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Research Article
Hankuil Yi
Chungnam National University School of Biosciences and Biotechnology: Chungnam National University College of Biological Sciences and Biotechnology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9951-7856
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This work is licensed under a CC BY 4.0 License. Read Full License
Toll/interleukin -1 receptor (TIR) domains, which have NAD+ cleavage activity, are used as signaling modules in NOD-like receptors for defense responses. It has been shown that TIR domains not only form homo- or heterodimers with TIR domain-containing proteins but also interact with various proteins. A previous study showed that overexpression of Arabidopsis thaliana (Arabidopsis) AtTX14, encoding an N-terminal TIR domain and a C-terminal domain with unknown function, resulted in dwarfism and constitutive defense signaling or autoimmunity. Transgenic Arabidopsis overexpressing AtTX14 displays enhanced defense responses and associated dwarf phenotypes at 28 °C compared with those at 22 °C, which differs from other mutant or transgenic Arabidopsis with constitutive defense responses. We found that AtTX14 is alternatively spliced to encode three different proteins, and the TIR domain itself can induce autoimmunity and elevated defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato. In addition, we revealed that the transcription of AtTX14 is regulated by a positive feedback mechanism. With transient overexpression of three AtTX14 protein forms in tobacco leaves, providing a heterologous system free from the positive feedback of AtTX14 in Arabidopsis, we demonstrated that expression of a splicing variant encoding the TIR domain-only protein is sufficient to activate defense signaling. A deeper understanding of interaction networks involving AtTX14 will broaden our knowledge on how plant defense signaling is regulated in response to pathogen infection and ambient temperature changes.
Keywords
Toll/interleukin-1 receptor, TIR, defense, disease resistance, alternative splicing
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This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.TIR.tif
Figure S1. Sequence alignment of TIR domains of AtTX14, RPP1, RPS4, and SNC1. Glutamate residue, which is catalytically important for NADase activity in SNC1, RPP1, and RPS4, is indicated by an arrow (Horsefield et al. 2019; Wan et al. 2019).
- FigureS2.DUF641.tif
Figure S2. The C-terminal extension in AtTX14 includes a C-terminally truncated DUF641 domain. Sequence alignment of DUF641 sequences in AtTX14, CHIQ1, and CHIQL (CHIQ1-like) families DUF641 shows a DUF641 consensus sequence. AtTX14: AT2G32140, CHIQL7: AT2G32130, CHIQ1: AT2G45260, CHIQL1: AT4G34080, CHIQL2: AT4G33320, CHIQL3: AT4G36100, CHIQL4: AT3G14870, CHIQL5: AT1G53380, CHIQL6: TAIR: AT1G29300, CHIQL8: AT2G30380, CHIQL9: AT3G60680, CHIQL10: AT5G58960.
- FigureS3.Y2Hwestern.tif
Figure S3. Western blot results showing the expression of AtTX14 proteins in yeast cells. GAL4 BD- and AD-fused AtTX14 proteins expressed in yeast cells are indicated as 1IR, 2IR, or Full. Anti-Myc and anti-HA antibodies were used to detect GAL4 BD (bait)- and GAL4 AD (prey)-fused proteins, respectively.
- FigureS4.balFullRT210301.tif
Figure S4. AtTX14 and SNC1 transcriptionally activate each other. Agarose gel electrophoresis results of PCR products amplified with cDNA templates synthesized in the absence (-) or presence of (+) reverse transcriptase (RTase). Retained and Spliced indicate PCR products with or without introns.
- FigureS5.RTPCRPrimerPosition210301.tif
Figure S5. Locations of primers specifically detecting endogenous AtTX14 transcripts (A) and AtTX14 transgene transcripts (B). 5U or 3U: untranslated region (UTR) at the 5′ or 3′ part of the transcripts. p35S: CaMV 35S promoter. tN: NOS terminator. White and gray boxes indicate exons and UTRs, respectively, while lines indicate introns.
