Figure 1.
CsCyp complements the cpr1Δ/cpr1Δ and ess1H164R yeast mutants.
(A) Expression of CsCyp as a GST fusion in the yeast cpr1Δ/cpr1Δ (33513) and ess1H164R mutants, under the control of a copper-induced promoter. The recombinant proteins were induced with 0.5 mM copper sulfate for 2 h and probed with the anti-GST serum. Induced (I) and non-induced (NI) samples are shown. (B) The cpr1Δ/cpr1Δ mutant expressing GST-CsCyp (▴) but not GST alone (♦) increased the rates of sporulation of the 33513 mutant, relative to non-transformed cells (▪). Wild type cells used as reference are indicated (•). Sporulation was measured at different time points after transfer of the cells to sporulation medium and plotted as the percentage of asci formed. Measurements are the means of ten independent countings and the error bars represent standard deviations. (C) Growth of W303-1A wild-type (WT) and ess1H164R mutant (H164R) in YPD medium supplemented with 0.1 mM copper sulfate at the permissive (21°C) and non-permissive (37°C) temperatures. Cells were grown to the mid-log phase and serial dilutions were spotted onto plates and incubated for 24 h at the indicated temperatures. The growth of the ess1H164R mutant at 37°C was rescued by GST-CsCyp but not GST alone. Non-transformed WT and ess1H164R cells served as controls.
Figure 2.
Interactions of the CTD of the citrus RNA polymerase II with CsCyp.
(A) Yeast two-hybrid assay showing the interaction between CsCyp (pOAD-CsCyp) and the C. sinensis CTD (pOBD-CTD). Control yeast cells co-transformed with the bait (pOBD-CTD) plus empty pOAD or the prey (pOAD-CsCyp) plus empty pOBD are indicated. (B) Yeast two-hybrid assay showing interactions between the CTD with CsTdx (pOAD-CsTdx) and CsUev (pOAD-CsUev), but not CsUbc13 (pOAD-CsUbc13). Yeast cells were co-transformed with the bait plus empty pOAD (CTD+pOAD) or the preys CsTdx, CsUev, CsUbc13 plus empty pOBD as controls. (C) Western blot of immunoprecipitation (IP) reactions showing that the anti-human RNA pol II (anti-pol II) cross-reacted with the recombinant citrus CTD (CsCTD) and with proteins from the citrus cell extracts treated with DNase I (Input 1) or not (Input 2), including a major band of ∼110 kDa. The anti-pol II serum detected various bands in the protein fractions that were immunoprecipitated by the anti-CsCyp and anti-pol II sera, but not by the CsCyp pre-immune serum (control). The anti-CsCyp serum (Anti-Cyp) also detected the recombinant CsCyp and CsCyp that were immunoprecipitated by the anti-CsCyp and anti-pol II sera, but not by the CsCyp pre-immune serum. (D) GST-pulldown assay using the GST-CTD as bait and purified CsCyp without the 6×His tag as prey. Protein samples were electrophoresed and probed with the anti-GST and anti-CsCyp sera. CsCyp bound to the GST-CTD but not to GST alone. The cyclophilin inhibitor CsA prevented CsCyp from interacting with the CTD. Soluble cell extracts of GST-CTD, GST alone and the purified CsCyp used as inputs are indicated and the molecular sizes of the corresponding proteins are shown on the left.
Figure 3.
PthA variants interact with the CTD through their LRR region.
(A) Yeast two-hybrid assay showing the interaction between the CTD and the four PthA variants (1–4, respectively). Yeast cells co-transformed with the individual pOAD-PthA 1–4 constructs plus empty pOBD (5–8, respectively) were used as controls. (B) No interactions were observed between each of the repeat domains (RDs) of the PthA variants 1–4 (1–4, respectively) with the CTD. Yeast cells co-transformed with the bait pOBD-CTD plus empty pOAD (5) or the preys pOAD-RD 1–4 plus empty pOBD (6–9, respectively), as controls, are indicated. (C) Yeast two-hybrid interactions of the CTD with the truncated version of PthA2 carrying 5.5 internal repeat units plus the C-terminal domain (1) or the LRR region alone (2). Control yeast cells co-transformed with empty pOBD plus the prey constructs pOAD-5.5rep+CT (3) and pOAD-LRR (4) are indicated. (D) GST-pulldown assay using the GST-CTD as bait and purified PthA2 as prey. Protein samples were electrophoresed and probed with the anti-GST and anti-PthA sera. PthA2 bound to the GST-CTD but not to GST alone. Soluble cell extracts of GST-CTD, GST alone and the purified PthA2 used as inputs are indicated and the molecular sizes of the corresponding proteins are shown on the left.
Figure 4.
Interaction of PthA2 with CsCyp in plant cells.
(A) Immunoprecipitation (IP) assay of PthA2 with the anti-CsCyp serum. Cell extracts of citrus epicotyls transiently expressing PthA2 (input) were incubated with the anti-CsCyp (Anti-Cyp) or the pre-immune serum (control) and protein-A Sepharose. The beads were washed and the bound proteins were resolved on a 13% SDS-polyacrylamide gel and probed with the indicated antibodies. PthA2 was detected in the IP reaction performed with the anti-CsCyp but not with the control pre-immune serum. (B) Co-localization of PthA2 and CsCyp in the nucleus of N. benthamiana cells (arrows). Leaf sectors transiently co-expressing PthA2-GFP (PthA2) and CsCyp-DsRed (CsCyp) were treated with Dapi for 10 min before visualization. Pictures were taken in a Nikon fluorescence microscopy at a 1.000× magnification and merged using the software provided by the instrument.
