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Publikation

Mittelberger, C.; Hause, B.; Janik, K.; The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection PLOS ONE 17, e0272467, (2022) DOI: 10.1371/journal.pone.0272467

’Candidatus Phytoplasma mali’, is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from ’Candidatus Phytoplasma asteris’. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.
Publikation

Mittelberger, C.; Stellmach, H.; Hause, B.; Kerschbamer, C.; Schlink, K.; Letschka, T.; Janik, K.; A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts Int. J. Mol. Sci. 20, 4613, (2019) DOI: 10.3390/ijms20184613

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.
Bücher und Buchkapitel

Janik, K.; Stellmach, H.; Mittelberger, C.; Hause, B.; Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC) (Musetti, R. & Pagliari, L., eds.). Methods Mol. Biol. 1875, 321-331, (2019) ISBN: 978-1-4939-8837-2 DOI: 10.1007/978-1-4939-8837-2_24

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.
Publikation

Janik, K.; Mithöfer, A.; Raffeiner, M.; Stellmach, H.; Hause, B.; Schlink, K.; An effector of apple proliferation phytoplasma targets TCP transcription factors—a generalized virulence strategy of phytoplasma? Mol. Plant Pathol. 18, 435-442, (2017) DOI: 10.1111/mpp.12409

The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple‐growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11‐like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11‐like proteins seem to be key players in phytoplasmal infection.
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