Genetic Requirements for Signaling from an Autoactive Plant NB-LRR Intracellular Innate Immune Receptor

<div><p>Plants react to pathogen attack via recognition of, and response to, pathogen-specific molecules at the cell surface and inside the cell. Pathogen effectors (virulence factors) are monitored by intracellular nucleotide-binding leucine-rich repeat (NB-LRR) sensor proteins in plants and mammals. Here, we study the genetic requirements for defense responses of an autoactive mutant of ADR1-L2, an Arabidopsis coiled-coil (CC)-NB-LRR protein. ADR1-L2 functions upstream of salicylic acid (SA) accumulation in several defense contexts, and it can act in this context as a “helper” to transduce specific microbial activation signals from “sensor” NB-LRRs. This helper activity does not require an intact P-loop. ADR1-L2 and another of two closely related members of this small NB-LRR family are also required for propagation of unregulated runaway cell death (rcd) in an <i>lsd1</i> mutant. We demonstrate here that, in this particular context, ADR1-L2 function is P-loop dependent. We generated an autoactive missense mutation, ADR1-L2_D484V, in a small homology motif termed MHD. Expression of ADR1-L2_D848V leads to dwarfed plants that exhibit increased disease resistance and constitutively high SA levels. The morphological phenotype also requires an intact P-loop, suggesting that these ADR1-L2_D484V phenotypes reflect canonical activation of this NB-LRR protein. We used ADR1-L2_D484V to define genetic requirements for signaling. Signaling from ADR1-L2_D484V does not require NADPH oxidase and is negatively regulated by <i>EDS1</i> and <i>AtMC1</i>. Transcriptional regulation of <i>ADR1-L2_D484V</i> is correlated with its phenotypic outputs; these outputs are both SA–dependent and –independent. The genetic requirements for ADR1-L2_D484V activity resemble those that regulate an SA–gradient-dependent signal amplification of defense and cell death signaling initially observed in the absence of LSD1. Importantly, <i>ADR1-L2_D484V</i> autoactivation signaling is controlled by both <i>EDS1</i> and SA in separable, but linked pathways. These data allows us to propose a genetic model that provides insight into an SA–dependent feedback regulation loop, which, surprisingly, includes ADR1-L2.</p></div

An intact P-loop catalytic domain is required for the <i>ADR1-L2_D484V</i> morphological phenotype.

<p>(A) Pictures of five-week-old Col-0, <i>ADR1-L2_D484V</i>, and <i>ADR1-L2_{AAA D484V}</i> plants show relative morphology. (B) Western blot of Col-0 and HA-tagged ADR1-L2_D484V and ADR1-L2_{AAA D484V} protein from plants in (A). Relative loading indicated by Ponceau stained blot.</p

<i>eds1 lsd1 ADR1-L2_D484V</i> plants lose ectopic activation phenotypes.

<p>(A) Pictures of five-week-old plants of the indicated genotypes show suppression of the <i>eds1-2 ADR1-L2_D484V</i> phenotype in an <i>lsd1</i>-2 background. (B) Western blot of HA-tagged ADR1-L2_D484V protein from the indicated genotypes. Ponceau stain shows relative loading. (C) Total SA amounts (mean ±2× SE) were measured from plants of the indicated genotypes. Values are average µg of total SA from 4 replicates. Error bar represents ±2× SE. Controls here are from same experiment as data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003465#pgen-1003465-g006" target="_blank">Figure 6D</a>. (D) Quantitative real time PCR for the transcript amounts of <i>ADR1-L2</i> in the indicated genotypes. Error bars represent ±2× SE.</p

ADR1-L2_D484V autoactivity signaling requires both SA and EDS1.

<p>A) Pictures of five-week-old plants representative of the indicated genotypes. <i>SID2 eds1 ADR1-L2_D484V</i> is a segregating F2 derived from the <i>eds1 ADR1-L2_D484V</i>×<i>sid2 ADR1-L2_D484V</i> cross. (B) Western blot of HA-tagged ADR1-L2_D484V protein from plants in (A). Coomassie stain shows relative loading. (C) Quantitative real-time PCR for the transcript amounts of <i>ADR1-L2</i> in the indicated genotypes. Error bar represents ±2×SE. (D) Ten-day-old seedlings were inoculated with 5×10⁴ sporangia/mL <i>Hpa</i> Emco5. At 4 dpi, sporangiophores were counted and classified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003465#pgen-1003465-g004" target="_blank">Figure 4</a>. Means per cotyledon are listed below the graph.</p

<i>ADR1-L2_D484V</i> ectopically activates basal defense.

