Source: UNIV OF MARYLAND submitted to NRP
DECIPHERING THE MECHANISM OF A BROAD-SPECTRUM RESISTANCE GENE AGAINST FUSARIUM GRAMIN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1023904
Grant No.
2020-67013-32558
Cumulative Award Amt.
$499,998.00
Proposal No.
2020-08218
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2025
Grant Year
2020
Program Code
[A1171]- Plant Biotic Interactions
Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
PSLA
Non Technical Summary
Plant diseases cause huge losses to global agricultural production. Understanding the mechanism of resistance to major pathogens of crop plants is essential to ensure food security for the ever-increasing human population. Fusarium graminearum, regarded as one of the top 5 fungal pathogens of plants by plant pathologists worldwide, is a serious threat to global food security, and food safety. Overarching goal of this proposal is to investigate the mechanism of plant resistance against necrotrophic and hemi-biotrophic pathogens, which, to date, is poorly understood. Using a 'Pore-forming toxin-like' (PFT) protein, the molecular components of plant quantitative resistance against this category of pathogens will be deciphered. Mechanistic investigations on PFT will be carried out using Arabidopsis, Nicotiana, and wheat plant systems with the following objectives: 1. Study of the unconventional trafficking of PFT in plants: PFT is a leaderless protein without any secretory peptide signal, but is found in the apoplast. We will characterize the non-traditional trafficking pathway of PFT using co-localization with cell organelles and chemicals affecting cellular trafficking of proteins. 2. Temporal and spatial dimension of PFT-F. graminearum interaction will be studied by correlating the transcript levels with protein levels of PFT in developmental stages of wheat spikes: the infection court of F. graminearum. 3. Interaction of PFT with other pathogens will be studied for analyzing the specificity of recognition of Fusarium spp. With PFT. The knowledge generated in this proposal on the mechanism of a broad-spectrum resistance protein will facilitate development of plant resistance against hemi-biotrophic and necrotrophic pathogens.
Animal Health Component
5%
Research Effort Categories
Basic
90%
Applied
5%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011549103040%
2021549116060%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1030 - Cellular biology; 1160 - Pathology;
Goals / Objectives
In this proposal, we will investigate the physiological, spatial and temporal dimensions of the interaction of a broad-spectrum resistance gene (PFT) with fungal pathogen Fusarium graminearum to gain understanding of plant resistance mechanism against hemibiotrophic/necrophic pathogens. The major goals of the project are:Study of the unconventional trafficking of PFT in plants: PFT does not contain any secretory peptide signal, however, our preliminary results suggest that it is synthesized in the cytoplasm and is secreted outside the cells in the apoplast. This classifies PFT in the interesting category of leaderless proteins, which have not yet been fully explored in plants[58]. In this objective we will characterize exact mode of the unconventional trafficking of PFT.Temporal and spatial dimension of PFT-F. graminearum interaction: Based on qPCR studies PFT expression was found to be highest in spike tissues at pre-emergence and falls sharply (~1000 times) thereafter. This stage precedes anthesis, at which wheat plants are most susceptible to infection by F. graminearum. In this objective we will determine if the high levels of mRNA before emergence translate to high protein levels at anthesis, when the growth of the fungal pathogen needs to be arrested. The temporal dimension of this interaction will also be investigated at sub-cellular level.Interaction of PFT with other pathogens: Gene ontology searches define the function of PFT as carbohydrate binding and pathogenesis. In silico analysis showed specific residues in the protein bind to glycan monomers of fungal membrane as potential ligands. However, the specificity of this interaction is to Fusarium is not known. In this objective we will use transgenic Arabidopsis plants to study the response of various fungal pathogens (ranging from strictly obligate powdery mildew to necrotrophic Botrytis cinerea) against PFT. This goal will be helpful in determining if PFT can be used to design resistance in other plants against different fungal pathogens in future.
