|HUANG, H - University Of South Florida|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 5/4/2012
Publication Date: 8/4/2012
Citation: Chen, J., Huang, H. 2012. Detection of small RNAs in Xylella fastidiosa. Phytopathology. 102:S4.22.
Technical Abstract: Non-coding small RNAs (sRNAs) are regarded as ubiquitous regulatory elements in bacteria. For Xylella fastidiosa, a plant pathogen causing many economically important crop diseases, research attention to sRNAs has been limited. With the availability of whole genome sequences and increasing bioinformatic knowledge, putative sRNA genes can be identified based on computational analyses. In the genome of X. fastidiosa strain M23, 49 sRNA genes were predicted. The goal of this study was to experimentally verify the presence of sRNAs in the bacterium. Due to the low expression levels of sRNAs, compounded by the nutritional fastidiousness of X. fastidiosa, implementation of the commonly used techniques such as Northern-blotting has been problematic. An alternative method was developed that took advantage of the sensitivity of PCR technology. Primers were designed within (internal) and outside (external) the putative sRNA genes. sRNAs in bacterial cultures were verified by real-time quantitative reverse transcriptase PCR (qRT-PCR) when internal primer sets yielded positive amplifications and external primer sets yielded weak or no products. Nine sRNAs have been detected in X. fastidiosa strain M23 so far. Profiles of sRNA types varied depending on culture media. Results from this study provide the first experimental proof of sRNAs in X. fastidiosa. The developed technique also has a potential to be implemented in sRNA detection of other microbes, including fastidious prokaryotes.