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. 2014 Sep 2;166(2):1009–1021. doi: 10.1104/pp.114.248757

Arabidopsis Triphosphate Tunnel Metalloenzyme2 Is a Negative Regulator of the Salicylic Acid-Mediated Feedback Amplification Loop for Defense Responses1,[W],[OPEN]

Huoi Ung 1,2, Wolfgang Moeder 1,2, Keiko Yoshioka 1,2,*
PMCID: PMC4213072  PMID: 25185123

A triphosphate tunnel metalloenzyme is a negative regulator in amplification of SA-dependent defense responses.

Abstract

The triphosphate tunnel metalloenzyme (TTM) superfamily represents a group of enzymes that is characterized by their ability to hydrolyze a range of tripolyphosphate substrates. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes, AtTTM1, AtTTM2, and AtTTM3. Although AtTTM3 has previously been reported to have tripolyphosphatase activity, recombinantly expressed AtTTM2 unexpectedly exhibited pyrophosphatase activity. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. In addition, stronger systemic acquired resistance compared with wild-type plants was observed. These enhanced defense responses are dependent on SA, PHYTOALEXIN-DEFICIENT4, and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. Despite their enhanced pathogen resistance, ttm2 plants did not display constitutively active defense responses, suggesting that AtTTM2 is not a conventional negative regulator but a negative regulator of the amplification of defense responses. The transcriptional suppression of AtTTM2 by pathogen infection or treatment with SA or the systemic acquired resistance activator benzothiadiazole further supports this notion. Such transcriptional regulation is conserved among TTM2 orthologs in the crop plants soybean (Glycine max) and canola (Brassica napus), suggesting that TTM2 is involved in immunity in a wide variety of plant species. This indicates the possible usage of TTM2 knockout mutants for agricultural applications to generate pathogen-resistant crop plants.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that are characterized by their ability to hydrolyze a range of tripolyphosphate (PPPi) substrates. All members of this superfamily use triphosphate substrates and require a divalent cation cofactor for their activity, usually Mg2+ or Mn2+ (Bettendorff and Wins, 2013). This superfamily contains two previously characterized groups of proteins: RNA triphosphatases and CYTH domain proteins (Iyer and Aravind, 2002; Gong et al., 2006). The CYTH domain was named after its two founding members, the adenylate cyclase CyaB from Aeromonas hydrophila and the mammalian thiamine triphosphatase (Iyer and Aravind, 2002). Despite low overall amino acid sequence similarity, all TTM family members possess a tunnel structure composed of eight antiparallel β-strands (β-barrel; Gallagher et al., 2006; Gong et al., 2006; Song et al., 2008; Moeder et al., 2013). The signature EXEXK motif (where X is any amino acid) located in the β-barrel has been shown to be important for catalytic activity (Lima et al., 1999; Gallagher et al., 2006).

The enzymatic and biological functions of most TTM family members are unknown. However, they seem to act on nucleotide and organophosphate substrates (Bettendorff and Wins, 2013) and acquired divergent biological functions in different taxonomic lineages (Iyer and Aravind, 2002). Known functions include adenylate cyclase for CyaB from A. hydrophila and Yersinia pestis adenylate cyclase IV from Y. pestis (Sismeiro et al., 1998; Gallagher et al., 2006), thiamine triphosphatase in mammals (Lakaye et al., 2004), and RNA triphosphatase in fungi, protozoa, and some viruses (Shuman, 2002). In some instances, TTM proteins are fused to additional domains, such as a nucleotide kinase domain (Iyer and Aravind, 2002).

Plants possess two types of TTM proteins: one type that comprises only the CYTH domain and another type with a CYTH domain fused to a phosphate-binding (P-loop) kinase domain (Iyer and Aravind, 2002). Arabidopsis (Arabidopsis thaliana), like most other plant species, codes for three TTM genes. We termed them AtTTM1, AtTTM2, and AtTTM3. AtTTM3 possesses only a CYTH domain, whereas AtTTM1 and AtTTM2 encode a nucleotide/uridine kinase domain fused to the CYTH domain (Moeder et al., 2013). So far, the exact biological function of TTM proteins in plants is not clear. We previously analyzed AtTTM3 and found that it does not display adenylate cyclase activity, despite its annotation, but it does act on PPPi and with lower affinity, nucleotide triphosphates, releasing inorganic phosphate, similar to the TTM proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM; Keppetipola et al., 2007; Delvaux et al., 2011; Bettendorff and Wins, 2013; Moeder et al., 2013). Additionally, a transfer DNA (T-DNA) insertion knockout (KO) line of AtTTM3 displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development.

To gain insight into the biological function of AtTTM1 and AtTTM2, we surveyed the Bio-Analytic resource (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi; Winter et al., 2007) for any publicly available expression analysis data that might provide clues for the biological role of these AtTTMs. The expression of AtTTM2 was suppressed almost 2-fold after treatment with flg22, the well-studied pathogen-associated molecular pattern (PAMP) peptide, and infection with various virulent and avirulent strains of Pseudomonas syringae (Supplemental Fig. S1). These data suggest the possible involvement of AtTTM2 in pathogen defense responses in plants.

