Toll/interleukin-1 Receptor Domain-Only Protein of Arabidopsis Thaliana AtTX14 2IR, Produced By Alternative Splicing, Can Induce Defense Responses


 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.


Introduction
Plants have developed a multilayered immune system to deploy defense mechanisms in response to pathogen attacks appropriately. Two major types of receptors are utilized for surveillance of invading pathogens: surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding (NB) oligomerization domain (NOD)-like receptors (NLRs) (Jones and Dangl 2006; Barragan and Weigel 2020). Pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns, such as g22 and chitin oligomers, are recognized by PRRs, triggering an immune response referred to as PAMP-triggered immunity (PTI) (Macho and Zipfel 2014;Yoon et al. 2018). Damage-associated molecular patterns of host origin are also perceived by PRR-like cellular surface receptors that modulate defense responses (Albert et al. 2019). However, pathogens secreting effector proteins, encoded by Avirulence genes, can neutralize PTIs and compromise plant resistance (Jones and Dangl 2006). In this case, the successful resistance of hosts can be mounted by effector-triggered immunity using NLRs, which monitor modi cations in proteins targeted by pathogen effectors. Once activated, NLRs induce robust defense responses, which are associated with transcriptional boosting and reprogramming of defense-related genes, hypersensitive responses (HR) causing localized cell death, and systemic acquired resistance (Balint-Kurti 2019; Cui et al. 2015;Fu and Dong 2013). Depending on the domains located at the N-terminus, two major classes are found among NLRs with C-terminal leucine-rich repeats (LRR): Toll/interleukin-1 (TIR)-NB-LRR (TNL) and coiled-coil (CC) -NB-LRR (Dangl and Jones 2001;Cui et al. 2015). N-terminal TIR or CC domains in NLRs are important for defense signal transduction, and NB domains in NLRs are used as molecular switches, which turn on NLR-mediated signaling when ATP is bound . LRR  In plants, TIR domains are also found in various proteins whose domain compositions are different from those of classical TNL NLRs. For example, TIR domains are found in TIR-NB (TN) proteins without LRR domains and TIR-X (TX) proteins, which have a TIR domain and another domain (X) that is neither NB nor LRR (Meyers et al. 2002). In a model plant, Arabidopsis thaliana (Arabidopsis), a total of 145 genes encoding TIR domain-containing proteins were identi ed (Meyers et al. 2003 ): 83 TNLs, 5 TNLXs, 2  TNTNLs, 2 TTNLs, 21 TNs including one pseudogene, 30 TXs including 3 pseudogenes, and 2 XTNXs. It has been reported that overexpression of TX or TIR alone, such as AtTX12 and AtRBA1, can also induce elevated defense responses or cell death (Nandety et al. 2013;Nishimura et al. 2017;Song 2016).
Alternative splicing (AS) can produce multiple transcripts from a single precursor messenger RNA (pre-mRNA) (Nilsen and Graveley 2010). In plants, AS appears to occur in most multiexonic genes (Chamala et al. 2015). At least one AS event was identi ed in 50.2% Arabidopsis multi-exonic genes, even when only four types of AS-intron retention, alternative acceptor site, exon skipping, and alternative donor site -were considered. Another study revealed that intron retention is most common among different splicing types and is found in approximately 40% of AS events, singly or combined (Zhang et al. 2017b). AS plays an important role in reprogramming defense-related genes, both qualitatively by expressing distinct splicing variants and quantitatively by modulating the ratio of splicing variants (Rigo et al. 2019). It is known that complete resistance to tobacco mosaic virus requires two alternative transcripts of the tobacco N gene, encoding a full TNL protein and a truncated protein with TN, and only the rst LRR (Dinesh- Kumar et al. 2000). In addition, removing Arabidopsis RPS4 (resistance to pseudomonas syringae 4) introns with in-frame stop codons, which can be included by intron retention, abolishes these functions (Zhang and Gassmann. 2003;Zhang and Gassmann. 2007). Only partial resistance was observed in transgenic plants expressing intron-de cient and truncated RPS4 transcripts. In this study, we demonstrate that AtTX14 encodes three proteins with distinct domain compositions, as a result of intron retention-type AS: (1) a full-length TX protein with a truncated domain of unknown function (DUF) 641 in addition to the TIR domain encoded by a fully spliced transcript (AtTX14 Full), (2) a TIR-only protein encoded by a splicing variant with the 2nd intron retained (AtTX14 2IR), and (3) a protein with a truncated TIR encoded by another splicing variant with the rst intron retained (AtTX14 1IR). We found that AtTX14 Full and 2IR with intact TIR can form homo-and heterodimers with each other, but AtTX14 1IR cannot. Defense responses, including positive feedback of AtTX14, are induced by overexpression of AtTX14Full and 2IR in Arabidopsis, but not by AtTX14 1IR. Results obtained with tobacco, a heterologous plant system allowing an assessment of phenotypic effects in the absence of endogenous AtTX14 feedback regulation, con rmed that the TIR domain in AtTX14 2IR and Full is su cient to induce defense responses in plants.

