Functional Analysis of Paired NLR Immune Receptors in Plant Immunity
von Qiqi Yan
Datum der mündl. Prüfung:2023-11-13
Erschienen:2024-03-21
Betreuer:Prof. Dr. Marcel Wiermer
Gutachter:Prof. Dr. Marcel Wiermer
Gutachter:Prof. Dr. Volker Lipka
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-10408
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Diese Datei ist bis 12.11.2025 gesperrt.
Zusammenfassung
Englisch
The Arabidopsis gene AT3G04210 encodes a truncated Toll/interleukin-1 receptor nucleotide-binding leucine-rich repeat (TIR-NB-LRR or TNL) protein that is lacking the canonical C-terminal LRR domain of NLRs, and is named TIR-NB13 (TN13). The gene directly linked to TN13 on chromosome 3 encodes a full-length TNL protein, named TNL LINKED TO TN13 (TLT13). Immunophenotypic analysis revealed that both TN13 and TLT13 contribute to the basal defense of Arabidopsis against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 ΔAvrPto/AvrPtoB. TN13 and TLT13 co-localize to the endoplasmic reticulum (ER) membrane and the nuclear envelope via their N-terminal transmembrane domains, and form homo- and hetero-oligomers in planta. The interaction of TN13 and TLT13 with a group of genomically duplicated NLRs suggests that this NLR pair functions as part of a larger NLR signaling network to mediate immune response in Arabidopsis. In an approach to investigate the TN13-TLT13 interaction in vivo at sub-cellular resolution, confocal laser scanning microscopy (CLSM)-based fluorescence lifetime imaging-Förster resonance energy transfer (FLIM-FRET) analyses were conducted and revealed that TN13 and TLT13 interact at the ER and the nuclear envelope when expressed transiently in Nicotiana benthamiana (N. benthamiana) leaves. A generated double deletion CRISPR/Cas9 mutant C-tn13 tlt13 shows a level of susceptibility to Pst DC3000 ΔAvrPto/AvrPtoB that is similar to the level observed for tn13-1 and tlt13-1 single mutants, indicating that these two NLRs function in the same defense pathway against mildly virulent Pseudomonas bacteria. When co-expressed to equal levels with TLT13 in N. benthamiana, TN13 can partially inhibit the hypersensitive cell death response induced by TLT13, suggesting that TN13 negatively regulates the cell death-inducing activity of TLT13. The major objective of this study was the identification and characterization of interaction partners of TN13 and TLT13. Initial in silico investigations and subsequent co-immunoprecipitation (Co-IP) interaction analyses revealed that TN13 interacts with the transcription factor ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN13 (ANAC013), whereas TLT13 interacts with both, ANAC013 and its closest homolog ANAC017. Consistent with the interaction, ANAC013 and ANAC017 localize to the ER as well as the nucleus in N. benthamiana. The protein interaction and localization analyses suggest that TN13, TLT13, ANAC013, and ANAC017 may form a larger protein complex at the cytoplasmic side of the ER. Pathogen infection experiments using the hemibiotrophic Pst DC3000 ΔAvrPto/AvrPtoB and the necrotrophic fungal pathogen Botrytis cinerea (B. cinerea) B05.10 indicate that both ANAC013 and ANAC017 are involved in the regulation of plant immunity. Infection of B. cinerea led to an increased nuclear accumulation of ANAC017, suggesting that the nuclear pool of ANAC017 is involved in mediating defense responses against B. cinerea. Untargeted proteomic approaches including immunoprecipitation coupled to mass spectrometry (IP-MS) and TurboID-base proximity labelling-MS were utilized to identify additional interactors of TN13 and TLT13. Among the interacting candidates that were revealed from both approaches, RESISTANCE TO LEPTOSPHAERIA MACULANS3 (RLM3), a TIR-NB protein with three BREVIS RADIX (BRX) domains integrated at the C-terminus, was investigated in more detail based on its high co-expression with TN13. Immunophenotypic analyses of rlm3 mutants showed that RLM3 contributes to Arabidopsis resistance against both Pst DC3000 ΔAvrPto/AvrPtoB and B. cinerea B05.10. Transient expression of RLM3 induced an EDS1-dependent cell death response in Nicotiana benthamiana, and subcellular localization analysis revealed that RLM3 localizes to the nucleus, the cytoplasm, and chloroplasts. Co-IP assays for the independent validation of the interactions between RLM3 and TN13 as well as TLT13 did not result in conclusive data. However, indirect evidence supports potential associations of RLM3 with TN13 and TLT13 because TN13 partially suppressed the RLM3-induced cell death in N. benthamiana and RLM3 showed increased protein accumulation when co-expression with TLT13 in N. benthamiana. Together, the results presented in this thesis indicate a complex interplay of the NLR proteins TN13, TLT13, and RLM3 and the transcription factors ANAC0013 and ANAC017 in the regulation of plant immunity and cell death.
Keywords: NLR; plant immunity
