article: The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system.
  Programmed cell death (PCD) has crucial roles in development and immunity in multicellular organisms [1]. In plants, hypersensitive response (HR) cell death is one of most studied forms of PCD. The HR is a part of effector-triggered immunity (ETI), the second layer of the plant immune system, and plays an important role in restricting pathogen growth. ETI is primarily effective against biotrophic and hemi-biotrophic pathogens [2], which obtain nutrients from live host cells and actively suppress the first layer of the plant immune system, pathogen-associated molecular pattern (PAMP) -triggered immunity (PTI). By contrast, ETI-associated HR cell death may benefit necrotrophic pathogens, which often secret toxic compounds to kill host cells and obtain nutrients from dead cells [3]. Indeed, some necrotrophic pathogens promote virulence by hijacking the plant’s HR machinery [3]. The contribution of HR cell death to resistance against hemi-biotrophic pathogens, which switch from a biotrophic phase to a necrotrophic one [4], is still under debate [5–7]. Under this premise, minimizing the induction of cell death, as part of a defense response, would result in an advantage for plants against pathogens that can benefit from dead cells. Identification of so called ‘lesion mimic mutants (LMMs) ’ that display spontaneous HR-like cell death, has greatly advanced our understanding of HR cell death regulation [8]. The phytohormone SA promotes HR cell death induction, and LMM phenotypes are often compromised in SA-deficient mutants background such as the sid2 mutant, which are not able to accumulate SA upon immune activation [9–12]. Several LMMs initiate lesion formation under specific growth conditions and/or upon chemical treatments [1]. Lesion formation of the lsd1 mutant can be triggered by shifting plants from short day conditions to long day conditions [13]. Upon pathogen inoculation, the lsd1 mutant displays runaway cell death (RCD) phenotype that forms lesions beyond the inoculation site [14]. LSD1 encodes a zinc finger protein, and negatively regulate initiation of PCD and RCD, partly via maintenance of ROS homeostasis [11,15,16]. The basic region leucine zipper (bZIP) transcription factor, bZIP10, and the type I metacaspase, MC1, interact with LSD1 and regulate the PCD [6,17]. HR cell death can be regulated both positively and negatively by ROS [18–21]. Likewise, autophagy can act as both a positive and negative regulator of HR cell death, which was proposed to be dependent on plant age [22–26]. Infection of the avirulent bacterial strain, Pseudomonas syringae pv. tomato DC3000 (Pto) carrying AvrRPM1 or AvrRPS4, triggers the autophagic activity [23]. Application of an SA agonist, benzo (1,2, 3) thiadiazole-7-carbothioic acid (BTH), induces autophagosome formation [26]. The atg5 mutant displays an RCD phenotype that depends on SA accumulation and signaling [26]. Similarly, the autophagic component BECLIN1 is required to prevent RCD [27,28], suggesting that autophagy negatively regulates RCD. In contrast, the autophagic components positively regulates HR cell death induction upon Pto AvrRPS4 or the avirulent oomycete Hyaloperonospora arabidopsidis (Hpa) infection [23]. PAMP receptors also play roles in HR-like cell death regulation. BRASSINOSTEROID (BR) INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), which is a co-receptor of PAMP and BR receptors, negatively regulates cell death together with its close homologue SOMATIC EMBRYOGENESIS RECEPTOR KINASE 4 (SERK4) [29–32]. The bak1-3 and bak1-4 mutant, null alleles of BAK1, are impaired in PAMP- and BR-signaling, and the bak1-4 serk4 double mutant displays spontaneous cell death [30]. N-glycosylation and components of endoplasmic reticulum (ER) quality control contribute to activate the bak1-4 serk4-depdendent cell death [33]. The bak1-3 and bak1-4 alleles display RCD phenotype upon hemi-biotrophic bacterial pathogen Pto or necrotrophic fungal pathogen Alternaria brassiciola infection [32]. By contrast, the bak1-5 allele is only impaired in PAMP-signaling, but not in BR-signaling or the SERK4-dependent cell death regulation [34]. The cell death induction in the bak1-5 allele upon pathogen inoculation has not been characterized yet. A mechanistic link between PCD and immune system is also suggested by the observation that the activation of PTI suppresses Fumonisin B1 (FB1) -triggered PCD. Fumonisin B1, is a mycotoxin produced by the necrotrophic fungal pathogen Fusarium moniliforme that induces host PCD and promotes fungal virulence [35]. This suggesting the existence of a pathway capable of restricting cell death induction [35]. Recently, a signaling sector that mediates ETI and is inhibited by PTI, namely ETI-mediating and PTI-inhibited sector (EMPIS), has been identified [36]. However, the molecular mechanisms underlying the restriction of cell death remain obscure. The glycine-tyrosine-phenylalanine (GYF) motif represents the conserved signature of the GYF domain, which was first identified in human CD2-binding protein 2 (CD2BP2), where it was required for binding to the cytoplasmic tail of CD2 [37]. CD2BP2 plays a role in CD2-triggered T lymphocyte activation and spliceosomal protein functions [37,38]. The GYF domain is highly conserved across eukaryotic species and can be roughly classified into two subfamilies, the CD2BP2-type GYF domain and the suppressor of myosin 2 (SMY2) -type GYF domain [38,39]. Structures of the GYF domains have been determined [39,40], and the recognition motifs have been characterized in detail [41]. The GYF domain of PSIG1 (At5g42950) recognizes proline-rich sequences as do other GYF domains found in human and yeast proteins [41]. Recently, PSIG1 was reported to be indispensable for plantago asiatica mosaic virus infection and designated as Essential for poteXvirus Accumulation 1 (EXA1) [42]. Furthermore, MUTANT, SNC1-ENHANCING 11 (MUSE11) was found to be EXA1/PSIG1 [43]. EXA1/MUSE11/PSIG1 was proposed to regulate levels of plant immune receptors via translational repression, and thereby negatively regulate plant immunity [43]. Nevertheless, significance of the GYF domain for functions of PSIG1 and other plant GYF domain proteins has not been addressed yet. Here, we identify PSIG1, a plant-specific protein with the GYF domain, as a key player in the restriction of pathogen-induced PCD. Analyses of psig1 mutants suggested that the PCD-restriction system is crucial for resistance against hemi-biotrophic bacterial pathogens.
