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The Plant Journal (2006) 48, 647–656 doi: 10.1111/j.1365-313X.2006.02903.x Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs Yuelin Zhang1,2,*, Yu Ti Cheng2, Na Qu1, Qingguo Zhao1, Donglin Bi1 and Xin Li2,3 National Institute of Biological Sciences, #7 Science Park Road, Zhongguancun Life Science Park, Beijing, People’s Republic of China 102206, 2 Michael Smith Laboratories, Room 301, 2185 East Mall, University of British Columbia, Vancouver, BC V6T 1Z4, Canada, and 3 Department of Botany, Room 3529, 6270 Univ. Blvd., University of British Columbia, Vancouver, BC V6T 1Z4, Canada 1 Received 9 May 2006; revised 7 July 2006; accepted 24 July 2006. *For correspondence (fax þ86 10 807 28646; e-mail [email protected]). Summary NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1 expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts. Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant, we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through their association with TGA2 and its paralogs. Keywords: plant disease resistance, PR genes, NPR3, NPR4, TGA transcription factors. Introduction Plants have evolved sophisticated defense mechanisms to deal with constant exposure to a wide range of microbial pathogens. Systemic acquired resistance (SAR) is a secondary resistance response that is effective against a broad spectrum of pathogens (Durrant and Dong, 2004). One important signal molecule in SAR is salicylic acid (SA). The endogenous level of SA increases upon pathogen infection (Malamy et al., 1990; Metraux et al., 1990; Rasmussen et al., 1991). Exogenous application of SA or SA analogs induces Pathogenesis-related (PR) genes and pathogen resistance, suggesting that SA is sufficient to induce SAR (Gorlach et al., 1996; Metraux et al., 1991; White, 1979). Salicylic acid is also essential for SAR since blocking its accumulation by expressing the bacterial gene NahG, which encodes the SAdegrading enzyme salicylate hydroxylase, leads to reduced expression of PR genes and enhanced susceptibility to both compatible and incompatible pathogens (Delaney et al., 1994; Gaffney et al., 1993; Nawrath and Metraux, 1999). In Arabidopsis, mutants unable to synthesize SA after infection ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd are also more susceptible to both virulent and avirulent pathogens (Nawrath and Metraux, 1999; Wildermuth et al., 2001). NPR1 (also known as NIM1 and SAI1) is required for SAinduced PR gene expression and pathogen resistance in Arabidopsis (Dong, 2004). It encodes a protein with two protein–protein interaction domains, a BTB/POZ domain at the N-terminus and an ankyrin-repeat domain in the central region (Aravind and Koonin, 1999; Cao et al., 1997; Ryals et al., 1997). NPR1 accumulates in the nucleus after induction by SA treatment or pathogen infection (Kinkema et al., 2000). Nuclear localization of NPR1 is required for the activation of PR-1 expression. An increase in the level of SA induces redox changes, which appear to regulate the translocation of the NPR1 protein from the cytoplasm to the nucleus (Mou et al., 2003). Increasing evidence indicates that NPR1 functions through its association with TGA transcription factors. NPR1 interacts with various TGA transcription factors in the yeast two-hybrid assay as well 647 648 Yuelin Zhang et al. Results Identification of knockout mutants for NPR3 and NPR4 Real-time RT-PCR analysis of NPR3 and NPR4 expression showed that both genes were induced after treatment with the SA analog INA (Figure 1a,b) or pathogen infection (Figure 1c,d). Both NPR3 and NPR4 have high sequence similarity to NPR1 throughout the protein (Figure S1). The most conserved regions are the four ankyrin repeat domains corresponding to amino acids 265–393 in NPR1. Both the Nterminal BTB/POZ domain and the C-terminal nuclear localization signatures are also present in NPR3 and NPR4. In addition, the intron–exon structures are conserved among NPR1, NPR3 and NPR4 (Figure 2 and Figure S1). Sequence analysis indicates that there are orthologs of NPR3 and NPR4 in rice (data not shown). To identify the functions of NPR3 and NPR4, deletion mutants of NPR3 and NPR4 were obtained by screening a fast neutron-mutagenized Arabidopsis population (Li et al., 2001b). These were named npr3-1 and npr4-3, respectively. The coding sequences of NPR3 and NPR4 were completely