Project Methods
Objective 1: Study of unconventional trafficking of PFT in plants:For co-localization studies in Nicotiana, Agrobacterium transformants with labelled proteins of trans-golgi network (SYP61 and SYP42), vacuole (VIT1, Vac1), endosomes (syp121), and plasma-membrane (Lti6b) (available from Dr. Shunyuan Xiao, please see letter of collaboration) will be used in conjunction with GFP/RFP labeled PFT. The extra-cellular localization of PFT will be confirmed by studying its co-localization with RFP-fused to signal peptide. Transformed Agrobacterium colonies with labelled organelle proteins will be grown in LB medium amended with antibiotics followed by centrifugation. Cell pellets from both the cultures will be dispended in infiltration media and will used for co-infiltration on six weeks old Nicotiana leaves using a needle-less syringe. Leaves will be examined 36 h post infiltration using Zeiss LSM 710 laser spectral scanning confocal microscope available in UMD Core Imaging Facility (See letter of support). GFP and RFP will be excited at 488 and 543 nm, respectively and emissions will be captured at 505-530 nm (GFP) and 565- 615 nm filter (RFP). The chloroplast autofluorescence will be recorded at 650-750 nm. The samples will be scanned at 8-40 optical sections. All the images will be processed with ZEN Lite 2012 software and Adobe photoshop.Objective 2: Temporal and spatial dimension of PFT-F. graminearum interaction:Correlation between protein levels and the transcript levels of PFT will be studied in the wheat spikes with and without F. graminearum infection. Total mRNA and total proteins will be extracted from F. graminearum inoculated versus non-inoculated spikes of R-NIL and PFT1958 knock-out mutants at the seven stages shown in Figure 8 from initiation of spike all the way up to anthesis. Spikes from these seven stages will be cut with a chilled pair of sharp scissors and flash frozen in liquid nitrogen. Tissue will be ground into fine powder using mortar-pestle with liquid nitrogen and will be divided into two fractions: 1) RNA fraction and 2) Protein fraction.RNA will be extracted from first fraction using Trizol reagent following Rawat et al.[12] and clean-up will be done using RNeasy Mini Kit followed by quantification using Spectrostar Nano. cDNA synthesis will be done using High-Capacity cDNA Reverse Transcription Kit followed by quantitative real-time PCRs on a Bio-Rad CFX96 Real-Time PCR Detection System. Actin and β-tubulin will be used as internal controls. All samples will have at least three biological replicates and will be run in three technical replicates. Three independent qPCR runs will be performed. The expression levels of PFT will be calculated using the 2−ΔCT method.Total protein from will be extracted from the protein fraction powder prepared above using FOCUS™ Plant Proteome extraction kit (G Biosciences). Briefly, solubilization buffer will be mixed with 100mg tissue powder, followed by centrifugation at 20,000g for 30 minutes. Lysate will be collected and used for total protein quantification using Bradford assay. Quantification of PFT protein will be done by western blotting with polyclonal antibodies developed in Objective 1. The patterns of RNA and protein levels will give us insight into the temporal dynamics of PFT- F. graminearum interaction in wheat plants.Objective 3. Interaction of PFT with other pathogens:We will use transgenic GFP-PFT and RFP-PFT Arabidopsis plants stably expressing PFT and already tested to show resistance response against F. graminearum. Selected plants will be inoculated with different fungal pathogens of Arabidopsis, including biotrophic powdery mildew fungus Golovinomyces cichoracearum (Gc) and necrotrophic fungus Botrytis cinerea. For powdery mildew inoculation Gc isolate UCSC1 will be obtained from collaborator Dr. Shunyuan Xiao and his lab protocol will be used. Briefly, Arabidopsis plants at six to eight leaf stage will be inoculated by brushing conidia of Gc- UCSC1 maintained on infected eds1-2 plants to ensure uniform spread of conidia on the leaf surfaces. Plants will be watered before inoculation to avoid running off of spores from leaves. Plants will be scored visually at 8, 12, and 16 days after inoculation. Microscopic examination will be done following Xiao lab protocol. Control treatments will include non-transgenic Arabidopsis plants. Statistical analysis of results will be performed to study the patterns of response of plants with PFT versus without PFT.Necrotrophic fungus Botrytis cinerea was obtained from Dr. Mengjun Hu, small fruit pathologist at UMD. Inoculation with B. cinerea will be performed according to Camera et al.