  To explore for novel components in PTI, we performed a differential phosphoproteome analysis and found that the phosphorylation status of the N-terminal region of PSIG1 (At5g42950) was modulated within 10 minutes after treatment of Arabidopsis seedlings with PAMP flg22, the conserved immunogenic epitope of bacterial flagellin (Fig 1A and 1B, S19 Fig and S4 Table). PSIG1 contains the GYF domain that is highly conserved among a variety of proteins from diverse eukaryotic species (Fig 1C and S1A Fig). However, the predicted PSIG1 sequence was found to be plant specific (Fig 1D and S1B Fig). The functions of plants proteins containing the GYF-domain are largely unknown. We therefore isolated three independent T-DNA insertion mutants to study PSIG1’s functions (S2 Fig). Treatment with flg22 enhanced oxidative bursts for all three psig1 mutant alleles when compared to wild-type (WT) Col-8 plants (Fig 2A and S3A Fig). Similarly, mitogen-activated protein kinase activation and callose deposition were also enhanced in the psig1-1 and psig1-2 alleles, but were similar to WT for psig1-3 (Fig 2B and 2C and S3B–S3D Fig). Both the psig1-1 and psig1-2 alleles displayed a weak dwarf phenotype mostly absent in psig1-3 (S2A Fig). Dwarf phenotypes are often associated with the inappropriate activation of the SA pathway [44]. Indeed, expression of the SA-related marker gene PATHOGENESIS-RELATED 1 (PR1) was upregulated in the psig1 mutants under normal growth conditions (Fig 2E and S4A and S4B Fig). Therefore, to investigate the contribution of SA signaling to the phenotypes, the mutant having the strongest phenotype, psig1-1, was crossed with the SA biosynthesis-deficient sid2-2 mutant [45]. Unexpectedly, the enhanced oxidative burst phenotype was not suppressed in the psig1-1 sid2-2 double mutant. Instead, the oxidative burst was delayed (Fig 2F and S5C Fig), and in addition, the dwarf phenotype was unaffected by the sid2-2 introgression (Fig 2D). The expected decrease in SA levels caused by sid2-2 in the double mutant, was confirmed by analyzing PR1 expression (Fig 2E). Phenotypes of psig1-1 were complemented by the expression of PSIG1 driven by its native promoter (S6A–S6C Fig), indicating that PSIG1 negatively regulates PAMP responses and positively plant growth in an SA-independent manner. Because the psig1 mutants displayed enhanced responsiveness to flg22, we hypothesized that this would result in increased pathogen resistance. To our surprise, the psig1 mutants displayed enhanced susceptibility against the virulent hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) (Fig 3A and 3B and S5A, S5B and S6D Figs). The enhanced susceptible phenotype may reflect stomatal defects, as plants were spray inoculated. To test this possibility, we analyzed stomata density and flg22-induced stomatal closure. However, psig1-1 responses were comparable to that of WT plants (S7 Fig). To investigate the role of PSIG1 in plant immunity, we challenged the mutant plants with different types of pathogens. Bacterial growth was unaffected in the psig1 mutants spray inoculated with the avirulent strain Pto carrying AvrRPM1 (Pto AvrRPM1) (Fig 3D). Instead, we observed the development of visible cell death symptoms 4 days after Pto AvrRPM1 inoculation in the psig1 mutants, which were absent in WT plants (Fig 3C). Infected leaves were stained with trypan blue to visualize dead cells, revealing that cell death induction was significantly increased in the psig1 mutants compared to WT plants (S8A and S8B Fig). Similar levels of cell death induction were observed in the psig1-1 mutant after inoculation with Pto carrying AvrRPS4 (Pto AvrRPS4) that activates effectual ETI in Col-0 without macroscopically visible HR cell death (Fig 4A–4D and S8F and S8G Fig) [46]. Cell death was not observed in the psig1 mutants under normal growth conditions without pathogen inoculation (Fig 4A–4D and S8–S11 Figs), in difference to the typical LMMs that develop spontaneous cell death without pathogen attack [8]. It suggests that PSIG1 function is dedicated to the restriction of cell death after activation. The Col-8 ecotype is susceptible to obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) isolate Noco2, meaning that penetrated cells generally do not trigger HR cell death (Figs 3E and 4E). However, infection with the virulent Hpa Noco2, resulted in the induction of cell death in the psig1 mutants (Figs 3E and 4E and S6E Fig). In spite of the cell death, the psig1 mutants were not fully resistant against Hpa Noco2 (Figs 3E, 3F and 4E and S6E and S6F Fig). This observation contrasts to incompatible interactions, where penetrated cells trigger a fast HR cell death response, thereby limiting further pathogen growth [47]. Of note, RCD was not observed in the psig1 mutants, and cell death induction was restricted to cells in contact with the Hpa Noco2 hyphae, and therefore, potentially penetrated (Figs 3E and 4E). Our observations that Hpa Noco2 grew in spite of HR-like cell death may be reconciled by the possibility that pathogen growth was faster than the cell death. Together, these results suggest that PSIG1 negatively regulates the induction of cell death in pathogen-targeted cells. We found that the