was organized, coordinated and hosted by the UMass team with 50-80 on-line participants depending on the session. The agenda and feedback from the workshop are included in the Appendix. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Kelly Allen: a graduate student who submitted a revised USDA fellowship application and a Lotta Crabtree Graduate Fellowship proposal using preliminary data collected though this project. Jacob Mamam: a graduate student who submitted a proposal to Lotta Crabtree Graduate Fellowship using some preliminary results collected through this research. Courtney McConnell, Hailey Aguiar, Marykate Rosano, Nathan Wilson, Peishan Xie, Sean Sullivan and Vista Sohrab: these undergraduates preformed research and received independent study credit. Biochem426, a Course-Based Undergraduate Research Experience (CURE): This laboratory based course is designed to provide BMB majors with an opportunity to conduct original research in a biochemistry laboratory. The ultimate goal of this research is to generate Fusarium strains expressing effector proteins tagged with green fluorescent protein (GFP) allowing researchers to use fluorescence microscopy techniques to characterize effectors and image how effectors are trafficked when Fusarium infects host plants. The course started with an intensive, 4 weeks boot-camp covering many of the basic approaches used in the biochemistry laboratory. For the rest of the semester, students worked on independent research projects to clone and characterize candidate Fusarium effectors. Several teams successfully transfected Fusarium with GFP tagged effectors. Equity & Inclusion in Plant Biology: Graduate student Kelly Allen was part of an ad-hoc group of graduate students in the interdepartmental graduate programs at UMass that worked over the summer to craft an actionable strategy to promote diversity, equity, and inclusion in the graduate programs and beyond. Kelly continues to serve on the Academic Equity subcommittee. In recognition of her work, Kelly was nominated for the CNS Excellence in Diversity and Inclusion Award, but she has chosen to decline the nomination so that a student from a marginalized community may instead be celebrated for their contributions. How have the results been disseminated to communities of interest?In 2020 this project continued the efforts to target two types of audiences. Increase the awareness of wilt diseases in academia: PI Ma presented knowledge gained through this project at Clemson University and at SC20, the international conference for high performance computing, networking, storage and analysis held virtually Nov. 9-19, 2020. Disseminate genomics to vegetable growers, seed companies and other stakeholders: UMass served as host for the USDA/NIFA 2020 Basil Workshop: Managing Basil Under Increasingly Challenging Conditions. This workshop was originally conceived as a 30-40 in-person meeting focusing on basil research being performed at UMass, Rutgers University, Cornell University and the University of Florida. However, due to the pandemic, the meeting morphed into a virtual 2-day meeting held on Zoom December 10-11, 2020. This free workshop had over 200 registrants from across the US including home gardeners, commercial basil growers, extension agents, seed and chemical company representatives, and basil researchers. The meeting was organized, coordinated and hosted by the UMass team with 50-80 on-line participants depending on the session. The agenda and feedback from the workshop are included in the Appendix. UMass talks at the 2020 Basil Workshop: Kelly S. Allen: Developing molecular markers for basil downy mildew resistance. Jacob Maman: Identification of a Fusarium isolate that bypasses resistance in the sweet basil cultivar 'Newton' While not part of this Hatch project, we also note that UMass Prof. Robert Wick also presented a talk titled "Reflections on Fusarium Wilt Disease on Sweet Basil". And Prof. Wick was honored for his decades of work on basil diseases beginning in the 1990s with his discovery of the first documented instance of Fusarium wilt on basil in the US. What do you plan to do during the next reporting period to accomplish the goals?We have hired a highly experenced postdoc Domingo Martinez Soto to lead this project.

