Expression Patterns of Nt CMPG1 and Sl Cmpg1 after Avr9 Elicitation.
Cf9 tobacco cell cultures (A) were treated with Avr9 peptide (+Avr9) or untreated (control), and samples were harvested at the time points indicated. Cf9 tobacco plants (B) or Cf9 tomato plants (C) were infiltrated with IF that contains Avr9 (+Avr9) or that does not contain Avr9 (−Avr9), and plant leaf discs were harvested at the time points indicated. Total RNA was isolated and used for RT-PCR with specific primers for Nt CMPG1, Sl Cmpg1, and Actin.

Nt CMPG1 Is Required for Efficient HR Mediated by Cf-9 in N. benthamiana.
(A) VIGS in N. benthamiana using TRV. Seedlings were inoculated with TRV:Nt CMPG1, TRV:Nb SGT1, or empty vector as a control (TRV:00). Three weeks after inoculation, leaves were infiltrated with agrobacteria delivering Cf-9 and Avr9. Pictures were taken 4 d after agrobacteria infiltration.
(B) RT-PCR from silenced plants. N. benthamiana leaves silenced with the indicated TRV construct were infiltrated with water to induce Nb CMPG1 expression. Samples were collected 30 min after infiltration, and RNA was isolated and used for RT-PCR. Nb ACIK1 was used as a control for induction by flooding (bottom panel).

Overexpression of Nt CMPG1 Dominant-Negative Mutants.
(A) Leaf panels from Cf9 tobacco control plants (control) and Cf9 tobacco plants transgenic for Nt CMPG1 wild type and mutants (C37A line I and W64A line B) were infiltrated with IF(+Avr9) for HR induction. Pictures were taken 3 d after infiltration.
(B) Tobacco leaf discs were harvested from the same leaves shown in (A), and Nt CMPG1:TAP protein levels were determined as described in Figure 5B.
(C) The presence of high levels of Nt CMPG1C37A correlates with a reduced HR production. Two leaves of Cf9 tobacco control plants (left panels) and of six different plants of the segregating population of transgenic Nt CMPG1C37A tobacco (right panels) were infiltrated with IF(+Avr9) for HR induction. Pictures were taken 3 d after infiltration.
(D) Nt CMPG1C37A protein levels were analyzed by protein gel blots. Tobacco leaf discs were harvested from the same leaves shown in (C), and Nt CMPG1C37A-TAP protein levels were determined as described in Figure 5B. Numbers on top of the lanes correspond to the numbers of the leaves shown in (C). C, control leaf.
(E) Leaf panels of Cf9 tobacco control and Cf9 tobacco overexpressing Nt CMPG1 wild type or Nt CMPG1C37A were infiltrated with agrobacteria delivering Pto and AvrPto at the indicated cell densities. Pictures were taken 4 d after infiltration.

Sequence of Nt CMPG1 Protein and Alignment with Homologs.
(A) Protein sequence comparison between tobacco Nt CMPG1, tomato Sl Cmpg1, parsley Pc CMPG1, and Arabidopsis PUB20 and PUB21 was performed using ClustalW software. Position of the U-box domain is indicated. Shading represents the conservation of the residues among the sequences of the alignment: black, the residue is conserved in all the sequences; dark gray, the residue is conserved in four proteins of the alignment; light gray, the residue is conserved in three proteins of the alignment.
(B) Protein sequence alignment of the U-box domain of Nt CMPG1 homologs and comparison with U-box and CMPG consensus sequences. Asterisks show the amino acid residues mutated in this work to generate the dominant-negative versions of Nt CMPG1.
(C) Protein sequence comparison between the ARM repeats of Nt CMPG1, Sl Cmpg1, Pc CMPG1, and β-catenin (from human). The gray boxes indicate the protein region where an α-helix secondary structure was predicted. The consensus sequence is indicated, where p indicates polar residue and h indicates hydrophobic residue.

Nt CMPG1 Is an E3 Ligase, and Mutations in Its U-Box Domain Abolish Its Activity.
GST-Nt CMPG1, GST-Nt CMPG1C37A, and GST-Nt CMPG1W64A proteins were expressed and purified from E. coli and tested for E3 ligase activity using yeast E1 and three differents E2s. GST-PUB26 with the E2 Ubc8 was used as a positive control (line 12). Lines 1 to 5 and 11 are negative controls. The reactions were analyzed by protein gel blots using anti-ubiquitin antibody (A) or anti-GST antibody (B).