frequency of cell death after inoculation with the avirulent bacteria Pto AvrRPS4 markedly increased in the psig1 mutants compared to WT plants (Fig 4A–4D). It was not clear, however, whether the observed cell death in the psig1 mutants was associated with the activation of AvrRPS4-dependent ETI. AvrRPS4 is recognized by the TIR-NB-LRR-type receptor pair RRS1/RPS4 [48], and EDS1 is required for RRS1/RPS4-triggered ETI [49]. Therefore, we generated psig1-1 eds1-2 double mutant and inoculated it with Pto AvrRPS4. As expected, AvrRPS4-triggered cell death was suppressed in the psig1-1 eds1-2 mutant (Fig 4A and 4B). In addition, AvrRPM1-triggered cell death, which does not require EDS1, was unaffected in the psig1-1 eds1-2 mutant (S9 Fig). These results indicate that the induction of ETI-triggered cell death is increased in the psig1 mutants. Since SA potentiates the HR [9], increased SA levels in the psig1 mutants could result in the enhanced induction of ETI-triggered cell death. To examine this possibility, the psig1-1 sid2-2 mutant was challenged with Pto AvrRPS4 or Pto AvrRPM1. The enhanced cell death phenotype of psig1-1 was retained in the sid2-2 background (Fig 4A and 4B), indicating that PSIG1 negatively regulates the induction of ETI-triggered cell death in an SA-independent manner. Similarly, enhanced ROS production in response to flg22 treatment in the psig1 mutants occurred in an SA-independent manner (Fig 2F and S5C Fig). ROS accumulation is also known to regulate the induction of cell death in both positive and negative manners [21]. Therefore, we crossed psig1-1 with the rbohD mutant, which has a defect in pathogen-induced ROS production [19], and investigated the contribution of ROS accumulation to the psig1-1 phenotypes. There was no detectable flg22-induced ROS production in the psig1-1 rbohD double mutant (Fig 2G and S5D Fig), and the dwarf phenotype of psig1-1 was retained in the rbohD background (Fig 2D). In addition, enhanced ETI-triggered cell death phenotype of psig1-1 was also unaffected by the rbohD introgression (Fig 4C and 4D and S8C Fig), indicating that PSIG1 negatively regulates the induction of ETI-triggered cell death in a ROS-independent manner. Collectively, these results suggest that the induction of cell death by avirulent bacterial pathogen infection is limited to a certain level in WT plants, and PSIG1 is involved in a mechanism required for the restriction of cell death in an SA- and ROS-independent manner. We additionally investigated the contribution of SA and ROS to the Hpa Noco2-induced cell death. Similarly, the induction of cell death observed in the psig1-1 mutant for cells in contact with Hpa Noco2 hyphae was still present in both genetic backgrounds containing sid2-2 or rbohD mutations (Fig 4E). However, the induction of cell death was clearly compromised in the sid2-2 background (Fig 4E), indicating that elevated SA levels in psig1-1 partly contributed to the Hpa Noco2-induced cell death. Interestingly, we also observed a clear reduction in cell death in the eds1-2 background (Fig 4E). This result may suggest that Hpa Noco2 effectors are weakly recognized by TIR-NB-LRR-type receptor proteins, and the remaining cell death is triggered by alternative NB-LRR-type receptors, or the result of reduced SA level. As PSIG1 was originally identified as an early PAMP-responsive phosphoprotein, it is possible that PSIG1 is a PAMP-signaling component that regulates the plant immune system. If this is the case, there may be other PAMP-signaling mutants that phenocopy the psig1 mutant cell death phenotype. BAK1 is a co-receptor of PAMP and BR receptors, and the bak1-5 mutant is only impaired in PAMP-signaling [34]. BIK1 and PBL1 are highly homologous receptor-like cytoplasmic kinases that directly interact with PAMP receptors, and the double mutant is impaired in PAMP-induced resistance [50]. We found that flg22-induced phosphoregulation of PSIG1 was compromised in the bak1-4, bak1-5, and bik1 pbl1 mutants (Fig 5A). In addition, the bak1-4, bak1-5, and bik1 pbl1 mutants displayed the psig1-like cell death phenotype upon Pto AvrRPS4 or Hpa Noco2 inoculation (Fig 5B and 5C and S10 Fig). These results suggest that activation of the PAMP-signaling pathway suppresses the induction of cell death through an elusive mechanism that is independent of the reported BAK1/SERK4-regulated cell death mechanism [30,32,33]. Activation of the PAMP-signaling pathway has been reported to inhibit HR cell death induced by avirulent bacterial pathogens by restricting the ability of type III protein secretion system to inject effectors [51]. Therefore, impaired PAMP-signaling may result in enhanced effector injection and thus excess HR cell death. To test whether PSIG1 is involved in the PAMP-induced restriction of effector injection by Pto, we inoculated mutant plants with Pto AvrRPM1. As reported, WT plants displayed inhibition of cell death by the flg22 pretreatment, in contrast to the fls2 mutant (Fig 5D and S11 Fig). As in WT plants, the induction of HR cell death was also inhibited in the psig1-1 mutant by flg22 pretreatment, suggesting that the PAMP-induced restriction of effector injection by Pto is intact in the psig1 mutants. These