This grant proposal aims to test the effects of expressing quorum sensing inhibitors (QSIs) in Arabidopsis thaliana on resistance to food crop pathogens. The researchers hypothesize that A. thaliana transformed to express farnesol and QsdH will show increased resistance to Pseudomonas syringae and Puccinia triticina compared to wild-type plants. They plan to use CRISPR/Cas9 to generate plants expressing QSIs from different promoters and expose them to pathogens. If successful, this research could help reduce crop losses from disease without pesticides and provide insights into clinical applications of quorum sensing inhibition.

1. Effects of quorum sensing inhibitor expression by
Arabidopsis Thaliana on food crop associated
fungal and bacterial pathogenesis: a model for food
crop QSI GMOs
A Grant Proposal by
Beau Smith & Jacob Harris

2. Background - Quorum Sensing
● Bacteria used to be thought of as individualistic organisms
- They are not
● Use of autoinducers to “talk” to each other & coordinate cellular activities
● Quorum sensing (QS) - epigenetic pathway which modifies the expression of
genes based on the presence of conspecific and interspecific molecules and their
concentrations
● Many different types of QS molecules used for different activities
● Often used for activation of population virulence (Kalia 2013)

3. Background - QSI and Agriculture
● Many organisms naturally produce quorum sensing
inhibitors (QSIs) that cripple communications
between QS organisms (Kalia 2013)
● Agricultural crops have to deal with pests that use QS
- Many don’t naturally produce QSIs to fight
them
● 20 - 40% loss in worldwide agricultural production
due to diseases (Savary et al. 2012)
- Pesticide resistance increasing
● Genetic engineering may allow these crops to
produce QSIs
P. syringae
Puccinia triticina

4. Background - Arabidopsis thaliana
● One of the most well-studied model plant organisms
(Bortesi & Fischer 2015)
○ Seed is transparent, allowing developmental studies
○ Self polinates
● Used in CRISPR/Cas9 genetic engineering
● Test the efficacy of QSI expression in plants

5. Research Design and Plan
Hypothesis: Arabidopsis thaliana transformed to express and co-express farnesol and
QsdH will be more resistant to Pseudomonas syringae and Puccinia triticina than
wild-type A. thaliana.
Specific Aims:
● CRISPR/Cas9 and Plaques
● Surface Promoter + Variable CMV35s: Single, Double, Triple = 9 Cohorts
● QsdH and Farnesol cDNA
● Exposed to pathogen from leaf of infected A. Thaliana (Why do we do this?)
● Scored for phenotypic markers of stress vs untransformed control

6. Cohort Construct:

7. Scoring for Phenotypic Markers:
P. syringae Puccinia triticina

8. Perspectives
● Expected results
- Decreased virulence & biofilm development
- Increased plant resistance, even when exposed to pathogens
● Test QSIs on murine gut microbiota health
● QSI production may produce undesirable crop traits due to resource shifts
● Applications in agriculture - P. syringae affects wheat and fruit trees (Moore
1988)
● Research can be used as stepping stone for future QS research

9. Significance
● Quickly increasing world population = need for more agricultural production
- Est. 8.5 billion people by 2030 (United Nations 2015)
● Still high usage of pesticides even with increasing pesticide resistance
- US: 1 billion lbs per year (Alavanja 2009)
● Less food waste, fewer chemicals, more bountiful harvest
● QS is still a relatively new biological discovery
● Applicable future QS clinical studies
- P. aeruginosa: respiratory infections, developed drug resistance (Smith &
Iglewski 2003)
- Candida albicans: farnesol to prevent hyphal development, 1st QS eukaryote
(Shareck & Belhumeur 2011)
- Both affect immunocompromised persons, i.e. HIV infected

10. References:
Alavanja, M. C. R. (2009). Pesticides Use and Exposure Extensive Worldwide. Reviews on Environmental Health, 24(4), 303–309.
Albuquerque, P., & Casadevall, A. (2012). Quorum sensing in fungi–a review. Medical mycology, 50(4), 337-345.
Bortesi, L., & Fischer, R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33(1), 41-52. doi:10.1016/j.biotechadv.2014.12.006
Ciofu, O., Giwercman, B., Høiby, N. and Pedersen, S. S. (1994), Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center.
APMIS, 102: 674–680. doi: 10.1111/j.1699-0463.1994.tb05219.x
Dong, Y. H., & Zhang, L. H. (2005). Quorum sensing and quorum-quenching enzymes. J Microbiol, 43(1), 101-109.
Hogan, Deborah A. "Talking to themselves: autoregulation and quorum sensing in fungi." Eukaryotic cell 5.4 (2006): 613-619.
Huang, W., Lin, Y., Yi, S., Liu, P., Shen, J., Shao, Z., & Liu, Z. (2012). QsdH, a novel AHL lactonase in the RND-type inner membrane of marine Pseudoalteromonas byunsanensis strain 1A01261. PLoS One,
7(10), e46587.
Kalia, V. (2013). Quorum sensing inhibitors: An overview. Biotechnology Advances, 31(2), 224-245. doi:10.1016/j.biotechadv.2012.10.004
L.W. Moore. (1988). Pseudomonas syringae: disease and ice nucleation activity. Ornamentals Northwest Newsletter. 12:4-16.
Moore, L. W., Chilton, W. S., & Canfield, M. L. (1997). Diversity of Opines and Opine-Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors. Applied and Environmental Microbiology, 63(1),
201–207.
Savary, S., Ficke, A., Aubertot, J., & Hollier, C. (2012). Crop losses due to diseases and their implications for global food production losses and food security. Food Sec. Food Security, 4(4), 519-537.
doi:10.1007/s12571-012-0200-5
Shareck, J., & Belhumeur, P. (2011). Modulation of Morphogenesis in Candida albicans by Various Small Molecules . Eukaryotic Cell, 10(8), 1004–1012. http://doi.org/10.1128/EC.05030-11
Smith, R. S., & Iglewski, B. H. (2003). Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. Journal of Clinical Investigation, 112(10), 1460–1465. http://doi.org/10.1172/JCI200320364
United Nations, Department of Economic and Social Affairs, Population Division(2015). World Population Prospects: The 2015 Revision, Key Findings and Advance Tables. Working Paper No. ESA/P/WP.241.
Xavier, Karina B., and Bonnie L. Bassler. "LuxS quorum sensing: more than just a numbers game." Current opinion in microbiology 6.2 (2003): 191-197.