Molecular analysis of importin-α-mediated nucleocytoplasmic signaling in plant innate immunity

by Charlotte Roth
Doctoral thesis
Date of Examination:2015-04-23
Date of issue:2015-04-30
Advisor:Dr. Marcel Wiermer
Referee:Prof. Dr. Volker Lipka
Referee:PD Dr. Thomas Teichmann
Referee:Prof. Dr. Ivo Feußner
Referee:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Cynthia Gleason
Referee:Prof. Dr. Andrea Polle
Persistent Address: http://dx.doi.org/10.53846/goediss-5045

 

 

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Abstract

English

IMPORTIN-αs are a family of nuclear transport receptors that mediate the translocation of nuclear localization signal (NLS)-containing cargo proteins from the cytoplasm into the nucleus through nuclear pores. The IMPORTIN-α3, MOS6 (MODIFIER OF SNC1, 6), is one of nine putative IMPORTIN-αs encoded by the Arabidopsis genome. MOS6 was identified as an essential component of auto-immune responses and related growth inhibition caused by the constitutively active TIR-NB-LRR Resistance protein variant, snc1 (suppressor of npr1-1, constitutive 1). In addition, MOS6 is required for basal resistance. This suggests that MOS6 is required for specific or preferential nuclear import of unknown cargo proteins involved in defense signaling. In this study, the contribution of the nine IMPORTIN-α family members to basal resistance and snc1-mediated auto-immunity was investigated and potential functional redundancies within the nuclear import receptor family in plant defense signaling were addressed. For reverse-genetic analyses, a collection of importin-α single, double and triple mutant combinations was generated and a prominent role of MOS6 in plant immunity and snc1 auto-immunity could be demonstrated. To identify defense-related cargo substrates and interaction partners of MOS6, in planta affinity purification of functional epitope-tagged MOS6 coupled with mass spectrometry was performed in addition to analyses of an established Arabidopsis interactome database. From these approaches, thirteen candidate MOS6 interactors were selected for further characterization. Transient expression in Nicotiana benthamiana and subsequent co-immunoprecipitation was used to validate these interactions. Several interactors were shown to selectively bind MOS6 as they did not interact with its closest homolog IMPORTIN-α6, reinforcing the idea of MOS6 substrate specificity. T-DNA insertion mutants of MOS6-interactor candidates were isolated and subjected to functional analyses. Interestingly, a mutation in the gene encoding the TIR-NBS protein TN13 leads to impaired resistance against Pseudomonas syringae pv. tomato DC3000 with an incomplete effector repertoire (ΔAvrPto/AvrPtoB). This finding demonstrates a so far unknown involvement of the novel MOS6-interacting protein TN13 in plant innate immunity. Confocal laser scanning microscopy revealed that TN13 localizes to the endoplasmic reticulum when transiently expressed in N. benthamiana. This may suggest release of TN13 from the ER-membrane upon pathogen attack and subsequent MOS6-mediated nuclear translocation for maintenance of basal resistance.
Keywords: importin alpha; MOS6
 
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