results suggest that PAMP-signaling pathways suppress effector-triggered HR cell death through at least two different mechanisms at different stages of infection. Next, we investigated whether PSIG1 suppresses cell death induced by different mechanisms involved in plant-pathogen interactions. Necrotrophic pathogens actively induce cell death of host cells by secreting toxins that support infections. Fumonisin B1 (FB1) is a mycotoxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme. FB1 induces a type of PCD with some similarities to HR cell death [47]. Of note, FB1-induced PCD can be suppressed by PAMP pretreatment in Arabidopsis [35]. FB1 treatment resulted in leaf chlorosis and cell death, which was confirmed by trypan blue staining, in both psig1-1 and WT plants (Fig 5E). The pronounced induction of cell death in the psig1-1 mutant is most likely the result of elevated SA levels [9]. Pretreatment with flg22 less effectively suppressed the cell death in the psig1-1 mutant, irrespective of the enhanced FB1-triggered cell death, as compared to WT plants (Fig 5E). These results indicate that PSIG1 participates in the PAMP signaling-dependent suppression of FB1-induced PCD. To investigate the role of the GYF domain in the function of PSIG1, we complemented the psig1-1 mutant with a construct driven by its native promoter and carrying Y575A mutated version of the GYF domain (Fig 6B). The Y575A mutation was shown to abrogate interaction with proline-rich sequences of interacting proteins [41,52]. Even though expression of PSIG1Y575A complemented the dwarf phenotype (Fig 6A), it failed to suppress the enhanced cell death phenotype triggered by Pto AvrRPS4 inoculation (Fig 6C and 6D). These results uncouple the GYF domain function in the regulation of cell death from other functions responsible for the dwarf phenotype. The GYF domain of PSIG1 was shown to recognize the proline-rich sequence proline-proline-glycine-phenylalanine (PPGF) [41]. Therefore, proteins that have the PPGF sequence expected to regulate the cell death along with PSIG1. To explore the molecular function of PSIG1, we assessed the subcellular localization of PSIG1. The C-terminus of PSIG1 was tagged with GFP, and the labelled protein was transiently expressed in Nicotiana benthamiana leaves. Interestingly, PSIG1-GFP localized to cytoplasmic foci and co-localized with DCP1-mCherry, a processing body (P-body) marker (Fig 7D and S12 Fig). The GYF domain of PSIG1 was shown to recognize the proline-rich sequence PPGF [41]. Therefore, we searched for PPGF sequences in known plant P-body components and found the PPGF sequence at the C-terminal region of SMG7 (At5g19400) (Fig 7A and S13 Fig). Importantly, mutations in SMG7 were previously shown to cause autoimmune phenotypes, including the induction of spontaneous cell death in Arabidopsis [53]. We therefore investigated whether PSIG1 interacts with SMG7. GST-tagged PSIG1 and HisMBP-tagged SMG7 recombinant proteins were prepared, and the interaction was assessed by a GST-pull down assay. We found that GST-PSIG1 interacts with HisMBP-SMG7 in vitro (Fig 7B). The C-terminal half of SMG7 (SMG7-C), which contains the PPGF sequence, was required for the interaction, while the N-terminal half of SMG7 (SMG7-N) was dispensable (Fig 7B). Interestingly, PSIG1 possesses the PPGF sequence (Fig 7A), opening the possibility of an self-inhibitory intramolecular interaction [41]. Although the full-length PSIG1 interacted with SMG7 in vitro, their interaction could be fine-tuned through the intramolecular interaction in planta. In spite of interacting in vitro, the PPGF sequence in PSIG1 could nevertheless affect the interaction with SMG7 and complicate interpretation of the results. Therefore, we decided to use the N-terminal region of PSIG1 that contains the GYF domain but not the PPGF sequence for the further analyses. To investigate the significance of the GYF domain in PSIG1 and the PPGF sequence in SMG7, we mutated residues important for the interaction [41]. Mutations in the GYF domain (Y575A and W570A/Y575A) or PPGF sequence (G933A) indeed abolished the interaction (Fig 7C and S14 Fig). Moreover, we found that PSIG1 co-localizes with SMG7 in Nicotiana benthamiana (Fig 7D and S12B Fig). Collectively, these results suggest that PSIG1 directly interacts with SMG7 through its GYF domain and functions within P-bodies. As in the case of the psig1 mutants, two smg7 mutant alleles, smg7-4 and smg7-6, that have T-DNA insertions in the C-terminal region, do not show autoimmune phenotypes and are indistinguishable from WT plants (Fig 7E) [53]. The PPGF sequence that mediates interaction with the GYF domain is located downstream of the T-DNA insertion sites of smg7-4 and smg7-6 (Fig 7E). Therefore, if SMG7 plays a role in the regulation of PSIG1-dependent cell death, the smg7-4 allele is expected to display the enhanced cell death phenotype as seen in the psig1 mutants. To test this, we challenged the smg7-4 allele with Pto AvrRPS4 or Pto AvrRPM1 and found that the smg7-4 allele phenocopied the psig1’s enhanced cell death (Fig 7F and S15 Fig). These results suggest that PSIG1 regulates the induction of cell death by interacting via the C-terminal region of SMG7.