deleted in the mutants (Figure 2a,c), suggesting that they are null mutations. The double mutant npr3-1 npr4-3 was (a) (b) 1.0 Relative NPR4/ACTIN1 20 10 0 0.8 0.6 0.4 0.2 0 –INA +INA (c) –INA +INA (d) 15 Relative NPR4/ACTIN1 0.4 10 5 0.3 0.2 0.1 6 32 .E S4 M S4 32 6 oc k M P. s. m .E oc k 0 0 P. s. m Relative NPR3/ACTIN1 30 Relative NPR3/ACTIN1 as in planta (Despres et al., 2000; Kim and Delaney, 2002; Subramaniam et al., 2001; Zhang et al., 1999; Zhou et al., 2000). In addition, the triple knockout mutant of TGA2, TGA5 and TGA6 has phenotypes similar to npr1 (Zhang et al., 2003b). Induction of PR-1 expression and pathogen resistance by the SA analog 2,6-dichloroisonicotinic acid (INA) was blocked in tga6-1 tga2-1 tga5-1, but not in tga6-1 or tga21 tga5-1 plants, suggesting that TGA2, TGA5 and TGA6 encode proteins with redundant and essential functions in SA signaling. In the Arabidopsis genome, there are five paralogs of NPR1 (At4g26120/NPR2, At5g45110/NPR3, At4g19660/NPR4, At3g57130/BOP1 and At2g41370/BOP2). Phylogenetic analysis of the NPR1 protein family revealed that NPR2 is closest to NPR1 and forms a subgroup with NPR1; NPR3 and NPR4 form a distinctive pair; and BOP1 and BOP2 form another pair farthest from NPR1 (Hepworth et al., 2005; Liu et al., 2005). It was previously reported that At3g57130 and At2g41370 encode BOP1 (Blade-on-petiole, 1) and BOP2, respectively, which function in the control of growth symmetry (Ha et al., 2004; Hepworth et al., 2005; Norberg et al., 2005). Phenotypes in the bop1 bop2 double mutant include leafy petioles, loss of abscission of floral organs and asymmetric flowers subtended by a bract. BOP1 and BOP2 interact with PERIANTHIA (AtbZip46), a bZip transcription factor in the TGA subfamily (Hepworth et al., 2005). Here we report the knockout analysis of At5g45110 (NPR3) and At4g19660 (NPR4) and their overlapping roles in negative regulation of plant defense responses. Figure 1. Expression of NPR3 and NPR4. (a, b) Real-time RT-PCR analysis of the expression of NPR3 and NPR4 in wildtype plants with or without INA treatment. (c, d) Real-time RT-PCR analysis of NPR3 and NPR4 expression in wild-type plants inoculated with P.s.m. ES4326. For RT-PCR analysis, RNA was extracted 2 days after the treatments from control plants and plants sprayed with 0.33 mM INA or inoculated with P.s.m. ES4326 (OD600 ¼ 0.001). The cDNA was reverse transcribed and normalized to the expression of Actin1. Error bars represent standard deviation from three measurements. Each experiment was repeated once with similar results. generated by crossing the two single mutants and screening for lines carrying both homozygous mutations in the F2 population. In the double deletion mutant, no transcripts of NPR3 and NPR4 were detected by RT-PCR (data not shown). Neither the single nor the double mutant plants have obvious developmental phenotypes. Insertion mutants for NPR3 and NPR4 were also obtained from the Arabidopsis Stock Center (Alonso et al., 2003). SALK_043055 was named npr3-2 and SALK_098460 was previously named npr4-2 (Liu et al., 2005). The locations of the T-DNA insertions in npr3-2 and npr4-2 are shown in Figure 2(b) and (d). Both T-DNA insertions are in exons. npr3 and npr4 mutants have elevated basal PR gene expression To determine whether basal PR-1 expression was altered in the npr3 and npr4 mutants, seedlings of wild-type and mutant plants were grown on MS medium and their PR-1 expression was determined with real-time PCR. As shown in Figure 3(a), single mutants for NPR3 have slightly increased ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 Negative regulation of defense responses in Arabidopsis 649 (a) NPR3 At5g45110 At5g45120 TAC clone K17O22 Deletion 49832–56001 (b) ATG NPR3 At5g45110 SALK_043055 Insertion (c) NPR4 At4g19650 At4g1966 0 BAC clone T16H5 Deletion 311–9188 (d) ATG NPR4 At4g19660 SALK_098460 Insertion Figure 2. Molecular lesions in the NPR3 and NPR4 mutants. The white boxes represent exons, and the gray boxes represent genes. (a) Deletion in npr3-1. (b) Location of T-DNA insertion in npr3-2. (c) Deletion in npr4-3. (d) Location of T-DNA insertion in npr4-2. basal PR-1 expression. PR-1 expression in the npr3-1 npr4-3 double mutant is dramatically increased (Figure 3a). Previously the tga2-1 tga5-1 tga6-1 triple mutant was also reported to have elevated PR-1 expression. Because background PR-1 expression is often very