Abstract
BackgroundThe Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce′s disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers.MethodsSite-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations.ResultsWe have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present.ConclusionsWe have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.
Highlights
The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops
The gene was deleted from X. fastidiosa Temecula 1 using site directed replacement with a kanamycin resistant marker [10]
Transformants were selected on antibiotic plates at 10 μg/mL since the minimum inhibitory concentration of kanamycin for X. fastidiosa Temecula 1 is 4 μg/mL [11]
Summary
The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. Xylella fastidiosa is a Gram-negative, xylem-limited, insect-vectored bacterium that is a causal agent of many economically important plant diseases, including Pierce’s disease of grapevines [1]. Once in the plant xylem, X. fastidiosa is postulated to migrate, attach, aggregate, and form biofilm that clogs the vessels leading to disease development. X. fastidiosa cells are proposed to attach to the xylem wall mainly using non fimbrial adesins, such as XadA (Xanthomonas adhesin-like protein A) and hemagglutinin proteins HxfB (hemagglutinin Xylella fastidiosa B) [4]. Cell-to-cell adhesion occurs via non fimbrial adhesins HxfA, HxfB, XatA (Xylella fastidiosa autotransporter A) and the X. fastidiosa type I pili [4,5,6,7]. Biofilm formation commences [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
