|Posada, Martha Lucia|
Submitted to: Intercollegiate Student Chemists Convention
Publication Type: Abstract Only
Publication Acceptance Date: 4/3/2003
Publication Date: 4/3/2003
Citation: Ewing, L.J., Posada, M., Smith, S., Frederick, R.D. 2003. Real time rt-pcr analysis of gene expression during the first 14 days post inoculation of the soybean rust fungus, phakopsora pachyrhizi. Intercollegiate Student Chemists Convention. Abstract Booklet at meeting (on 04/05/2003) at Villanova University, Villanova, PA.
Technical Abstract: Soybean rust, caused by the fungus, Phakopsora pachyrhizi, is responsible for significant losses of soybean crop in Africa, Asia, Australia and South America. If it enters the continental United States it could decrease yields in southeastern states. An understanding of early pathogen-host interactions between P. pachyrhizi and soybean is crucial for development of strategies to prevent and combat soybean rust. In this study, Real Time RT-PCR was used to examine the expression of five putative genes: heat-induced catalase, ATP-binding cassette (ABC) transporter, plasma membrane (H+) ATPase and alpha and beta-tubulin obtained from an EST library of P. pachyrhizi germinating spores (Posada and Frederick, manuscript in preparation). Primers and fluorogenic probes were designed for each of the five genes and used to determine their relative levels of expression in germinating spores and infected leaf tissue at 1,2,4,6,8,10,12,14 days post inoculation (dpi). Gene expression was monitored using the threshold cycle (CT) which is the first cycle in which a significant increase in normalized Reporter (Rn) is detected. All five genes showed similar expression patterns during the first 14 dpi. Expression levels of the genes change during the course of infection; however, the variation is likely due to increasing fungal growth. Current studies are being conducted using in vitro transcription to produce RNA template for generating standard curves which can be used to quantify the amount of original transcript at each time point. The relative expression of the heat-induced catalase, ABC transporter and ATPase genes will be normalized against the two constitutive genes, alpha and beta-tubulin.