The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

Abstract

Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Deltaubc13 and Deltamms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Figure 1

PthA proteins localize to the nucleus and form homo‐ and heterodimers. (A) Fluorescence of PthA–green fluorescent protein (GFP) fusions in the nucleus of transiently transfected Nicotiana benthamiana cells 72 h after Agrobacterium tumefaciens infiltration. The top panels show the PthA4–GFP and 4′,6‐diamidino‐2‐phenylindole (DAPI) fluorescence of the same cell at ×400 magnification. The bottom panels show the localization of PthA1– and PthA2–GFP fusions in nuclear dots at ×1000 magnification. (B) Yeast two‐hybrid assays showing that PthAs 2 and 3 form homodimers but also heterodimerize with each other and with PthAs 1 and 4. Yeast cells co‐transformed with the bait and prey constructs are shown at the top of the plate, whereas cells carrying the bait construct plus the empty pOAD or the prey construct plus the empty pOBD are shown at the sides, as indicated. (C) Under less stringent conditions of interaction (synthetic complete medium lacking histidine but containing adenine, SC − His + Ade), PthAs 1 and 4 homodimerize but do not self‐interact. (D) The repeat domain (RD2) as well as the 5.5rep + CT2 of PthA2 were sufficient for homo‐ and heterodimerization with PthAs 2 and 3, respectively. In (B–D), bait and prey are indicated horizontally and vertically, respectively.

Figure 2

Differential interactions between PthA proteins with the citrus α‐importin (α‐Imp), cyclophilin (Cyp), thioredoxin (TDX) and ubiquitin‐conjugating enzyme variant (Uev). (A) Yeast two‐hybrid assays showing that PthAs 2 and 3 interact most strongly with the citrus proteins, whereas PthAs 1 and 4 interact with the prey under less stringent conditions (synthetic complete medium lacking histidine but containing adenine, SC − His + Ade). The disposition of the yeast co‐transformants in the plates is the same as shown in Fig. 1. (B) Schematic representation of the tetratricopeptide repeat (TPR) and thioredoxin (TRX) domains of TDX with the numbers representing the positions of the amino acid residues. The TPR (residues 1–178) and TRX (residues 179–328) domains were subcloned into the pOAD vector for two‐hybrid assays. (C) Differential interactions between the four PthA proteins with the TDX domains, showing the preferential interaction of PthAs 2 and 3 with both TPR and TRX domains, relative to PthAs 1 and 4. PthA 4 and the TRX domain of TDX self‐interacted in the presence of adenine. (D, E) Glutathione transferase (GST) pulldown assays with PthAs and the citrus proteins. 6 × His–PthAs 2 and 4 were expressed in Escherichia coli and purified by affinity chromatography. The purified proteins were subjected to GST pulldown assays, as described in Experimental procedures, and the resulting samples were separated by electrophoresis and probed with the anti‐PthA and anti‐GST sera. (D) PthA2 binds to all citrus proteins (GST fusions), but more strongly to α‐Imp and TDX. (E) Pronounced interaction of PthA4 with α‐Imp. No interactions were detected between PthA 4 and Cyp or the TPR domain of TDX. Purified PthA proteins used as controls are shown on the left. The GST fusions are indicated by asterisks and the molecular sizes of the proteins are shown on the left. The corresponding sodium dodecylsulphate (SDS) gels are shown in Fig. S2 (see Supporting Information).

Figure 3

The citrus proteins cyclophilin (Cyp), thioredoxin (TDX) and ubiquitin‐conjugating enzyme variant (Uev) interact with each other. (A) Yeast two‐hybrid assays showing interactions between TDX, Cyp and Uev, as well as between Cyp and Uev. The tetratricopeptide repeat (TPR) and thioredoxin (TRX) domains of TDX also interact with Cyp and Uev. The disposition of the yeast co‐transformants in the plates is the same as shown in Fig. 1. (B) Western blot of glutathione transferase (GST) pulldown assays using GST–TDX or GST–Cyp as bait and 6 × His–Cyp and 6 × His–Uev as prey. Consistent with the in vivo assay shown in (A), stronger interactions between TDX and Cyp are observed in the presence of Uev, suggesting the existence of a protein complex formed by TDX, Cyp and Uev. The corresponding sodium dodecylsulphate (SDS) gel is shown in Fig. S2 (see Supporting Information).

