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. 2011 Oct;23(10):3798-811.
doi: 10.1105/tpc.111.088708. Epub 2011 Oct 7.

Separable fragments and membrane tethering of Arabidopsis RIN4 regulate its suppression of PAMP-triggered immunity

Ahmed J Afzal  1 , Luis da Cunha, David Mackey
Affiliations

Separable fragments and membrane tethering of Arabidopsis RIN4 regulate its suppression of PAMP-triggered immunity

Ahmed J Afzal et al. Plant Cell. 2011 Oct.

Abstract

RPM1-interacting protein 4 (RIN4) is a multifunctional Arabidopsis thaliana protein that regulates plant immune responses to pathogen-associated molecular patterns (PAMPs) and bacterial type III effector proteins (T3Es). RIN4, which is targeted by multiple defense-suppressing T3Es, provides a mechanistic link between PAMP-triggered immunity (PTI) and effector-triggered immunity and effector suppression of plant defense. Here we report on a structure-function analysis of RIN4-mediated suppression of PTI. Separable fragments of RIN4, including those produced when the T3E AvrRpt2 cleaves RIN4 and each containing a plant-specific nitrate-induced (NOI) domain, suppress PTI. The N-terminal and C-terminal NOIs each contribute to PTI suppression and are evolutionarily conserved. Native RIN4 is anchored to the plasma membrane by C-terminal acylation. Nonmembrane-tethered derivatives of RIN4 activate a cell death response in wild-type Arabidopsis and are hyperactive PTI suppressors in a mutant background that lacks the cell death response. Our results indicate that RIN4 is a multifunctional suppressor of PTI and that a virulence function of AvrRpt2 may include cleaving RIN4 into active defense-suppressing fragments.

