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Common scab, a bacterial disease of potato causes significant losses to the Australian potato industry through rejected seed and/or increased processing costs. Disease symptoms caused by plant pathogenic Streptomyces sp., are attributable to a key phytotoxin it produces, namely thaxtomin A. The development of resistance to this disease, thrqugh selection of somaclonal varieties tolerant to thaxtomin A is a current key industry supported program. However, mechanisms relating to resistance and patterns of toxicity produced by thaxtomin A are not well understood and represent the key objectives of this project. The purpose of this study was to initially assess the impact of thaxtomin A on various plants and plant cell systems from an electrophysiological, morphological and pathological perspective. The effect of thaxtomin A in combination with various auxin sources and analogues was also examined. Further work aimed to quantify resistance to thaxtomin A within mutant strains of potato plants and calli, and whether any specific change to a known thaxtomin A susceptible gene may be responsible for altered levels of resistance to thaxtomin A. Electrophysiological data obtained using ion-selective microelectrode ion flux estimation (the MIFE) technique showed that interaction between plant and toxin was characterised by a rapid and short-lived Ca2 + influx and activation of the plasma membrane proton pump. Thaxtomin A was more effective in young, physiologically active tissues (root elongation zone or pollen tube apex), suggesting higher density of thaxtomin A-binding sites in these regions. This provided the first evidence that thaxtomin A triggers an early signalling cascade, which may be crucial in plantpathogen interactions. Glasshouse trials showed that foliar application of sublethal concentrations of 2,4-D sprays on potato foliage reduced severity and occurrence of common scab. Lenticel numbers, lenticel external dimensions and periderm structure (key features critical to S. scabiei entry and penetration into a tuber) were generally not changed by 2,4-D sprays, suggesting no direct effect of 2,4-D on these morphological structures. In contrast, tubers harvested from 2,4-D treated plants had a decreased sensitivity to th~xtomin A compared with the controls, which may explain enhanced resistance. This suggests an indirect effect of2,4-D impacting on the toxin, thaxtomin A, rather than morphological changes to the developing tuber. Further experimental evidence confirmed an interaction between thaxtomin A and auxin. fuhibition of tomato pollen tube growth by thaxtomin A was ameliorated by addition of NAA. Also, auxin/auxin transport inhibitor sensitive 'ucu2-2/gi2 'A. thaliana mutant showed significantly greater sensitivity to thaxtomin A, observed as root growth suppression, increased rates of necrosis ( chlorosis ), plant death, and more severely altered W flux profiles ( electrophysiological data) in the mutant compared to the wild-type. Moreover, inhibition root growth assays with the thaxtomin A-resistant 'txr I' A. thaliana mutant showed a 3 fold increase in resistance to the polar auxin transport inhibitor, 1-NPA, suggesting an interaction between thaxtomin A and the auxin efflux carrier associated with the NP A binding 'protein. Cross-resistances to 1-NPA and isoxaben of 'txrl' and the isoxaben resistant 'ixr 1 'A. thaliana mutant suggests a similarity of function between isoxaben and thaxtomin A, and also 'txrl' and 'ixrl '. The 'txrl' gene homolog from potato has been successfully cloned and sequenced from a series of mutant potato lines, selected for resistance to thaxtomin A. Compared with the parent (control) there were no mutations within the 'txr 1 ' gene examined suggesting the resistance phenotype is due to some other genetic change. These studies have contributed to a better understanding of mechanisms of toxicity of thaxtomin A in plant cells and advanced our knowledge of pathogen: host interactions within the common scab disease pathosystem. |