Figure 4

The citrus Ubc13 interacts with ubiquitin‐conjugating enzyme variant (Uev) and PthA proteins. (A) Sweet orange Ubc13 interacts with Uev and with PthAs 2 and 3. The disposition of the yeast co‐transformants in the plates is the same as shown in Fig. 1. (B) Western blots of glutathione transferase (GST) pulldown assays using GST–Ubc13 as bait and 6 × His–Uev and 6 × His–PthAs 2 and 4 as prey. According to the assay shown in (A), Ubc13 binds to Uev and PthA2, but not to PthA 4. Purified PthA proteins used as controls and the molecular sizes of the proteins are shown on the left. The corresponding sodium dodecylsulphate (SDS) gels are shown in Fig. S2 (see Supporting Information).

Figure 5

The repeat domain and leucine‐rich repeat (LRR) are important for protein–protein interactions. (A) Yeast two‐hybrid assays showing the interactions of the internal repeat domain of PthA2 (RD2) and the 5.5rep + CT2 construct with all citrus proteins. LRR interacted with α‐importin (α‐Imp) only. (B) Western blot of glutathione transferase (GST) pulldown assays showing that both RD2 and 5.5rep + CT2 constructs bind to citrus proteins (GST fusions). The C‐terminus of PthA2 (CT2), carrying LRR and the nuclear localization signal (NLS), binds to α‐Imp only. Purified PthA proteins used as controls and the molecular sizes of the proteins are shown on the left. The corresponding sodium dodecylsulphate (SDS) gels are shown in Fig. S2 (see Supporting Information).

Figure 6

The citrus Ubc13 and ubiquitin‐conjugating enzyme variant (Uev) proteins complement yeast Δubc13 and Δmms2/uev1a mutants. (A) The Citrus sinensis Ubc13 and Uev genes under the control of a copper‐induced promoter were expressed in the yeast mutants Δubc13 and Δmms2/uev1a as glutathione transferase (GST) fusions. The proteins (43 kDa) were induced with 0.5 mm copper sulphate for 2 h, purified by affinity chromatography and probed with anti‐GST serum. Induced (I) and noninduced (NI) samples are shown. The amounts of protein loaded on the gels are shown in Fig. S2 (see Supporting Information). (B) Growth of the yeast strain SUB62 and its derivative mutants Δubc13 and Δmms2/uev1a in synthetic complete (SC) medium in the presence of increasing amounts of methylmethanesulphonate (MMS). The growth of both mutants in MMS gradient plates can be rescued by the corresponding citrus Ubc13 and Uev genes.

Figure 7

PthA proteins are not K63‐linked ubiquitinated in vivo. (A) PthAs 2 and 4 were expressed in the wild‐type yeast strain SUB62 and its derivative mutants Δubc13 and Δmms2/uev1a as glutathione transferase (GST) fusions (∼140 and 147 kDa, respectively). The proteins were purified by affinity tag and subjected to sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blot detection with anti‐PthA or anti‐ubiquitin sera. Major PthA bands of the expected size for the GST fusions are indicated by the arrows. (B) PthAs 2 and 4 were expressed in sweet orange epicotyl sections using Agrobacterium tumefaciens transformation. A β‐glucuronidase (GUS) construct was used as control. Both PthA proteins (∼116 kDa) were detected by anti‐PthA serum (arrow), but not by anti‐ubiquitin serum. (C) Growth of yeast strain SUB62 (control expressing GST only) and SUB62 separately transformed with PthA 2 and 4 GST fusions in yeast peptone dextrose (YPD) medium in the presence of a methylmethanesulphonate (MMS) gradient. (D) The anti‐PthA serum confirms the presence of PthA proteins in the yeast extracts (arrow), whereas the anti‐ubiquitin serum reveals a missing band in the yeast extract expressing PthA2 (arrow). The amounts of proteins loaded on the sodium dodecylsulphate (SDS) gels are shown in Fig. S2 (see Supporting Information).