low for plate-grown seedlings, and varies depending on the growth conditions, the npr3-1 npr4-3 double mutant and the tga2-1 tga5-1 tga6-1 triple mutant were grown on the same MS plate to directly compare the PR-1 level in these plants. As shown in Figure 3(b), PR-1 expression in the npr3-1 npr4-3 double mutant was comparable with that in the tga2-1 tga5-1 tga6-1 triple mutant. We also found that PR-2 and PR-5 expression levels in the npr3-1 npr4-3 double mutant plants were significantly higher than those in the wild-type plants (Figure 3c,d). To test whether PR-1 expression in the npr3-1 npr4-3 double mutant can be induced by SA, we grew the mutant and wild-type plants on MS plates with or without the SA analog INA. As shown in Figure 3(b), treatment with INA further induced the expression of PR-1 in the double mutant. The expression level of PR-1 in the INA-treated double mutant is very similar to that in the wild type. We also tested the soil-grown plants for the induction of PR-1 by INA. One day after induction, PR-1 expression increased about sevenfold in the npr3-1 npr4-3 double mutant and less than twofold in the wild-type plants (Figure 3e,f), suggesting that induction of PR-1 in the double mutant is quicker than that in the wild-type plants. Two days after INA induction, the PR-1 level is similar in the npr3-1 npr4-3 and wild-type plants. The npr3-1 npr4-3 double mutant shows enhanced resistance against virulent pathogens To test whether NPR3 and NPR4 affect the bacterial disease resistance response, single and double mutants were challenged with Pseudomonas syringae pv. maculicola (P.s.m.) ES4326, which is virulent on the Columbia ecotype. Figure 4 shows that while there is no significant difference in bacterial growth between wild type and npr3-1 or npr4-3, about tenfold less bacterial growth was observed in the npr3-1 npr4-3 double mutant. Similar results were obtained when the plants were challenged with Pseudomonas syringae pv. tomato (P.s.t.) DC3000 (Figure S2). To confirm whether the enhanced resistance phenotype observed in the double mutant is caused by mutations in NPR3 and NPR4, a cDNA clone of NPR3 under the control of the CaMV 35S promoter and a genomic clone of NPR4 were constructed and transformed into the npr3-1 npr4-3 double mutant. As shown in Figure 4, both the NPR3 cDNA clone and the NPR4 genomic clone can suppress the enhanced resistance phenotype in the npr3-1 npr4-3 double mutant, indicating that the enhanced resistance phenotype observed in the double mutant is caused by the mutations in both NPR3 and NPR4. In addition, we constructed another double mutant, npr3-2 npr4-2, using the T-DNA insertion mutant alleles. Similar to npr3-1 npr4-3, the npr3-2 npr4-2 double mutant also displayed enhanced resistance to P.s.m. ES4326 (Figure S3), further confirming that NPR3 and NPR4 perform overlapping functions and that loss of the function in both genes leads to enhanced resistance to P.s.m. ES4326. To determine whether mutations in NPR3 and NPR4 confer enhanced resistance against the virulent oomycete pathogen Hyaloperonospora parasitica Noco2, the single and double mutants were infected with H. parasitica Noco2 conidiospores at a concentration of 5000 spores ml)1. To assess disease severity, conidiospores were collected from the plants 6 days post-inoculation, and quantified with a hemocytometer. As shown in Figure 5, npr3-1 and npr3-2 single mutants are significantly more resistant to H. parasitica Noco2. Resistance in the npr3-1 npr4-3 double mutant is further enhanced compared to the npr3 single mutants, indicating that both the npr3-1 and the npr4-3 mutation contribute to the enhanced resistance. In addition, the enhanced disease resistance phenotype in the npr3-1 npr43 double mutant can be partially rescued by the wild-type NPR3 or NPR4 genes (Figure 5). