Arabidopsis MORC1 and MED9 Interact to Regulate Defense Gene Expression and Plant Fitness

Abstract

Arabidopsis MORC1 (Microrchidia) is required for multiple levels of immunity. We identified 14 MORC1-interacting proteins (MIPs) via yeast two-hybrid screening, eight of which have confirmed or putative nuclear-associated functions. While a few MIP mutants displayed altered bacterial resistance, MIP13 was unusual. The MIP13 mutant was susceptible to Pseudomonas syringae, but when combined with morc1/2, it regained wild-type resistance; notably, morc1/2 is susceptible to the same pathogen. MIP13 encodes MED9, a mediator complex component that interfaces with RNA polymerase II and transcription factors. Expression analysis of defense genes PR1, PR2, and PR5 in response to avirulent P. syringae revealed that morc1/2 med9 expressed these genes in a slow but sustained manner, unlike its lower-order mutants. This expression pattern may explain the restored resistance and suggests that the interplay of MORC1/2 and MED9 might be important in curbing defense responses to maintain fitness. Indeed, repeated challenges with avirulent P. syringae triggered significant growth inhibition in morc1/2 med9, indicating that MED9 and MORC1 may play an important role in balancing defense and growth. Furthermore, the in planta interaction of MED9 and MORC1 occurred 24 h, not 6 h, postinfection, suggesting that the interaction functions late in the defense signaling. Our study reveals a complex interplay between MORC1 and MED9 in maintaining an optimal balance between defense and growth in Arabidopsis.

Keywords: Arabidopsis; MORC1; defense; growth; mediator.

Fig. 1

Verification of the interaction between MORC1 and its interacting proteins in yeast two-hybrid assay. MORC1 and 14 MORC1-interacting protein (MIP) clones identified from the yeast two-hybrid screening were reconfirmed by a targeted yeast two-hybrid assay. pB27 plasmid with a LexA DNA binding domain (Y187) and pP6 plasmid with a GAL4 activating domain (L40) were used as a bait and a prey vector, respectively. The plasmids were transformed into S. cerevisiae carrying the HIS3 reporter gene under the control of the LexA DNA binding sites. Transformants were plated onto minimal media lacking uracil, methionine, tryptophan, and leucine (-UMTL) or lacking uracil, methionine, tryptophan, leucine, and histidine (-UMTLH), with or without 0.1 mM 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of the HIS3 gene product.

Fig. 2

Interaction of MORC1 with MORC1-interacting proteins (MIPs) in planta. Physical interaction of MORC1 and MIPs were tested in Nicotiana benthamiana plants transiently expressing 35S-Myc-MORC1 and 35S-HA-MIPs. Proteins extracted were immunoprecipitated with anti-HA antibody. The asterisks indicate the expected sizes of MIP and MORC1 proteins. Expression of Myc-MORC1 and HA-MIPs were examined by western analysis with anti-Myc and anti-HA antibodies, respectively. Co-immunoprecipitated proteins were detected by western analysis with anti-Myc antibody, indicating that MORC1 physically interacts with the MIPs in plant cells. IB, immunoblot; IP, immunoprecipitation.

Fig. 3

Bacterial resistance of mips and morc1/2 mutants against Pseudomonas syringae pv. tomato (Pst). Bacterial growth of Pst carrying a luminescent luxCDABE reporter gene was measured at 2 days post-inoculation (dpi) with the indicated genetic backgrounds; initial inoculum was at 1 × 10⁵ cfu/ml. The luminescence was monitored by an electron-multiplying charge-coupled device (EMCCD) camera to indirectly measure the bacterial concentration. The mean ± standard error is presented (n = 10). Statistical difference from wild-type (WT) is indicated: *P < 0.05, **P < 0.01 (t-test).

Fig. 4

Resistance phenotypes of med9, morc1/2, and morc1/2 med9 mutants. Bacterial growth in the indicated plants was measured at 0 and 3 days post-inoculation (dpi) with virulent Pseudomonas syringae pv. tomato (VirPst) and Pseudomonas syringae pv. tomato carrying AvrRpt2 (AvrPst). Initial inoculum of VirPst and AvrPst was at 1 × 10⁵ and 5 × 10⁵ cfu/ml, respectively; the mean ± standard deviation (n = 3) is presented. Statistical difference from wild-type (WT) is indicated: *P < 0.05, **P < 0.01 (t-test).

Fig. 5

MED9 regulates the expression of select defense genes in response to virulent Pseudomonas syringae pv. tomato (VirPst) and Pseudomonas syringae pv. tomato carrying AvrRpt2 (AvrPst). Total RNA was isolated from 3.5-week-old plants of the indicated Arabidopsis lines that were infected with VirPst, AvrPst, or mock treatment at 1 × 10⁶ cfu/ml for the indicated time points. Transcript levels were examined using real-time quantitative reverse transcription PCR with primers specific for PR1 (A), PR2 (B), and PR5 (C). The Tip41-like gene was used as a reference gene for normalization. The mean ± standard error (n = 6) of two biological replicates is shown. Each biological replicate includes three technical replicates, with each dot representing one of these six technical replicates. 10 mM of MgCl₂ was used as mock treatment.

Fig. 6

Late interaction of MED9 with MORC1 during effector-triggered immunity. Three point five-week-old transgenic Arabidopsis line (first four lanes) carrying Myc-MORC1 and MED9-Flag were used; both transgenes were under their native promoters. Plants were infected with Pseudomonas syringae pv. tomato carrying AvrRpt2 (AvrPst) at 10⁶ cfu/ml for the indicated times; a MED9-Flag transgeneic line was used as a negative control (the last two lanes). Proteins extracted were immunoprecipitated with anti-Myc antibody. Expression of Myc-MORC1 and MED9-Flag was examined by immunoblot (IB) analysis with anti-Myc and anti-Flag antibodies, respectively. Proteins co-immunoprecipitated by anti-Myc antibody were detected by IB with anti-Myc and anti-Flag antibodies. IP, immunoprecipitation.

Fig. 7

Fitness cost assessment by repeated pathogen challenges. Three point five-week-old WT, morc1/2, med9, and morc1/2 med9 were infected with Pseudomonas syringae pv. tomato carrying AvrRpt2 (AvrPst) at 10⁵ cfu/ml every 2 days for 3 weeks. (A) Representative plants before (upper) and after (lower) the repeated infection. (B) The weight of 3.5 weeks-old plants was measured. (C) Several growth and development characteristics were measured after the serial infection. To this end, the number of inflorescences, leaves, and siliques were counted, while the weight of inflorescences and leaves were measured. A minimum of ten plants in each line were examined. Statistical difference between mock and AvrPst treatment is indicated: *P < 0.05 (t-test).