  Activation of cell death during the interaction of plants with biotrophic or necrotrophic pathogens, can lead to antagonistic results [4,54]. The ability to fine-tune PCD activation during plant-pathogen interactions is critical for plants to effectively fend off pathogens with different infection strategies [3,50]. However, the biological significance of the systems regulating cell death activation remains unclear because a general framework and the molecular components of the system are poorly understood. With the initial aim of understanding PAMP-signaling pathways, we identified PSIG1 as an early PAMP-responsive protein by differential phosphoproteome analysis (S4 Table). Through our approach, we were also able to detected phospho-regulation of MAPK cascade components, SERK2, CDPKs, MPK1, RbohD, and WRKY1 (S4 Table). By isolating T-DNA insertion mutants of PSIG1, we found that PSIG1 was required for resistance against the hemi-biotrophic bacterial pathogen Pto (Fig 3B). However, detailed analyses with well-described PAMP responses in the psig1 mutants, did not reveal major differences to WT plants. Instead, we discovered that the induction of cell death was accelerated in the psig1 mutants upon inoculation with avirulent bacterial pathogens (Fig 4 and S8 Fig). This observation led us to hypothesize that PSIG1 has a role in adjusting the induction of ETI-triggered cell death by potentially monitoring the active state of PTI. The significance of SA and ROS pathways in the regulation of cell death has been well documented. However, our genetic analyses with SA biosynthesis- and ROS production-deficient mutants indicated that PSIG1 regulates the induction of cell death in an SA- and ROS-independent manner (Fig 4A–4D). In this respect, the psig1 mutants differ strikingly from the lsd1 and autophagy mutants that require SA accumulation for their uncontrolled PCD phenotypes [11,26,55], suggesting that PSIG1 controls cell death through an alternative pathway. Moreover, we addressed whether PSIG1 regulates the induction of cell death in a PAMP signal-dependent manner. Up to now, there has been no experimental system available to evaluate effect of PTI on the induction of HR cell death [35]. Therefore, we focused on the inhibitory effect of PTI on PCD induction by the fungal toxin FB1 [35], and reveal that the PTI-induced inhibition was compromised in the psig1 mutant (Fig 5E). Collectively, these results suggest that plants possess a pathway to restrict cell death induction during PTI. A potential reason may be to override cell death activation during an effective PTI response to avoid unnecessary tissue damage and infection by necrotrophic pathogens. It will be important to address how PSIG1 suppresses the cell death triggered by ETI activation and the fungal toxin FB1. To that purpose, however, molecular mechanisms how ETI and FB1 lead to the cell death must be unraveled. With regards to the enhanced cell death phenotype of the psig1 mutants, they expectedly displayed enhanced resistance against the biotrophic pathogen Hpa Noco2, and enhanced susceptibility to the necrotrophic pathogen Alternaria brassiciola O-264 (Fig 3G). Necrotrophic pathogens could kill host cells in a variety of ways, some of which may not utilize PCD machinery of host cells. Therefore, the psig1 mutants may not necessarily display susceptible phenotype against all necrotrophic pathogens. Again, an intriguing observation was that the psig1 mutants were more susceptible to the hemi-biotrophic pathogen Pto compared to WT plants. These results suggest that the PTI-dependent PCD regulation system may be crucial for coping with hemi-biotrophic pathogens that can switch their infection strategies during colonization. In addition, we found that resistance against the avirulent bacteria strains was not enhanced in the psig1 mutants (Fig 3D and S17 Fig), supporting the idea that the host plants do not benefit much by maximizing the induction of HR cell death. Taken together, these results have allowed us to identify PSIG1 as a unique component in plant immunity that connects PTI and PCD, highlighting the biological significance of the PTI-dependent PCD regulation. Our findings may also offer an explanation why toxin-producing necrotrophic pathogens potentially suppress PTI for their successful infection [56]. The psig1 mutants, especially the psig1-1 and psig1-2 alleles, seem to be sensitive to environmental conditions. Recently, exa1-1/muse11-1/psig1-1 was reported to display auto-immune phenotype, which was characterized by elevated PR1 gene expression and reduced plant size [43]. However, these phenotypes were not observed in another study [36]. Under our experimental conditions, the psig1-1 allele displays intermediate phenotype based on the PR1 gene expression and plant size. Importantly, the psig1-3 allele displays the cell death phenotype but is less sensitive to growth conditions compared to the psig1-1 and psig1-2 alleles. The difference between these alleles may result from the expression of a C-terminally truncated PSIG1 in psig1-3 allele (S2C Fig). The psig1-3 allele is a useful tool to assess significance of the cell death regulation in disease resistance. Indeed, the psig1-3 allele clearly displayed susceptible phenotype against Pto also when it was inoculated by syringe infiltration, while the psig1-1 and psig1-2 alleles gave inconsistent results (S16 Fig). The psig1-1 and psig1-2 alleles are more likely to be affected by stress responses caused by the syringe infiltration. Interestingly, the psig1-3 allele became susceptible 3 days post inoculation (dpi) but displayed no difference 2 dpi compared to WT plants, which is different from