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. 2009 Aug;21(8):2458-72.
doi: 10.1105/tpc.107.056044. Epub 2009 Aug 11.

Proteolysis of a negative regulator of innate immunity is dependent on resistance genes in tomato and Nicotiana benthamiana and induced by multiple bacterial effectors

Yao Luo  1 , Katherine S Caldwell, Tadeusz Wroblewski, Michael E Wright, Richard W Michelmore
Affiliations

Proteolysis of a negative regulator of innate immunity is dependent on resistance genes in tomato and Nicotiana benthamiana and induced by multiple bacterial effectors

Yao Luo et al. Plant Cell. 2009 Aug.

Abstract

RPM1-interacting protein 4 (RIN4), a negative regulator of the basal defense response in plants, is targeted by multiple bacterial virulence effectors. We show that RIN4 degradation is induced by the effector AvrPto from Pseudomonas syringae and that this degradation in Solanaceous plants is dependent on the resistance protein, Pto, a protein kinase, and Prf, a nucleotide binding site-leucine-rich repeat protein. Our data demonstrate overlap between two of the best-characterized pathways for recognition of pathogen virulence effectors in plants. RIN4 interacts with multiple plant signaling components and bacterial effectors in yeast and in planta. AvrPto induces an endogenous proteolytic activity in both tomato (Solanum lycopersicum) and Nicotiana benthamiana that degrades RIN4 and requires the consensus site cleaved by the protease effector AvrRpt2. The interaction between AvrPto and Pto, but not the kinase activity of Pto, is required for proteolysis of RIN4. Analysis of many of the effectors comprising the secretome of P. syringae pv tomato DC3000 led to the identification of two additional sequence-unrelated effectors that can also induce degradation of RIN4. Therefore, multiple bacterial effectors besides AvrRpt2 elicit proteolysis of RIN4 in planta.