Impacts
What was accomplished under these goals? Based on the defined course of infection, we have generated transcriptomics data to compare pathogenic versus non-pathogenic interactions using twoF. oxysporumstrains, Fo5176 and Fo47 and Arabidopsis, to investigate plant immunity againstFusariumwilts. Arabidopsis (Col-0) is highly susceptible to the Fo5176 strain, but is asymptomatic when inoculated with Fo47. Using next-generation sequencing, we have sequenced and analyzed the transcriptomes ofA. thalianaplants infected with these two strains respectively. Hierarchical clustering based onA. thalianagene expression identified 30 gene clusters responding toF. oxysporuminfection. Modules 3 (C3) and 14 (C14) are significantly enriched for genes involved in plant immunity and biotic stress responses. Particularly, module C14 has a distinctive expression pattern in responding to the two different strains. We have collected sufficient evident to support the conclusion that the biocontrol F. oxysporum strain induces a programmatic response that promote plant growth and defense through the nitrate signaling pathway. Based on this conclusion, we could develop molecular markers for the functional dissection of the fungal-plant interactions. In 2020 we also published the genome ofF. oxysporumf. sp. matthiolae a pathogen that infects Arabidopsis and other Brassicaceae species (reference 3 below). We have recently taken a similar approach toF. oxysporumstrains that infect sweet basil (Fob). We screened Fob strains collected by UMass Prof. Robert Wick to determine if any of them are capable of infecting sweet basil cultivars that are resistant to Fob. We recently identified a single strain that infects both a susceptible and a resistant sweet basil cultivar. We are currently collecting samples and submitting genomic DNA and RNA for genomics and transcriptomics analysis, respectively. These datasets will allow us to perform similar analyses to those described above for Arabidopsis. Goal 1:Reverse genetics to confirm the functional importance of key effectors Due to Covid, we were not able to make any progress toward this goal.? Goal 2:biochemical and biophysical characterization of effector proteinsin vitro This year, theBiochem 426 (see below) were extremely successful -- a total of 8 effectors were cloned usingeither mox-green fluorescent protein (mox-GFP), a GFP variant optimized for folding in the endoplasmic reticulum (ER), or mox-GFP-HDEL, a variant to be captured within ER or both.we have successfully expressed mox-GFP tagged protein in Fusarium usingan Agrobacterium based transformation system and visualized usingfluorescent microscope.Note that this research was performed by the teaching assistants and undergraduates. The next steps are to confirm that the tagged effectors are successfully secreted andto monitor effector trafficking and localization during plant infection and to biophysically characterize the tagged effectors. We have also established aPichia pastorisbased system for secretion and purification of effectors containing 6XHis tags. This yeast-based system will allow us to purify and biochemically characterize effectors. Goal 3: in vivocharacterization of effector-plant interactions Developing a live-image system is the most critical step toward the goal of this aim. We are moving forward with two directions: 1) We have collected several organelle-specific Arabidopsis marker lines to see co-localization signals; 2) we have aquired a confocal microscope for easy access.?

Publications

• Type: Book Chapters Status: Published Year Published: 2020 Citation: Viljoen, A., Ma, L.-J., & Molina, A. B. 2020. Fusarium wilt (Panama disease) and monoculture banana production: Resurgence of a century-old disease. In A. Records & J. Ristaino (Eds.), Emerging plant diseases and global food security. St Paul: APS Press.159-184.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yang H, Yu H, Ma L.-J. 2020. Accessory Chromosomes in Fusarium oxysporum. Phytopathology 110:1488-1496.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yu H!, Ayhan DH!, Diener A., Ma L-J. 2020. Genome Sequence of Fusarium oxysporum f. sp. matthiolae, a Brassicaceae Pathogen. Molecular Plant-Microbe Interactions 33(4): 569-572. https://doi.org/10.1094/MPMI-11-19-0324-A

