Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7

Abstract

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants.

Figure 1.

RT-PCR Analysis for Group I Genes and NUDT7 in Wild-Type and Mutant Plants. (A) Relative transcript levels were determined by quantitative real-time PCR as described in Methods. Expression levels were normalized with respect to the internal control ACTIN and displayed relative to the expression in mock-treated wild-type samples (WT MgCl₂, with a relative expression value set at 1). Data bars represent the mean levels of transcripts (±sd, n = 3). (B) Semiquantitative RT-PCR analysis for At5g55450. UBIQUITIN expression was used to standardize transcript levels in each sample. A second RT-PCR experiment with independent RNA samples gave similar results.

Figure 2.

Defects in FMO1 Compromise TIR-NB-LRR–Triggered Resistance. (A) Schematic representation of FMO1 and NUDT7 genomic structures with exons represented as black boxes. The positions of T-DNA and Ds insertions (fmo1-2) are indicated. (B) RT-PCR of RNA isolated from leaves of the indicate plant genotypes. ACTIN mRNA was used to normalize transcript levels in each sample (see Methods). (C) Resistance to H. parasitica isolate Cala2 conferred by the TIR-NB-LRR–type R gene RPP2 in Col-0 wild-type and defense mutant lines. Lactophenol trypan blue–stained leaves were viewed under a light microscope for pathogen structures and plant cell death 6 d after inoculation. HR, tightly delimited plant cell death; eHR, extended HR; TN, trailing necrosis; fH, free pathogen hyphae; O, oospores. Images are representative of at least 18 plants per genotype. Similar results were obtained in two independent experiments. (D) Response phenotypes of the wild type (Ler-0), pad4-2, and fmo1-2 to avirulent H. parasitica isolate Noco2 (recognized by RPP5). Inoculation and staining procedures were as described in (C).

Figure 3.

Pathogen Growth in Arabidopsis fmo1 Mutants. (A) RPM1 (CC-NB-LRR) resistance to Pst avrRpm1 is not compromised in fmo1-1, whereas RPS4 (TIR-NB-LRR) resistance to Pst avrRps4 is reduced. Leaves of 5-week-old plants were vacuum infiltrated with bacterial suspensions at 5 × 10⁵ cfu/mL and bacterial titers determined in triplicate at 0 and 3 d after inoculation (dpi). Data points are the average of three replicate samples (±sd). (B) Basal resistance of Ler-0 leaves to virulent H. parasitica isolate Cala2 is reduced in fmo1-2. Numbers of pathogen conidiospores were measured on leaves 6 d after inoculation. Values are the average of four replicate samples (±sd).

Figure 4.

Attenuated Resistance in Arabidopsis fmo1 Plants Is Uncoupled from Impaired SA Accumulation. (A) Total SA levels were measured in leaves of 4-week-old plants that were either untreated (NT) or 24 h after vacuum infiltration with 10 mM MgCl₂, 5 × 10⁶ cfu/mL Pst avrRpm1, or Pst avrRps4 in 10 mM MgCl₂. Data represent the average of three replicate samples (±sd). (B) Response phenotypes of wild-type and mutant plants 5 d after inoculation of leaves with avirulent H. parasitica Cala2 (4 × 10⁵ spores/mL; recognized by RPP2 in Col-0). Leaves were viewed on a binocular microscope under UV light to visualize cell death–associated fluorescence indicative of plant trailing necrosis. (C) Spore production on leaves of single and double mutant lines 6 d after inoculation with avirulent H. parasitica isolate Cala2 (recognized by RPP2 in Col-0 and virulent on Ler-0). Data points are the average of four replicate samples (±sd).

Figure 5.

FMO1 Catalytic Domains Are Required for Defense Signaling. (A) Alignment of amino acid sequences from Arabidopsis (At FMO1 and YUCCA; Zhao et al., 2001), rice (Os FMO), Methylophaga sp Strain SK1 (bFMO; Choi et al., 2003), yeast (yFMO; Zhang and Robertus, 2002), and human (hFMO1; Lawton et al., 1994) was performed, and the N-terminal sequences are shown here. FMO-defining motifs and the conserved Gly residues exchanged by site-directed mutagenesis are indicated above the top line: I, FAD binding motif GXGXXG; II, FMO identifying sequence motif FXGXXXHXXX(Y/F); and III, NADPH binding domain GXGXX(G/A). Multiple alignments were visualized using GeneDoc (Nicholas et al., 1997) with conserved residue shading mode set to level 4 using default settings and enabled similarity groups function. Amino acids with 100, 80, and 60% conservation are presented as white letters on black background, white letters on dark-gray background, and black letters on light-gray background, respectively. (B) Expression of wild-type and mutant forms of FMO1-StrepII in independent fmo1-1 transformants. Protein gel blot analysis was performed after StrepII affinity purification. Equal amounts of the input fraction are shown by Coomassie blue staining. (C) Intact FAD and NADPH binding sites in FMO1 are required for basal resistance to virulent strain H. parasitica. The plant lines correspond to those tested in (B). Numbers of pathogen conidiospores were measured on leaves 6 d after inoculation. Values are the average of four replicate samples (±sd).

Figure 6.

Phylogeny of Arabidopsis Nudix Hydrolase Family Members. A phylogenetic tree drawn from neighbor-joining analysis using Mega 3.0 software (Kumar et al., 2004). Bootstrap values (1000 replicates) are shown. Annotations of Nudix hydrolase–like proteins were taken from Ogawa et al. (2005).

Figure 7.

Developmental and Basal Resistance Phenotypes of nudt7 Single and Double Mutants. (A) Attenuated growth of nudt7-1 is suppressed by eds1-2 but exacerbated by sid2-1. Four-week-old soil-grown plants representative of single or double mutants are shown. Bar = 1 cm. (B) Average fresh weight (FW) of 4-week-old plants (±sd) calculated from the aerial tissue weight of six plants per genotype. (C) Enhanced basal resistance to H. parasitica isolate Noco2 in nudt7 mutants is dependent on EDS1 and partially requires SID2. Pathogen spores were counted as in Figure 3B. (D) Levels of total SA in leaves of healthy 4-week-old plants. Data points are the average of three replicate samples (±sd). (E) Visualization of dead cells in leaves of 4-week-old nudt7 single and double mutants after staining with lactophenol trypan blue. The scale bar unit is in micrometers. (F) Quantification of leaf cell death. Numbers of dead cells were determined in leaves of 3-week-old plants after staining with lactophenol trypan blue. Data represent samplings from 10 leaves from at least five plants per genotype (±sd). Single cell death did not occur in Col eds1-2 or sid2-1 (data not shown).