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Figures

Figure 1.
Figure 1.
RIN4 Contains Separable Domains with Distinct Defense Suppressing Activities. (A) Schematic diagram of the 211 amino acid RIN4 protein and its derivatives. RIN4 contains two conserved NOI domains (black bars), two AvrRpt2 cleavage sites (triangles), and C-terminal cysteines required for acylation and membrane targeting of RIN4 (CCC, amino acids 203, 204, and 205). Full-length RIN4 (RIN4FL) and other derivatives all contain an N-terminal T7-tag (black rectangle). AvrRpt2 cleaves RIN4 between amino acids 10/11 and 152/153 and the derivatives ACP2 and ACP3 (AvrRpt2-cleavage products) consist of RIN4 amino acids 11 to 152 and 153 to 211, respectively. w.t., wild type. (B) and (C) Callose induced by flg22 infiltration (B) and growth of hrcC (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives. Expression was induced with 20 μM dexamethasone 48 h before infiltration with (B) 30 μM flg22 or (C) 1 × 105 CFU/mL of hrcC. Numbers in (B) are average and sd of the number of callose deposits per 1.1 mm2. Bacterial numbers and sd (C) are plotted at the time of infiltration (day 0) in Col-0 and after 4 d. *P = 0.008; **P < 0.0002 [two-tailed Student’s t test for comparison with Col-0 plants]. (D) Immunoblots with anti-T7 antisera to compare levels of RIN4 derivatives and with anti-RIN4 antisera to compare levels of native RIN4 and RIN4FL. Numbers are band intensities with the signal in plants expressing RIN4FL normalized to 1. Panels below are ponceau stains for total protein loading.
Figure 2.
Figure 2.
NOI Domains Are Required for Defense-Suppressing Activity of RIN4. (A) Schematic diagram of RIN4 derivatives as in Figure 1. ΔΔNOI lacks amino acids 1 to 64 and 149 to 176. w.t., wild type. (B) and (C) Callose induced by flg22 infiltration (B) and growth of hrcC (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives as in Figure 1. *P < 0.005; **P < 0.05 [two-tailed Student’s t test for comparison with Col-0 plants]. (D) Immunoblots with anti-T7 antisera to compare levels of RIN4 derivatives as in Figure 1.
Figure 3.
Figure 3.
The N-NOI and C-NOI of RIN4 Are from Evolutionarily Distinct Clades. The NOI domains from RIN4, its closest BLAST homolog from a variety of plants, and other NOI-containing proteins from Arabidopsis (NOI1 to NOI14) were analyzed. The NOI domains were aligned by ClustalW, and their evolutionary relationship was inferred by the Neighbor-Joining method using MEGA4. The confidence probability (×100) that the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates), is shown next to the branches. Branch lengths represent evolutionary distances. Number ranges are the amino acid positions of each NOI within its respective protein. The N-NOIs and C-NOIs from RIN4 homologs from different plant species are indicated by red squares and blue diamonds, respectively. Orange squares and purple diamonds indicate the N-NOIs and C-NOIs from Arabidopsis NOI proteins with a structure similar to RIN4 (NOI10 and NOI11). Black triangles indicate the NOIs from Arabidopsis proteins with only a single NOI domain. All NOIs start with an AvrRpt2 cleavage site, with the exception of the C-NOIs of NOI10 and NOI11 from Arabidopsis, which have a Pro insertion and Trp substitution predicted to disrupt cleavage by AvrRpt2. NOI8 (At5G18310) is excluded from the analysis, because ambiguous gene models exist in TAIR9. RIN4 is At3G25070. For other Arabidopsis proteins containing NOIs, the AGI numbers are shown. RIN4 homologs are from the following plant species: A. lyrata, B. juncea, M. truncatula, L. saligna, S. lycopersicum, P. trichocarpa, V. vinifera, O. sativa, Z. mays, B. distachyon, and P. patens.
Figure 4.
Figure 4.
Nonmembrane-Tethered Derivatives of RIN4 Cause a Cell Death Response in Col-0. (A) Schematic representation of RIN4FL and derivatives as in Figure 1. CCC>AAA has RIN4 Cys203/204/205 substituted by Ala203/204/205. w.t., wild type. (B) Immunoblot showing PR-1 expression in Col-0 and transgenic plants expressing RIN4FL, CCC>AAA, and 177Δ211 at 48 h after Dex treatment. (C) CCC>AAA and 177Δ211 cause a cell death–like response in Col-0 but not in rpm1 rps2 rin4. Pictures show plant phenotypes 72 h after RIN4FL or the indicated derivatives were induced by Dex in either Col-0 or rpm1 rps2 rin4. Macroscopic cell death was evident in plants expressing either CCC>AAA or 177Δ211 in Col-0, whereas mild symptoms were observed in Col-0 plants expressing RIN4FL. Cell death symptoms were not observed for any of the RIN4 derivatives in rpm1 rps2 rin4 plants. (D) Trypan blue staining of leaves from plants shown in (C). Leaves of plants were collected 72 h after Dex spray and were stained with trypan blue. Consistent with results obtained in (C), CCC>AAA and 177Δ211 show strong cell death in Col-0 but not in rpm1 rps2 rin4. (E) Anti-T7 immunoblot showing that CCC>AAA and 177Δ211 accumulate to similar levels in Col-0 and rpm1 rps2 rin4 plants 48 h after mock (−) or Dex (+) spray. Col-0 plants expressing the CCC>AAA derivative are not included, because the tissue is completely collapsed by 48 h. (F) Total protein extracts (T) of Col-0 leaves expressing indicated derivatives of RIN4 were separated into soluble (S) and microsomal (M) fractions and were subjected to anti-T7 immunoblotting. The microsomal fractions are overloaded by approximately fivefold relative to the total and soluble fractions. Ponceau red staining (shown below T7 blots) demonstrates that RuBisCo partitions into the soluble fraction.
Figure 5.
Figure 5.
Derivatives of RIN4 Suppress PTI More Potently When Not Membrane-Tethered. (A) Expression of RIN4 derivatives in rpm1 rps2 rin4 plants was induced by spraying with Dex 48 h before infiltration with 30 μM flg22. Callose numbers were counted from plants expressing comparable levels of RIN4FL, CCC>AAA, and 177Δ211 proteins (see Supplemental Figure 8 online). The results shown are averaged from three independent biological replicates, with the average number of callose deposits induced in plants expressing RIN4FL in each experiment normalized to 1. Error bars represent sd. *, P = 0.001; **P < 0.0001 [two-tailed Student’s t test for comparison with rpm1 rps2 rin4 plants expressing RIN4FL]. (B) Expression of RIN4 derivatives in Col-0 or rpm1 rps2 rin4 plants was induced by spraying with Dex 48 h before infiltration with 105 CFU/mL of hrcC. Growth data in rpm1 rps2 rin4 was obtained from plants expressing similar, low levels of RIN4FL and derivatives (see Supplemental Figure 9 online). Numbers of hrcC were determined 0 (gray bars), 2 (white bars), and 4 (black bars) d after infiltration. Error bars represent sd. *P < 0.05 [two-tailed t test for comparison with rpm1 rps2 rin4 plants]; **P < 0.005 [two-tailed Student’s t test for pairwise comparison with Col-0 plants].
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