Source: UNIVERSITY OF KENTUCKY submitted to
LOCOWEED AND ITS FUNGAL ENDOPHYTE: IMPACT, ECOLOGY, AND MANAGEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1008881
Grant No.
(N/A)
Project No.
KY012041
Proposal No.
(N/A)
Multistate No.
W-1193
Program Code
(N/A)
Project Start Date
Jan 1, 2016
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Project Director
Schardl, CH, LE.
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Plant Pathology
Non Technical Summary
Locoweeds are leguminous plants that contain symbiotic (endophytic) fungi that, in turn, produce the toxic alkaloid, swainsonine. Locoweeds commonly occur in rangelands in the western United States, where they pose a hazard to livestock. Although many species of legumes in genera Astragalus and Oxytropis can contain swainsonine, other species may lack or have very little swainsonine. In such instances, it is not known if the plants lack or have very little endophyte, or their endophytes lack the ability to produce substantial levels of swainsonine. It is also unclear what environmental or agroecological factors might promote swainsonine synthesis or favor plants with swainsonine-producing endophytes, or even whether swainsonine somehow protects or enhances fitness of the plant that contains it. Experiments to address these questions will be facilitated by the identification of genes for biosynthesis of this alkaloid, as well as linked genes and sequences governing regulation of swainsonine expression and where the swainsonine may be localized within fungal or host plant. Complete genome sequences will be determined for swainsonine producers, and bioinformatic tools will be employed to identy genes and their likely functions, and to compare genomes of different fungi, to help identify swainsonine biosythesis genes. Knowledge and tools gained will enhance prospects for determining the potential for toxin production and aid in developing, testing and implementing strategies to manage locoweeds and reduce risks of livestock poisoning.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31440201040100%
Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Determine the biochemistry of swainsonine biosynthesis and metabolism.
Project Methods
Total DNA will be prepared from fungi of interest and submitted to the Genomics Core on campus for sequencing on Illumina platforms. Other sequencing platforms on or off campus may be employed to refine the sequences. Available bioinformatic tools for genome annotation will be used to predict gene structures throughout the sequenced genomes, and to identify likely functions of the gene products. Orthology search programs such as OrthoMCL and COCO-CL will be employed to compare genomes and identify candidate biosynthetic genes, particularly those for swainsonine. Efforts include provision of genome sequences and annotations to public repositories such as NCBI, publication of results in peer-reviewed scientific journals, and incorporation of the new information into formal classes and informal instructional opportunities. Progress will be evaluated primarily on the basis of peer reviewed publications, their citations as primary literature in other publications, and invitations to present findings at meetings or in written reviews.

Progress 01/01/16 to 09/30/20

Outputs
Target Audience:Ranchers. Scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Elucidation of the swainsonine biosynthesis gene cluster (SWN)in the locoweed endophyteAlternaria oxytropisled to the identification of SWN in other fungal symbionts, fungal pathogens of humans and animals, and fungal pathogens of plants. The discovery of swainsonine in the dermataceous pathogens causing ringworm or other tinea diseases in humans and animals (particularly horses and cats) provides information relevant to further research in those systems. Furthermore, the identification and sequence determination of SWN in the fungal agents of locoweed toxicity can allow molecular diagnostic tools such as PCR to assess risk of toxicity to livestock. Finally, the identification of additional SWN homologs in the legume black patch pathogen Slafractonia leguminicola enhances future prospects of characterizing the genetics and biochemistry of both swainsonine and the related toxin, slaframine.

Publications


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

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Balansia epichloe isolates from various grass hosts will be screened for the swnK paralog. Those grasses symbiotic with B. epichloe strains with the swnK paralog will be assayed for slaframine, swainsonine and related indolizidines.