The plant defense system has been studied extensively in the last two decades, and two levels of resistance responses have been reported. The first line of defense is basal immunity, which is triggered by the recognition of molecules that are conserved among many pathogens (the above-mentioned PAMPs), and thus, it is referred to as PAMP-triggered immunity. The second line of defense is a stronger response to pathogen infection, which is mediated by resistance (R) genes that can recognize their cognate effectors from the pathogen either directly or indirectly. This is known as effector-triggered immunity (Bent and Mackey, 2007). The hypersensitive response (HR), which is characterized by apoptosis-like cell death at and around the site of pathogen entry, is one common defense mechanism activated by R gene-mediated pathogen recognition (Hammond-Kosack and Jones, 1996; Heath, 2000). During HR development, an increase in salicylic acid (SA) and the accumulation of pathogenesis-related (PR) proteins are observed (Vlot et al., 2008). Later, resistance against virulent pathogens can also be seen in uninoculated systemic leaves. This phenomenon is called systemic acquired resistance (SAR) and confers a long-lasting, broad-range resistance to subsequent infection (Vlot et al., 2008; Shah and Zeier, 2013). Elevated SA levels and PR gene expression can also be detected in uninoculated leaves that exhibit SAR. Treatment with SA or synthetic SAR activators, such as benzothiadiazole (BTH), can also trigger SAR (Lawton et al., 1996; Vlot et al., 2008). Recently, a number of metabolites that are involved in long-distance signaling have been identified, such as methyl salicylate, dehydroabietinal, azelaic acid, glycerol-3-P, and the Lys catabolite pipecolic acid (Shah and Zeier, 2013).

Over the last two decades, significant effort has been made to identify components in the pathogen resistance signal transduction pathway. For instance, ISOCHORISMATE SYNTHASE1 (ICS1) has been revealed to play a critical role in the biosynthesis of pathogen-induced SA. salicylic acid induction-deficient2 (sid2)/ics1 mutants fail to produce elevated levels of SA after pathogen infection and thus, are hypersensitive to pathogens (Nawrath and Métraux, 1999; Wildermuth et al., 2001). NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is a key regulator of SA-mediated resistance, and npr1 mutant plants fail to respond to exogenously supplied SA (Cao et al., 1994). The lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN-DEFICIENT4 (PAD4; Glazebrook et al., 1996; Parker et al., 1996) participate in both basal and R protein-mediated defense responses (Falk et al., 1999; Jirage et al., 1999). EDS1 interacts with PAD4 and SENESCENCE ASSOCIATED GENE101, and both EDS1 and PAD4 are required for HR formation and restriction of pathogen growth (Feys et al., 2001, 2005). A screen of mutants exhibiting constitutive activation of resistance responses also identified components in defense. They show heightened resistance, usually accompanied by elevated levels of SA and PR genes. These autoimmune mutants also frequently display spontaneous HR-like lesions, and thus, they are referred to as lesion mimic mutants (Moeder and Yoshioka, 2008; Hofius et al., 2009).

Here, we show that AtTTM2 acts as a negative regulator of plant immunity, likely at the positive amplification loop of defense responses. KO mutants for AtTTM2 show enhanced pathogen resistance, whereas overexpressors display enhanced susceptibility. The KO mutants do not show constitutive activation of defense responses like most autoimmune mutants but exhibit enhanced SAR on treatments with pathogens, suggesting that they are in a primed state. Furthermore, the expression of TTM2 orthologs in canola (Brassica napus) and soybean (Glycine max) displays the same transcriptional down-regulation after BTH treatment, suggesting that the biological function of TTM2 in pathogen defense is conserved among agriculturally important crop plants.

RESULTS

AtTTM2 Is Down-Regulated after Pathogen Infection

Three genes, At1g73980, At1g26190, and At2g11890, are annotated as CYTH domain proteins in the Arabidopsis genome and have been named AtTTM1, AtTTM2, and AtTTM3 (Moeder et al., 2013). Two allelic homozygous T-DNA insertion KO lines were obtained for AtTTM2 (Salk_145897 [ttm2-1] and Salk_114669 [ttm2-2]). The T-DNA insertion positions were found to be located in exon 3 and intron 5 in ttm2-1 and ttm2-2, respectively (Supplemental Fig. S2A). Reverse transcription (RT) -PCR analysis showed that both lines are, indeed, KO mutants (Supplemental Fig. S2B). A morphological comparison showed no detectable difference in the size or shape of both ttm2 KO lines compared with wild-type Columbia-0 (Col-0; Supplemental Fig. S2C).

As mentioned, public microarray data revealed the down-regulation of AtTTM2 during pathogen infection (Supplemental Fig. S1). To confirm these results, quantitative real-time PCR (qPCR) was conducted on Col-0 wild-type plants that were infected with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), isolate Emwa1. We observed a 2-fold reduction in AtTTM2 transcript levels in infected cotyledons compared with mock treatment (Fig. 1A), indicating the involvement of AtTTM2 in pathogen defense. Interestingly, AtTTM2 was also down-regulated in uninfected systemic tissue of the same seedlings, indicating a role for AtTTM2 in SAR as well (Fig. 1B).

Figure 1.

Figure 1.

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AtTTM2 is down-regulated after pathogen infection. A, qPCR analysis of AtTTM2 expression in H. arabidopsidis, isolate Emwa1-infected (Emwa1) or water-treated cotyledons (H2O) of 10-d-old Col-0 wild-type plants 7 d after infection. B, qPCR analysis of AtTTM2 expression in uninfected true leaves of the same plants. Transcripts were normalized to AtEF1A. Each bar represents the mean of three independent experiments ± se. Each sample is a mix of 16 seedlings. *, Statistical significance (Student’s t test) at P < 0.05. **, Statistical significance (Student’s t test) at P < 0.001.

ttm2 Exhibits Enhanced Resistance against Hpa

Because AtTTM2 is down-regulated after pathogen infection, we asked whether ttm2 mutants show alterations in defense-related phenotypes. Cotyledons of 7-d-old to 10-d-old seedlings were infected with the Hpa isolate, Emwa1, which is avirulent to the Col-0 ecotype. It is notable that, although the Emwa1 isolate is considered to have an incompatible interaction with the Col-0 ecotype, the resistance in this ecotype is not perfect, and initial layers of mesophyll cells may show the emergence of some hyphae (Fig. 2A, Cot infected). ttm2 lines, in addition to having fewer or no hyphae, also exhibited a greater manifestation of HR cell death on infected tissue compared with the wild type, suggesting enhanced resistance (Fig. 2A, Cot infected). qPCR analysis also showed approximately 2-fold less internal transcribed spacer2 (ITS2) transcript levels, a marker to quantify oomycete infection (Quentin et al., 2009; Fig. 2B; Supplemental Fig. S3A), indicating less growth of pathogens in ttm2 plants. We frequently observed the formation of micro-HR-like cell death in uninfected systemic leaves of wild-type plants after avirulent infection on cotyledons (Fig. 2A, TL uninfected systemic), similar to the findings of Alvarez et al. (1998). Interestingly, ttm2 plants displayed significantly enhanced HR cell death on the uninfected systemic true leaves (Fig. 2A, TL uninfected systemic).

