Initiation of RPS2-Specified Disease Resistance in Arabidopsis Is Coupled to the AvrRpt2-Directed Elimination of RIN4  Michael J. Axtell, Brian J. Staskawicz  Cell  Volume 112, Issue 3, Pages 369-377 (February 2003) DOI: 10.1016/S0092-8674(03)00036-9

Figure 1 RPS2-HA and AvrRpt2-HA Transgenic Plants Express Functional, Detectable Proteins (A) Schematic diagrams of transgene constructs. P(RPS2): RPS2 native 5′ untranslated region. T(RPS2): RPS2 native 3′ untranslated region. T(OCS): Octopine synthase terminator. (B) RPS2-HA complements the rps2-101C null mutant. Quantification of virulent Pst (left image) and Pst-avrRpt2 (right image) growth in Arabidopsis seedlings after vacuum infiltration. Two independent RPS2-HA transgenic lines complement the rps2-101C mutation by restriction of Pst-avrRpt2 growth. (C) AvrRpt2-HA is able to compromise the function of the RPM1 resistance gene. Representative leaf symptoms 5 days after low-density pathogen inoculation with Pst-avrRpm1. AvrRpt2-HA expressing transgenic plants show a defect in RPM1 function. (D) RPS2-HA and AvrRpt2-HA are detectable from transgenic plants. HA immunoblot analysis against total crude lysates (20 μg protein/lane) derived from two RPS2-HA and one AvrRpt2-HA transgenic plant line. Numbers on left and marks on blot indicate size standards in kDa. Cell 2003 112, 369-377DOI: (10.1016/S0092-8674(03)00036-9)

Figure 2 RPS2-HA and AvrRpt2-HA Are Membrane-Associated Proteins (A) RPS2-HA is a membrane-associated protein. Immunoblots of total protein (T) from RPS2-HA plants fractionated into soluble (S) and membrane (M) samples by ultracentrifugation at 150,000 × gmax. SEC12: control integral membrane protein; NptII: control soluble protein. The membrane fraction is 5× concentrated relative to soluble and total. (B) RPS2-HA is a plasma membrane protein. Immunoblots of fractions derived from aqueous two-phase partitioning of total membranes (Input) derived from RPS2-HA plants. Endo: plasma membrane depleted fraction. PM: Plasma membrane enriched fraction; SKU5: Plasma membrane control protein; SEC12: Endoplasmic reticulum control protein; VPPase: Tonoplast control protein; Mannosidase: Golgi control protein. 5 μg total protein loaded per fraction. (C) RPS2-HA remains membrane bound during activation and pathogenesis. RPS2-HA transgenic plants were vacuum infiltrated with a high-density of the indicated pathogen treatment and sampled at 0 and 24 hr post-infiltration. Total protein extracts from each sample were fractionated into soluble (S) and total membrane (M) fractions by ultracentrifugation at 150,000 × gmax. Anti-HA immunoblots were then performed against each sample. Each sample was prepared from an aggregate of 15–20 identically treated leaves. Membrane samples are 5× concentrated relative to the soluble. Note that RPS2-HA functions only in the Pst-avrRpt2 treatment, while the NB-LRR proteins RPM1 and RPS5 function in the Pst-avrRpm1 and Pst-avrPphB treatments, respectively. (D) RPS2-HA behaves as an integral membrane protein. Immunoblots of soluble (S) and membrane (M) fractions derived from RPS2-HA total membranes after treatment with the indicated buffers. SEC12: control integral membrane protein; ATPase F1b: control peripheral membrane protein. Soluble and membrane fractions are loaded at an equal ratio. (E) AvrRpt2 is a membrane-associated protein. HA Immunoblots of total protein (T) from AvrRpt2-HA plants fractionated into soluble (S) and membrane (M) samples by ultracentrifugation at 150,000 × gmax. SEC12: control integral membrane protein; NptII: control soluble protein. The membrane fraction is 5× concentrated relative to soluble and total. Cell 2003 112, 369-377DOI: (10.1016/S0092-8674(03)00036-9)

Figure 3 AvrRpt2 Eliminates RIN4 Independent of the RPS2 Pathway (A) RIN4 is absent in AvrRpt2-expressing transgenic plants. RIN4 immunoblot of crude extracts from AvrRpt2 and AvrRpt2-HA transgenic plants and controls. Sixty μg total protein was loaded per lane. (B) RIN4 mRNA levels are similar to wild-type in AvrRpt2-expressing transgenic plants. RNA blot probed with a radiolabeled RIN4 cDNA probe upon 12 μg total RNA from each genotype using standard procedures (Ausubel et al., 1994). rRNA: ethidium bromide staining of total RNA samples as loading control. (C) RIN4 is eliminated by AvrRpt2 during pathogen infection. RIN4 immunoblots of total protein samples derived from leaves of the indicated plant genotypes 0 and 24 hr after high-density syringe inoculation of either Pst or Pst-avrRpt2. >: RIN4; *: induced anti-RIN4 cross-reactive band. Numbers on the right indicate size standards in kDa. Each sample combines at least 4 identically treated leaves and contains 30 μg total protein. (D) RIN4 is not eliminated by alleles of avrRpt2 that are not recognized by RPS2. RIN4 immunoblots of total protein samples derived from wild-type Col-0 leaves 0, 12, and 24 hr after high-density syringe inoculation with the indicated pathogen strain. Each sample combines at least 9 identically treated leaves and contains 30 μg total protein. Cell 2003 112, 369-377DOI: (10.1016/S0092-8674(03)00036-9)

Figure 4 RPS2-HA and T7-RIN4 Interact In Vivo Immunoblots of immunoprecipitated proteins from the indicated tissue sources. IP indicates antibody used for immunoprecipitation. Top image: HA-HRP immunoblot; Bottom image: T7-HRP immunoblot. Note that anti-T7 precipitates RPS2-HA and anti-HA precipitates T7-RIN4 only in RPS2-HA/T7-RIN4 hybrid tissue. Cell 2003 112, 369-377DOI: (10.1016/S0092-8674(03)00036-9)

Figure 5 Model for Indirect Recognition of Pathogens by RPS2 and RPM1 (A) TTSS delivery of AvrRpt2 by Pst results in the elimination of RIN4 at the plasma membrane. This in turn is recognized by the previously RIN4-bound RPS2 causing the activation of RPS2-dependent disease resistance responses. A second consequence of RIN4 elimination is elimination of RPM1, accounting for the AvrRpt2-mediated destruction of RPM1 function. (B) Delivery of either AvrRpm1 or AvrB by Pst results in the phosphorylation of RIN4, which is in turn recognized by the RIN4-bound RPM1 resistance protein, resulting in the activation of RPM1-dependent disease resistance responses. Cell 2003 112, 369-377DOI: (10.1016/S0092-8674(03)00036-9)