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Figures

Figure 1.
Figure 1.
AvrPto Caused the Proteolysis of Sl RIN4 in Tomato. (A) AvrPto- and AvrPtoB-triggered degradation of RIN4 was dependent on Pto and Prf in tomato. 3HA:SlRIN4 and AvrPto, AvrPtoB, or empty vector (EV) were transiently expressed using A. tumefaciens in RG-76R tomato plants and RG-76R plants with mutations in Pto (RG-pto11) or Prf (RG-prf3). Twenty-four hours after infiltration, samples were collected and immunoblotted using anti-HA (α-HA) antibody to detect the presence of Sl RIN4. LC, silver staining of the membrane to show equal loading. (B) AvrPto caused the proteolysis of Sl RIN4 expressed from its native promoter in tomato. AvrPto (+) or empty vector (−) were transiently expressed by A. tumefaciens in RG-76R tomato plants. Twenty-four hours after infiltration, samples were collected and immunoblotted using anti-At RIN4 (α-RIN4) antibody to detect native Sl RIN4. LC, Ponceau S staining of the membrane to show equal loading.
Figure 2.
Figure 2.
Degradation of RIN4 Elicited by AvrPto Involves the Proteosomal Pathway. In both experiments, 1 d after infiltration, leaves were infused with protease inhibitor (lane 1), MG132 proteosomal inhibitor (lane 2), or DMSO (lanes 3 and 4); DEX was applied to induce expression of avrPto 2 h later. Tissues were harvested 4 h after induction with DEX, and immunoblotting was performed using anti-HA antibody (α-HA) or anti-GFP antibody (α-GFP). LC, Ponceau S staining of the membrane to show equal loading. (A) The proteosomal inhibitor MG132 partially blocked the degradation of RIN4 elicited by AvrPto. A. tumefaciens carrying a gene encoding HA-tagged Sl RIN4 was simultaneously coinfiltrated with either A. tumefaciens carrying HA-tagged avrPto expressed from a DEX-inducible promoter (+) or with A. tumefaciens carrying the empty vector (−) into N. benthamiana expressing Pto. (B) MG132 partially blocked the proteolysis of GFP after its being cleaved at the RCS. A. tumefaciens carrying a gene to express an artificial substrate (see Figure 5A) was simultaneously coinfiltrated either with A. tumefaciens carrying avrPto expressed from a DEX-inducible promoter (+) or with A. tumefaciens carrying the empty vector (−) into N. benthamiana expressing Pto. A weak band of GFP was detected as marked by an asterisk.
Figure 3.
Figure 3.
RIN4 Degradation Is Not Due to General Proteolysis. (A) The proteolysis of Sl RIN4 is not correlated with the HR phenotype. MKK2T215D/S221D, RPP8, HopAM1DC3000, HopQ1-1DC3000, RPS2, AvrB (+) or empty vector (−), and HA-tagged Sl RIN4 were transiently coexpressed in wild-type N. benthamiana. The leaf tissues were collected 22 h after infiltration (hai), and immunoblotting was performed using anti-HA antibody. The macroscopic necrosis (HR) phenotype was photographed 48 h after infiltration. (B) Proteolysis of Sl RIN4 elicited by AvrPto was not the result of general proteolysis due to a HR. HA-tagged Sl RIN4 and GFP were coexpressed with AvrPto under a DEX-inducible promoter (DEX-AvrPto) in transgenic N. benthamiana leaves that overexpressed Pto (N.b.35S:Pto). After 24 h, expression of avrPto was induced by applying DEX to the leaves. Plant proteins were extracted at 0, 3, 6, and 24 h after DEX induction and assayed by immunoblotting with anti-HA (α-HA) or anti-GFP (α-GFP) antibodies. No Sl RIN4 was detected 24 h after the application of DEX, but GFP was readily detected.
Figure 4.
Figure 4.
Pto and AvrPto Interaction but Not Pto Kinase Activity Is Required for RIN4 Proteolysis. (A) AvrPtoI96T cannot trigger HRs in N. benthamiana expressing Pto. AvrPtoI96T has a polar amino acid substitution in the GINP Ω loop. This mutant does not interact with Pto in the yeast two-hybrid assay. A. tumefaciens carrying genes encoding AvrPtoI96T, AvrPto, or empty vector (EV) were infiltrated into the leaves of N. benthamiana expressing Pto. Photographs of the necrosis phenotype were taken 3 d after infiltration. (B) Interaction between AvrPto and Pto was required to trigger proteolysis of RIN4 in N. benthamiana expressing Pto. HA-tagged Sl RIN4 and AvrPtoI96T (+) or empty vector (−) were transiently coexpressed in transgenic N. benthamiana that constitutively overexpressed Pto (N.b.35S:Pto), and immunoblotting was performed using anti-HA antibody (α-HA). AvrPtoI96T was unable to elicit RIN4 degradation. (C) Kinase activity of Pto is not required for the proteolysis of RIN4. The constitutively active but kinase-deficient mutant PtoD164N,L205D and HA-tagged Sl RIN4 were transiently coexpressed in wild-type N. benthamiana, RG-76R tomato plants, or an isogenic mutant RG-prf3. Total protein was extracted 22 h after coinfiltration, and immunoblotting was performed using anti-HA antibody (α-HA). PtoD164N,L205D elicited RIN4 degradation in a Prf-dependent manner.
Figure 5.
Figure 5.