Figure 2.

Figure 2.

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ttm2 exhibits enhanced resistance against Hpa. A, Infection phenotype of Col-0 wild-type and ttm2 mutant plants 10 d after infection with avirulent Hpa, isolate Emwa1. Shown is Trypan Blue staining of infected cotyledons (Cots) and uninfected true leaves (TLs) revealing some hyphae (Hy) in the wild type (white arrows) and enhanced HR cell death in the ttm2 mutant lines (red arrows). Uninfected TLs also displayed enhanced HR-like cell death (red arrows). B, Quantification of Hpa, isolate Emwa1 infection by qPCR of the oomycete marker ITS2. Transcripts were normalized to AtEF1A. Each bar represents the mean of three technical replicates ± se. Each sample is a mix of 16 seedlings. Data from an independent experiment with the same result are shown in Supplemental Figure S3A. C, Infection phenotype of Col-0 wild-type and ttm2 mutant plants 12 d after infection with virulent Hpa, isolate Emco5. Shown is Trypan Blue staining of infected Cots and uninfected TLs revealing Hy and oospores (Oo) in the wild type (white arrows) and reduced hyphal growth in the ttm2 mutant lines. Uninfected TLs of ttm2 mutants also displayed some HR-like cell death along veins (red arrow). D, Quantification of Hpa, isolate Emco5 infection by qPCR of the oomycete marker ITS2. Transcripts were normalized to AtEF1A. Each bar represents the mean of three technical replicates ± se. Each sample is a mix of 16 seedlings. Data from an independent experiment with the same result are shown in Supplemental Figure S3B. E and F, Free SA (E) and conjugated SA (SAG; F) levels in Hpa, isolate Emwa1-infected Cots 5 d after infection. Each bar represents the mean of three biological replicates ± se. Experiments were repeated three times with similar results; 10-d-old seedlings were used for all infections. FW, Fresh weight. Bars = 250 µm. *, Statistical significance (Student’s t test) at P < 0.05.

To determine whether this enhanced resistance was specific to effector-triggered immunity or whether it also affected PAMP-triggered immunity, infection with the virulent Hpa isolate, Emco5, was conducted. Trypan Blue analysis revealed little to no hyphae on infected tissue of ttm2, whereas in wild-type plants, hyphal structures and oospore formation were clearly visible throughout the leaf (Fig. 2C, Cot infected). Consistent with this observation, ITS2 transcript levels in infected cotyledons of ttm2 seedlings were more than 2-fold lower compared with the wild type (Fig. 2D; Supplemental Fig. S3B). Interestingly, we also observed enhanced HR-like cell death along the veins of uninfected systemic leaves of ttm2 seedlings (Fig. 2C).

Supplemental Figure S3C shows that ttm2 plants also displayed enhanced resistance to the bacterial pathogen P. syringae DC3000 (AvrRps4). These data indicate that ttm2 plants exhibited enhanced resistance against both avirulent and virulent pathogens.

SA has been shown to be a critical signaling molecule in pathogen defense. In line with the resistance phenotype, a significant increase in free SA and its conjugated form, salicylic acid glucoside (SAG), was observed in ttm2 plants upon pathogen infection compared with the wild type (Fig. 2, E and F). Taken together, these data suggest that AtTTM2 is likely involved in SA-mediated defense signaling.

ttm2 Is Not a Lesion Mimic Mutant

To date, various autoimmune mutants have been reported. They show enhanced resistance against various pathogens and often exhibit activation of resistance responses, such as accumulation of SA and constitutive PR gene expression without pathogen infection. One well-studied class of autoimmune mutants, called lesion mimic mutants, additionally exhibits spontaneous cell death formation without pathogen infection (Moeder and Yoshioka, 2008). To test whether resistance responses are activated without pathogen infection in ttm2, Trypan Blue analysis on uninfected ttm2 seedlings was conducted and revealed no spontaneous cell death formation (Supplemental Fig. S4A). Additionally, no elevated expression of the defense marker gene PR1 (Laird et al., 2004) was observed in ttm2 seedlings without pathogen infection (Supplemental Fig. S4B). These data suggest that ttm2 is not a lesion mimic or conventional autoimmune mutant but likely, a priming mutant that exhibits enhanced resistance upon pathogen infection.

ttm2 Exhibits Enhanced SAR

The observation that AtTTM2 was also down-regulated in uninfected systemic leaves (Fig. 1B) combined with the enhanced HR cell death in ttm2 seedlings (Fig. 2A) prompted us to investigate whether ttm2 is also affected in its SAR response. To assess SAR, we first treated cotyledons of wild-type and ttm2 plants with either water (SAR−) or the avirulent Hpa isolate, Emwa1 (SAR+). We then performed challenge inoculation using the aggressive virulent Hpa isolate, Noco2, on the upper systemic leaves (Fig. 3A; Supplemental Fig. S5A). We used very strong infection conditions (i.e. 1 × 105 conidiospores) of the aggressive isolate, Noco2, to see a clear difference between SAR-induced and noninduced groups. Thus, both wild-type and ttm2 plants displayed comparable hyphae growth in water-treated plants (Fig. 3A, SAR−; Supplemental Fig. S5A, lower). In contrast, Hpa-treated ttm2 plants (Fig. 3A, SAR+; Supplemental Fig. S5A, upper) revealed a stronger reduction in pathogen growth in systemic leaves compared with SAR+ wild-type plants. Stained leaves were microscopically examined and assigned to different classes (Fig. 3B; Supplemental Fig. S5B). Fisher exact probability test indicated a significant difference between the ttm2 KO lines and the Col-0 wild type (P < 0.0001). These data suggest that ttm2 mutants exhibit enhanced SAR.

