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. 2012 Oct;24(10):4220-35.
doi: 10.1105/tpc.112.103028. Epub 2012 Oct 12.

Non-recognition-of-BTH4, an Arabidopsis mediator subunit homolog, is necessary for development and response to salicylic acid

Juan Vicente Canet  1 , Albor Dobón, Pablo Tornero
Affiliations

Non-recognition-of-BTH4, an Arabidopsis mediator subunit homolog, is necessary for development and response to salicylic acid

Juan Vicente Canet et al. Plant Cell. 2012 Oct.

Abstract

Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana non-expresser of pathogenesis-related gene 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.

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Figures

Figure 1.
Figure 1.
SA-Related Phenotypes of nrb4 Plants. (A) Plants were treated with either mock or 350 µM BTH four times over 3 weeks, their weights recorded, and the ratio between the BTH and mock-treated plants calculated (15 plants in three groups of five). The ratio is expressed as percentage of fresh weight (%FW). (B) Plants were grown on Murashige and Skoog plates supplied with 0, 400, and 500 µM SA; the picture shows the 500 µM SA plate at day 14. (C) The chlorophyll content of plants growing in the plates described in (B) was measured as an indication of the response to SA (30 plants in three groups of 10). The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05). In (C), the differences were evaluated between genotypes grown at either 400 µM SA or 500 µM SA (marked with a prime symbol). [See online article for color version of this figure.]
Figure 2.
Figure 2.
Pathogenic Phenotypes of nrb4 Plants. (A) Seventeen-day-old plants were treated with either 500 µM SA, 350 µM BTH, or a mock solution. One day later, the plants were inoculated with Pto at an OD600 of 0.1. Three days after inoculation, the growth of Pto was evaluated as logarithm of colony-forming units per plant. (B) PR1 immunoblot of the indicated genotypes 3 d after mock or a Pto inoculation (top) and 1 d after mock or 350 µM BTH treatment (bottom). The genotypes are abbreviated as in (A). The arrow indicates the position of PR1 (14 kD). (C) Plants (32 days old) were treated with Pto as in (A). In these plants, only a sample of the surface area was measured, so the units are log(cfu/cm2). (D) Three leaves of 30-d-old plants were hand-infiltrated with either Pto(avrRpm1) or a mock solution. Two days later, Pto was inoculated and its growth in systemic leaves measured as in (C). (E) Pto(avrRpm1) was inoculated in nrb4 as in (A). Resistance to Pseudomonas maculicola 1 (rpm1) is included as a control. (F) Inoculations with Pto(avrRpt2), with rps2 added as a control. (G) P. syringae pv phaseolicola isolate NPS3121 was inoculated as in (A), with nho1 used as a control. (H) Inoculations with P. syringae pv tabaci was inoculated as in (A), with nho1 used as a control. The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd of 15 plants in three groups of five in (A) and (E) to (H). In (C) and (D), 12 samples of known size from three plants were taken in three groups of four. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05). Asterisks indicate statistically significant differences from the mock treatment (*P < 0.05; **P < 0.01) using the Student’s t test (one tail).
Figure 3.
Figure 3.
Epistasis of NRB4 with NPR1. (A) Three double mutants nrb4-1 npr1-70 were tested as in Figure 1A. FW, fresh weight; Laer-0, Landsberg erecta-0. (B) 35S:NPR1-HBD (NPR1HBD) in an nrb4-2 background was tested with and without dexamethasone (DEX) for its response to BTH. The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd of 15 plants in three groups of five. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05).
Figure 4.
Figure 4.
Predicted Structure of NRB4. (A) Drawing of the predicted NRB4 protein, showing the conserved KIX domain and the region rich in Gln. AA, amino acids. (B) Magnification of the KIX domain, showing the introns (horizontal lines), the point mutations (arrows), and the T-DNA insertions (triangles) found in NRB4 (At1g15780). The number above the mutation indicates the number of alleles. Only a section of the NRB4 gene is shown; the region shown corresponds to the gray rectangle in (A). (C) Sequence of the first 100 amino acids of NRB4, indicating the point mutations.
Figure 5.
Figure 5.