Proteolysis of an Artificial Substrate Induced in N. benthamiana and Tomato. (A) The artificial substrates comprising either the native or mutated RCS between an HA-tag and GFP. Eight amino acids from the first AvrRpt2 cleavage site in RIN4 were placed between an HA-tag and GFP (HA-RCS-GFP). Similarly, a mutated cleavage site with three Ala substitutions at positions known to be important for cleavage by AvrRpt2 was placed between an HA tag and GFP (HA-RCS*-GFP). (B) AvrPto induced proteolysis of artificial substrates in a Pto-dependent manner in N. benthamiana. Panel 1: HA-RCS-GFP was transiently coexpressed with AvrRpt2, AvrPto, or empty vector (EV) in wild-type N. benthamiana. Total proteins were extracted before the HR caused by AvrRpt2 or AvrPto was evident, and immunoblotting was performed using anti-HA (α-HA) or anti-GFP (α -GFP) antibody. Panel 2: HA-RCS-GFP was transiently coexpressed with AvrPto (+) or empty vector (−) in transgenic N. benthamiana expressing Pto. Total proteins were extracted and immunoblotted against anti-HA (α-HA) antibody or anti-GFP (α-GFP) antibody. The lack of a detectable HA tag or GFP indicated that the cleavage site in AS is sufficient for AvrPto-induced proteolysis. Panel 3: Constitutively active PtoL205D induced proteolysis in N. benthamiana. HA-RCS-GFP was coexpressed with PtoL205D (+) or empty vector (−) in wild-type N. benthamiana, and immunoblotting was performed using anti-HA (α-HA) or anti-GFP (α-GFP) antibody. (C) Proteolysis induced by AvrPto or PtoL205D is dependent on Prf in tomato. HA-RCS-GFP was coexpressed in RG-76R tomato plants or an isogenic mutant at the Prf locus (RG-prf3) with AvrPto (+), PtoL205D (+), or empty vector (EV), and immunoblotting was performed using anti-HA (α-HA) antibody. (D) and (E) Amino acid substitutions in the cleavage site prevented cleavage caused by AvrRpt2, AvrPto, or PtoL205D in N. benthamiana. HA-RCS*-GFP was coexpressed with AvrRpt2, AvrPto, PtoL205D, or empty vector (EV) in wild-type or transgenic N. benthamiana expressing Pto, and immunoblotting was performed using anti-HA (α-HA) or anti-GFP (α-GFP) antibody. LC, Ponceau S staining of the membrane to show equal loading.
Figure 6.
Figure 6.
Degradation of RIN4 Is Important for AvrPto-Dependent Inhibition of Bacterial Growth. (A) Growth of P. syringae pv tomato T1 expressing avrPto was reduced in RIN4 RNAi-silenced T2 tomato plants. T2 plants derived from original (T1) transgenic plants 415 (one plant) and 412 (three plants) were inoculated by infiltrating the pathogen at 103 cfu/mL and assayed after 0 (white), 2 (gray), and 4 (black) d; error bars indicate the sd from three biological replications. (B) Wild-type P. syringae pv tomato T1 exhibited normal levels of growth in RIN4 RNAi-silenced T2 plants. Three transgenic RNAi T2 plants derived from one RNAi-silenced T1 line (412) with reduced RIN4 expression relative to their isogenic wild-type tomato RG-76R were inoculated by infiltrating with pathogen at 103 cfu/mL and assayed 0, 2, and 4 d after infiltration. Error bars indicate the sd from three measurements.
Figure 7.
Figure 7.
Degradation of RIN4 by Pseudomonas spp. (A) P. syringae pv tomato T1 with or without AvrPto caused proteolysis of Sl RIN4 in tomato in a Pto- and Prf-independent manner. P. syringae pv tomato strain T1 (PtoT1) with and without avrPto (avrPto and EV, respectively) were coinoculated with A. tumefaciens strain C58C1 with a binary vector that provided expression of HA-tagged Sl RIN4 in planta. The negative control (Mock) was infiltrated with 10 mM MgCl2. Coinoculations were made with A. tumefaciens at OD600 = 0.5 and P. syringae pv tomato at OD600 = 10−2 into wild-type RG-76R, RG-pto11, and RG-prf3 tomato leaves, and total proteins were extracted 24 h after infiltration and the presence of RIN4 assayed using anti-HA antibody (α-HA). LC, Ponceau S staining of the membrane to show equal loading. (B) P. syringae pv tomato DC3000 and its derivative lacking AvrPto caused T3SS-dependent proteolysis of RIN4 in tomato and in N. benthamiana. Wild-type P. syringae pv tomato DC3000 (PtoDC3000), a knockout derivative lacking AvrPto (PtoDC3000ΔavrPto), or a T3SS-deficient mutant (PtoDC3000hrcC) were individually inoculated at OD600 = 10−2 into leaves of tomato RG-76R, RG-prf3, or transgenic N. benthamiana constitutively expressing Pto and expressing HA-tagged Sl RIN4 under the DEX-inducible promoter. In tomato, total protein was extracted and detected using anti-At RIN4 (α-RIN4) antibody (Panel 1). In N. benthamiana, 24 h after inoculation, Sl RIN4 expression was induced by applying DEX onto the leaves. Total plant protein was extracted 24 h after induction and the presence of RIN4 assayed using anti-HA antibody (α-HA) (Panel 2). LC, Ponceau S staining of the membrane to show equal loading. (C) Effectors HopAM1 and HopQ1-1 from PtoDC3000 cleaved the artificial substrate (HA-RCS-GFP) containing the native RCS independent of Pto. HA-RCS-GFP and HopAM1DC3000 (lane 1), HopQ1-1DC3000 (lane 2), or empty vector (lane 3) were coexpressed in wild-type N. benthamiana leaves, and immunoblotting was performed using anti-HA antibody (α-HA) or anti-GFP antibody (α-GFP). LC, Ponceau S staining of the membrane to show equal loading.
Figure 8.
Figure 8.
AvrPto-Specific Degradation of Sl RIN4 by P. fluorescens in N. benthamiana. (A) AvrPto-specific degradation of Sl RIN4 by P. fluorescens. Strains of P. fluorescens 55 (pHIR11) (OD600 = 0.1) expressing (PflavrPto) or not expressing (Pfl) avrPto were coinoculated with A. tumefaciens carrying Sl RIN4 (OD600 = 1.0) or GFP (as a control) into leaves of transgenic N. benthamiana that expressed Pto. After 24 h, leaf samples were harvested and immunoblotted using anti-HA (α-HA) or anti-GFP (α-GFP) antibodies. The mock treatment (Mock) was infiltration with 10 mM MgCl2 solution. LC, Ponceau S staining of the membrane to show equal loading. (B) P. fluorescens at a high inoculum concentration elicited necrosis in an avrPto-dependent manner. Strains of P. fluorescens 55 (pHIR11) (OD600 = 1.0) expressing (PflavrPto) or not expressing (Pfl) avrPto were inoculated individually into leaves of transgenic N. benthamiana that expressed Pto. Only P. fluorescens expressing avrPto elicited macroscopic necrosis indicative of a HR 48 h after the inoculation.
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