    Impacts
    What was accomplished under these goals? The gene swnK in the locoweed endophyte Alternaria oxytropis, and in the related plant pathogen Slafractonia leguminicola, is required for biosynthesis of the indolizidine alkaloid swainsonine, and SwnK is a multifunctional protein likely to carry out the first several steps in conversion of pipecolic acid and malonic acid to the precursor of swainsonine. Previously swnK was identified in sequenced genomes of those and other fungi, but the S. leguminicola genome sequence also included two paralogs. Potentially one or both of the swnK paralogs are involved in biosynthesis of another indolizidine alkaloid, slaframine, which has so far only been found as a product of S. leguminicola. Elucidating function of the swnK paralogs would provide insight into the basis for indolizidine diversity. Newly sequenced genomes of three isolates of Balansia epichloe (a systemic symbiont of several warm-season grasses) revealed a swnK paralog in one of those isolates. Among sequences available in the public databases, that paralog is most closely related to one of the paralogs in S. leguminicola, even though Balansia and Slafractonia are in different fungal orders.

    Publications


      Progress 10/01/17 to 09/30/18

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The collaborators will participate in the annual meeting, anda resubmission of the grant proposal is planned for February 2019.

      Impacts
      What was accomplished under these goals? Collaborators met in a virtual meeting in November, 2017, and submitted a joint grant proposal to NSF in March 2018. The proposal was declined.

      Publications


        Progress 10/01/16 to 09/30/17

        Outputs
        Target Audience:The target audience was the research community with interest in fungal biology and specialized (secondary) metabolism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results were reported at the 2016 W-1193 Multistate Project meeting, and were published in G3 (Bethesda), a journal of the Genetics Society of America. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

        Impacts
        What was accomplished under these goals? Swainsonine is a cytotoxic indolizidine alkaloid responsible for locoism, a debilitating toxicity experienced by livestock grazing on locoweeds (Astragalus and Oxytropis species) with symbiotic fungi (endophytes) of genus Alternaria section Undifilum. Swainsonine inhibits alpha-mannosidase II and disrupts the endomembrane system, causing cell death. Previously there was very limited information about the enzymes and none about the genetics of swainsonine biosynthesis. Genomes were sequenced for three swainsonine-producing fungi: the locoweed endophyte Alternaria oxytropis, the legume black patch pathogen Slafractonia leguminicola, and an unnamed species from the pink morning glory Ipomoea carnea. The assembled genome sequences were annotated by ab initio gene prediction, as was the published genome sequence of another known swainsonine producer, Metarhizium robertsii, and the four genomes were searched for orthologous candidate genes and gene clusters predicted to encode biosynthetic enzymes. One such cluster of genes was identified in each, and a collaborator verified its role by molecular genetic methods to inactivate one of the M. robertsii genes and eliminate swainsonine production, and then to reintroduce the wild-type gene and restore swainsonine production. Searches of public databases of sequenced fungal genomes also identified orthologs of swainsonine biosynthesis genes in most sequenced Metarhizium species, which are insect pathogens, and all sequenced members of the family Arthrodermataceae, which are mammalian pathogens that cause tinea dermatophytoses (skin diseases) such as ringworm, jock itch and athelete's foot. Collaborators tested isolates of Metarhizium species and Arthrodermataceae and verified that those with swainsonine biosynthesis gene orthologs are capable of producing swainsonine.

        Publications

        • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cook D, Donzelli BGG, Creamer R, Baucom DL, Gardner DR, Pan J, Moore N, Krasnoff SB, Jaromczyk JW, Schardl CL (2017) Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3: Genes - Genomes - Genetics 7: 1791-1797. doi 10.1534/g3.117.041384


        Progress 01/01/16 to 09/30/16

        Outputs
        Target Audience:Fellow agricultural, biological and molecular biological scientists were targeted in this multistate collaboration. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Targeted gene knockouts of swainsonine genes in Metarhizium species will generate strains that can be tested to determine if swainsonine is required for, or involved in, pathogenicity to insects.

        Impacts
        What was accomplished under these goals? Genome sequences were determined from the plant pathogen, Rhizoctonia leguminicola, the seed-transmitted endophytes Alternaria sect. Undifilum oxytropis, and an unnamed symbiont of the morning glory plant, Ipomoea carnea. Comparative genomic analysis identified the swainsonine biosynthesis gene clusters.

        Publications