|Huang, H. - University Of South Florida|
Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 12/14/2012
Publication Date: 12/15/2012
Citation: Chen, J., Huang, H. 2012. Validation of small RNAs in Xylella fastidiosa by qRT-PCR. In: CDFA Pierce's Disease Control Program Research Symposium. p.84.
Technical Abstract: Xylella fastidiosa causes many economically important crop diseases including almond leaf scorch disease and Pierce’ disease of grapevine. Although non-coding small RNAs (sRNAs) are regarded as ubiquitous regulatory elements in bacteria, research attention to sRNAs in X. fastidiosa has been limited. In X. fastidiosa strain M23, 49 sRNA genes were predicted based on the availability of its whole genome sequence and computational analyses. X. fastidiosa is, however, nutritionally fastidious and grows very slowly even in complex culture media. Compounded by low expression levels of sRNAs, implementation of commonly used techniques such as Northern-blotting to detect sRNAs has been problematic. In this study, an alternative PCR method was developed to experimentally verify the presence of sRNAs in X. fastidiosa. Primers were designed within (internal) and flanking (external) 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. Amplification of 16S rRNA was used as an internal control. A total of nine sRNAs have been detected by qRT-PCR in X. fastidiosa strain M23. Different sRNA types were observed, depending on culture media. Results from this study provide the first experimental proof of sRNAs in X. fastidiosa. The developed technique also has potential to be used in sRNA detection of other bacteria.