Figure 5.
PthA2 inhibits the PPIase activity of CsCyp.
(A) The PPIase activity of recombinant CsCyp protein was evaluated by the α-chymotrypsin-coupled assay. Enzyme activity was measured immediately after the addition of α-chymotrypsin and the substrate into the reaction mixture. The PPIase activity of CsCyp reached a plateau within 30 to 60 s after the start of the reaction (red). This activity was drastically reduced in the presence of 30 nM CsA (light blue), as compared to buffer only, as control (black). PthA2 (green), its internal repetitive DNA-binding domain RD2 (brown), or its C-terminal domain+5.5 internal repeats (purple) but not BSA (dark blue), added into the reaction mixture reduced the PPIase activity of CsCyp in a time-course measurement. (B) PPIase activity of CsCyp in the absence and presence of CsA (30 nM), PthA2 or BSA (15 nM). Activities were the mean of three independent measurements recorded 30 s after the start of the reaction, and the asterisks indicate statistically different means relative to that of normal activity. (C) GST-pulldown assay using the GST-CsCyp as bait and purified PthA2 as prey. Protein samples were electrophoresed and probed with the anti-GST and anti-PthA sera. PthA2 bound to the GST-CsCyp only and the cyclophilin inhibitor CsA abrogated the interaction. Purified PthA2 used as input and the molecular sizes of the corresponding proteins are shown on the left.
Figure 6.
Effect of CsA on citrus canker development.
Sweet orange leaves were infiltrated with X. citri (approximately 2.0×104 cells) in the absence or presence of CsA at 0.1 or 0.5 mM. Canker lesions started to develop one week after bacterial infiltration and were pronounced 14 days after bacterial inoculation, when the pictures were taken (20× magnification). Left panels show representative leaf sectors that were infiltrated with water suspensions of X. citri plus CsA at 0.1 or 0.5 mM final concentrations, or X. citri alone, as control (dotted lines). CsA substantially increased pustule formation and symptom development including tissue hypertrophy and water-soaking, relative to control. The effect of CsA on canker development was dose-dependent as 0.5 mM CsA induced more confluent pustules than 0.1 mM CsA or no CsA (right panels).
Figure 7.
Silencing of CsCyp enhanced canker symptoms.
(A) Schematic representation of the RNAi hairpin construct used to transform sweet orange plants. The construct carries the whole cyclophilin domain of CsCyp in an inverted orientation and separated by the intron of the pHANNIBAL vector. (B) Transgenic plants carrying the CsCyp RNAi hairpin construct were assayed for GUS activity and analyzed by western blot using the anti-CsCyp serum (Anti-Cyp). Examples of CsCyp RNAi plants (GUS positive) showing significantly lower levels of CsCyp relative to untransformed controls are shown. Protein loads were controlled by Coomassie Brilliant Blue (CBB) staining of the bands corresponding to the large subunit of Ribulose-1,5-bisphosphate Carboxylase (RBCL). Recombinant CsCyp with no tags was loaded in the first lane of the blot as a positive control. (C) Sweet orange leaves of control and three RNAi plants (Cyp RNAi) challenged with X. citri (approximately 2.0×104 cells). Pictures (10× magnifications) were taken 14 days after bacterial infiltration. Canker lesions were substantially enhanced in the RNAi plants compared to controls. (D) Sweet orange leaves of control and RNAi plants infiltrated with X. citri at a lower density (approximately 103 cells). Canker pustules in the RNAi plants developed much faster and produced more rupture of the epidermis than in control plants. Pictures (20× magnifications) were taken 20 days after bacterial infiltration.
Figure 8.
Protein-protein interactions involving cyclophilins and the CTD.
(A) Cartoon model of protein-protein interactions illustrating how TAL effectors (PthAs) might associate with components of the host basal transcription machinery. The unfolded CTD of RNA pol II (purple) with phosphorylated heptapeptides (P) may function as a protein scaffold for the assembly of a multiprotein complex, including the citrus CsCyp (blue), CsTdx (orange) and the CsUev/Ubc13 pair (red/green). The N-terminal (N-term), DNA-binding domain, leucine-rich repeat region (LRR) and activation domain (AD) of PthA are shown in light blue. Upon binding to its target DNA, the effector protein associates with the CsCyp/CsTdx/CsUev/Ubc13 complex through its DNA-binding domain [15], and with the CTD through its LRR. The effector protein then inhibits the PPIase activity of CsCyp causing changes in the phosphorylation status of the CTD, allowing the CTD to recruit other co-factors for the progress of transcription. (B) Schematic representation of protein-protein associations found in an Arabidopsis interactome study, depicting the interactions between ROC4 (Cyp20-3) with the CTD and thioredoxin H3, and between Uev and Ubc13 [39]. The Arabidopsis AtCyp59 is also a CTD interactor [40]. Sweet orange and Arabidopsis proteins sharing similar domains are represented by the same colors in A and B.