the eds1-2 mutant that displayed susceptible phenotype 2 dpi (S16B Fig). A similar trend was observed by spray inoculation (S5B Fig). In contrast, growth of the avirulent strain Pto AvrRPS4 was unaffected in the psig1 mutants (S17 Fig). These observations, especially the Pto growth kinetics in the psig1-3 allele, support our hypothesis that the cell death regulation becomes crucial when hemi-biotrophic pathogens switch their infection strategies. The GYF domains found in plant proteins are all classified into the SMY2-type GYF domain family (S1 Fig). Although the structure and recognition properties of the GYF domains in plant proteins are highly conserved, the overall structure of GYF domain-containing proteins in plants is rather plant specific (Fig 1D, S1B Fig and S1 and S2 Tables) [41]. PSIG1 gene family is well conserved in land plants but not in other organisms. No additional domains with known function are predicted on PSIG1, which would help elucidate further molecular function (s). In many cases, the GYF domain proteins modulate signaling pathways through their binding to key regulatory components. Therefore, identification of PSIG1 interactors are expected to facilitate an understanding of the molecular mechanisms by which PSIG1 inhibits PCD. A previous study identified the PPGF sequence containing fragments of RNA binding proteins in a Y2H screen using the GYF domain of PSIG1 as a bait [41]. Interestingly, PSIG1 itself has the PPGF sequence (Fig 7A), and the intramolecular interaction could happen and affect interaction with other PPGF sequence containing proteins [41]. In another study, PSIG1 was identified in 5′-cap complexes [57]. These findings suggest that PSIG1 might be involved in regulating RNA metabolism and/or translation. Indeed, we found that PSIG1 localizes to P-bodies that are involved in post-transcriptional regulation [58] (Fig 7D). Moreover, we found that PSIG1 interacts with the NMD factor SMG7 through the GYF domain (Fig 7C). In general, SMG7 is functionally conserved among different organisms [53,59,60]. However, the PPGF sequence recognized by the GYF domain was only found in the less conserved C-terminal region of plant SMG7 homologs (S13A Fig). Additionally, the GYF domain of yeast Smy2 was shown to interact with P-body components other than the SMG7 homolog [61]. These results suggest that the GYF domain proteins function within P-bodies in general but cooperate with different components in different organisms. Disruption of the NMD-related genes often results in autoimmune phenotypes [62]. In addition, PAMP treatment was shown to modulate NMD efficiency [63]. Moreover, the C-terminal region of SMG7 was suggested to play a role in the target degradation step of plant NMD [64]. The observations that PSIG1 interacts with the C-terminal region of SMG7 (Fig 7B) and that the C-terminal truncated smg7-4 allele phenocopies the psig1 cell death phenotype (Fig 7F) suggest that PSIG1 regulates the induction of cell death by modulating an SMG7-dependent NMD. It will be important to identify PSIG1- and SMG7-dependent NMD targets to understand how NMD contributes to the induction of cell death.
  Arabidopsis plants were grown in soil at 22°C in 8 h light /16 h dark or 12 h light /12 h photoperiods. Mutant seeds for psig1-1 (SALK_005994), psig1-2 (SALK_135013), psig1-3 (SAIL_1282_B9), smg7-4 (SAIL_63F08) were obtained from the Nottingham Arabidopsis Stock Center (NASC). Homozygous insertion mutants were identified by PCR. The fls2 (SALK_141277), rbohD [19] and sid2-2 [45] seeds were kindly provided by Kohki Yoshimoto (Meiji University, Japan). The seeds for the null eds1-2 mutation in the Col-0 background (referred to as Col eds1-2) [65] were kindly provided by Shigeyuki Betsuyaku (University of Tokyo, Japan) and Jane Parker (MPI for Plant Breeding Research, Germany). The bik1 pbl1 [44], bak1-4 (SALK_116202) and bak1-5 [34] seeds were kindly provided by Cyril Zipfel (The Sainsbury Laboratory, UK) and Jian-Min Zhou (National Institute of Biological Sciences, Beijing, China). Double mutants were generated by crossing individual mutants and were identified by PCR. Primer sets used in this study are listed in S3 Table. Phylogenetic analysis was performed using MEGA6. 06 software (www. megasoftware. net). Full-length amino acid sequences were used to generate the phylogenetic tree. The bootstrap value was set at 10,000 replicate samples. Arabidopsis seedlings were grown in liquid MGRL medium with 0. 1% (w/v) sucrose [66] at 22°C under continuous light for 10 days. Phosphoproteome analysis was performed as described previously with minor modifications [67]. MS peaks were detected using an in-house 2DICAL software package [68] that identified all MS/MS spectra using Mascot software, adjusted the retention times of each LC-MS data point utilizing the similarity index of the mass spectrum pattern, and grouped peaks from different samples that were derived from same peptides in the direction of acquiring time. The peak intensities of MS chromatograms were used for quantitative values. Three biological replicates were analyzed for each condition, and the significance of differences was tested. Statistical relevance was determined using a two-tailed Student’s t-test (p-value < 0. 05). Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Netherlands), and cDNA was prepared using the ReverTra Ace Reverse Transcription Kit (Toyobo, Japan). Quantitative reverse transcription PCR (qRT-PCR) was performed using the Mx3000P QPCR system (Agilent Technologies, USA) with the Thunderbird SYBR qPCR Mix (Toyobo, Japan). Data were analyzed using an in-house script written in the R language (S1 Information) as described previously [69]. Arabidopsis seedlings were grown in liquid MGRL medium with 0. 