[77]. Briefly, B. cinerea will be cultured on PDA, incubated for 2 weeks in dark at 25 oC and spores will be harvested in water. Spores will be filtered using glass wool and suspended in PDB. Six µl spore solution (5x104 spores/ml) will be used to inoculate four leaves of 6 week old plants each. Plants will be kept in high humidity condition and disease will be scored as area of necrosis at 8, 12 and 16 days after inoculation. Non-transgenic Arabidopsis plants will be included as controls in all the experiments. Statistical analysis of results will be performed to study the patterns of plant reaction to PFT.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Target audience includes: plant scientists, researchers, students, farmers, and stakeholders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Six high school students, four undergraduate students, one PhD student, one visiting scientist, and a postdoctoral scientist were trained in the project. The PhD student and postdoctoral researcher gained experience as mentors tohigh school and undergraduate students. All participants had opportunities to present their work as posters or oral presentations at various platforms, includinglocal/regional symposia, and national and international conferences. How have the results been disseminated to communities of interest?The results have been communicated through oral and poster presentations, as well as through publications. What do you plan to do during the next reporting period to accomplish the goals?We plan to test the transgenic plants with a diverse set of pathogens in replicated experiments

Impacts
What was accomplished under these goals? Arabidopsis plants expressing PFT were challenged with 4 different fungal pathogens, including Fusarium graminearum, Botrytis cinerea, Colletotrichum higginsianum, and Sclerotinia sclerotiorum. The T1, T2 and T3 plants expressing the PFT protein were found to be resistant to all four fungal pathogens. However, the plants did not show resistance to either bacterial or Oomycete pathogens. To investigate the reason for the specific response to the fungal pathogens, we expressed the protein with an N-terminal His-tag in Nicotiana andisolated the protein using affinity chromatography. The purified protein was hybridized to a 300-glycan microarray. The protein was found to specifically bind with oligomers containing N-acetylglucosamine, which is the monomer of chitin, indicating specific resistance to fungal pathogens in transgenic plants. These results were published in MPMI (Singh et al. 2023). Further, the PFT gene was transgenically expressed in tomato and strawberry plants. The tomato transgenic plants were challenged with two soil-borne pathogens, Fusarium oxysporum and Verticillium dahliae, and two foliar pathogens, Alternaria linariae and Colletotrichum gloeosporioides, in T1 and T2 generations.The plants stably expressing PFT were found to be significantly resistant to all four pathogens tested. Strawberry plants were challenged with two foliar fungal pathogens, Botrytis cinerea and Colletotrichum acutatum, and were found to be resistant to both fungal pathogens. In conclusion, wheat PFT provides resistance against a broad spectrum of fungal pathogens transgenically independent of the plant background. We are currently preparing this manuscript for submission. His-tagged PFT protein was transiently expressed in Nicotiana, followed by its extraction and purification. The purified PFT protein was incubated with fungal spores, and it was found to inhibit spore germination, ultimately leading to their disintegration. The concentration of the purified PFT protein was directly related to the degree of spore germination inhibition and subsequent spore death. Multiple fungal pathogens were tested for their response to the PFT fungicidal activity, and all were found to be disintegrated at different protein concentrations. These experiments showed that PFT has a direct broad-spectrum fungicidal activity.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Yadav, I.S., Rawat, N., Chhuneja, P., Kaur, S., Uauy, C., Lazo, G., Gu, Y.Q., Dole~el, J., Tiwari, V.K., 2023. Comparative genomic analysis of 5Mg chromosome of Aegilops geniculata and 5Uu chromosome of Aegilops umbellulata reveal genic diversity in the tertiary gene pool. Frontiers in Plant Science. 14.