Construction of plant overexpression vector
PCR products and a pCAMBIA3300m vector were digested using BamHI and PstI (Enzynomics Inc.) and puri ed using the EZ-Pure™ PCR Puri cation Kit ver.2 (Enzynomics Inc.). The pCAMBIA3300m vector was generated from pCAMBIA3300 by adding (1) an extra CaMV 35S promoter and (2) multiple cloning sites made up of BamHI, KpnI, SacI, XbaI, and PstI recognition sequences downstream of the additional CaMV 35S promoter. AtTX14 1IR transgenic plants were generated by cloning AtTX14 genomic DNA from the start codon to the in-frame stop codon in the rst intron at the multiple cloning site. In the case of AtTX14 2IR and Full transgenic plants, coding sequences ampli ed from cDNA were used for cloning.

Agrobacteriumtumefaciens (Agrobacterium) and plant transformation
Plant overexpression vectors were introduced into Agrobacterium strain GV3101 by the freeze-thaw method (Holsters et al. 1978). Transgenic plants were generated by the oral dipping method of WT plants using transformed Agrobacterium (Clough and Bent 1998). T1 transformants and their selfpollinated progenies were selected based on their resistance to glufosinate-ammonium (BASTA ® ; BASF, Ludwigshafen, Germany).
Yeast two-hybrid (Y2H) analyses pGBKT7 and pGADT7 (Takara Bio., Inc., Shiga, Japan), containing GAL4 DNA binding domain (BD) and activation domains (AD), respectively, were used as bait and prey cloning vectors to investigate proteinprotein interactions in the Y2H system. pGBKT7 and pGADT7 with the genes of interest were cotransformed into yeast AH109 strain, following the manufacturer's protocol for Yeastmaker™ Yeast Transformation System 2 (Clontech Laboratories, Mount View, CA, USA). Transformants were grown at 30°C for 4-5 days (d) on synthetic dropout (SD) without leucine and tryptophan (-LT) or without leucine, tryptophan, and histidine (-LTH).

Western blotting of proteins expressed in yeast
Yeast total proteins were extracted from cells grown in YPDA medium (10 g Bacto yeast extract, 20 g Bacto peptone, and 40 mg adenine hemisulfate in 1 L water) at 30 °C overnight (O/N), using Urea/SDS protein extraction buffer (40 mM Tris-HCl [pH 6.8], 5% sodium dodecyl sulfate [SDS], 30% glycerol, 2% βmercaptoethanol, and 8 M urea). The proteins were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene di uoride membrane (Merck Group, Darmstadt, Germany) with a transfer buffer (4.5 g Tris, 7.765 g glycine, 0.05 g SDS, 175 mL methanol in 1 L water) at 100 V for 2 hours (h). After rinsing with water, the membranes were blocked using General-Block Solution (TransLab, Daejeon, Korea) for 1 h.
Membranes were washed once with PBS-T buffer (8 g NaCl, 0.

