Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Wirthmueller, Lennart; Roth, Charlotte; Fabro, Georgina; Caillaud, Marie‐Cécile; Rallapalli, Ghanasyam; Asai, Shuji; Sklenář, Jan; Jones, Alexandra M. E.; Wiermer, Marcel; Jones, Jonathan D. G.; Banfield, Mark J.

doi:10.1111/tpj.12691

Cited by 44 publications

(66 citation statements)

References 55 publications

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“…Like TN13, neither SNC1 nor EDS1 or other known defense regulators were identified in MOS6 affinity purifications from Hpa Noco2‐infected tissues. We also did not identify the known MOS6 interactor HaRxL106 (Wirthmueller et al ., ) or other Hpa Noco2 effector proteins. Generally, this might be because of the fact that the interactions of α‐importins with their cargos are transient (e.g.…”

Section: Discussionmentioning

confidence: 99%

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Roth¹,

Lüdke²,

Klenke³

et al. 2017

The Plant Journal

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Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

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Section: Discussionmentioning

confidence: 99%

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Roth¹,

Lüdke²,

Klenke³

et al. 2017

The Plant Journal

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“…Transgenic Arabidopsis plants expressing yellow fluorescent protein (YFP)-tagged HaRxL106 have been described (Wirthmueller et al, 2015). To generate transgenic lines expressing 3xHA-StrepII (HS):HaRxL106, RFP:HaRxL106, RFP:NLS: HaRxL106DC, and RFP:HaRxL106-Cterm58 (all Hpa Emoy2) we used the following previously described pENTR plasmids: pENTR4-HaRxL106, M followed by HaRxL106 amino acids I 25 -S 285 (Fabro et al, 2011), pENTR/D-TOPO-SV40NLS: HaRxL106DC, sequence APKKKRKV followed by HaRxL106 amino acids I 25 -G 229 , and pENTR/D-TOPO-HaRxL106-Cterm58, HaRxL106 amino acids G 228 -S 285 (Wirthmueller et al, 2015). Plasmids pXCSG-HS and pH7WGR2 were recombined with the aforementioned pENTR plasmids using Gateway ® LR clonase II to generate HS-and red fluorescent protein (RFP)-tagged versions of the HaRxL106 constructs respectively.…”

Section: Generation Of Transgenic Arabidopsis Linesmentioning

confidence: 99%

“…HaRxL106 is actively transported into nuclei of plant cells (Wirthmueller et al, 2015). Given that NPR1 is an important nuclear signal integrator of the SA pathway, we tested whether HaRxL106 affects NPR1 localization or protein levels.…”

Section: Effector Harxl106 Attenuates Npr1-dependent Defense Activationmentioning

confidence: 99%

“…RCD1 interacts with the Hpa effector HaRxL106 in a yeasttwo-hybrid (Y2H) assay, and this effector renders Arabidopsis more susceptible to biotrophic pathogens when expressed as a transgene (Fabro et al, 2011;Mukhtar et al, 2011). In plant cells, HaRxL106 is actively transported into the nucleus, indicative of a nuclear virulence-promoting activity of the effector (Wirthmueller et al, 2015). Here, we report that HaRxL106, when expressed as a transgene, affects both SA signaling and light-regulated developmental processes.…”

Section: Introductionmentioning

confidence: 99%

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Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL‐INDUCED CELL DEATH1

et al. 2018

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Summary The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL‐INDUCED CELL DEATH1 (RCD1).We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes.We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)‐induced defense genes and alters plant growth responses to light. HaRxL106‐mediated suppression of immunity is abolished in RCD1 loss‐of‐function mutants. We report that RCD1‐type proteins are phosphorylated, and we identified Mut9‐like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1‐interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA‐induced defense marker gene expression compared with wild‐type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling.Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

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“…Nuclear import occurs in a highly dynamic and rapid manner, thus the interactions of importin α with cargoes in the cytoplasm are usually not easily visualized or detected in cells. In addition, importin α superfamily contains several distinct isoforms (seven in human) that have both specific and partially redundant functions, and it remains unclear how cargoes are preferentially recognized by a distinct importin α . Thus, to test the efficiency and application of Mito‐docking, we tried to determine whether the method can capture this kind of dynamic interactions occurring in nuclear transport by detecting the interaction of six isoforms of human importin α (α1, α3, α4, α5, α6, and α7) with the nuclear localization signal (NLS) of wild‐type simian virus 40 (SV40) (NLS wt ).…”

Section: The Interactions Of Different Importin α Isoforms With Sv40‐mentioning

confidence: 99%

Mito‐docking: A Novel In Vivo Method to Detect Protein–Protein Interactions

Shao

Deng

et al. 2019

Small Methods

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Many methods have been developed to detect protein–protein interactions (PPIs) and explore cellular processes. However, effective methods for detecting complicated PPIs under physical conditions are still in demand. Here, a simple and efficient mitochondria‐docking (Mito‐docking) method for PPI detection in vivo is developed. The strategy is to anchor a “bait” protein to mitochondrial outer membrane (MOM), and then trap the “prey” protein onto MOM. In this way, interacting signals are enriched to allow easy detection. This method efficiently detects the well‐known interaction between two G protein subunits (Gγ2 with Gβ1) and is successfully applied to investigate the recognition of importin α superfamily members for the classical nuclear localization signal (NLS) of simian virus 40 (SV40) large T antigen, which are highly dynamic and not easily visualized by conventional methods. As far as is known, this is the first time that the interaction between human importin α receptors with NLSs has been visualized. The results prove that Mito‐docking can be used as a simple, straightforward, and intuitive method to study PPIs qualitatively and quantitatively in vivo.

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Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Cited by 44 publications

References 55 publications

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL‐INDUCED CELL DEATH1

Mito‐docking: A Novel In Vivo Method to Detect Protein–Protein Interactions

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