  • Type: Other Status: Published Year Published: 2024 Citation: Rawat, N. Designing new strategies for disease resistance in crop plants. Invited talk at Motilal Nehru National Institute of Technology, Allahabad, UP India Aug 6, 2024.
  • Type: Other Status: Published Year Published: 2023 Citation: Rawat, N. Designing novel genetic solutions to the classic fungal pathogens of wheat. Plant Biology core seminar series. Invited talk at Rutgers University, New Brunswick, NJ. Sep 29, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rawat, N. Making sense of non-sense: Using mutations for wheat and barley improvement. Plant and Animal Genome Conference, San Diego, CA. Jan 16, 2023.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience: Scientific community: We published 8papers in thisannual reporting period. In addition, we gave 6academic talks about the work in the project at universities and national and regional conferences. One female undergraduate student was trained in the project. Four female minority high school students receivedsummer training in the project. Small grain farmers and stakeholders: The PD delivered talks (3) to the farmers and stakeholders informing them about theresearch. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This year, the project provided hands-on training as well as professional development opportunities to five minority high school students, and two PostDocs. One PhD student has already completed his degree working on the project last year. How have the results been disseminated to communities of interest?The results were communicated as peer-review journal articles, invited talks and poster presentations. 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 of the unconventional trafficking of PFT in plants:PFT RFP and GFP labelled PFT constructs were developed in E. coli and transferred into Agrobacterium. Transient expression was performed in Nicotiana and stable Arabidopsis transgenic lines were developed. Confocal microscopy localized majority of the protein expression in the cytoplasm and nucleus and some signal in the aploplastic spaces. After plasmolysis RFP/GFP labelled strands, characterizing PFT protein were observed in the apoplastic spaces. To robustly distinguish them from Hechtian strands, new vectors with known apoplastic and membrane-bound protein (Mlo locus) labelled with fluorescent tags were procured to use as control in the analysis. Transient expression analysis with the new tracking markers showed PFT being released as membrane-bound vesicles pinching off from the cytoplasm into the apoplast. Stable transgenic lines with GFP-PFT and RFP-Mlo were developed. Further work on systematic visualization using these labelled control proteins in Nicotiana and Arabidopsis (apoplastic and membrane-bound) is underway. Temporal and spatial dimension of PFT-F. graminearuminteraction:To quantify protein levels of PFT in wheat spikes at different time points, we collected tissues from resistant wheat plants at different stages. Three polyclonal antibodies with conserved PFT epitope were generated in mice, and the one providing highest binding with PFT from E.coli was selected for carrying out protein quantification on wheat tissue. Wheat tissue was collected at seven growth stages after flowering. However, the western blotting experiments revealed non-specific binding with RUBISCO in the plant samples. To quantify the PFT protein levels, and simultaneously correlate it with PFT mRNA levels, we are using an LC-MS-Ms based strategy now. We are running pilot experiments to test if the strategy will work specifically for out protein of interest. After, which the actual samples will be analyzed in multiple replications. Interaction of PFT with other pathogens:Wheat PFT was ectopically expressed in model plant Arabidopsis. Stable homozygous lines expressing PFT were found to resist fungal pathogens Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, and Collectotrichum higginsianum. However, these lines were not resistant to bacterial pathogen Pseudomonas syringae, or Oomycete pathogen Phytophthora capsici, indicating that PFT protein is effective only against fungal pathogen. Glycan micro-array binding experiment showed that PFT specifically binds to the chitin monomer N-Acetyl Glucosamine (NAG). The resultswere published in Molecular Plant Microbe Interactions journal. Work on expressing the PFT protein in other plants such as tomato and Strawberry is in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Arora, G., Steed, A., Goddard, R., Gaurav, K., O'Hara, T., Schoen, A., Rawat, N., Elkot, A.F., Chinoy, C., Nicholson, M.H., Asuke, S., Steuernagel, B., Yu, G., Awal, R., Forner-Martinez, M., Wingen, L., Baggs, E., Clarke, J., Krasileva, K.V., Tosa, Y., Jones, J.D.G., Tiwari V.K., Wulff, B.B.H., Nicholson, P. (2023). A wheat kinase and immune receptor form the host-specificity barrier against the blast fungus. Nature Plants DOI: https://doi.org/10.1038/s41477-023-01357-5.