Reprogramming the expression of the double-stranded RNA mitovirus OnuMV1c from the mitochondria to the cytoplasm in the fungal pathogen Ophiostoma novo-ulmi
Bibliographic record
Abstract
Dutch elm disease (DED) is a debilitating wilt disease that has decimated elm populations globally. The current pandemic of this disease is caused by the ascomycete fungal pathogen Ophiostoma novo-ulmi. A number of strategies have been used to attempt to mitigate the effects of DED but none have met any sustainable success, and the disease continues to have severe ecological and economic impacts. Consequently, research focus has turned to the development of control strategies at the genetic level. One such genetic strategy is the use of naturally occurring fungal viruses (mycoviruses) to induce hypovirulence in their fungal hosts. Hypovirulence, or attenuation of fungal pathogenicity using mycoviruses, has been well studied in other systems but has yet to be developed for O. novo-ulmi. \nA candidate virus, OnuMV1c, was found in an isolate of O. novo-ulmi (93-1224) at the western Canadian disease front and its genome was sequenced. OnuMV1c is a mitochondrial virus and has a 3.1 kb single-stranded positive RNA genome that encodes an RNA-dependent RNA polymerase (RdRp) involved in its replication as a double-stranded RNA molecule. It exists in O. novo-ulmi mitochondria in both its single-stranded and double-stranded forms. \nOur research group identified OnuMV1c as a potential candidate for biological control of Dutch elm disease. Our long-term research goal is to use the virus as a means to activate the RNA interference pathway of O. novo-ulmi, leading to down-regulation of genes involved in pathogenicity. If OnuMV1c is engineered such that it carries an RNA interference cassette in addition to its own complement of genes, it could act as an enhanced hypovirus. RNA interference (RNAi) is a cytoplasmic process, and therefore in order to use OnuMV1c for RNAi the viral genome needed to be reprogrammed such that it could be expressed in the cytoplasm rather than the mitochondria. The objectives of my master’s research were to 1) genetically engineer OnuMV1c to express in the cytoplasm using a cDNA reverse genetics approach, and 2) test the functionality of the re-engineered cDNA OnuMV1c virus (MV1cCyt). \nThe first objective was accomplished by modifying codons in the RdRp sequence of OnuMV1c such that the sequence could be translated in the cytoplasm. This genetically engineered cytoplasmic version of OnuMV1c, named MV1cCyt, was flanked with exogenous promoter and terminator sequences to drive its transcription. The entire construct was engineered as a cDNA molecule and was cloned into the fungal transformation vector pAN7-1, which was used to transform O. novo-ulmi protoplasts. \nThe second objective was achieved through the use of strand-specific RT-PCR, a technique that allowed the detection of both the positive and negative strands of MV1cCyt. Results indicated that while four individual cell lineages contained MV1cCyt cDNA stably integrated into the nuclear genome, only one transformant was able to produce double-stranded MV1cCyt RNA. These results have important implications for the use of OnuMV1c as an engineered hypovirus and represent the first step towards the development of a biological control strategy for Dutch elm disease.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.002 | 0.000 |
| Research integrity | 0.000 | 0.002 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".