An Examination of the Solanum-Erwinia Host-Pathogen Interaction Using Protoplast and Tissue Culture Techniques.

Abstract

Four questions formed the basis of the objectives of this study : a) Is it possible to develop an in-vitro assay capable of reliably and quantitatively assessing the level of susceptibility of Solanum lines to soft-rot infection ? b) Could in-vitro regeneration, hybridisation and selection techniques be used to introduce new resistance characteristics into the gene pool of commercial potato cultivars within New Zealand ? c) Can the differences identified by assay be detected within the plant lines on cytological and biochemical examination ? d) what can be learned from analysing the in-vitro host-pathogen interaction that could shed light on aspects of the Solanum-Erwinia host-pathogen interaction in the field ? The following conclusions concerning these questions were drawn from the results of a number of experiments. a) In-vitro plantlets, established from four potato lines, a potato/Solanum brevidens hybrid line and a S. brevidens line, all with predetermined levels of soft-rot susceptibility, as well as protoplasts and tissue segments taken from the in-vitro plantlets, were exposed in a series of experiments to the bacterial soft-rot pathogen Erwinia carotovora subspecies atroseptica by using prefabricated bacterial media. The effect of the bacterial media on the viability of the various tissues was then gauged using quantitative and qualitative measures' and comparisons were made between these effects on lines known to be either susceptible or resistant. In the case of the experiments involving the exposure of leaf and stem tissue segments, by using a quantitative measure of callus growth it was possible to show that the tissues were responding to bacterial exposure, however uneven growth of control tissue on the media made comparisons between lines difficult. By measuring the proportion of viable protoplasts in the protoplast exposure experiments it was possible to identify a clear response of each line to the bacterial solution. However, it was not possible to establish a correlation between the response (or susceptibility) of these lines to bacterial exposure as isolated protoplasts and their level of susceptibility as whole plants. By measuring the weight of in-vitro plantlets from the lines listed above after 3 weeks of exposure to the bacteria it was possible to show that growth was significantly inhibited when compared with a control medium. while no correlation could be made between the responses of the potato lines to the bacteria as in-vitro plantlets and their level of susceptibility as whole plants, an inverse correlation between these factors could tentatively be shown for the lines containing the s. brevidens genome. In other words, the more resistant S. brevidens line seemed to be growing less when exposed to Erwinia in vitro than the more susceptible lines. b) Using the inverse correlation between lower in-vitro plantlet growth and higher whole plant resistance, established for the S. brevidens line above on exposure to Erwinia, 28 lines regenerated from two potato lines via protoplast and tissue regeneration techniques were assayed as in-vitro plantlets. The growth of three of these regenerants was shown to be significantly less than the growth of the parent lines when they were exposed to bacteria, as had been the case for plantlets from the S. brevidens line. Further assessment of the resistance levels of all of these regenerants would be required at the whole plant level before this assay method could be verified. However, the possibility that the number of regenerants required for further screening could be reduced by as much as 90% suggests that in the future this assay may prove to be a useful tool in screening out unwanted regenerants before beginning the more extensive field trials, should potato express resistance at the in-vitro level. c) While most of the regenerant lines showed phenotypic differences from their parent lines in vitro and four lines had been established from protoplasts that had gone through a somatic fusion treatment, no differences were detected in chromosome number between the regenerant and parent lines. In only one case were differences detected in their protein composition using one-dimensional polyacrylamide gel electrophoresis (PAGE). For the one exception, gross phenotypic changes were detected in vitro suggesting that in most cases only minor genotypic alterations had occurred to produce small changes in phenotype. However, a number of quantitative changes in protein content were detected between bacterially exposed and unexposed tissues indicating that the plants were making significant changes in protein expression in response to the bacteria. While the potato lines lost several proteins in response to Erwinia exposure, the S. brevidens line both lost and gained a protein suggesting that in this case a new protein had been produced in response to the bacteria. A hybrid line containing both the potato and the S. brevidens genomes both lost and gained these same proteins. d) The bacterial medium, or the method of exposure to the bacterial medium by the tissues, was altered in a number of experiments in an attempt to better understand the interaction between pathogen and host. The results from these experiments tentatively suggested that mechanisms involved in the hypersensitive response of an organised tissue may, at least in S. brevidens lines, be primarily responsible for the resistance expressed in field-grown plants. Interestingly it was also shown that, at least in the case of in-vitro plantlets, salicylic acid inhibited growth to the same extent as a bacterial medium. As salicylic acid is a known endogenous transduction signal molecule of systemic acquired resistance it is possible that the effect of the bacteria on plantlet growth is also related to mechanisms involved with systemic acquired resistance. Evidence from these experiments also suggested that the bacteria produced a heat labile toxin that may play a significant role in the infection process by possibly affecting protein metabolism within the host cells. Neither salicylic acid nor the oligogalacturonates, both of which are known to elicit a host defence response, could themselves be the toxin as they are relatively heat stable. The bacterial exoenzymes, while being heat labile, are also unlikely to be toxic because protoplasts that were regenerated back into plants were themselves exposed to similar enzymes during their isolation.