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Designing novel genetic solutions to the classic fungal pathogens of wheat. Plant Biology core seminar series. Rutgers University, New Brunswick, NJ. Sep 29, 2023
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Schoen, A., Yadav, I., Wu, S., Poland, J., Rawat, N., Tiwari, V.K. (2023). Identification and high-resolution mapping of a novel tiller inhibition gene (tin6) by combining forward genetics screen and MutMap approach in bread wheat. Functional & Integrative Genomics. 23:157. https://doi.org/10.1007/s10142-023-01084-2
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yadav, I.S., Singh, N., Wu, S., Raupp, J., Wilson, D.L., Rawat, N., Gill, B.S., Poland, J., Tiwari, V.K., 2023. Exploring genetic diversity of wild and related tetraploid wheat species Triticum turgidum and Triticum timopheevii. J. Adv. Res. 48, 4760. https://doi.org/10.1016/j.jare.2022.08.020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Making sense of non-sense: Using mutations for wheat and barley improvement. Plant and Animal Genome Conference, San Diego, CA. Jan 16, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Genome-wide discovery of susceptibility factors for Fusarium head blight in wheat. APS Potomac Division meeting, Fairfax, Virginia. Mar 23, 2023.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Singh, L., Sinha, A., Gupta, M., Xiao, S., Hammond, R., Rawat, N.* (2023). Wheat Pore-forming toxin-like protein confers broad-spectrum resistance to fungal pathogens in Arabidopsis. Molecular Plant-Microbe Interactions, 36:489501.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ahmed, H.I., Heuberger, M., Schoen, A., Koo, D.-H., Quiroz-Chavez, J., Adhikari, L., Raupp, J., Cauet, S., Rodde, N., Cravero, C., Callot, C., Lazo, G.R., Kathiresan, N., Sharma, P.K., Moot, I., Yadav, I.S., Singh, L., Saripalli, G., Rawat, N., Datla, R., Athiyannan, N., Ramirez-Gonzalez, R.H., Uauy, C., Wicker, T., Tiwari, V.K., Abrouk, M., Poland, J., Krattinger, S.G., 2023. Einkorn genomics sheds light on history of the oldest domesticated wheat. Nature 19. https://doi.org/10.1038/s41586-023-06389-7
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kajla, A., Schoen, A., Paulson, C., Yadav, I.S., Neelam, K., Riera-Lizarazu, O., Leonard, J., Gill, B.S., Venglat, P., Datla, R., Poland, J., Coleman, G., Rawat, N., Tiwari, V., 2023. Physical mapping of the wheat genes in low-recombination regions: radiation hybrid mapping of the C-locus. Theor. Appl. Genet. 136, 159. https://doi.org/10.1007/s00122-023-04403-0
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Schoen, A., Wallace, S., Holbert, M.F., Brown-Guidera, G., Harrison, S., Murphy, P., Sanantonio, N., Van Sanford, D., Boyles, R., Mergoum, M., Rawat, N., Tiwari, V., 2023. Reducing the generation time in winter wheat cultivars using speed breeding. Crop Sci. 63, 20792090. https://doi.org/10.1002/csc2.20989
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Saripalli, G., Adhikari, L., Amos, C., Kibriya, A., Ahmed, H.I., Heuberger, M., Raupp, J., Athiyannan, N., Wicker, T., Abrouk, M., Wallace, S., Hosseinirad, S., Chhuneja, P., Livesay, J., Rawat, N., Krattinger, S.G., Poland, J., Tiwari, V., 2023. Integration of genetic and genomics resources in einkorn wheat enables precision mapping of important traits. Commun. Biol. 6, 114. https://doi.org/10.1038/s42003-023-05189-z


