Author:

Abstract:

Apple scab, caused by the ascomycete Venturia inaequalis (Cooke) G. Wint., is the most detrimental disease of cultivated apple (Malus x domestica) worldwide, causing severe reductions in fruit quality and yield. In case of insufficient control of apple scab, the economic losses can increase up to 70 % of the production value (Gupta, 1992). The main strategy used for scab control is still the frequent application of fungicides throughout the season. However, selection pressure has resulted in the evolution of fungicide-resistant strains of scab that represent a threat to the industry. Therefore, all major Western breeding programs currently view the selection of disease-resistant cultivars as a priority. Most of today's scab-resistant cultivars rely on the introduction of a single gene for scab resistance from the ornamental apple Malus floribunda 821, referred to as Rvi6 (Vf) resistance. However, Rvi6 resistance has been overcome by new pathotypes of V. inaequalis. This has aroused interest in other major resistance genes, in polygenically controlled resistance, effected by several minor resistance genes, and in ‘pyramiding’ of different sources of resistance, to confer more durable resistance. However, there is still a lack of fundamental knowledge on the apple – V. inaequalis interaction with regard to the resistance mechanism. Even the mechanism of action of Rvi6 resistance is still largely unknown. These insights are essential for the development and application of new markers in breeding programs, and thus for the development of new resistant cultivars and for attaining durable resistance. Therefore, the aim of the research presented in this thesis was to gain a better insight in these resistance and defense mechanisms of apple against V. inaequalis.To this end, a high-throughput proteomics technology, two-dimensional gel electrophoresis (2-DE), was applied. First, we needed to fine-tune some experimental conditions and procedures. The scab infection procedure was optimized and a monogenic Rvi6/Vf-resistant cultivar (‘Topaz’), a polygenic resistant cultivar (‘Discovery’) and a scab race 1 susceptible cultivar (‘Golden Delicious’) were chosen for further research. The 2-DE conditions, including protein extraction, first and second dimension separation, and protein visualization, also needed optimization. To optimize the resolution of the protein separation, we decided to use 24 cm IPG strips of pH-range 4-7 for the first dimension separation of the proteins extracted from the complete cells of the apple leaves. To visualize the proteins, they were CyDyeTM labeled prior to a two-dimensional difference-in-gel electrophoresis (2D-DIGE) in most experiments.To study plant-fungus interactions for resistance breeding purposes, early and accurate detection and quantification of the plant pathogen is often required for proper evaluation of fungal virulence and host resistance. So far, the evaluation of scab resistance in apple was mainly based on artificial inoculation and a disease rating that takes into account foliar chlorosis, necrosis and sporulation symptoms (Chevalier et al., 1991). However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. Therefore, we developed a real-time PCR assay that amplifies the internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene of Venturia spp. only. Results were compared with the classical phenotypic disease rating scores and the assay proved to be a fast, sensitive and objective method to monitor fungal growth and to evaluate host resistance.Further, we compared the proteomes of the V. inaequalis inoculated, mock-inoculated and control leaves of the Rvi6-resistant cultivar ‘Topaz’. At 2 and 3 days post-inoculation (dpi), differences appeared not to be present yet, were less distinct, or at most comparable to 5 dpi. At 5dpi the V. inaequalis inoculated leaves showed an up-regulation of proteins mainly known to be involved in stress responses (34 %) and/or in catabolism (glycolysis and TCA pathway; 24 %). On the other hand, 60 % of the down-regulated proteins were involved in anabolic processes, i.e. in carbon fixation (both light-dependent reactions of photosynthesis and light-independent reactions of the Calvin cycle; 40 %) and in gluconeogenesis (20 %). Besides, we detected different isoforms of proteins involved in some of these pathways that were differentially accumulated. In summary, the energy metabolism of the Rvi6-resistant ‘Topaz’ leaves was altered upon infection. When comparing the protein profiles of ‘Topaz’ and the susceptible cultivar ‘Golden Delicious’, we could identify the same (categories of) proteins that were differentially accumulated exsclusively after infection. The established response of ‘Topaz’ seemed less explicit in ‘Golden Delicious’, which could explain the differences in susceptibility of both cultivars. Finally, the responses upon infection of the monogenic resistant cultivar ‘Topaz’ with and without preceding treatment with fosetyl-Al (Aliette®) were compared. This preventative, systemic fungicide caused a reduction in foliar disease symptoms and in hyphal growth as measured by real-time PCR. The effect of Aliette® was more clear using the real-time PCR technique as compared to the phenotypic disease rating score. When we analyzed the 2-DE protein profiles of Aliette®-treated ‘Topaz’ leaves, we could verify that fosetyl-Al intensifies the apple response upon scab infection that was already observed in the prior experiment (i.e. up- and down-regulation of the same (catgeories of) proteins in the Aliette®-treated leaves compared to the non-treated leaves). Interestingly, the observed Aliette®-induced reduction in degree of infection was notably less distinct in the scab-susceptible cultivar ‘Golden Delicious’ (~20 % compared to ~60 % in ‘Topaz’). This suggests that, for cultivating purposes, it seems useful to combine the use of (monogenic) resistance sources with the application of systemic fungicides such as Aliette®.In summary, we can state that the use of the 2D-DIGE technology and our developed Venturia specific real-time PCR protocol delivered some interesting insights in the response of apple upon V. inaequalis infection and allowed us to formulate several new working hypotheses, such as:· the infection-induced up-regulation of proteins involved in catabolic pathways such as glycolysis and TCA cycle in order to provide an extra energy supply necessary for stress response;· the infection-induced down-regulation of proteins involved in the anabolic carbon fixation pathways, i.e. photosynthesis and Calvin cycle;· the infection-induced down-regulation of ROS scavenging mechanisms in a monogenic resistant cultivar compared to a susceptible cultivar;· the similarity in cellular responses to different stresses, including abiotic stresses (such as mock-infection) and biotic stresses (such as fungal infection), and the hypothesis that plant acclimation to different stresses is controlled by sophisticated quantitative rather than qualitative effects;· the hypothesis that the differences in the changes in accumulation of proteins (involved in primary metabolism as well as defense response) between compatible and incompatible interactions are rather quantitative than qualitative.Further research is needed to validate these hypotheses and to determine whether interfering with the involved proteins and pathways could represent a strategy for developing new resistant cultivars and for attaining durable resistance.