Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-031-028-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Jun 30, 2021
Objective:
Mycoviruses hold great potential in controlling plant pathogenic fungi. The overall goal of this project is to develop methods to efficiently deliver debilitating mycoviruses in the field. The objectives include: 1) evaluating the feasibility of expressing in transgenic plants a proven mycovirus biopesticide and another related but uncharacterized mycovirus triggered by Sclerotinia sclerotiorum infection; 2) comparing in vitro expression systems for production of self-assembled virions as biopesticides; 3) optimizing the conditions for scale-up of the production of the biopesticides. Proposed mycovirus candidates include a recently discovered ssDNA mycovirus, named as soybean leaf-associated gemycircularvirus 1 (SlaGemV1; KT598248) and a demonstrated field-effective biopesticide, Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV1; NC_03116, KM598382~4). SlaGemV1 was recently discovered via a metatranscriptomic search of soybean leaves infected by fungal pathogens in Southern Illinois that is 55% identical to SsHADV1 in the amino acid sequence of the replicase. SsHADV1 has been shown to be effective either as a suspension of virus-infected hyphal fragments or purified virus particles applied directly on fungal hyphal mats or on plant leaves.
Approach:
To attain the proposed objectives, steps taken will include: 1) confirming the bioactivity of the clones by transfecting S. sclerotiorum with infectious clones of SlaGemV1 and SsHADV1; 2) transform Arabidopsis thaliana with constructs derived from the infectious clones to express both of the ssDNA viruses; 3) express and assemble the mycoviruses in Agrobacterium and insect cells based-systems; 4) scale virion production and purify the virions from the expression system. Two hypotheses will be tested: H1) SlaGemV1 and SsHADV1 can be integrated into plant genomes and induced to produce extrachromosomal virus particles that are infectious to S. Sclerotiorum; H2) SlaGemV1 and SsHADV1 can replicate and self-assemble in Agrobacterium tumefaciens or E. coli to produce stable virions. To test these hypotheses, self-circularizing plasmid clones will be used to transfect virus-free S. sclerotiorum. Once the infectivity of the clones are confirmed, the viral cassettes will be inserted between the left and right T-DNA border regions of an Agrobacterium tumefaciens binary plasmid allowing for the integration to the plant genome. Floral dip method of A. thaliana will be used for Agrobacterium-mediated transformation. Transgenic Arabidopsis plants will be assessed for the production of episomal copies of viral genomes, production of virus particles and their abilities to transmit the viruses to S. sclerotiorum. Further, because the lack of fitness of S. sclerotiorum strains infected with SlaGemV1 or SsHADV1 could limit the scale-up production of the fungicide, bacterial and existing insect cell line expression systems will be evaluated for production of virions.