Figure 3.

Figure 3.

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ttm2 exhibits enhanced SAR. A, Primary infection of 10-d-old cotyledons of Col-0 wild-type and ttm2 mutant plants was performed with the avirulent Hpa isolate Emwa1 (SAR+) or water (SAR−). After 7 d, a challenge infection was performed on systemic true leaves with Hpa, Noco2 (virulent). Hyphal structures were visualized 10 d later by Trypan Blue staining. B, Stained leaves were microscopically examined and assigned to different classes. Data shown are from two independent experiments and taken from 50 plants each; Fisher exact probability test indicates a significant difference between SAR+ ttm2 lines and Col-0 (P < 0.0001). The experiment was repeated three times with similar results. Data from an independent experiment with a similar result are shown in Supplemental Figure S5. Bars = 250 µm.

The Enhanced Resistance Phenotype of ttm2 Requires PAD4, ICS1, and NPR1

It has been shown that PAD4, SID2 (ICS1), and NPR1 play key roles in SA-dependent defense responses (Glazebrook et al., 1996; Cao et al., 1997; Jirage et al., 1999; Nawrath and Métraux, 1999; Wildermuth et al., 2001). To investigate whether AtTTM2-mediated resistance requires these signaling components, we performed epistatic analyses using double mutants of ttm2-2 and pad4-1, sid2-1, or npr1-1. Col-0 and Wassilewskija (Ws) ecotypes are resistant and susceptible, respectively, to the Hpa isolate, Emwa1 (Fig. 4). As expected, the Col-0 wild type exhibited resistance with some hyphae present on the infected tissue along with punctate areas of HR cell death in both infected and uninfected systemic tissue, whereas the Ws wild type exhibited susceptibility with massive hyphal growth and oospore formation in infected tissue and no visible signs of HR in the uninfected systemic leaves (Fig. 4, TL uninfected). pad4-1, sid2-1, and npr1-1 single mutants also exhibited susceptibility with little or no visible HR (Fig. 4; Supplemental Fig. S6) but a great presence of hyphae and in some cases, oospores (Fig. 4), as expected. All double mutants with ttm2 exhibited similar susceptibility as pad4-1, sid2-1, and npr1-1 single mutants (Fig. 4; Supplemental Fig. S6). These data indicate that PAD4, ICS1, and NPR1 are all required for the enhanced resistance phenotype of ttm2.

Figure 4.

Figure 4.

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Involvement of PAD4, NPR1, and SA in ttm2-mediated resistance. Infection phenotype of the Col-0 wild type, the Ws wild type, pad4-1, sid2-1, npr1-1, and ttm2 mutants, and corresponding double mutants 10 d after infection with avirulent Hpa, isolate Emwa1. Shown is Trypan Blue staining of infected cotyledons (Cots) and uninfected true leaves (TLs). White arrows indicate hyphal (Hy) growth, and red arrows indicate HR cell death. Experiments were repeated three times with similar results; 10-d-old seedlings were used for infection. Oo, Oospore. Bars = 250 µm.

AtTTM2 Expression Is Negatively Regulated by SA and PAMP Treatment

Because pathogen infection down-regulates the transcription of AtTTM2 (Fig. 1), the effect of SA on AtTTM2 expression was tested. Col-0 wild-type plants were sprayed with 100 μm SA and assessed 24 h later for changes in expression levels. AtTTM2 was down-regulated by more than 2-fold after SA treatment (Fig. 5A; Supplemental Fig. S7A). This down-regulation was also observed after treatment with the SAR activator BTH (200 µm; Fig. 5B; Supplemental Fig. S7B). This was correlated with an increase in PR1 gene expression (Fig. 5, A and B; Supplemental Fig. S7, A, bottom and B, bottom). Publicly available microarray data indicated that AtTTM2 is also down-regulated after treatment with the PAMP flg22 (Supplemental Fig. S1). Our qPCR confirmed that, 4 h after treatment with the flg22 peptide (5 µm), AtTTM2 was down-regulated by 70% (Fig. 5C; Supplemental Fig. S7C).

Figure 5.

Figure 5.

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AtTTM2 expression is suppressed by SA and flg22 treatment. qPCR analysis of Col-0 wild-type plants. A, Twenty-four hours after treatment with 100 µm SA or water. B, Forty-eight hours after treatment with 200 µm BTH or water. Shown are AtTTM2 and PR1 gene expressions relative to AtEF1A. C, qPCR analysis of AtTTM2 in Col-0 wild-type, sid2, pad4, and npr1 plants 4 h after treatment with flg22 or water. Transcripts were normalized to AtEF1A. Each bar represents the mean of three technical replicates ± se. Each sample is a mix of 16 seedlings (A and B) or four leaves (C). Data from an independent experiment with the same result are shown in Supplemental Figure S7. For A and B, 10-d-old seedlings were used; for C, 4-week-old plants were syringe infiltrated.

The fact that AtTTM2 gene expression was down-regulated upon pathogen infection (Fig. 1) as well as SA/BTH treatment and flg22 treatment (Fig. 5) made us assess the requirement of key components in SA-mediated resistance for the transcriptional regulation of AtTTM2. Interestingly, after treatment with flg22, sid2, pad4, and npr1 plants displayed the same level of AtTTM2 down-regulation as wild-type plants (Fig. 5C; Supplemental Fig. S7C). A similar result was seen after infection with P. syringae ES4326 (Supplemental Fig. S8; Wang et al., 2008). Taken together, these data suggest that SA, PAD4, and NPR1 are not required for the transcriptional down-regulation of AtTTM2 but are required for the resistance phenotype of the ttm2 mutants.