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Target audience was the scientific community and wheat commodity boards. The disseminationwas achieved through peer-reviewed research publications,invited talks and poster presentations. Talks: Fighting the fungal foes of wheat. Punjab Agricultural University, Ludhiana, India. Aug 4, 2022. Fighting the fungal foes of wheat. Regional Center for Biotechnology, Faridabad, India. Aug 2, 2022. Investigation of the resistance provided by a wheat pore-forming toxin-like protein against fungal pathogens. College Park, MD. May 26, 2022. An approach for fine mapping genes from the tertiary gene pool members of wheat. Plant and Animal Genome Conference, San Diego, CA. (held virtually) Jan 9, 2022. Determine the best systems approach to managing Fusarium head blight and vomitoxin levels in wheat and barley. Maryland Grain Producers and Utilization Board.Virtual. Jan 6, 2022 Peer-reviewed Poster and Abstract: 1. Wheat Pore-forming toxin-like protein confers a broad-spectrum resistance against multiple fungal pathogens in Arabidopsis. In The Annual National Fusarium Head Blight forum, Tampa, Forida Dec 6, 2022. The Post Doc Megha Gupta won best poster award for this presentation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided hands-on training as well as professional development opportunities to five minority high school students, one PhD student, and two PostDocs. The PhD student completed his degree working on the project. How have the results been disseminated to communities of interest?The results were communicated as peer-review journal articles, invited talks and poster presentations. 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. Study of the unconventional trafficking of PFT in plants:RFP and GFP labelled PFT constructs weredeveloped in E. coli and transferred into Agrobacterium. Transient expression was performed in Nicotiana ans stable Arabidopsis transgenic lines were developed.Confocal microscopy localized majority of the protein expression in the cytoplasm and nucleus and some signal in the aploplastic spaces. After plasmolysis RFP/GFP labelled strands, characterizing PFT proteinwere observed in the apoplastic spaces. However, to robustly distinguish them from Hechtian strands, new vectors with known apoplastic and membrane-bound proteins labelled with fluorsecent tagswere procured to use as control in the analysis. Further work on the visualization using these labelled control proteins(apoplastic and membrane-bound) is underway. 2. Temporal and spatial dimension of PFT-F. graminearuminteraction:Three polyclonal antibodies with conserved PFT epitope were generated in mice, and the one providing highest binding withPFT from E.coli was selected for carrying out protein quantification on wheat tissue. Wheat tissue was collected at seven growth stages after flowering. However, the western blotting experiments revealed non-specific binding with RUBISCO in the plant samples. Therefore, new monoclonal antibodies have been ordered now, which are expected to provide highly specific binding to the protein of interest. Confirmation of specific binding will be performed on plant extracted PFT samples. RNA and protein expression levels will be analyzed in different stages of wheat samples with the new specificmonoclonal antibody. 3.Interaction of PFT with other pathogens:Wheat PFT was ectopically expressed in model dicot plant Arabidopsis. The heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including, F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. For exploring the reason for the resistance response to exclusively the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-Acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or Oomycetes. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Arora, G., Steed, A., Goddard, R., Gaurav, K., O'Hara, T., Schoen, A., Rawat, N., Elkot, A.F., Chinoy, C., Nicholson, M.H., Asuke, S., Steuernagel, B., Yu, G., Awal, R., Forner-Martinez, M., Wingen, L., Baggs, E., Clarke, J., Krasileva, K.V., Tosa, Y., Jones, J.D.G., Tiwari V.K., Wulff, B.B.H., Nicholson, P. (2022). A wheat kinase and immune receptor form the host-specificity barrier against the blast fungus. BioRxiv DOI: https://doi.org/10.1101/2022.01.27.477927
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Multifaceted aproaches to control Fusarium head bligfht in wheat: Genetuc mapping, mechanistic studies and fungicide efficacy analyses
