Fig 1.
NPR2 complements npr1-1 in Arabidopsis.
(a) Homozygous transgenic plants 35S:GFP-NPR2 of Arabidopsis in an npr1-1 background were tested for their response to benzothiadizole (BTH, an analogue of SA), along with control genotypes. The response to BTH was measured as weight, and plants were treated with either mock or 350 μM BTH. The ratio is expressed as percentage of fresh weight (%FW). The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD Test, P < 0.05). In all the figures that give numerical information, the data represent the average, with the error bars plotting the standard deviation. All the experiments were repeated at least three times with similar results. (b) The indicated genotypes (14-day-old plants) were treated with either 1 mM SA, 350 μM BTH or a mock solution. One day later the plants were inoculated with Pseudomonas syringae pv. tomato isolate DC3000 (Pto) at 5*107 colony forming units (cfu) per mL. Three days after inoculation, the growth of Pto was evaluated as the Logarithm of cfu per plant. Asterisks indicate statistically significant differences from the mock treatment in each genotype (P < 0.05 one asterisk, P < 0.01 two) using the Student’s t test (one tail). (c) The indicated genotypes were grown on MS plates, and the picture was taken at day 7 after germination. (d) The same experiment as in (a), with 200 μM SA. (e) The same experiment as in (a), with 300 μM SA.
Fig 2.
Phenotypes of the NPR1 paralogs knockouts.
(a) SA perception of combinations of KOs, measured by fresh weight after BTH treatment, as in Fig 1a. The abbreviations used are: n1 = npr1-1; n2 = npr2; n3 = npr3; n4 = npr4; n5 = bop2; n6 = bop1. The combinations of mutants are indicated by the letter “n” and the corresponding numbers, e.g. n1234 correspond to the quadruple npr1-1 npr2 npr3 npr4. (b) Enhanced SA perception of the quadruple npr3 npr4 bop1 bop2. Plants were treated and measured as in (a), but with a ten-fold reduction in the BTH, to visualize increased sensitivity to SA. 35S:NPR1 and 35S:GFP-NRB4 are included as controls of enhanced perception ([31] and [4] respectively). (c) SA perception, measured as pathogen growth as in Fig 1b. (d) eds-like phenotype. Seven-week-old plants were hand infiltrated with Pto at 5*104 cfu/mL. Three days after inoculation, the growth of Pto was evaluated as the Logarithm of cfus per g of fresh weight.
Fig 3.
Gene expression depending on NPR1 paralogs.
RNA was extracted from two week old plants of the indicated genotypes. In the (a) to (d) panels, the samples were taken one day after treatments of mock or 1 mM SA, while in the (e) to (h) panels, the samples were taken 2.5 h after treatment of 1 mM 4-hydroxybenzoic (HBA, a isomer of SA with no biological activity) or 1 mM SA. (a) Fold enrichment of α -Dioxygenase 1, one day after treatment. (b) Fold enrichment of Glutaredoxin 480, one day after treatment. (c) Fold enrichment of PR1 one day after treatment, in logarithmic scale. (d) Fold enrichment of 12-Oxophytodienoate reductase 11, one day after treatment. (e) Fold enrichment of Glutaredoxin 480, 2.5 h after treatment. (f) Fold enrichment of 12-Oxophytodienoate reductase 11, 2.5 h after treatment. (g) Fold enrichment of NAC domain containing protein 102, 2.5 h after treatment. (h) Fold enrichment of UDP-glucose transferase 1, 2.5 h after treatment. The transcript levels of the indicated genes were measured by RT-qPCR, and the levels of expression are normalized to three reference genes and to the level of Col-0 in mock or HBA treatment.
Fig 4.
(a) Interactions of NPR2 with proteins that interact with NPR1. Yeast transformed with the indicated plasmids and inserts were grown on three different sets of plates, by depositing a 0.5 μL drop of OD600s 1, 0.1, and 0.01 (indicated in the top of the first plate). The first plate contained minimal media supplemented with histidine (+His). The second had the same minimal media with no histidine (-His), 100 μM SA, and 5 mM 3-Amino-1,2,4-triazole (3AT), while the third plate is -His, 100 μM HBA, and 5 mM 3AT. (b) Interactions of NPR2 with β carbonic anhydrases (βCAs), as in (a) but without 3AT.
Fig 5.
NPR1 paralogs in yeast two hybrid.
(a) Interactions of NPR1 with the paralogs. (b) Interactions of NPR2 with the paralogs. (c) Interactions of NPR3 with the paralogs. (d) Interactions of NPR4 with the paralogs. (e) Controls of empty plasmids.
Fig 6.
In planta interactions among NPR1 paralogs.
Bimolecular fluorescence complementation (BiFC) among NPR1 paralogs. The constructs were agroinfiltrated with 1mM SA treatment one day before visualization. The first protein is fused with the N-terminal part of GFP, and the second with the C-terminal. (a) NPR1 and NPR1. (b) NPR2 and NPR1. (c) NPR1 and NPR2. (d) NPR2 and NPR2. (e) NPR1 and NPR3. (f) NPR2 and NPR3. (g) NPR1 and NPR4. (h) NPR2 and NPR4. (i) NPR1 and BOP1. (j) NPR2 and BOP1. (k) NPR1 and BOP2. (l) NPR2 and BOP2. (m) NPR1 and AKIN10, as a negative control[37]. (n) NPR2 and AKIN10, as a negative control. The bars in these pictures represent 20 μm.
Fig 7.
Co-sedimentation among paralogs.
(a) GFP-NPR2 and the MBP fused to some NPR1 paralogs were transiently expressed in N. benthamiana by agroinfiltration and pulled-down with amylose resin. MBP-AKIN10 [37] is an unrelated protein, used as negative control with a similar size to the MBP-NPRs. The panel shows GFP-NPR2 detected by immunoblot before treatment with resin. Ponceau-S staining of the nitrocellulose membrane is shown as a loading control. (b) Expression of MBP-NPRs detected by immunoblot analysis before treatment with resin. (c) The panel shows the eluted fraction from the resin detecting GFP-NPR2 by immunoblot analysis. (d) The same eluted fraction detecting MBP-NPRs.
Fig 8.
Stability of NPR1 and NPR2 in the presence of the NPR1 paralogs.
(a) Stability of GFP-NPR1 when co-expressed with the NPR1 paralogs fused to MBP. The constructs were agroinfiltrated with 1mM SA treatment one day before sampling. (b) Stability of GFP-NPR2 when co-expressed with the NPR1 paralogs, as in (a). Ponceau-S staining is shown below as a loading control.
Fig 9.
SA binding of the NPR1 paralogs.
(a) Purified recombinant proteins were incubated with 4-AzSA, then treated with UV light. 4-AzSA-cross-linked proteins were detected by immunoblot analysis with anti-SA antibody. The first line corresponds to a negative control, where the UV light was omitted. (b) Loading control, the same amount of protein used in A was detected with an anti-MBP. The image was cut to show the MBP empty vector, as indicated by the vertical line.