Overexpression of AtTTM2 Confers Enhanced Susceptibility to Pathogens

The observation that AtTTM2 is down-regulated upon pathogen infection and SA/flg22 treatment combined with the fact that ttm2 plants display enhanced disease resistance strongly suggest that AtTTM2 is a negative regulator of disease resistance. Therefore, constitutive expression of AtTTM2 may lead to enhanced disease susceptibility. Thus, we created AtTTM2 overexpressor lines, where AtTTM2 expression is driven by the strong Cauliflower mosaic virus35S promoter. To detect differences in disease outcome, we used relatively moderate infection conditions with the virulent Hpa isolate, Emco5. We observed elevated expression of AtTTM2 in three independent transgenic lines, even after pathogen infection (Fig. 6A; Supplemental Fig. S9). Although only 60% of Col-0 wild-type plants and 30% of ttm2 plants exhibited heavy hyphal growth 10 d after infection, 100% of the plants of the three overexpression lines showed strong infection (Fig. 6, B and C; Supplemental Fig. S9C). Fisher exact probability test indicated a significant difference between the overexpressor lines and the Col-0 wild type (P < 0.001). This was also confirmed quantitatively by measuring the expression of the oomycete marker, ITS2 (Fig. 6D; Supplemental Fig. S9). These data strongly suggest that down-regulation of AtTTM2 is, indeed, required for normal levels of disease resistance.

Figure 6.

Figure 6.

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Overexpression of AtTTM2 causes enhanced susceptibility. A, qPCR analysis of AtTTM2 in Hpa-infected cotyledons 10 d after infection. Transcripts were normalized to AtEF1A. Each bar represents the mean of three technical replicates ± se. Each sample is a mix of 15 seedlings. Data from an independent experiment are shown in Supplemental Figure S9. B, Trypan Blue staining of the Col-0 wild type, ttm2, and two independent 35S:AtTTM2 overexpressor lines (35S-2 and 35S-5) 13 d after infection with Hpa, Emco5. Bars = 250 µm. C, Quantitative assessment of infection. Stained leaves were microscopically examined and assigned to different classes. Data shown were taken from 15 to 16 plants; Fisher exact probability test indicates a significant difference between overexpressor lines and Col-0 (P < 0.001). The experiment was repeated three times with similar results. D, qPCR analysis of ITS2 in Hpa-infected cotyledons 10 d after infection. Transcripts were normalized to AtEF1A. Each bar represents the mean of three technical replicates ± se. Each sample is a mix of 15 seedlings. Data from an independent experiment are shown in Supplemental Figure S9. The analysis of a third independent line is shown in Supplemental Figure S9, B and C; 10-d-old seedlings were used for all infections.

AtTTM2 Function Is Likely Conserved among Different Plant Species

Data from Phytozome (www.phytozome.net) indicated that TTM2 is highly conserved in a wide variety of plant species. This may indicate that these orthologs are also involved in pathogen defense responses. Similarities in the transcriptional expression pattern of TTM2 orthologs can serve as an indication of functional conservation. Thus, the expression of AtTTM2 orthologs of soybean and canola was analyzed by qPCR after treatment with BTH. Interestingly, the TTM2 orthologs in canola (BnTTM2a and BnTTM2b; Fig. 7A; Supplemental Fig. S10A) and soybean (GmTTM2a/b; note that the two isoforms could not be distinguished because of high sequence identity; Fig. 7B; Supplemental Fig. S10B) were similarly down-regulated in response to BTH as their Arabidopsis orthologs. These data combined with the high sequence identity (BnTTM2a, 94%; BnTTM2b, 92%; GmTTM2a, 75%; GmTTM2b, 75%; Supplemental Fig. S11) suggest that the function of TTM2 as a negative regulator of defense responses is likely evolutionarily conserved in other plant species as well.

Figure 7.

Figure 7.

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AtTTM2 function is conserved in crop species. qPCR analysis of canola (var Westar; A) and soybean (var Harasoy; B) plants treated with 200 µm BTH or water 48 h after treatment. A, qPCR analysis of canola BnTTM2a, BnTTMb, and BnPR1. Transcripts were normalized to BnUBC21. B, qPCR analysis of soybean GmTTM2a/GmTTM2b and BnPR1. Transcripts were normalized to GmEF1B. (Note that primers could not distinguish between the two soybean paralogs because of high sequence homology.) Each bar represents the mean of three technical replicates ± se. Data from an independent experiment with the same result are shown in Supplemental Figure S10; 3- to 4-week-old plants were used for treatments.

AtTTM2 Displays Pyrophosphatase Activity

The three TTM genes in Arabidopsis are annotated as adenylate cyclases. However, we recently reported that AtTTM3 does not produce cAMP (Moeder et al., 2013). Similarly, recombinantly expressed AtTTM2 also was not able to produce cAMP (Supplemental Fig. S12). Because AtTTM3 displayed strong tripolyphosphatase activity, we assessed the enzymatic properties of AtTTM2 on several organophosphate substrates. Although AtTTM3 showed strong affinity for tripolyphosphate (PPPi), weaker affinity for ATP, and no affinity for pyrophosphate (PPi; Moeder et al., 2013), AtTTM2 surprisingly displayed stronger affinity for PPi, weaker activity for ATP, and almost no affinity for PPPi (Fig. 8). AtTTM2 was expressed as a glutathione S-transferase (GST)-fusion protein. Protein extracted from Escherichia coli expressing the GST tag alone confirmed that the observed activities are not caused by contaminating bacterial proteins (Fig. 8). These data suggest divergent biological functions of the AtTTM genes, which is consistent with the different phenotypes observed in ttm2 and ttm3.