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 650 Yuelin Zhang et al. Relative PR-2/ACTIN1 MS+INA 160 10000 120 0 (e) Relative PR-1/ACTIN1 400 300 200 100 np r4 -3 200 100 0 Col 800 600 400 200 0 Day 0 Day 1 Day 2 C 600 npr3-1 npr4-3 400 200 0 Day 0 Day 1 Day 2 np r3 -1 C ol 0 300 (f) Relative PR-1/ACTIN1 (d) 400 np r3 -1 tg a2 np r3 -1 tg a5 C np ol r4 np 3 r4 np 2 r4 np -3 r3 -2 np r3 -1 1 40 Co l np r4 -3 10 80 np r3 -1 100 tg np a6 r3 -1 np r4 -3 1000 Relative PR-5/ACTIN1 (c) MS ol np r4 -3 (b) Relative PR-1/ACTIN1 Relative PR-1/ACTIN1 (a) Figure 3. Analysis of PR gene expression in npr3 and npr4 mutants. (a) PR-1 expression in npr3 single, npr4 single and npr3-1 npr4-3 double mutants. The relative expression levels of PR-1 were normalized to the expression of Actin1 (multiplied by 1000 for clarity). (b) Induction of PR-1 expression in wild-type and npr3-1 npr4-3 plants grown on MS plates with or without INA. The relative expression levels of PR-1 were normalized to the expression of Actin1. (c) PR-2 expression in the npr3-1 npr4-3 double mutant. The relative expression levels of PR-2 were normalized to the expression of Actin1 (multiplied by 1000 for clarity). (d) PR-5 expression in the npr3-1 npr4-3 double mutant. The relative expression levels of PR-5 were normalized to the expression of Actin1 (multiplied by 1000 for clarity). (e, f) Induction of PR-1 expression in wild-type and npr3-1 npr4-3 plants grown on soil. The relative expression levels of PR-1 were normalized to the expression of Actin1. Plants were grown on soil and 20-day-old seedlings were treated with 0.33 mM INA. Samples were taken 0 h (day 0), 24 h (day 1) and 48 h (day 2) after the application of INA. The RNAs were reverse transcribed to obtain total cDNA and the cDNAs were used to determine the relative expression levels of PR-1, PR-2 and PR-5 expression in wild-type and mutant plants by real-time PCR using SYBR Green I chemistry. Error bars represent the standard deviation from three measurements. Each experiment was repeated once with similar results. For (a–d), total RNA was extracted from 20-day-old seedlings grown on MS plates with or without 50 lM INA under a 16-h day/8-h night cycle. The level of SA is not elevated in the npr3-1 npr4-3 double mutant Most mutants with constitutive PR gene expression have increased SA levels (Bowling et al., 1994, 1997; Clarke et al., 1998; Li et al., 2001a; Maleck et al., 2002; Rate et al., 1999; Shirano et al., 2002). To test whether the constitutive PR-1 expression and pathogen resistance in the npr3-1 npr4-3 double mutant is due to an elevated level of SA, the level of SA was determined in the double mutant. As shown in Figure 6(a), the total SA level of npr3-1 npr4-3 plants is not significantly higher than that of wild-type plants. To determine whether accumulation of SA in the npr3-1 npr4-3 double mutant is affected after avirulent pathogen induction, leaves of wild-type and mutant plants were challenged with P.s.t. DC3000 carrying AvrRpt2 and collected for SA measurement. As shown in Figure 6(b), the double mutant accumulated a similar amount of total SA as the wild-type plants. The levels of free SA in the npr3-1 npr4-3 plants were also not increased compared with those in the wild-type plants under induced or uninduced conditions (data not shown). Thus the enhanced resistance in npr3-1 npr4-3 is not caused by increased endogenous SA. Disease resistance in npr3-1 npr4-3 is partially dependent on NPR1 To test the relationship between NPR3, NPR4 and NPR1, a triple mutant of npr3-1 npr4-3 npr1-1 was constructed. As shown in Figure 7(a), constitutive PR-1 expression in the npr3-1 npr4-3 double mutant was only partially affected by the npr1-1 mutation. When triple mutant plants were infected with P.s.m. ES4326, the resistance in npr3-1 npr4-3 was ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 Negative regulation of defense responses in Arabidopsis 651 P.s.m. ES4326 Day 0 P.p. Noco2 8 Day 3 5 7 4.5 6 Spores per plant (x104) Log (cfu mg FW–1) 4 3.5 3 2.5 2 1.5 1 5 4 3 2 0.5 1 Figure 4. Growth of P.s.m. ES4326 in wild type, npr3-1, npr4-3, npr3-1 npr4-3 double mutant and npr3-1 npr4-3 transformed with either NPR4 genomic clone (NPR4g#15) or 35S-NPR3 cDNA clone (NPR3c#6). The complementing lines are BASTA-resistant T2 plants from the designated lines. The plants were infiltrated with a bacterial suspension of OD600 ¼ 0.0001. The error bars represent the standard deviation of four replicates. The experiment was repeated three times with similar results. Cfu, colony-forming units. also partly compromised (Figure 7b). These data suggest that the enhanced resistance mechanisms being activated in npr3-1 npr4-3 are partially dependent on NPR1, thus both NPR1-dependent and NPR1-independent pathways are activated in the npr3-1 npr4-3 double mutant. NPR3 and NPR4 interact with TGA transcription factors in the yeast two-hybrid assay To identify proteins that interact with NPR4, a yeast two-hybrid screen