Results
AtTX14 transcripts produced by distinct intron retentions encode three AtTX14 proteins A previous study reported that AtTX14 has an alternatively spliced form generated by the retention of the second intron and encodes a protein with a TIR domain (Fig. S1) and a short C-terminal extension (Kato et al. 2014). It was found that the rst intron of AtTX14 could also be retained, yet another alternatively spliced mRNA transcript was produced (Fig. 1A). The presence of an mRNA transcript containing the rst intron of AtTX14 is supported by the Reference Transcript Dataset for Arabidopsis, in which three differently processed mRNAs of AtTX14-Spliced mRNA 1, 2, and 3 have been reported (Zhang et al. 2017b) (Fig. 1B). Spliced mRNA 1, containing the rst intron but lacking the second intron, encodes a protein with 185 amino acids (aa) made of a truncated TIR domain (aa 21-166) and 19-aa C-terminal extension produced from the rst intron sequence. In contrast, Spliced mRNA 2, in which the rst intron is spliced out, but the second intron is retained, encodes a 220 aa-long protein with an intact TIR domain (aa 21-188) and 32-aa C-terminal extension, as previously reported (Kato et al. 2014). Spliced mRNA 3 without intron sequences encodes a 353 aa-long protein made of an intact TIR domain (aa 21-188) and 165 aa-long C-terminal extension. We named the translational products of Spliced mRNA 1, 2, and 3 as AtTX14 1IR ( rst intron retention form), AtTX14 2IR (second intron retention form), and AtTX14 Full (fulllength form), respectively (Fig. 1B).
Sequence alignment of the C-terminal part of AtTX14 Full and related proteins, a transcriptional regulator CHIQ1 and ten CHIQLs, revealed that the C-terminal half of AtTX14 Full contains a truncated DUF641 domain (Bossi et al. 2017) (Fig. S2). CHIQL7 (AT2G32130), located downstream of AtTX14 and transcribed in the same direction as AtTX14, was predicted to encode a protein with an intact DUF641 domain. Our results show that the upregulation of AtTX14, which activates defense signaling, produces three different proteins with truncated TIR, intact TIR only, and intact TIR plus a truncated DUF641.