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

Outputs
Target Audience:This functional genomics project "Molecular characterization of plant-microbe interactions" investigate molecular interaction between plant wilt pathogen Fusarium oxysporum and two plant hosts: Arabidopsis andsweet basil (Ocimum basilicum). WhileArabidopsis represents the best studied model plant,sweet basilone of the most important culinary herbs grown in the US and worldwide. The goals are to use cutting edge genetic, biochemical and biophysical techniques to understand molecular mechanisms that underpin the disease development of plant pathogens; to use these results to develop molecular markers for the functional dissection of fungal-plant interactions; and to characterize the molecular mechanisms of the identified fungal effectors and plant resistance proteins. These studies will also establish a pipeline for evaluating the molecular mechanisms of effectors, provide new insight into how plant pathogens infect host plants and have the potential to reveal new avenues for plant disease control. If accomplished, this project will not only advance scientific knowledge; the knowledge gained through such studies would also provide new avenues for disease management and the development of wilt resistance plants. This period of time, this project continued the efforts to target three groups of audiences. * Increase the awareness of wilt diseases in academia:PI Ma presented the knowledge gained through this project at regional, national and international meeting, including a plenary talk at the30thFungal Genetic Conferenceand visited several college campuses includingHarvard University. Cambridge,MA, USA. , Smith College, Purdue university and University of Helsinki, Helsinki Finland. * Disseminate genomics to vegetable growers:Graduate student Kelly Allan supported by this Hatch project presented her research at New Jersey Vegetable growers annual meeting and reached out to this group of audience. * Reach out to high school students:PI Ma organized a 2-week long summer high school course to disseminate knowledge to high schoolers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PI Ma developed a teaching model derived from this project to teach 24 undergraduate student and taughtEleven high school students who participated in the summer pre-college course. Specifically: Kelly Allan: a graduate student who submitted a USDA fellowship proposal using preliminary data collected though this project; Jacob Mamam: a graduate student who submitted a proposal to Lotta Crabtree Graduate Fellowship using some preliminary results collected through this research; Mladen Petreš: a Fulbright visiting graduate student from University of Novi Sad in Serbia conducted research outlined in this project; Peishan Xie: an undergraduate student supported by the CAFÉ scholar fund to work on this research over the summer Hailey Aguiar, Nathan Wilson and Sean Sullivan: undergraduate students work on this research project through independent study. How have the results been disseminated to communities of interest?We have established a long term collaboration with a local organic grower DeWitt at happy valley organics. What do you plan to do during the next reporting period to accomplish the goals?More emphasis will be given to clone candidate effectors.

Impacts
What was accomplished under these goals? Goal 1:Reverse genetics to confirm the functional importance of key effectors This year, we have taken several approaches to accomplish this objective. In addition to generate additional mutants using established transformation system, we adopted CRISPR-Cas9 protocol (Pham et al unpublished), which will allow us to target out multiple effectors to overcome potential functional redundancy. Focusing on regulators of the candidate effectors, we started our focus on kinases (DeIulio et al 2018)/ We will continue this effort into transcription factors. To increase the sensitivity, we are in the mode of assay development. We have tried multiple soil-independent methods and are still fine tuning parameters to finalize on a robust and revealing phenotyping assay. Goal 2:biochemical and biophysical characterization of effector proteinsin vitro We are testing both Fusarium and bacterial expression systems. The transformation of GFP tagged effector usingAgrobacterium was successful, unfortunately no fluorescent signal. We are improving the construct. Concerning the correct folding of cysteine-rich effectors using bacterial expression system, we are exploring other eukaryotic expression systems at the moment. Goal 3: in vivocharacterization of effector-plant interactions Developing a live-image system is the most critical step toward the goal of this aim. We are moving forward with this aim, but made minimal progress.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Yu H., Ayhan DH, Diener AC, and Ma L.-J. 2020, Genome Sequence of Fusarium oxysporum f. sp. matthiolae, a Brassicaceae Pathogen. MPMI submitted.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Liu S, Lin J, Zhang Y!, Liu N, Viljoen A, Mostert D, Zuo C, Hu C, Bi F, Gao H, Sheng O, Deng G, Yang Q, Dong T, Dou Tongxin, Yi G*, Ma L-J* and Li C*. Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4. New Phytologist (available online: doi.org/10.1111/nph.16193
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ma, L-J* and Xu JR. 2019. Shuttling effector genes through mini-chromosomes. PLoS Genet 15(9): e1008345. https://doi.org/10.1371/journal.pgen.1008345
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gao S, Gold SE, Wisecaver JH, Zhang Y!, Guo L!, Ma L-J, Rokas A, Glenn AE*. 2019. Genome-wide analysis of Fusarium verticillioides reveals inter-kingdom contribution of horizontal gene transfer to the expansion of metabolism. Fungal Genetics and Biology 128, 60-73.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Milo-Cochavi S, Pareek M, Delulio G!, Almog Y, Anand G, Ma L-J, Covo S*. 2019. The response to the DNA damaging agent methyl methanesulfonate in a fungal plant pathogen. Fungal Biology 123 (5), 408-422
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Anand G, Waiger D, Vital N, Maman J!, Ma L-J, Covo S*. 2019. How does Fusarium oxysporum sense and respond to nicotinaldehyde, an inhibitor of the NAD+ salvage biosynthesis pathway? Frontiers in Microbiology 10, 329