of an Arabidopsis cDNA library was performed using NPR4 as bait. Among the positive clones identified and sequenced, three contained truncated cDNAs of TGA2, TGA3 or TGA6. A comparison of the sequences of the recovered cDNA clones with the full-length cDNAs showed that the TGA2 clone starts from nucleotide 265 of At5g06950 (accession number BT006134), the TGA3 clone starts from nucleotide 588 of At1g22070 (accession number NM_102057) and the TGA6 clone starts from nucleotide 466 of At3g12250 (accession number NM_112061). Yeast two-hybrid assays were subsequently performed using NPR3 or NPR4 as bait and TGA1 to TGA6 as prey. As shown in Figure 8, NPR3 interacts with TGA2, TGA3, TGA5 and TGA6, but not TGA1 or TGA4 in the yeast two-hybrid assay. Similar results were observed when NPR4 were used as bait except that weak interaction was observed between NPR4 and TGA1. C ol r3 -1 np r3 -2 np r4 -3 n np pr 4r3 2 -1 np r4 N PR 3 3c #6 N PR 4g #1 3 0 np Col npr 3-1 npr npr 43-1 npr 3 4- 3 NP R4g #15 NP R3c #6 Col npr 3-1 npr npr 4 -3 3-1 npr 4-3 NP R4g #15 NP R3c #6 0 Figure 5. Growth of H. parasitica Noco2 on npr3 single, npr4 single, npr3-1 npr4-3, and npr3-1 npr4-3 double mutant plants transformed with the 35SNPR3 cDNA clone (NPR3c#6) or npr3-1 npr4-3 plants transformed with the genomic clone of NPR4 (NPR4g#13). The complementing lines are BASTA-resistant T2 plants from the designated lines. Two-week-old soil-grown seedlings were sprayed with H. parasitica Noco2 spores at a concentration of 5000 spores ml)1 of water. After 6 days of incubation under 90% humidity and 16-h day/8-h night cycles, five plants were randomly harvested into 1 ml of water and conidiospores on the plants were vortexed off and counted using a hemocytometer. Each treatment contains four replicates, and the numbers represent the average spore counts on one plant. The error bars represent the standard deviation of four replicates. The experiment was repeated twice with similar results. Bimolecular fluorescence complementation analysis of NPR3 and TGA2 interaction To test whether NPR3 and TGA2 associate with each other in vivo, we used a bimolecular fluorescence complementation (BiFC) approach for visualization of protein–protein interactions in plant cells. In this method, two non-fluorescent fragments (YFPN and YFPC) of yellow fluorescent protein (YFP) are fused to two different proteins separately (Walter et al., 2004). When these two proteins associate with each other, a fluorescent YFP complex is formed. We first made constructs to express NPR3–YFPN and TGA2–YFPC fusion proteins. In both cases, the YFP fragments are located at the C-terminus of the fusion proteins. When the NPR3–YFPN and TGA2–YFPC constructs were co-bombarded to onion epidermal cells, YFP fluorescence was observed, predominantly in the nucleus (Figure 9a). When Arabidopsis mesophyll protoplasts were transiently transfected with the NPR3–YFPN and TGA2–YFPC constructs, YFP fluorescence was also observed in the nucleus of the transfected protoplasts (Figure 9b), suggesting that NPR3 and TGA2 associate with each other in vivo in the nucleus. No yellow fluorescence was observed when NPR3–YFPN or ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 652 Yuelin Zhang et al. (a) (a) Uninduced total SA 0.8 100000 Relative PR-1/ACTIN1 0.7 0.5 0.4 0.3 0.2 10000 1000 100 10 0.1 Local total SA (b) 6 npr 1-1 npr 4-3 Day 0 6 Day 3 5 4 TGA2–YFPC was co-transfected with the empty YFPC or YFPN vectors (data not shown). Discussion Systemic acquired resistance is an important pathway in plant innate immunity, and NPR1 is a key positive regulator of SAR (Cao et al., 1997). Since there are five paralogs of NPR1 in the Arabidopsis genome, their functions were intriguing. Here we present the knockout analysis of NPR3 and NPR4. Our data suggest that these genes function as negative regulators of plant defense. The npr3-1 single mutant displayed elevated PR-1 level and enhanced resistance to H. parasitica Noco2. In npr4-3 plants, no obvious enhanced resistance phenotype was observed. The combination of npr3-1 and npr4-3 leads to much higher PR-1 expression and a dramatic increase in resistance to both the virulent bacterial pathogen P.s.m. ES4326 and the oomycete pathogen H. parasitica Noco2. 