AtTX14 2IR and AtTX14 Full can form homo-and heterodimers
Modeling of the AtTX14 Full structure based on the crystal structure of the RPS4 TIR domain (Protein Data Bank ID: 4C6R) using SWISS-MODEL revealed that AtTX14 1IR has an incomplete TIR domain with a modi ed αE helix and a loop leading to the αE helix (EE loop), compared with the TIR domains in AtTX14 Full and AtTX14 2IR (Fig. 2)  , we tested whether the three proteins translated from AtTX14-spliced mRNAs can form homo-or heterodimers using the Y2H system. In the Y2H analyses, AtTX14 2IR or Full was found to form a homodimer when fused to GAL4 BD and AD, allowing the growth of co-transformed yeasts on selective media (Fig. 3). Furthermore, AtTX14 2IR and Full were found to form a heterodimer. In contrast to the other forms, AtTX14 1IR with a modi ed αE helix and an EE loop could not form a homodimer, and heterodimers with AtTX14 2IR or Full, although AtTX14 1IR fused to BD or AD is expressed as expected in yeast cells (Fig. S3) To resolve this discrepancy, we generated transgenic plants, gAtTX14, in which 1.3 kb genomic DNA covering from the start to the stop codon of AtTX14 is regulated by the constitutive CaMV 35S promoter, and investigated the phenotypes. In line with the Kato group's observation but different from that of the Meyers group, we found that upregulation of AtTX14 induces stunted growth leading to dwar sm (Fig. 4A). To unequivocally determine whether the expression of one speci c spliced form of AtTX14 or a combination of AtTX14 spliced forms is necessary for dwar sm, we generated transgenic plants, in which AtTX14 1IR, 2IR, or Full was speci cally overexpressed by transgenes. Morphological observation of AtTX14 1IR, 2IR, or Full transgenic plants revealed that dwar sm was induced by transgenic expression of AtTX14 2IR or Full, but not by overexpression of AtTX14 1IR (Fig. 4A).
AtTX14 is regulated by a positive feedback mechanism to induce defense responses Some NLR genes are regulated by a positive feedback mechanism that requires accumulation of SA, a plant hormone playing important roles in plant disease resistance (Delaney et al. 1994;Xiao et al. 2003).
Expression analyses showed that AtTX14 is upregulated by biotic stress conditions, such as pathogen infection and elicitor treatment, resulting in SA accumulation (Nandety et al. 2013), raising the possibility that the ampli cation circuit also regulates AtTX14 transcription through SA accumulation. When the expression levels of AtTX14 were compared between WT and bal variants, in which constitutive defense responses caused by the duplication of SNC1 are regulated by the SA-mediated positive feedback mechanism Richards 2007, 2009), a stronger expression of AtTX14 was observed in the bal variant (Fig. S4A). Moreover, SNC1 expression was found to be elevated in AtTX14Full transgenic plants (Fig. S4B). To address the question of whether AtTX14 is controlled by a positive feedback mechanism, we determined the expression levels of endogenous AtTX14 using primers that differentiate endogenous and transgenic AtTX14 transcripts (Fig. S5). Expression levels of alternatively spliced forms with or without introns originating from the endogenous AtTX14 gene were increased in AtTX14 2IR and Full transgenic plants showing dwar sm (Fig. 4B), whereas feedback induction of endogenous AtTX14 transcription was not observed in AtTX14 1IR transgenic plants.
The expression of Pathogenesis related 1 (PR1), a marker for SA-dependent defense responses (Uknes et al. 1992;Delaney et al. 1994), was found to be elevated in AtTX14 2IR and Full transgenic plants, compared with WT plants (Fig. 5A). In general, stronger induction of PR1 was observed in AtTX14 Full transgenic plants than in AtTX14 2IR plants (Fig. 5B), when expression levels of PR1 were normalized to those of the transgenes expressed in Fig. 4B. We also tested whether overexpression of the TIR domain in AtTX14 2IR increases the resistance of transgenic plants to the bacterial pathogen, P. syringae pv. tomato (Pst). While no signi cant difference in the growth of Pst was observed between WT plants and AtTX14 1IR plants at 3 d post-infection (dpi), the growth of the bacteria was suppressed in the AtTX14 2IR plants compared with that in WT plants at 3 dpi (Fig. 5C). AtTX14 2IR plants were found to be more resistant to both virulent Pst, DC3000 without the AvrRpm1 effector, and avirulent Pst, DC3000 (AvrRpm1) with AvrRpm1 effector.
Elevated temperature enhances dwar sm in AtTX14 2IR transgenic plants For gAtTX14 transgenic plants, a more dramatic decrease in the length and width of the largest leaves, as well as more severe inhibition of stem elongation, was observed in growth at 28 °C than at 22 °C (Kato et al. 2014). Because multiple splicing variants were detected with slightly different ratios in the gAtTX14 transgenic plants at different temperatures, it was unclear whether a speci c splicing variant could induce high temperature-dependent phenotypic enhancement. We found that stunting phenotypes in AtTX14 2IR plants, which overexpress the AtTX14 TIR domain without the C-terminal-truncated DUF641 domain, were more severe at 28 than at 22 °C (Fig. 6A). 2IR transgenic plants grown for 14 d at 28 °C had less than 20% fresh weight compared with 2IR transgenic plants grown for 18 d at 22 °C (Fig. 6B). In contrast, WT plants grown for 14 d at 28 °C had about 50% fresh weight compared with WT plants grown at 22 °C.