nrb4-4 Is a Null Allele. (A) From left to right, wild-type plant, NRB4/nrb4-4 plants, and nrb4-4 homozygous plants. Picture was taken after 6 weeks in short-day conditions. (B) Cryo-scanning electron microscopy pictures of wild-type trichomes. The leaves sampled were ∼7 mm, and plants were 5 weeks old. (C) Cryo-scanning electron microscopy of nrb4-4 trichomes, although plants were 7 weeks old to sample leaves of roughly the same size. Bar = 100 µm. (D) Picture of nrb4-4 plants taken after 18 weeks (seven in short day and 11 in long day). (E) Plants were tested as in Figure 1A, but with one more week of growth and two more treatments. FW, fresh weight. (F) Response of nrb4-4 plants to SA and BTH in growth curves. The inoculations were done as in Figure 2C, except that the nrb4-4 plants were 7 weeks old. (G) P. syringae pv maculicola CR299 was inoculated and its growth measured as in (F). (H) The amount of SA (both free and total) was measured 3 days after a mock or a Pto inoculation. The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd of 15 plants in three groups of five, except in the case of (H), where three samples of 100 mg were taken. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05). The differences in free SA and total SA (marked with the prime symbol) were evaluated between genotypes. Asterisks indicate statistically significant differences from the mock treatment (*P < 0.05; **P < 0.01) using the Student’s t test (one tail). [See online article for color version of this figure.]
Figure 6.
Figure 6.
Transcriptome Analysis of nrb4 Mutants. (A) The transcriptomes of nrb4-2, nrb4-4, and Col-0 plants were determined and then compared with different transcriptomic experiments by means of hierarchical clustering with Cluster 3.0 (de Hoon et al., 2004) and visualized as a dendogram with JavaTreeView (Saldanha, 2004). The references of the experiments used are specified in Methods, and the parameters used were the default settings. (B) Growth response to cytokinins. Seeds of the indicated genotypes were grown in rock wool imbibed with 5 µM trans-zeatin. This picture was taken after 21 d of growth. arr1,10,12 stands for the triple mutant arr1 arr10 arr12, used as a control for lack of response to cytokinins. [See online article for color version of this figure.]
Figure 7.
Figure 7.
Expression of NRB4 and Subcellular Localization. (A) NRB4 expression was measured 1 d after treatment with mock, 350 µM BTH, 500 µM SA, and 100 µM MeJA or 3 d after a Pto inoculation (RNA extracted and qRT-PCR from three independent samples of 100 mg each). The levels of expression are normalized to three reference genes and to the level of Col-0. (B) RNA was extracted from 3-week-old plants (five weeks for nrb4-4), and transcript levels for NRB4 were measured by RT-qPCR as in (A). (C) Agrobacterium tumefaciens with the construct 35S:NRB4-GFP was infiltrated into leaves of N. benthamiana, and the expression was detected by confocal microscopy 4 d later. (D) Similar to (C) with the construct 35S:GFP-NRB4. The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd of three samples. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05).
Figure 8.
Figure 8.
Phenotypes of Transgenic Lines. (A) Transgenic plants homozygous for the construct 35S:NRB4-GFP (NRB4F) or for the equivalent construct with only the first 670 amino acids (NRB4M) were obtained in the mutant alleles. The panel shows the response to BTH in fresh weight (FW) of the mentioned lines tested as in Figure 1A. (B) The constructs described in (A) were transformed into Col-0, and their response to BTH in fresh weight recorded. The number indicates an independent line. (C) Response of the transgenic lines described in (A) to SA and BTH in growth curves, as described in Figure 2A. (D) Response of the transgenic lines described in (B) to SA and BTH in growth curves, as described in Figure 2A. The experiments were repeated three times with similar results, and the data represent the average, with the error bars plotting the sd of 15 plants in three groups of five. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD test, P < 0.05). Asterisks indicate statistically significant differences from the mock treatment (*P < 0.05; **P < 0.01) using the Student’s t test (one tail).
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References

    1. Argueso C.T., Ferreira F.J., Epple P., To J.P., Hutchison C.E., Schaller G.E., Dangl J.L., Kieber J.J. (2012). Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity. PLoS Genet. 8: e1002448. - PMC - PubMed
    1. Argyros R.D., Mathews D.E., Chiang Y.H., Palmer C.M., Thibault D.M., Etheridge N., Argyros D.A., Mason M.G., Kieber J.J., Schaller G.E. (2008). Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development. Plant Cell 20: 2102–2116 - PMC - PubMed
    1. Autran D., Jonak C., Belcram K., Beemster G.T., Kronenberger J., Grandjean O., Inzé D., Traas J. (2002). Cell numbers and leaf development in Arabidopsis: A functional analysis of the STRUWWELPETER gene. EMBO J. 21: 6036–6049 - PMC - PubMed
    1. Bäckström S., Elfving N., Nilsson R., Wingsle G., Björklund S. (2007). Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit. Mol. Cell 26: 717–729 - PubMed
    1. Benjamini Y., Hochberg B. (1995). Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B 57: 289–300

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