1 1-1 npr 1-1 3-1 npr 4-3 npr 4-3 npr Col 0 3-1 Figure 6. Total SA in uninfected and infected local leaves of Col, npr3-1, npr4-3 and the npr3-1 npr4-3 double mutant plants. (a) Total SA from uninfected leaf tissue of 4-week-old soil-grown plants. (b) Total SA from the local leaves of 4-week-old soil-grown plants infected with P.s.t. DC3000 AvrRpt2 at a dose of OD600 ¼ 0.2. Half of a leaf was inoculated and the whole leaf was collected 3 days after infection. The error bars represent the standard deviation of four replicates. The experiment was repeated once with similar results. 2 npr npr3-1 npr4-3 npr Col 3 Col 3-1 npr n p r 3-1 4-3 npr 4-3 npr 1-1 npr 1-1 0 4 npr 2 Log (cfu mg FW–1) µg SA / g tissue (b) npr 3-1 np r3 npr 3-1 -1 np r4 Col -3 np r4 -3 1 np r3 -1 Co l 0 npr 4-3 µg SA / g tissue 0.6 Figure 7. Analysis of the npr3-1 npr4-3 npr1-1 triple mutant. (a) PR-1 expression in npr3-1 npr4-3 npr1-1 mutant plants determined by realtime PCR. The relative expression levels of PR-1 were normalized to the expression of Actin1; error bars represent standard deviation from three measurements. The experiment was repeated once with similar results. (b) Growth of P.s.m. ES4326 in the npr3-1 npr4-3 npr1-1 triple mutant. The plants were infiltrated with a bacterial suspension of OD600 ¼ 0.0001. The error bars represent the standard deviation of four replicates. The experiment was repeated three times with similar results. Cfu, colony-forming units. Previously Liu et al. (2005) analyzed the npr4 single mutants for altered resistance to pathogens. Similar to our results, they did not observe any obvious difference in resistance or susceptibility to H. parasitica Noco2 in the npr4 single mutants compared to the wild-type plants. Interestingly, they found that the npr4 single mutants are more susceptible to P.s.t. DC3000. However, under our growth conditions, no enhanced susceptibility to P.s.m. ES4326 or P.s.t. DC3000 was observed in npr4-3 or npr4-2 in multiple repeated experiments, although npr4-2 is the same mutant ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 Negative regulation of defense responses in Arabidopsis 653 (a) (b) Figure 8. Yeast two-hybrid interactions between TGA transcription factors and NPR3 or NPR4. Yeast cells carrying the TGA cDNA clones and either pBI880 vector control or indicated bait were grown for 2 days at 30C on medium lacking tryptophan and leucine, and the b-galactosidase reporter expression was subsequently assayed using the X-gal filter assay. allele as used in the study by Liu et al. (2005). The effect of the npr4-2 mutation on disease resistance can only be observed when combined with the npr3-2 mutation. Since npr4 single mutants have an elevated PR-1 expression level and enhanced resistance to both H. parasitica and P.s.m. ES4326 when combined with npr3 knockout mutants, it is very unlikely that NPR4 is a positive regulator of disease resistance and required for disease resistance as suggested by Liu et al. (2005). In Arabidopsis, a large number of mutants that display constitutive PR gene expression and pathogen resistance have been identified (Bowling et al., 1994, 1997; Clarke et al., 1998; Li et al., 2001a; Maleck et al., 2002; Rate et al., 1999; Shirano et al., 2002). Most of these mutants constitutively accumulate high levels of SA in the absence of pathogen infection and this is often required for enhanced pathogen resistance. In contrast, the npr3-1 npr4-3 double mutant does not accumulate higher levels of SA than wild-type plants with or without pathogen induction, but still expresses elevated levels of PR genes and enhanced resistance to both P.s.m. ES4326 and H. parasitica Noco2. Since induction of PR-1 is quicker in the double mutant, the enhanced disease resistance phenotype is probably caused by an enhanced capability of the plants to activate infectioninduced defense responses, a process called priming (Conrath et al., 2002). As PR-1 is a hallmark of the SA signaling pathway (Uknes et al., 1992) and SA synthesis is not affected in the npr3-1 npr4-3 double mutant, our data suggest that NPR3 and NPR4 may regulate defense responses downstream of SA. Previous studies have suggested that both NPR1-dependent and NPR1-independent Figure 9. Bimolecular fluorescence complementation visualization of NPR3 and TGA2 interaction. (a) Epifluorescence (I) and bright field (II) images of onion epidermal cells cobombarded with constructs expressing the NPR3–YFPN and TGA2–YFPC fusion proteins. (b) Epifluorescence (I), bright field (II), chloroplast autofluorescence (III) and