- TableS1.docx
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Hankuil Yi
Chungnam National University School of Biosciences and Biotechnology: Chungnam National University College of Biological Sciences and Biotechnology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9951-7856
Badges
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Appropriate declaration statements
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History
CURRENT STATUS: Posted
Version 1
Posted 02 Apr, 2021
No community comments so far
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Comments: 0
PDF Downloads: ...
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
Toll/interleukin -1 receptor (TIR) domains, which have NAD+ cleavage activity, are used as signaling modules in NOD-like receptors for defense responses. It has been shown that TIR domains not only form homo- or heterodimers with TIR domain-containing proteins but also interact with various proteins. A previous study showed that overexpression of Arabidopsis thaliana (Arabidopsis) AtTX14, encoding an N-terminal TIR domain and a C-terminal domain with unknown function, resulted in dwarfism and constitutive defense signaling or autoimmunity. Transgenic Arabidopsis overexpressing AtTX14 displays enhanced defense responses and associated dwarf phenotypes at 28 °C compared with those at 22 °C, which differs from other mutant or transgenic Arabidopsis with constitutive defense responses. We found that AtTX14 is alternatively spliced to encode three different proteins, and the TIR domain itself can induce autoimmunity and elevated defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato. In addition, we revealed that the transcription of AtTX14 is regulated by a positive feedback mechanism. With transient overexpression of three AtTX14 protein forms in tobacco leaves, providing a heterologous system free from the positive feedback of AtTX14 in Arabidopsis, we demonstrated that expression of a splicing variant encoding the TIR domain-only protein is sufficient to activate defense signaling. A deeper understanding of interaction networks involving AtTX14 will broaden our knowledge on how plant defense signaling is regulated in response to pathogen infection and ambient temperature changes.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.TIR.tif
Figure S1. Sequence alignment of TIR domains of AtTX14, RPP1, RPS4, and SNC1. Glutamate residue, which is catalytically important for NADase activity in SNC1, RPP1, and RPS4, is indicated by an arrow (Horsefield et al. 2019; Wan et al. 2019).
- FigureS2.DUF641.tif
Figure S2. The C-terminal extension in AtTX14 includes a C-terminally truncated DUF641 domain. Sequence alignment of DUF641 sequences in AtTX14, CHIQ1, and CHIQL (CHIQ1-like) families DUF641 shows a DUF641 consensus sequence. AtTX14: AT2G32140, CHIQL7: AT2G32130, CHIQ1: AT2G45260, CHIQL1: AT4G34080, CHIQL2: AT4G33320, CHIQL3: AT4G36100, CHIQL4: AT3G14870, CHIQL5: AT1G53380, CHIQL6: TAIR: AT1G29300, CHIQL8: AT2G30380, CHIQL9: AT3G60680, CHIQL10: AT5G58960.
- FigureS3.Y2Hwestern.tif
Figure S3. Western blot results showing the expression of AtTX14 proteins in yeast cells. GAL4 BD- and AD-fused AtTX14 proteins expressed in yeast cells are indicated as 1IR, 2IR, or Full. Anti-Myc and anti-HA antibodies were used to detect GAL4 BD (bait)- and GAL4 AD (prey)-fused proteins, respectively.
- FigureS4.balFullRT210301.tif
Figure S4. AtTX14 and SNC1 transcriptionally activate each other. Agarose gel electrophoresis results of PCR products amplified with cDNA templates synthesized in the absence (-) or presence of (+) reverse transcriptase (RTase). Retained and Spliced indicate PCR products with or without introns.
- FigureS5.RTPCRPrimerPosition210301.tif
Figure S5. Locations of primers specifically detecting endogenous AtTX14 transcripts (A) and AtTX14 transgene transcripts (B). 5U or 3U: untranslated region (UTR) at the 5′ or 3′ part of the transcripts. p35S: CaMV 35S promoter. tN: NOS terminator. White and gray boxes indicate exons and UTRs, respectively, while lines indicate introns.
- TableS1.docx
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