Overexpression of Nt CMPG1 Induces a Stronger HR Response to Avr9 in Cf9 Tobacco Plants.
(A) HR in N. tabacum. Cf9 tobacco plants were transformed with 35S:Nt CMPG1:TAP and analyzed for Avr9-dependent HR production. Tobacco leaves from three independent transgenic lines and a control were infiltrated with a 1/16 dilution of IF(+Avr9). Pictures were taken 3 d after infiltration.
(B) Nt CMPG1 protein levels were analyzed by protein gel blots. Leaf discs were harvested from different Cf9 tobacco lines expressing 35S:Nt CMPG1:TAP or control leaves. Total proteins were separated in SDS gels and analyzed by inmunoblotting using PAP antibody for Nt CMPG1:TAP detection. Bottom panel shows Ponceau S Red staining of ribulose-1,5-biphosphate carboxylase/oxygenase for confirmation of equal loading.
(C) Growth inhibition assay. Cf9 tobacco seedlings expressing Nt CMPG1:TAP (line J) and control were transferred to MS medium containing the indicated amount of IF(+Avr9) (+) or IF(−Avr9) (−) (μL per mL of MS medium). Pictures were taken 10 d after transfer to MS medium.
(D) Growth inhibition assay. Cf9 tobacco seedlings were transferred to MS medium containing IF(−Avr9) or IF(+Avr9) as described in (C). Ten days after the transfer, the same number of seedlings was weighted for each treatment. The percentage was calculated based on the weight of seedling growth in IF(−Avr9) containing MS medium.

Tomato Cmpg1 Is Required for Full Cf-9–Mediated Resistance to C. fulvum Expressing Avr9.
(A) VIGS in tomato. Transgenic Cf9 tomato cotyledons were inoculated with agrobacteria carrying the indicated TRV construct. Three weeks after infiltration, the right half of the tomato leaf was infiltrated with IF(+Avr9), and pictures were taken 3 d after infiltration under daylight (top panel) or UV light (bottom panel).
(B) Transgenic Cf9 or Cf0 tomato seedlings were silenced with the indicated TRV construct as described in (A). Three weeks later, plants were infected with C. fulvum race 4 GUS. Leaves were stained with X-gluc 3 weeks after C. fulvum inoculation, when pictures were taken (top panel). The fungal cycle was complete, as indicated by the conidiophores extruding from the stomata observed under the microscope (bottom panel).
(C) Tomato Cmpg1 transcript levels were analyzed by RT-PCR. Leaf discs from the indicated silenced tomato plants were harvested 30 min after infiltration with water. Total RNA was extracted and used for RT-PCR analysis with Sl Cmpg1-specific primers. Equal amounts of cDNA were used, as shown by the amplification with the constitutively expressed Actin gene.
(D) RNAi in tomato. Cf9 tomato plants were transformed with pHellsgate containing a tomato Cmpg1 hairpin. One leaf half from the transgenic (Cmpg1 RNAi) and untransformed (control) Cf9 tomato plants was infiltrated with IF(+Avr9) for HR induction. Pictures were taken 3 d after infiltration under daylight (left leaves) and UV light (right leaves).
(E) Cf0 and Cf9 untransformed (control) and Cf9 transgenic (Cmpg1 RNAi) tomato plants were infected with C. fulvum race 4 GUS. Leaves were stained with X-gluc 3 weeks after infection (top panel). Fungal cycle was complete, as indicated by the conidiophores extruding from the stomata observed under the microscope (middle panel). Hyphal growth inside the leaves was confirmed under the microscope (bottom panel).
(F) Tomato Cmpg1 mRNA levels were analyzed by RT-PCR. Leaf discs from the indicated tomato lines were harvested 30 min after IF(+Avr9) infiltration and processed as described in (C). Additionally, equal induction treatment is shown by the amplification of Sl Acik1. From the same leaves, samples were harvested after 24 h, and transcript levels for the defense-related genes GlucB and PR5 were determined using gene-specific primers.