Biochemical and molecular studies of some aspects of disease resistance in potato (Solanum tuberosum L.)
Disease problems in crop plants are still a major threat to the agro-industry worldwide. Various strategies have been developed and evaluated in recent years. One strategy is to use naturally-occurring antipathogen factors such as lysozymes and chitinases in transgenic plants. In the present study, transgenic plants containing chick-egg white lysozyme (Lys 75) have been evaluated for lysozyme production in planta, subcellular localisation, and resistance to some potato pathogens, including Phytophthora infestans and Erwinia carotovora subsp. atroseptica, the two major potato pathogens worldwide. In addition, the evaluation of resistance was also undertaken for transgenic plants carrying other naturally-occurring antipathogen factors including a bean chitinase gene (BCH 35) and a snowdrop lectin gene (GNA 74). In order to accurately quantify the lysozyme production in Lys 75 plants, the turbidimetric lysozyme enzyme assay was optimised. Also, a modified substrate for the enzyme has been developed by covalently linked the Micrococcus lysodeikticus cell wall with a dye, remazol brilliant violet 5R to enable a colorimetric assay of the enzyme. In order to quantitatively assess resistance levels of the transgenic plant, a new method (leaf-bridge bioassay) for conducting and evaluating resistance in planta has been developed. All transgenic plants in tissue culture were tested for resistance using this technique. Evaluation of the progress of infection in detached leaves of Lys 75 showed that lysozyme gave some degree of protection against the bacterial pathogen, Erwinia carotovora subsp. atroseptica and the fungal pathogen, Fusarium sulphureum. Analysis of intercellular fluid from the Lys 75 leaves showed that more than 80% of the total lysozyme expressed in the leaf was located in the intercellular space which is a strategic place to combat pathogen attack. In contrast, the levels of protection in BCH 35 plants were relatively low compared with Lys 75. The progress of infection was delayed in BCH 35 leaves challenged with F. sulphureum only. No resistance at all was observed in GNA 74 to all the pathogens used. All the transgenic potato lines were susceptible to P. infestans. Recently, a new strategy to combat disease problems has been suggested based on a 'durable resistance'. Potato variety Stirling' which shows durable resistance in the field has been used to study the early biochemical and molecular events during elicitation of 'Stirling' cell suspension cultures with an elicitor mix derived from infective units of a compatible strain of P. infestans. For comparison, an elicitor mix from an incompatible strain of P. infestans was also prepared and used. The mixed elicitor comprising zoospore extract, culture filtrate and mycelium homogenate induced defence responses in 'Stirling' cell suspension cultures as judged by the increase in PAL enzyme activity. PAL activity in 'Stirling' ceUs elicited with an elicitor mix derived from an incompatible strain of P. infestans was twice the activity in the compatible interaction. The peak levels in both types of interaction were at 6 h post-elicitation. An oxidative burst was demonstrated also in both types of interactions indicated by rapid release of H(_2)O(_2) into the culture medium. The H(_2)O(_2) level peaked at 2 h post-elicitation in both interactions before being reduced to its normal level at 4 h. The H(_2)O(_2) released during incompatible interaction was twice the levels monitored in the compatible. A subtracted cDNA library of differentially expressed mRNAs during elicitation of 'Stirling' cell suspension cultures with the elicitor mix from a compatible strain of P. infestans was constructed using suppression subtractive hybridisation. Two cDNA clones, STS 42 and STS 52, relevant to the present study were identified and characterised. STS 42 showed high degree of similarity to potato leucine aminopeptidase gene which is induced in response to wounding. Gene expression studies using RT-PCR showed that the mRNA levels of STS 42 increased gradually throughout the 18 h elicitation. STS 51 was identified as a member of the ribonuclease T2 histidine proteins. It showed some degree of similarity to plant ribonucleases involved in self-incompatibility reactions during pollination. It has a site for tyrosine kinase phosphorylation at the hydrophilic region of the sequence and could possibly be involved in phosphorylation during signal transduction. mRNA levels of STS 51 were increased during the first 12 h of elicitation.