Figure 8.

Figure 8.

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AtTTM2 displays pyrophosphatase activity. Substrate specificity of AtTTM2 was tested with 0.5 mm PPi, ATP, or PPPi. Reactions were performed at pH 9.0 in the presence of 2.5 mm Mg2+; 2 μg of protein was used. Black bars, GST-TTM2; white bars, GST. Each bar represents the mean of three replicates ± se. Experiments were repeated more than three times with similar results.

DISCUSSION

To understand the biological function of the TTM, AtTTM2, we have characterized the AtTTM2 KO mutants ttm2-1 and ttm2-2. Both lines displayed enhanced resistance against both virulent and avirulent pathogens, because they exhibited lower growth of both types of pathogens combined with an enhancement of HR cell death. In addition, SAR was also enhanced in these mutants. The enhanced resistance was dependent on the well-known defense signaling components SA, PAD4, and NPR1, which indicates that AtTTM2 is involved in the bona fide defense signaling pathway and likely a negative regulator. Transcriptional suppression of AtTTM2 after pathogen infection, PAMP recognition, or SA/BTH treatment further supports this notion. Interestingly, the enhanced pathogen resistance is only observed upon pathogen infection—no significant autoactivation of defense responses, such as spontaneous cell death formation and elevated levels of basal SA or PR1 gene expression, were observed. This differentiates AtTTM2 mutants from the majority of conventional autoimmune mutants (Moeder and Yoshioka, 2008; Hofius et al., 2009).

A similar phenomenon was reported in the Arabidopsis mutants enhanced disease resistance1 (edr1) and edr2 (Frye and Innes, 1998; Tang et al., 2005a). EDR1 and EDR2 encode a CONSTITUTIVE TRIPLE RESPONSE1 family Mitogen-activated protein kinase kinase kinase and an unknown protein with a pleckstrin homology, a steroidogenic acute regulatory protein-related lipid-transfer, and a DOMAIN OF UNKNOWN FUNCTION1336 (DUF), respectively (Frye et al., 2001; Tang et al., 2005a, 2005b; Vorwerk et al., 2007). Both mutants were identified in the same screen for decreased susceptibility against P. syringae DC3000 without constitutive PR gene expression and also show enhanced resistance against other pathogens, such as Erysiphe cichoracearum. Interestingly, both mutants display stronger and faster defense responses upon pathogen infection; however, no obvious autoactivation of defense was observed, just like for ttm2. These phenotypes were suppressed in mutants with defects in the SA signal transduction pathway (e.g. sid2, pad4, npr1, and eds1) but not those with defects in the ethylene/jasmonate pathway, suggesting that they are hypersensitive to or have a lower threshold in activating the SA pathway (Frye et al., 2001; Tang et al., 2005a, 2005b; Vorwerk et al., 2007). The precise molecular mechanisms of these mutants are not yet clear; however, the reported phenotypes are remarkably similar to those of ttm2. The only outstanding difference between ttm2 and edr2 is the enhanced SAR phenotype in ttm2. As shown, ttm2 displayed strong enhancement of SAR, including HR cell death, in uninfected systemic leaves, but edr2-mediated enhancement of resistance does not occur in uninfected systemic leaves. This indicates that, although the mutant phenotypes are similar, the molecular mechanism behind the phenomena is fundamentally different.

In terms of SAR, AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) was shown to be involved in both local and systemic resistance (Song et al., 2004). ALD1 is transcriptionally induced by pathogen infection as well as BTH treatment in both inoculated and systemic tissues. ald1 mutant plants have increased susceptibility to avirulent pathogens and cannot activate SAR. The ALD1 aminotransferase is involved in the biosynthesis of the SAR regulator pipecolic acid, which accumulates in local and systemic tissue of SAR-induced plants (Návarová et al., 2012). Pipecolic acid has been shown to mediate signal amplification that enables systemic SA accumulation, SAR establishment, and defense priming responses in SAR-induced plants. Considering that ttm2 also does not show constitutive activation of resistance and displays an SAR phenotype, AtTTM2 may act by fine tuning the amplification of defense responses in both inoculated and uninoculated leaves. Indeed, an SA-mediated feedback amplification loop has been suggested for a long time (Shah, 2003). For instance, EDS1 and PAD4, which are important defense signaling components, are both regulators and effectors of SA signaling, strongly suggesting the existence of an SA-mediated feedback amplification loop (Dong, 2004). Likewise, ACCELERATED CELL DEATH6, which is believed to work upstream of SA biosynthesis, is transcriptionally induced by BTH (Lu et al., 2003).

Thus, it can be hypothesized that recognition of pathogen infection suppresses the expression of AtTTM2, which acts as a negative regulator of the amplification loop, to facilitate a quick and strong resistance response. At a later time point, SA accumulation induced by pathogen infection further suppresses the expression of AtTTM2 to boost the positive feedback amplification loop of defense responses. Transcriptional down-regulation of AtTTM2 can already be seen 4 h after treatment with flg22 and 24 h after infection with P. syringae (Fig. 5C; Supplemental Fig. S8). Interestingly, AtTTM2 down-regulation was also observed in flg22-treated plants as well as P. syringae-infected sid2, npr1, and pad4 mutant plants (Fig. 5C; Supplemental Figs. S7 and S8), indicating that the down-regulation is triggered upstream of PAD4. SA/BTH treatment causes AtTTM2 down-regulation through either an additional mechanism or feedback through the SA amplification loop (Fig. 9). In this scenario, AtTTM2 plays a role to prevent accidental activation of defense responses through the positive feedback amplification loop in the absence of pathogens. Thus, ttm2 exhibits a primed mutant phenotype: it can induce resistance responses stronger than wild-type plants, but no constitutive activation of defense responses is observed. A model of this concept is presented in Figure 9. Although an SA-mediated feedback amplification loop has been discussed for quite some time (Shah, 2003), only a few studies have identified components of this feedback loop (Song et al., 2004; Raffaele et al., 2006; Roberts et al., 2013). Whether AtTTM2 negatively regulates defense amplification by attenuating pipecolic acid biosynthesis remains to be determined. The molecular mechanism of AtTTM2 will further our understanding of the SA-mediated feedback amplification loop.

