Programmierter Zelltod in Arabidopsis: Funktion der KDEL-Peptidasen in Entwicklung und Pathogen-Abwehr, sowie ihr Transport innerhalb der Zelle
Final Report Abstract
Programmed cell death (PCD) is required for plant development and for the hyper-sensitive response, which restricts growth of biotrophic pathogens. KDEL-tailed cysteine endopeptidases (CEP) are a subgroup of papain-type peptidases found in senescing tissues undergoing PCD of all gymnosperms and angiosperms analyzed so far. CEPs are synthesized as pro-enzymes with a C-terminal KDEL endoplasmic reticulum retention signal, which is removed with the pro-sequence to activate enzyme activity. In order to localize AtCEP in cells by Confocal Laser Scanning Microscopy (CLSM) and detect them by western blot analysis, we constructed translational fusion proteins of AtCEP1 and AtCEP2 with different fluorescing proteins and three-fold hemaglutinin (HA)-tags. The fusion genes were cloned under the control of the respective endogenous promoters, thus obtaining PCEP1::pre-pro-3xHA-EGFP-CEP1-KDEL and PCEP2::pre-pro-3xHA-mCherry-CEP2-KDEL as functional reporters and these were transformed either into Columbia (Col)-0 wild type plants or into the respective knock out mutants. Furthermore, we cloned the reporters PCEP1::pre-pro-3xHA-EGFP-KDEL and PCEP2::pre-pro-3xHA-mCherry-KDEL without the mature protease subunit and transformed them into the respective knock out mutant plants. The pro-3xHA-mCherry-AtCEP2-KDEL fusion protein was isolated ex vivo and was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for CEP. mCherry-AtCEP2 was detected in the epidermal layers of leaves and hypocotyl and in roots in the root cap and at the upper end of the lateral root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: in 10 µm long spindle shaped organelles that appear to be ER-bodies, which are found specifically in Brassicaceae, and in round vesicles with a diameter of 1 µm that strongly resemble ricinosomes first described in castor bean. We demonstrated a function for AtCEP1 in pathogen-triggered epidermal cell death. atcep1 mutants showed enhanced susceptibility to the biotrophic fungus Erysiphe cruciferarum. The functional construct rescued the pathogenesis phenotype of atcep1 thus confirming the function of AtCEP1 in restricting this powdery mildew. The spatio-temporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Cells attacked by the fungus express EGFP- AtCEP1 in the endoplasmic reticulum and accumulate it around haustoria. Wild type plants exhibit significantly more dead epidermal cells and less established haustoria as compared to atcep1 mutants. Additionally, expression of biotic stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. In contrast to the role of AtCEP1 in pathogen defense, we observed a functional redundancy among AtCEPs in development, for instance between AtCEP1 and AtCEP2 at the upper end of the anther filament within the putative un-loading zone, thus avoiding a single mutant phenotype. On the other hand, in atcep2 single mutants and in triple atcep mutant lines (atcep1-knock-out_atcep3-knock-out_atcep2-knock-down), where impaired PCD at the upper end of the lateral root cap interferes with the elongation zone, we found significantly shortened primary roots in all mutants as compared to the wild type but not a complete impairment of root cap formation. In brief, our study of pathogen responses and development in single and multiple mutant combinations suggests that the members of the Arabidopsis gene family are differentially regulated and have some unique functions, but also a certain degree of functional redundancy. In addition, it is formally possible that other proteases, such as the metacaspases, are able to compensate for loss of CEP function in AtCEP triple mutants.
Publications
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(2012) Programmed cell death in Ricinus and Arabidopsis: the function of KDEL cysteine peptidases in development. Physiol. Plantarum 145: 103-113. GI154/14-1
Hierl, G., Vothknecht, U., Gietl, C.
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(2013) Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidases 2 (AtCEP2) pro- enzyme and its storage in endoplasmic reticulum derived organelles. Plant Mol. Biol. 84: 605-620
Hierl, G., Höwing, T., Isono, E., Lottspeich, F., Gietl, C.
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(2014) Endoplasmic reticulum KDEL-tailed cysteine endopeptidases 1of Arabidopsis(AtCEP1) is involved in pathogen defense. Frontiers in Plant Science 5: 58
Höwing, T., Huesmann, C., Hoefle, C., Nagel, M-K., Isono, E., Hückelhoven, R., Gietl, C.