Expression of AtTX14 2IR or AtTX14Full itself is su cient to induce defense responses
Since transgenic expression of either AtTX14 2IR or AtTX14 Full can activate the transcription of all possible spliced variants from endogenous AtTX14 via a positive feedback mechanism in Arabidopsis (Fig. 4B), it was unclear whether defense-associated phenotypic and gene expression changes observed in these transgenic plants are direct outcomes of the corresponding transgene expression. It is also possible that one or a combination of transcriptionally induced endogenous AtTX14-spliced mRNA(s) indirectly contributes to the defense responses. To test whether a speci c AtTX14 splicing variant can induce defense responses in the absence of the positive feedback mechanism in Arabidopsis, we took advantage of the Agrobacterium-mediated transient expression system using tobacco leaves, which are not interfered with by the feedback-regulated AtTX14 in Arabidopsis. Overexpression of AtTX14 2IR or Full with p19 RNA silencing suppressor in tobacco leaves induced HR-like chlorosis (Fig. 7A), but no phenotypic differences were observed in leaf parts where p19 and AtTX14 1IR were expressed together. Consistently, AtTX14 2IR and Full-in ltrated leaf areas were found to have lower amounts of chlorophyll than AtTX14 1IR-in ltrated leaf areas (Fig. 7B). Because both AtTX14 2IR and Full protein carry an intact AtTX14 TIR domain and transient expression of 2IR is su cient to induce HR and decrease chlorophyll content in the heterologous tobacco system, we concluded that expression of the AtTX14 TIR domain itself is su cient to activate defense responses.

Discussion
The TIR domain-only protein of AtTX14, which is naturally produced by AS, is su cient to induce defense responses ( Fig. 5 and 7). The observation that PR1 expression is elevated without pathogen infection in removing other domains such as NB and/or LRR (Bernoux et al. 2011;Swiderski et al. 2009). Based on the nding that RBA1 in the Ag-0 accession, which was previously thought to be TX but found to encode TIR-only protein, is su cient to induce defense responses (Nishimura et al. 2017), as well as AtTX14 2IR, we speculate that more TIR-only proteins, which play important roles in defense signaling, can be identi ed by re-annotation and close investigation of AS patterns of TIR domain-encoding genes. In addition, some Arabidopsis genes that were originally classi ed as TX need to be reclassi ed as TIR genes. AtTX12 (AT2G03300) encodes a 203 aa-long protein classi ed as TX, which results in the activation of defense signaling and growth defects (Song, 2016). The relatively short C-terminal extension (aa 165-204) in AtTX12 does not show any sequence similarity to known protein domains in the conserved domain database (https://www.ncbi.nlm.nih.gov/cdd) and is, therefore, more appropriate for classi cation as a TIR-only protein.
Given that AtTX14 2IR is not only capable of initiating a positive feedback loop (Fig. 4B) but also induces an HR-like phenotype by itself in tobacco (Fig. 7A), we conclude that the C-terminal region (aa 221-353) of AtTX14 Full with the truncated DUF641 domain is dispensable for the activation of defense responses. However, comparable levels of dwarf phenotypes and transcription of endogenous AtTX14 were observed between AtTX14 Full and 2IR transgenic plants in our experiments, when signi cantly smaller amounts of transgene transcripts were expressed in AtTX14 Full than in AtTX14 2IR (Fig. 4B). Although the C-terminal non-TIR part of AtTX14 Full, the truncated DUF641, is not necessary to induce dwar sm and HR, we do not rule out the possibility that the C-terminal part may increase the stability or activity of the AtTX14 TIR domain. Possible differences in protein translational e ciency and/or stability between AtTX14 2IR and Full requires further investigation. With the nding that both AtTX14 2IR and Full with intact TIR are positive components in plant defense signaling (Fig. 5-7), it needs to be determined which proteins can interact with AtTX14 proteins with intact TIR and how defense responses are activated by AtTX14 Full or 2IR expression. It also needs to be determined whether AtTX14 proteins, including AtTX14 1IR with the    levels of AtTX14 transcripts produced from endogenous or transgenic copies of AtTX14. TG: PCR products ampli ed from cDNA of AtTX14 transgene. 1IR AS: PCR products ampli ed from cDNA of endogenous AtTX14, with (Retained) or without (Spliced) the rst intron. 2IR AS: PCR products ampli ed from cDNA of endogenous AtTX14, with (Retained) or without (Spliced) the second intron. Actin7 (Act7) was used as a normalization control. Information on primers used here is found in Table S1 and Fig. S5.