Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2005
Publication Date: 10/1/2005
Citation: Gronwald, J.W., Plaisance, K.L., Marimanikkuppan, S., Ostrowski, B.G. 2005. Tagetitoxin purification and partial characterization. Physiological and Molecular Plant Pathology. 67:23-32. Interpretive Summary: Weed control strategies that do not involve using chemical herbicides are needed to reduce the environmental impact of weed control. Naturally occurring plant pathogens that infect weeds offer potential for controlling weeds without chemicals. However, the performance of plant pathogens as biocontrol agents for weeds is frequently inconsistent and as a result this environmentally friendly strategy has not been widely adopted. A better understanding of factors controlling efficacy of plant pathogens used for weed control is needed to improve their performance. Pseudomonas syringae pv. tagetis (Pst) is a naturally occurring bacterium that infects Canada thistle. Pst blocks growth of Canada thistle by producing a toxin called tagetitoxin which prevents the formation of chloroplasts -- small organelles in leaves that convert sunlight to sugar. Although Pst shows potential for controlling Canada thistle, the use of this biocontrol agent is limited because the bacterium does not consistently produce enough tagetitoxin to completely block growth of Canada thistle. Knowledge of the structure of tagetitoxin is needed so that weed scientists can determine how Pst makes this toxin and develop ways to increase its production in Canada thistle. Determining the structure of tagetitoxin requires that the compound be purified. Older purification protocols described in the literature do not yield pure tagetitoxin. We developed a new protocol that yielded pure tagetitoxin. The structure of the purfied tagetitoxin was analyzed using techniques called mass spectrometry and nuclear magnetic resonance spectrometry. Mass spectrometry analysis indicated that the molecular weight of the toxin is 678, not 416 as previously reported. Nuclear magnetic resonance spectroscopy indicated that previous reports regarding the arrangement of carbon atoms in the molecule are incorrect. The results of this research have corrected errors in the scientific literature regarding the properties of tagetitoxin. The protocol that we developed for purifying tagetitoxin and the new knowledge regarding its structure will be valuable to weed scientists who are investigating ways to increase the efficacy of Pst for controlling Canada thistle.
Technical Abstract: Tagetitoxin is a non-host specific toxin produced by Pseudomonas syringae pv. tagetis in host species (Asteraceae family) and in liquid culture by certain strains of the bacterium. A purification protocol involving anion exchange and partition chromatography was developed that yielded tagetitoxin purified to homogeneity. Based on dilution end-point (nanograms/plant causing just detectable apical chlorosis in sunflower), the protocol resulted in a 2000-fold purification of tagetitoxin. The dilution end-point of purified tagititoxin was 10 ng and the I50 for inhibition of E. coli RNA polymerase was 122 nM. Electrospray ionization mass spectrometry in 50% methanol indicated that the molecular wt of tagetitoxin was 678. Gated mass spectrometry (MS/MS) of the m/z 679 (MH+) ion produced fragmentation ions of m/z 453, m/z 435, and m/z 417. Preliminary characterization of tagetitoxin structure was performed using NMR spectrometry and elemental analysis. Although NMR spectra for one-dimensional 1H and 31P are consistent with the previously proposed structure (Mitchell et al. 1989), one-dimensional 13C spectra and two-dimensional NMR correlations contradict this structure. Elemental analysis of tagetitoxin indicated a ratio of carbon to nitrogen atoms of 2:1 in contrast to 6.5:1 proposed by Mitchell et al. (1989). Based on the combined evidence, the previously proposed structure of tagetitoxin with a molecular wt of 416 is incorrect.