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Sinha, A. , Singh, L. , Rawat, N.* (2022). Current understanding of atypical resistance against fungal pathogens in wheat. Current Opinion in Plant Biology, 68: 102247.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Rawat, N., CMJ Pieterse. (2022). Editorial overview: Dialogues with the good, the bad, and the ugly.Current Opinion in Plant Biology, 69: 102295
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lin, G., Chen, H., Tian, B., Sehgal, S.K., Singh, L. , Xie, J., Rawat, N., Juliana, P., Singh, N., Shrestha, S., Wilson, D.L., Shult, H., Lee, H., Schoen, A.W., Tiwari, V.K., Singh, R.P., Guttieri, M.J., Trick, H.N., Poland, J., Bowden, R.L., Bai, G., Gill, B.S., Liu, S. (2022). Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii. Nature Communications 13, 3044.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ayalew, H., Anderson, J.D., Krom, N., Tang, Y., Butler, T.K., Rawat, N., Tiwari, V.K., Ma, X.F. (2022) Genotyping-by-sequencing and genomic selection applications in hexaploid triticale. G3 Genes|Genomes|Genetics. DOI: 10.1093/g3journal/jkab413.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Target audience served within the report period are: Scientific community: We published 4 papers related to the scope of the work (Chhabra et al. 2021a; Chhabra et al. 2021b; Steadham et al. 2021; Wallace et al. 2021). In addition, the PD gave 7 academic talks about the work in the project in universities and national and regional conferences. One female undergraduate student was trained in the project. One high school student is receiving summer training in the project. Small grain farmers and stakeholders: The PD delivered talks (3) to the farmers and stakeholders informing them about the project. Changes/Problems:Covid-19 restrictions delayed hiring of the PostDoc in the project. Some experiments had to be stopped in the middle because of University closures. Strategies have been implemented by the PD to make up for the delays. What opportunities for training and professional development has the project provided?1. Undergraduate training 2. Graduate student training 3. Delivered invited talks to academic institutions on the project goals and outcomes that were attended by undergraduate, graduate, and postdoctoral scientists. How have the results been disseminated to communities of interest?The results were disseminated to communities of interest through publications and talks: For the Journal Articles, please refer to the Products section. Invited talks: 1. Fighting the fungal foes of wheat. Spring Semester Plant Sciences seminar series. Department of Plant Sciences and Landscape Architecture, University of Maryland, College Park, MD. Virtual. April 26, 2021. 2. Fighting the fungal foes of wheat. Plant sciences spring semester series. Department of Biology, University of Pennsylvania, Philadelphia, PA. Virtual. Jan 14, 2021. 3. Mechanistic Investigation into the PFT Fusarium graminearum Interactions. US Wheat and Barley Scab Initiative. Virtual. Dec 8, 2020. 4. Developing multi-pronged strategies to overcome fungal pathogens of wheat. Fall semester Plant Science Seminar series, Department of Crop and Soil Sciences, North Carolina State University, Raleigh, NC. Virtual. Sep 2, 2020. 5. Mechanistic insights into the broad-spectrum resistance of wheat Pore-forming toxin-like protein. Plant and Animal Genome Conference, San Diego, CA.2020. 6. Determine the best systems approach to managing Fusarium head blight in wheat. NJDelMArVa annual meeting- Annual regional meeting of Plant Pathologists from New Jersey, Delaware, Maryland and Virginia. Virtual. Feb 9, 2021. 7. Integrating the pieces together for managing Fusarium head blight of wheat and barley. MidAtlantic Crop Management School. Georgetown, DE. Virtual Nov 18, 2020. Extension talks: 1. Determine the best systems approach to managing Fusarium head blight in wheat and barley. Maryland Commodity Classic. Queen Anne's County MD USA. Jul 22, 2021. 2. Determine the best systems approach to managing Fusarium head blight in wheat and barley. Maryland Grain Producers and Utilization Board meeting. Virtual. Jan 7, 2021. 3. Determine the best systems approach to managing Fusarium head blight in wheat and barley. Maryland Grain Producers and Utilization Board meeting. Grasonville, MD. Jan 2, 2020. What do you plan to do during the next reporting period to accomplish the goals?Experiments toward investigation of the mechansim of action of the protein and its localization will be conducted.