merged (IV) images of Arabidopsis mesophyll protoplasts co-transfected with constructs expressing the NPR3–YFPN and TGA2–YFPC fusion proteins. resistance pathways exist downstream of SA synthesis (Li et al., 2001a; Zhang et al., 2003a). Our analysis of the npr3-1 npr4-3 npr1-1 triple mutant indicates that the enhanced resistance in the npr3-1 npr4-3 plants is a combination of both NPR1-dependent and NPR1-independent resistance. Like NPR1, NPR3 and NPR4 have no obvious biochemical functions except for the presence of two protein–protein interaction domains, suggesting that their functions may be to interact with other proteins as adaptors. NPR1 is located in the cytoplasm as an oligomer under non-inducing conditions and Cys216 is one of the cysteines that is required for keeping NPR1 in the cytoplasm (Mou et al., 2003). Sequence comparison between NPR1 and its paralogs indicate that this cysteine residue is not conserved in the NPR3 and NPR4 proteins, suggesting that NPR3 and NPR4 may be regulated differently. In the roots, the NPR4–GFP fusion protein was found to localize in the nucleus (Figure S4). It remains to be determined whether SA induction affects the localization of ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 654 Yuelin Zhang et al. NPR3 and NPR4. We found that TGA2, TGA3, TGA5 and TGA6 interact with NPR3 and NPR4 in yeast two-hybrid assays (Figure 8). It was further demonstrated that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts using BiFC analysis. Thus it is likely NPR3 regulates PR gene expression through interaction with TGA factors in the nucleus. We previously reported that the tga6-1 tga2-1 tga5-1 triple mutant displayed multiple phenotypes. The loss of SAR responses in the tga triple mutant resembles that in the npr1 mutant plants, but the elevated basal PR-1 expression in the tga triple mutant was not observed in the npr1 mutants, suggesting that suppression of basal PR-1 expression by these TGA transcription factors is facilitated by other factors (Zhang et al., 2003b). Since the npr3-1 npr4-3 double mutant also accumulates high levels of basal PR-1, and NPR3 interacts with TGA2, TGA5 and TGA6 in the yeast two-hybrid assays and associates with TGA2 in the nucleus of Arabidopsis mesophyll protoplasts, it is plausible to hypothesize that NPR3 and NPR4 negatively regulate PR-1 expression and pathogen resistance through association with TGA2 and its paralogs. Similar to the npr3-1 npr4-3 npr1-1 triple mutant, the tga triple mutant did not display enhanced disease susceptibility to P.s.m. ES4326. It is most likely that the enhanced disease susceptibility associated with the loss of response to SA was overcome by enhanced resistance associated with the elevated basal PR gene expression. Our study on the npr3 and npr4 single and double mutants provides strong evidence that NPR3 and NPR4 are negative regulators of plant defense responses. This is a major step toward understanding their functions in regulating plant defense responses downstream of SA synthesis. It remains to be determined how NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance. It is possible that NPR3 and NPR4 bind to TGA2, TGA5 and TGA6 and compete with NPR1 for interactions with the TGA factors under non-inducing conditions. When complexed with NPR3 or NPR4, these TGA factors cannot induce PR gene expression. Inactivating both NPR3 and NPR4 leads to activation of TGA2, TGA5 and TGA6 and expression of PR genes. Alternatively, NPR3 and NPR4 may function as cofactors of TGA2, TGA5 and TGA6, and are required for the function of these TGA factors. In this scenario, TGA2, TGA5 and TGA6 might be the transcription factors required for the expression of a negative regulator of PR gene expression and pathogen resistance. Experimental procedures Mutant isolation The npr3-1 and npr4-3 mutants were identified by screening a fast neutron-mutagenized population by PCR (Li et al., 2001b). The npr32 (SALK_043055) and npr4-2 (SALK_098460) mutants were obtained from the Arabidopsis Stock Center (ABRC). Homozygous plants were identified by PCR using primers flanking the insertion. The double mutants npr3-1 npr4-3 and npr3-2 npr4-4 were obtained by crossing the two single mutants, selfing F1, and screening in the F2 population for plants with an absence of both genes. The triple mutant npr3-1 npr4-3 npr1-1 was obtained by crossing npr1-1 with the double mutant