Figure 9.

Figure 9.

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AtTTM2 is a negative regulator of the SA-mediated defense amplification loop. Recognition of pathogens suppresses the transcription of AtTTM2 to amplify defense responses. At a later time point, production of SA leads to continuous transcriptional suppression of AtTTM2, further amplifying the feedback loop. The KO mutants of AtTTM2, thus, behave like in a primed state and show enhanced resistance on pathogen recognition. The mutant phenotype requires the known defense signaling components ICS1, PAD4, and NPR1. ETI, Effector-triggered immunity; PTI, pathogen-associated molecular pattern-triggered immunity.

All three Arabidopsis TTMs have been annotated as adenylate cyclases based on sequence similarity to CyaB from A. hydrophila (Iyer and Aravind, 2002). However, in this work and previous work, we have shown that recombinantly expressed AtTTM3 and AtTTM2 do not show adenylate cyclase activity (Moeder et al., 2013; Supplemental Fig. S12). Rather, AtTTM3 exhibits strong tripolyphosphatase activity with a strong affinity for PPPi. However, AtTTM2 showed the strongest affinity for PPi and only weak activities for ATP and PPPi. Although the actual in vivo substrates are currently unknown, the difference in the in vitro substrate preference between AtTTM3 and AtTTM2 indicates distinct biological functions of these two TTM family members. Furthermore, in addition to a CYTH domain, both AtTTM1 and AtTTM2, but not AtTTM3, possess a P-loop kinase domain in their N termini. It is annotated as a uridine/cytidine kinase and has conserved Walker A, Walker B, and lid module motifs (Supplemental Fig. S11; Leipe et al., 2003). This indicates the possibility that AtTTM1 and AtTTM2 have dual enzymatic activities (both phosphatase and kinase). Alternatively, the CYTH domain may have lost its catalytic function in AtTTM1 and AtTTM2, and its function might be to bind and position their specific in vivo substrate for the kinase domain (Iyer and Aravind, 2002). This idea is supported by the fact that many of the conserved catalytic residues of TTM proteins are altered in AtTTM1 and AtTTM2. The stereotypical EXEXK motif of CYTH proteins (including AtTTM3) is altered to TYILK. Furthermore, the majority of the conserved basic and acidic residues in the β-barrel is not conserved in AtTTM1 and AtTTM2 (Supplemental Fig. S11). These residue changes are conserved among the TTM2 orthologs in other plant species, indicating that they contribute to the unusual catalytic activity of AtTTM2. Unlike all other described TTM proteins, which act on triphosphate substrates, AtTTM2 prefers a diphosphate (PPi). In any case, the study of in vivo substrates for AtTTMs and the characterization of AtTTM1 will provide additional insights into this group of proteins in plants and the possible role of this phosphatase/kinase in pathogen defense responses. The analysis of AtTTM1 is currently in progress.

Genomic sequence analyses indicated that all three TTM family members are conserved among most plant species, further indicating the distinct function of all three TTMs in plants. Interestingly, transcriptional suppression of TTM2 by BTH was observed in soybean and canola, like in Arabidopsis, strongly indicating that the orthologs of TTM2 in these crop plants likely also work as negative regulators of defense responses. This raises the possibility that KO crop mutants for TTM2 will also show enhanced resistance similar to Arabidopsis ttm2 plants, providing a useful tool in agricultural biotechnology to generate pathogen-resistant crop plants.

MATERIALS AND METHODS

Plant Growth Conditions and Pathogen Assays

Arabidopsis (Arabidopsis thaliana; accession Col-0), canola (Brassica napus var Westar), and soybean (Glycine max var Harasoy) plants were grown in Sunshine Mix at 22°C, 60% relative humidity, and approximately 140 μE m−2 s−1 with a 9-h photoperiod; 7- to 10-d-old Arabidopsis plants were infected with Hyaloperonospora arabidopsidis. Spore counts of 1 × 105 conidiospores mL−1, 8 × 105 cells mL−1, and 2 × 105 cells mL−1 were used for Noco2, Emco5, and Emwa1 isolates, respectively. Seedlings were then infected by drop inoculation and left at 16°C and >90% relative humidity for 7 to 10 d before disease assessment. Four-week-old plants were infiltrated with 1 × 105 colony forming units mL−1 of bacterial pathogen Pseudomonas syringae DC3000 tomato (AvrRps4), and bacterial growth was assessed at 0 and 3 d postinfiltration.

Cauliflower Mosaic Virus35S Transgenic Lines

The coding sequence of AtTTM2 was amplified from Arabidopsis Col-0 complementary DNA (cDNA) using the primers 35S-TTM2-F and 35S-TTM2-R (Supplemental Table S1) and cloned into pBI121 (Clontech). The vector was transformed into Col-0 wild-type plants through Agrobacterium tumefaciens-mediated transformation using the floral dip method (Clough and Bent, 1998).

RNA Extraction and RT-PCR

RNA extraction was carried out using the TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RT-PCR was performed using cDNA generated by SuperScript II Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. Expression of PR1 was visualized by gel electrophoresis of samples after RT-PCR with PR1 primers (AtPR1-F and AtPR1-R).

qPCR

qPCR was performed using Fast SYBR Green Master Mix (Life Technologies). The expressions of Arabidopsis genes were normalized to the expression of elongation factor 1α (AtEF1A), whereas the expressions of soybean and canola genes were normalized to GmEF1B and ubiquitin conjugating enzyme21 (BnUBC21), respectively. All primer sequences are listed in Supplemental Table S1.