Impacts
What was accomplished under these goals? Hiring: A PostDoctoral scientist was supposed to be hired in the project. However, the selected candidate could not join by spring 2021 due to Covid-19. To make up for the time lost, two postdoctoral scientists are being hired jointly in this grant and the complementary NSF award. Drs. Arunima Sinha and Megha Gupta will be joining the lab at the University of Maryland, College Park, by the end of this summer. In the meanwhile, a PhD student is working on various aspects of the project. Progress made against project goals: For the study of location and cellular trafficking of PFT in the plant cells, PFT was expressed ectopically in Arabidopsis. Protein was tagged with RFP and GFP at the N or C terminus of the protein. Organelle tracker markers /dyes were procured. Preliminary studies in Arabidopsis and Nicotiana transient assays indicate that PFT is located in cytoplasm as well as the endoplasmic reticulum, and the apoplastic spaces. Work on further characterization of trafficking is in progress. In order to isolate the protein, we expressed it in E. coli system. Although the protein was expressed, but it accumulated in the inclusion bodies in the bacterial cells, which had to be treated with detergents to retrieve the protein. To overcome that limitation, we expressed it in Nicotiana using a Potato-Virus X system. In addition to the complete protein, different domains of the protein were expressed. It was found that the plants expressing the pore-forming toxin domain had cell death, whereas the other plants did not show obvious cell-death. This indicates that our hypothesis of action of the protein is true. Experiments on functional characterization of the agglutinin domain are in progress. To test the range of activity of the protein on fungal pathogens, biotrophic and necrotrophic pathogens were collected from different sources. Characterization of the activity of protein on different pathogens is being conducted currently. In addition to the work on characterization of the protein, we identified some novel susceptibility factors and resistance sources for wheat-F. graminearum interaction, mapped two new genes for resistance to obligate pathogens of wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Journal Articles: Chhabra, B., Tiwari V.K., Gill, B.S., Dong, Y., Rawat, N.* (2021). Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat. Theoretical and Applied Genetics 134(7):2273-2289. DOI: 10.1007/s00122-021-03825-y. Chhabra, B., Singh, L., Wallace, S., Schoen, A., Dong, Y., Tiwari, V.K., Rawat, N.* (2021). Screening of an EMS mutagenized population of a wheat cultivar susceptible to Fusarium head blight identifies resistant variants. Plant Disease DOI: 10.1094/PDIS-03-21-0670-RE. Steadham, J., Schulden, T., Kalia B., Gill, B.S., Bowden, L., Chhuneja, P., Erwin, J., Tiwari, V.K., Rawat, N.* (2021). An approach for high-resolution genetic mapping of distant wild relatives of bread wheat. Theoretical and Applied Genetics 134(8):2671-2686. DOI: 10.1007/s00122-021-03851-w. Wallace, S., Chhabra, B., Ma, X., Coleman, G., Tiwari, V., Rawat, N* (2021). Evaluation of a diverse Triticale collection to identify genetic resistance against Fusarium Head blight. Preprints-doi:0.20944/preprints202104.0300.v1.