Browsing by Subject "NEP1"

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Molekulare und biochemische Charakterisierung von NEP1 - ähnlichen Proteinen (NLPs) aus Plasmopara viticola
(2017) Schumacher, Stefan; Vögele, Ralf
Plasmopara viticola, the causal agent of grapevine downy mildew is one of the most important diseases in viticulture and leads to significant losses in crop in years with beneficial weather conditions. The molecular processes during the interaction between this pathogen and vine are yet poorly understood. Adopted pathogens achieve an infection by avoidance or suppression of plant innate immunity. This suppression takes place through pathogen secreted effector molecules, which can modulate plant defense mechanisms in all kinds of ways. One of these effector families are the necrosis and ethylene inducing peptide 1 – like proteins (NLP). These proteins occur in a vast variety of microorganisms and can on the one hand act as virulence factors and on the other hand induce a broad spectrum of defense responses in plants. In necrotrophic or hemibiotrophic pathogens these proteins are formed when the organism starts to feed from dead plant material. Beside these cytotoxic proteins many non-cytotoxic NLPs are known from hemibiotrophic or biotrophic microorganisms. However, the particular function of these NLPs is so far unknown. To date NLPs from Hyaloperonospora arabidopsidis, causal agent of downy mildew on the model plant Arabidopsis thaliana, are the only known example for these proteins from an obligate biotroph plant pathogen. These NLPs are not able to induce necrosis and their roll during infection by the pathogen is so far unknown. During this work two NLPs of complete size as well as one truncated version were identified in the genome of the obligate biotrophic oomycete Plasmopara viticola. During further experiments these proteins had been characterized by the use of molecular biological and biochemical techniques. The studies revealed a high degree of conservation of the corresponding genes isolated from several resistant and susceptible grapevine cultivars. Gene expression analysis showed high PvNLP expression during early time points of infection and even before first contact with host plant material, respectively. Necrosis-inducing activity of PvNLPs was neither observed in the model plant Nicotiana benthamiana nor in different susceptible and resistant Vitis species. To further investigate the reasons for the non-cytotoxic character of these proteins several experiments were conducted to clarify the relevance of different structural regions, their affinity to form homo- and hetero oligomers, as well as their subcellular localization. The crucial component for the lack of cytotoxicity was not identified. Neither the presence of a signal peptide nor a Nterminal region from another NLP with cytotoxic characteristics were able to form a necrosis inducing fusion protein with one of the identified NLPs from P. viticola. Formation of homodimers was observed for PvNLPs in vitro, but apparently does not occur during expression in planta. Furthermore PvNLPs are localized in the cytoplasm of N. benthamiana cells and show a possible association with the plant cell nucleus. This pattern of subcellular localization was also observed for a NLP with necrosis-inducing activity. The ability to induce plant innate immunity in Vitis could not be attested, suggesting a possible lack of the corresponding receptor in this plant genus. The results of this work further suggest a different role of non-cytotoxic NLPs which in P. viticola may fulfill a function during early infection stages ranging from zoospore release until the successful penetration of the host plant Vitis vinifera.