Viruses of Mutualistic Endophytic Fungi

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Viruses of Mutualistic Endophytic Fungi

• Drew Porter
• Dr. Marilyn Roossinck Lab

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Three Way Symbiotic Relationship

• Plant
• Fungus
• Virus
• Geothermal Tolerance

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Background

• 2002 Thermotolerance in Yellowstone requires
  plant/fungal symbiosis (Redman, etal, Science298,
  1581, 2002)
• 2007 Yellowstone plant/fungus symbiosis also
  requires mycovirus (fungal virus) (Márquez, etal
  Science315, 5811, 2007)

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Motivation for research

• Does the same three way symbiosis found in
  Yellowstone occur in other geothermal areas?
• MaybeCosta Rica?

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Volcán Rincón

• Geothermal area in Costa Rica.

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Methods

• In Costa Rica
• Collect plant
• Remove and grow fungi from plant
• Extract and enrich dsRNA
• Visualize on gel
• Send to America

• In Ardmore (me)
• Reverse transcription and PCR
• Ligation and transformation
• E-coli cloning
• Plasmid mini-preps
• Sequencing

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Host plant and fungus

• Plant Family Poaceae

Fungus Unknown
07-HACG-097
07-HACG-099
07 collected in 2007 HACG Hongas Area
Conservación Guanacaste 097, 099 sample
identification numbers
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Double Stranded RNA

• Fungal RNA is not double stranded, but viral RNA
  is often double stranded.
• dsRNA virus presence
• PhenolChloroform extraction followed by CF11
  cellulose dsRNA enrichment and DNase digestion

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Visualization of dsRNA

• Visualized on Agarose gel

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Reverse Transcription and PCR
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TA cloning
Ligation
Transformation
Cloning
http//www3.niaid.nih.gov/biodefense/Public/Images
.htm
E-coli with new plasmid
http//microbiology.scu.edu.tw/micro/microbe-exp/e
xp_website/Exp_2.htm
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Plasmid mini-preps

• Picked colonies into LB medium with antibiotic
  and let grow at 37oC in shaker overnight
• Performed plasmid mini-prep to isolate only the
  plasmid DNA, and not the host DNA
• Hypotonic solution used to swell the cell
• Alkaline solution used to lyse the cell
• Sodium acetate used to precipitate out everything
  but plasmid DNA.

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Plasmid mini-preps (cont.)

• Check for insertions using EcoRI digestion
• EcoRI sites located on both sides of insert
• Submit for sequencing those with inserts

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Sequencing Results

• 07-HACG-097 did not yield significant matches in
  a BLAST search
• Possible novel virus
• 07-HACG-099 was highly matched to both a
  mycovirus, as well as a hypovirus
• A virus was definitely involved in the
  plant/fungal relationship.

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07-HVACG-97
Conclusion Most hits were fungal. However, the
hits were all from mRNA sequences which could
mean that we have an uncharacterized virus. The
Curvularia thermal tolerance virus RNA2 has two
ORFs with no significant hits in GenBank (Márquez
etal, Science315, 5811, 2007).
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07-HVACG-99
Conclusion Closest known relative is the
Coniothyrium minitans virus, which is a
totivirus. Other BLAST hits show that fungal and
possibly plant RNA was also amplified from this
sample.
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Future Work

• Sequence entire virus genome
• Sequence other dsRNA from the Volcan Rincon and
  the same species from the non-geothermal areas to
  determine the viruses role in the endophytic
  relationship.
• Determine the biological role of the virus in the
  ecology of the geothermal plants

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Acknowledgements

• Dr. Marilyn Roossinck
• Roossinck Lab
• Tracy Feldman
• Ping Xu
• MASS lab
• Steve Rhines
• Emily Edwards
• The Samuel Roberts Noble Foundation
• Rhonda Schmidt
• Felipe Chavarría
• Dan Janzen