Die Plant Science Student Conference (PSSC) wird seit 20 Jahren im jährlichen Wechsel von Studierenden der beiden Leibniz-Institute IPK und IPB organisiert. Im Interview erläutern Christina Wäsch (IPK) und Carolin Apel (IPB),…
Über 600 Gäste kamen am 4. Juli ans IPB zur Langen Nacht, die Wissen schafft, um bei unserem Wissenschafts-Quiz-Parcours viel Neues zu erfahren und ihre Kenntnisse unter Beweis zu stellen. Unser Programm in diesem Jahr…
Sinelnikov, I. G.; Siedhoff, N. E.; Chulkin, A. M.; Zorov, I. N.; Schwaneberg, U.; Davari, M. D.; Sinitsyna, O. A.; Shcherbakova, L. A.; Sinitsyn, A. P.; Rozhkova, A. M.;Expression and refolding of the plant chitinase from Drosera capensis for applications as a sustainable and integrated pest managementFront. Bioeng. Biotechnol.9728501(2021)DOI: 10.3389/fbioe.2021.728501
Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution » protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.
Publikation
Brillada, C.; Teh, O.-K.; Ditengou, F. A.; Lee, C.-W.; Klecker, T.; Saeed, B.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M.;Exocyst subunit Exo70B2 is linked to immune signaling and autophagyPlant Cell33404-419(2021)DOI: 10.1093/plcell/koaa022
During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.