<p>(A) Schematic representation of ADR1-L2 showing the P-loop and MHD mutations used in this study. (B) Morphology of five-week-old <i>adr1-L2</i>, and <i>adr1-L2</i> complemented with <i>pADR1-L2::ADR1-L2-HA</i> or <i>pADR1-L2::ADR1-L2_D484V-HA</i>, showing relative size. White bar is 2 cm. (C) Western blot of HA-tagged protein from the indicated genotypes before and after BTH application. Protein was extracted from plants, run on a denaturing gel and probed with anti-HA antibody. Ponceau-stained blot shows relative loading. (D) Ten-day-old seedlings were inoculated with 5×10⁴ sporangia/mL <i>Hpa</i> Emco5 via spray inoculation. Sporangiophores per cotyledon were counted 4 dpi, with an average of 80 cotyledons per genotype counted. Sporangiophore counts were classified into: no sporulation (0 sporangiophores/cotyledon), light sporulation (1–5), medium sporulation (6–10), heavy sporulation (11–15), or very heavy sporulation (>15). Means of sporangiophore per cotyledon are listed below the graph. (E) Twenty-day-old seedlings were dip-inoculated with <i>Pto</i> DC3000(EV). Bacterial growth was assayed at 0 and 3 dpi. Values are mean cfu/mg ±2× SE, n = 4. Asterisk indicates significant difference (Post Hoc test, p<0.0001). (F) Trypan blue stained leaves from (D) and magnified sites (20×). Leaves were collected and stained 4 dpi. Red arrows indicate HR sites.</p

<i>lsd1</i> suppressors are regulators of <i>ADR1-L2_D484V</i> autoactivity.

<p>(A) Pictures of five-week-old Col-0, <i>ADR1-L2_D484V</i>, <i>atrbohD ADR1-L2_D484V</i>, <i>sid2-1 ADR1-L2_D484V</i>, <i>eds1-2 ADR1-L2_D484V</i>, or <i>atmc1-1 ADR1-L2_D484V</i> plants, showing morphological differences between the genotypes. White bar is 2 cm. (B) Western blots of HA-tagged ADR1-L2_D484V proteins from plants in (A). Ponceau staining shows relative loading. (C) Ten-day-old seedlings of the indicated genotypes were inoculated with 5×10⁴ sporangia/mL <i>Hpa</i> Emco5. At 4 dpi, sporangiophores were counted and classified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003465#pgen-1003465-g004" target="_blank">Figure 4</a>. Means per cotyledon are listed below the graph. (D) Steady-state total SA levels were measured from leaves of the indicated genotypes. Values are average µg of total SA from 4 replicates, ±2× SE.</p

A family of CC-NB-LRR proteins is required for <i>lsd1</i> runaway cell death.

<p>(A) Four-week-old plants were sprayed with 300 µM BTH or water. Pictures of plants were taken 5 days post-inoculation (dpi). (B) Leaves from plants in (A) were stained with trypan blue to visualize cell death. Leaves on the left are water-treated controls, leaves on the right are sprayed with 300 µM BTH. (C) Ion leakage measurements from (A), 5 days post-BTH treatment. Values are means ±2× SE (n = 5). (D) Ion leakage measurements for NANC. <i>adr1-L1 lsd1-2</i>×<i>lsd1-2</i>, <i>adr1-L2 lsd1-2</i>×<i>lsd1-2</i>, <i>adr1-L1 lsd1-2</i>×<i>adr1-L2 lsd1-2</i> represent F1 plants of the indicated crosses, and are thus <i>lsd1</i> homozygous and heterozygous for the indicated <i>adr</i> mutations. (E) Quantitative real time PCR for the transcript amounts of the three members of the <i>ADR</i> family in wild-type Col-0 plants, normalized to <i>UBQ5</i>.</p

A model for the regulation of ADR1-L2_D484V activity.

<p>ETI activates both an AtRbohD-dependent ROI burst and SID2-dependent SA accumulation via ADR1-L2. Activated ADR1-L2 initiates cell death and disease resistance via SA-dependent and -independent pathways. EDS1 functions downstream of activated ADR1-L2 as a positive regulator of both SA accumulation and of the SA-independent pathway. ADR1-L2 also triggers SA via a pathway that is controlled by LSD1 and antagonized by EDS1. Therefore, the spread of this SA accumulation is spatially down-regulated through a combined action of EDS1 and LSD1. Due to its position in these feedback loops, SA functions both up- and down-stream of ADR1-L2.</p

The requirement for ADR1-L2 in <i>lsd1</i> rcd is P-loop dependent.

<p>(A) Four-week-old plants of the indicated genotypes were sprayed with BTH or water. ADR1-L2 and AAA indicate <i>adr1-L2 lsd1</i> plants expressing C-terminally HA-tagged wild-type <i>ADR1-L2</i> or the mutated P-loop allele <i>ADR1-L2_AAA</i>, respectively. In both transgenics expression is driven by the native <i>ADR1-L2</i> promoter. Pictures of plants were taken 5 dpi. (B) Protein from the indicated genotypes was extracted before or after BTH treatment, run on a denaturing gel, and probed with anti-HA antibody. Ponceau-stained blot shows relative loading.</p

<i>coi1^rsp</i> alleles are insensitive to JA.

<p>(A) Seedlings of the indicated genotypes were grown on MS medium (control) or medium containing 10 or 50 µM MeJA. (B) Inhibition of root elongation by 50 µM MeJA in at least twenty seedlings of indicated genotypes. This assay was performed independently three times with similar results.</p