npr3-1 npr4-3, selfing F1 and screening for the triple mutant in the F2 population with primers specific for the three mutations. Two independent triple mutant lines were obtained from 72 F2 plants. Both lines have similar phenotypes and one line was used for detailed analysis. Expression analysis and pathogen infections All plants were grown at 22C under 16-h light/8-h dark cycles. Infection of plants with P.s.m. ES4326 and H. parasitica Noco2 was carried out as previously described (Li et al., 2001a). Ribonucleic acid expression was carried out as described earlier (Zhang et al., 2003b). Levels of SA were determined using a 1:5 scaled-down protocol as previously described (Li et al., 1999). Complementation of the npr3-1 npr4-3 double mutant A 3.8-kb fragment (T16H5, nucleotides 5136–8952, accession number AL024486) containing NPR4 was amplified by PCR from wild-type genomic DNA and cloned into the binary vector pGreen229 (Hellens et al., 2000) to create pG229NPR4g. The full-length cDNA of NPR3 was amplified by PCR and cloned into a modified pGreen229 vector under the control of the CaMV 35S promoter. To confirm that the phenotypes observed in the npr3-1 npr4-3 double mutant or the npr3-1 npr4-3 npr1-1 triple mutant were caused by mutations in NPR3 and NPR4, the cDNA clone of NPR3 and genomic clone of NPR4 were transformed into Agrobacterium and subsequently into the double or triple mutant by floral dipping (Clough and Bent, 1998). Yeast two-hybrid screen The plasmid vectors pBI880 and pBI881 and the prey Arabidopsis library were obtained from Dr William Crosby (University of Windsor, Ontario, Canada). The full-length NPR3 and NPR4 cDNAs were amplified by PCR and cloned into pBI880. The yeast two-hybrid screen was performed as previously described (Kohalmi et al., 1997) except that we used the yeast strain PJ694a. Transformants containing both the pBI880NPR4 bait plasmid and the library plasmids were plated on medium lacking tryptophan, leucine and histidine and supplemented with 5 mM 3-amino-1¢,2¢,4¢-triazole to select colonies that express the HIS3 reporter gene. The X-gal filter assay was subsequently performed to monitor the expression of the lacZ reporter gene. For the two-hybrid assays, cells were co-transformed with the bait and the prey plasmids, and the transformants were analyzed by X-gal filter assay. Bimolecular fluorescence complementation analysis of NPR3 and TGA2 The TGA2 cDNA was amplified by PCR and cloned into pUCSPYCE that contained the 35S promoter and C-terminal region of YFP to obtain pTGA2•YCE. Meanwhile, the NPR3 cDNA was amplified by PCR and cloned into pUC-SPYNE that contained the 35S promoter and N-terminal region of YFP to obtain pNPR3•YNE. Both plasmids were confirmed by sequencing and purified by CsCl gradient. ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656 Negative regulation of defense responses in Arabidopsis 655 For transient expression of the NPR3-YFPN and TGA2-YFPC fusion protein in onion epidermal cells, pNPR3•YNE and pTGA2•YCE were bombarded into onion epidermal cells according to a previously described protocol (von Arnim and Deng, 1994). For transient expression of the NPR3–YFPN and TGA2–YFPC fusion proteins, pNPR3•YNE and pTGA2•YCE were co-transfected into Arabidopsis mesophyll protoplasts according to a previously described protocol (Sheen, 2001). Yellow fluorescent protein fluorescence was observed by confocal microscopy. Acknowledgements We thank Gary Wong, Arthur Lau and Lu Qun for their excellent technical assistance; Zhang Jie, Quan Ruidang and Dr Yan Guo for help on BiFC experiments; Maxygen Inc. (especially Dr Mike Lassner) for providing us with the npr3-1 and npr4-3 mutants; ABRC for npr3-2 and npr4-2 mutant seeds; Dr William Crosby for providing the yeast two-hybrid library, Dr Phil Hieter for yeast strain PJ694a and Dr Jim Kronstad, Sandra Goritschnig and Kristoffer Palma for critical reading of the manuscript. We are grateful for financial support to Y.Z. from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Chinese Ministry of Science and Technology; and funds to X.L. from UBC Michael Smith Laboratories, NSERC, Canadian Foundation for Innovation (CFI), BCKDF and the UBC Blussum Fund. Supplementary Material The following supplementary material is available for this article online: Figure S1. Alignment of NPR1, NPR3 and NPR4. Figure S2. 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