Confirmation of T-DNA Insertion KO Lines

The SALK lines SALK_145897 (ttm2-1) and SALK_114669 (ttm2-2) were obtained from the SALK Institute (Alonso et al., 2003). Homozygous plants were isolated using gene-specific primers for ttm2-1 (897RP and 897LP) and ttm2-2 (244RP and 244LP) combined with the T-DNA-specific primer LBb1-F. RT-PCR was then performed on cDNA from both ttm2 lines to confirm the KO status using the full-length TTM2 primers (190RT-F and 244RT-R). Expression was normalized to the expression of β-tubulin. Primer sequences are listed in Supplemental Table S1.

Epistatic Analysis

ttm2-2 was crossed with pad4-1 (Glazebrook et al., 1996; Jirage et al., 1999), ics1-1 (Wildermuth et al., 2001), and npr1-1 (Cao et al., 1997). Homozygous double mutants were isolated in the F2 generation.

SA, BTH, and flg22 Treatments

Seven- to 10-d-old Arabidopsis seedlings were treated with 100 μm SA or 200 μm BTH. Treatments of canola and soybean plants were performed with the same concentrations but on 3- to 4-week-old plants, which were sprayed with the addition of 0.025% (v/v) Silwet. Treatment with 5 μm flg22 was performed on 3- to 4-week-old plants by syringe infiltration.

SAR Experiments

Seedlings were grown for 7 to 10 d and drop inoculated with either water or 2 × 105 conidiospores mL−1 of avirulent Hpa isolate, Emwa1. After true leaves emerged 7 d later, a secondary infection on upper systemic leaves with the virulent Hpa isolate, Noco2, was performed using 1 × 105 conidiospores mL−1 on all seedlings. Trypan Blue analysis was then performed 7 to 10 d later.

Trypan Blue Staining

Trypan Blue staining was performed as previously described (Yoshioka et al., 2001).

SA and SAG Measurements

Pooled tissue samples (n = 18) were collected 5 d after infection with the avirulent Hpa isolate, Emwa1, and frozen in liquid nitrogen. Endogenous SA and SAG were extracted and analyzed as previously described (Mosher et al., 2010).

Protein Expression in Escherichia coli

The coding region of AtTTM2 was cloned into pGEX-6P-1 from Arabidopsis Col-0 ecotype cDNA using the primers TTM2-TM-F and TTM2-TM-R, which excludes the annotated C-terminal transmembrane domain starting from D648. Plasmids were introduced into E. coli BL21 (DE3) and grown overnight in Luria-Bertani medium at 37°C. The overnight culture was used to seed a larger volume of autoinduction medium containing 1× NPS solution [25 mm (NH4)2SO4, 50 mm KH2PO4, and 50 mm Na2HPO4] and 1× 5052 solution (0.05% [w/v] Glc, 0.2% [w/v] α-lactose, and 0.5% [v/v] glycerol), and it was grown at 37°C for 3 to 4 h until optical density = 0.4. The temperature was then lowered to 18°C overnight before harvesting the cells by centrifugation at 4°C (Studier, 2005).

Protein Extraction

E. coli cultures were centrifuged, and pellets were resuspended in 1× phosphate-buffered saline (pH 7.5; 137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 1.8 mm KH2PO4) containing 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 10 μg mL−1 DNaseI. Cell suspensions were incubated on ice for 30 min before cell lysis by French press at 1,000 pounds per square inch. Soluble fractions were obtained by centrifugation and subjected to column purification using DE52 cellulose (Sigma) and GSH agarose (Sigma). Purified protein samples were eluted using 10 mm reduced glutathione.

Enzymatic Assays

Free phosphate released by AtTTM2 was measured with the Malachite Green assay (Bernal et al., 2005) as described by Moeder et al. (2013). The assay conditions were 0.5 mm PPi, ATP, or PPPi and 2.5 mm Mg2+, pH 9.0 at 37°C for 30 min. cAMP formation was assayed in 25 mm Tris, pH 8, 1 mm ATP, and 20 mm Mg2+ at 37°C for 30 min. HPLC analysis was an isocratic run with 20% (v/v) MeOH and 150 mm NaOAc, pH 5, on a Zorbax SB-C18 column (3.5 µm; Agilent).

Statistical Analysis

A two-tailed Student’s t test was performed for all comparisons between two sample groups. Fisher’s exact test was performed for all comparisons between two samples with multiple groups. A P value less than 0.05 was used to denote significance.

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AtTTM2 (At1g26190), Hpa-ITS2 (GU583836.1), PR1 (At2g14610), AtEF1A (At5g60390), β-tub (At5g23860), BnTTM2a (Bra011014), BnTTM2b (Bra012464), BnUBC21 (AC172883), BnPR1 (EF423806), GmTTM2a (Gm1g09660), GmTTM2b (Gm2g14110), GmEF1b (NM_001249608.1), and GmPR1 (XM_003545723.1).

Supplemental Data

The following materials are available in the online version of this article.

Supplementary Material

Supplemental Data
supp_166_2_1009__index.html (1.1KB, html)

Glossary

BTH

benzothiadiazole

cDNA

complementary DNA

Col-0

Columbia-0

GST

glutathione S-transferase

HR

hypersensitive response

KO

knockout

PAMP

pathogen-associated molecular pattern

PPi

pyrophosphate

PPPi

tripolyphosphate

qPCR

quantitative real-time PCR

RT

reverse transcription

SA

salicylic acid

SAG

salicylic acid glucoside

SAR

systemic acquired resistance

Ws

Wassilewskija

Footnotes

1

This work was supported by the Ontario Government (graduate student fellowships to H.U.) and by the Natural Science and Engineering Research Council of Canada, the Canadian Foundation for Innovation, and the Ontario Research Fund (Discovery Grant to K.Y.).

[W]

The online version of this article contains Web-only data.

[OPEN]

Articles can be viewed online without a subscription.

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