1% (w/v) sucrose [66] at 22°C under continuous light for 10 days. Ten 10-day-old Arabidopsis seedlings were incubated in liquid MGRL medium supplemented with 0. 1% (w/v) sucrose containing 100 μM 8-amino-5-chloro-7-phenylpyrido [3,4-d] pyridazine-1,4- (2H, 3H) (L-012) (Wako, Japan) for 2 h at 22°C under darkness, followed by transfer to liquid MGRL medium containing 100 nM flg22, a peptide that perceives bacterial flagellin. ROS production was determined by counting photons derived from L-012–mediated chemiluminescence using NightSHADE LB985 (Berthold Technologies, Germany). Arabidopsis seedlings were grown in liquid MGRL medium with 0. 1% (w/v) sucrose [66] at 22°C under continuous light for 10 days. Proteins were extracted from 100 nM flg22-treated or mock-treated seedlings in extraction buffer (50 mM Tris-HCl (pH 7. 5), 10 mM MgCl2,15 mM EGTA, 100 mM NaCl, 2 mM DTT, 1 mM sodium fluoride, 0. 5 mM Na3VO4,30 mM β-glycerophosphate, 0. 1% (v/v) NP-40 and one Complete tablet, EDTA-free per 50 ml (Roche, Germany) ). Phosphorylated MAPK proteins were detected by immunoblot analysis with anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13. 14. 4E) rabbit mAb (Cell Signaling Technology, USA) [61]. The blotted membrane was stained with Coomassie Brilliant blue (CBB) to verify equal loading. The callose deposition assay was performed as described previously with minor modifications [70]. Leaves from 6-week-old plants were syringe infiltrated with 100 nM flg22 or 10 mM MgCl2. Leaf discs were removed 20 h post-infiltration using an 8-mm-diameter cork borer and fixed in an acetic acid: ethanol (1: 3) solution for several h. Leaves were rehydrated in 70% (v/v) ethanol for 2 h, 50% (v/v) ethanol for 2 h, and overnight in water. Leaves were stained for 1 h in the dark in 150 mM sodium phosphate buffer (pH 9. 0) containing 0. 05% (w/v) aniline blue. Leaves were mounted in 10% glycerol (v/v) and the localization of fluorescently stained callose was determined by fluorescence microscopy. Callose deposition was quantified by Image-J software (http: //imagej. nih. gov/ij/). At least three leaves from three independent plants were used as biological replicates for the analysis. Pseudomonas syringae pv. tomato DC3000 (Pto), Pto AvrRPM1 or Pto AvrRPS4 were grown on King’s B medium at 28°C for 2 to 3 days. Six to 7-week-old plants grown in the 8 h light /16 h photoperiod were spray inoculated with bacterial suspensions of 5 x 107 to 5 x 108 c. f. u. (colony forming unit) ml-1 containing 0. 04% silwet-L77 (Bio medical science, Japan). For the infiltration assay, 5 to 6-weeks-old plants grown in the 12 h light /12 h photoperiod were syringe infiltrated with bacterial suspension of 5 x 104 c. f. u. ml-1. For bacterial growth experiments, inoculated plants were maintained at high humidity at 22°C in the 8 h light /16 h dark or 12 h light /12 h photoperiods. Hyaloperonospora arabidopsidis isolate Noco2 was maintained weekly by transferring spores onto WT Col-8 plants growing at 16°C in an 8 h light /16 h dark photoperiod. Fourteen-day-old plants were spray inoculated with 5 x 104 spores ml-1 of Hpa Noco2. Infected plants were maintained at high humidity at 16°C in an 8 h light /16 h dark photoperiod. Infection development was scored 6 days after infection by counting sporangiophores on true leaves. Alternaria brassicicola isolate O-264 was maintained on potato dextrose agar medium. The third to fourth leaves of 27-day-old plants were inoculated with 5 μl drops of a spore suspension (5 x 105 spores ml-1 in distilled water). Infected plants were maintained at high humidity at 16°C in an 8 h light /16 h dark photoperiod. Lesion sizes were measured at 6 days after inoculation using Image-J software (http: //imagej. nih. gov/ij/). Trypan blue staining was performed as described previously with minor modifications [71]. Leaves were stained in lactophenol-trypan blue solution (10 mg trypan blue dissolved in 80 ml of lactic acid: glycerol: phenol: distilled water: ethanol 1: 1: 1: 1: 4, v/v) and cleared in chloral hydrate solution (2. 5 g chloral hydrate in 1 ml distilled water). Leaves were mounted in chloral hydrate solution and dead cells were observed using a BX51 microscope (Olympus, Japan). Trypan blue staining area was measured using Photoshop CC software (Adobe, USA). Statistical analysis was performed using the Tukey HSD test (S2 Information) with R software version 3. 2. 3 (https: //www. r-project. org/). The fumonisin B1 assay was performed as described previously with minor modifications [35]. Leaves from 6 to 7-week-old plants were syringe infiltrated with 100 nM flg22 or distilled water (mock) 24 h prior to FB1 treatment. Pretreated leaves were then syringe infiltrated with 50 μM FB1 (Sigma-Aldrich, USA) or a mock solution (distilled water containing 1% (v/v) methanol) and plants were kept at 22°C in a 16 h light /8 h dark photoperiod. Photographs were taken 4 days after FB1 treatment, and leaves were stained with trypan blue to visualize dead cells. The open reading frame (ORF) of PSIG1 was cloned into pENTR4 dual-selection vector (Thermo Fisher SCIENTIFIC, USA) between SalI and NotI sites using an IN-FUSION HD Cloning Kit (Clontech Laboratories, USA). The 1. 5 kb promoter fragment of PSIG1 was cloned into the SalI site of the pENTR4-PSIG1 construct using IN-FUSION HD Cloning Kit. PSIG1pro: : PSIG1 stop codon deletion and PSIG1pro: : PSIG1Y570A were generated by PCR-based mutagenesis using the PSIG1pro: : PSIG1 construct as a template. PSIG1pro: : PSIG1 and PSIG1pro: : PSIG1Y570A were subcloned into binary vector pGWB1[72], and the stop codon-mutated PSIG1pro: : PSIG1 was subcloned into pGWB4 using LR clonase II enzyme mix (Thermo Fisher SCIENTIFIC, USA) [64]. The binary vectors were introduced into Agrobacterium tumefaciens C58C1. The psig1-1 mutant was transformed using a floral dip method [73]. T3 or T4 homozygous transformants were used in this study. The PSIG1 ORF was amplified by PCR and cloned into pGEX4T-3 vector (GE Healthcare) between SalI and NotI sites. PSIG11-606, not including its own PPGF motif, was cloned into the pGEX4T-3 vector between SalI and NotI sites. GST-PSIG11-606 Y575A and GST-PSIG11-606 W570A/Y575 were produced by PCR-based mutagenesis using PrimeSTAR Mutagenesis Basal Kit (TaKaRa, Japan). The NcoI site of the pENTR4 dual selection vector was mutagenized with the PrimeSTAR Mutagenesis Basal Kit to avoid the translation of additional amino acids from the NcoI site and was named pENTR4m. The open reading frame of AtSMG7, AtSMG7-N (1–548) and AtSMG7-C (549–1059) were amplified by PCR from Arabidopsis seedling (Col-8) cDNA and cloned into the pENTR4m vector between SalI and NotI sites using the IN-FUSION HD Cloning Kit. AtSMG7G933A were produced by PCR-based mutagenesis using PrimeSTAR Mutagenesis Basal Kit. AtSMG7, AtSMG7-N, AtSMG7-C and AtSMG7G933A were subcloned into pDEST-HisMBP vector [74] using the LR clonase II enzyme mix. GST-PSIG1, GST-PSIG11-606, GST-PSIG11-606 Y575A, GST-PSIG11-606 W570A/Y575, HisMBP-SMG7, HisMBP-SMG7-N, HisMBP-SMG7-C and HisMBP-SMG7G933A were expressed in Escherichia coli Rosetta-gamiTM 2 (DE3) (Merck Millipore, Germany) and were purified with Glutathione-Sepharose B columns (GE Healthcare) for GST-tagged proteins or Dextrin-Sepharose High Performance columns (GE Healthcare) for MBP-tagged proteins. The purified recombinant PSIG1 and SMG7 proteins were mixed in a binding buffer [50mM Tris-HCl pH7. 5,150 mM NaCl, 1 mM dithiothreitol (DTT), 0. 1% (v/v) Triton X-100 and one Complete tablet, EDTA-free per 50 mL (Roche) ]. The Glutathione-Sepharose B beads were mixed with the protein mixtures and incubated for 2 h at 4°C. The beads were washed three times with a washing buffer [50mM Tris-HCl pH7. 5,300 mM NaCl, 1 mM dithiothreitol (DTT), 0. 1% (v/v) Triton X-100 and one Complete tablet, EDTA-free per 50 mL (Roche) ]. Proteins were eluted from the beads with 2 x SDS buffer, and were detected by SDS-PAGE or immunoblotting. A Nicotiana benthamiana transient expression system was used for subcellular localization analysis. The stop codon-mutated PSIG1 ORF (PSIG1sm) was amplified by PCR and cloned into the pENTR4m between SalI and NotI sites using the IN-FUSION HD Cloning Kit. The PSIG1 and PSIG1sm fragments were subcloned into pGWB6 and pGWB5 binary vectors, respectively, using the LR clonase II enzyme mix. The SMG7 fragment was subcloned into pGWB45 or pGWB661 binary vectors [75]. The P-body marker construct pTA7002: : DCP1-mCherry was described previously [76]. The binary vectors were introduced into A. tumefaciens C58C1 strain carrying pCH32 plasmid. Agrobacterium cultures harboring each construct were incubated overnight at 28°C and subsequently resuspended in 10 mM MES pH 5. 6,10 mM MgCl2 buffer with 100 μM acetosyringone at an optical density of 0. 3 at 600 nm. Agrobacterium cultures were mixed and syringe infiltrated into N. benthamiana leaves. Agrobacterium carrying pJL3-p19 was co-infiltrated [69]. Two days after inoculation, agroinfiltrated leaves were sprayed with 30 μM dexamethasone (Dex). One day after Dex treatment, localization was observed using Leica TCS SP5II (Leica Microsystems, Germany). Yeast and human SMG7 homologs were described by Reichenbach, P. et al. 2003 [77]. SMG7 homologs in plants were identified using the Phytozome database (https: //phytozome. jgi. doe. gov/pz/portal. html): Arabidopsis thaliana TAIR10, Chlamydomonas reinhardtii v5. 5, Oryza sativa v7_JGI, Physcomitrella patens v1. 6 and Selaginella moellendorffii v1. 0.
summary: Programmed cell death (PCD) has crucial roles in development and immunity in multicellular organisms. In plants, rapid PCD induction, so-called hypersensitive response (HR) cell death, can be triggered as a part of immune system, and plays an important role in restricting pathogen growth. Despite its importance, cell death induction can backfire on plants because of the diversified infection strategies of plant pathogens. It is therefore assumed that plants have mechanisms by which they are able to minimize PCD induction during plant-pathogen interactions. However, their existence and biological significance are not clear yet. Here, we demonstrate that PSIG1, which has the GYF domain that is highly conserved among diverse eukaryotic species, restricts cell death induction during pathogen invasions. Importantly, psig1 mutants do not display autoimmune phenotypes, and are more susceptible to the virulent bacterial pathogen. Our findings suggest that the restriction of cell death can have benefits for plants to defend themselves against hemi-biotrophic bacterial pathogen infections. We further provide evidence suggesting a mechanism by which PSIG1 may contain cell death by regulating the RNA metabolism machinery.
keywords: cell death
  plant anatomy
  cell processes
  brassica
  plant science
  model organisms
  experimental organism systems
  plant pathology
  seedlings
  plants
  arabidopsis thaliana
  research and analysis methods
  specimen preparation and treatment
  staining
  necrotic cell death
  leaves
  cell staining
  eukaryota
  plant and algal models
  cell biology
  phenotypes
  plant pathogens
  genetics
  biology and life sciences
  organisms
title: The GYF domain protein PSIG1 dampens the induction of cell death during plant-pathogen interactions