Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector

Abstract

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.

Keywords: Arabidopsis thaliana; Hyaloperonospora arabidopsidis; importin-α; nuclear localization sequence; nucleo-cytoplasmic transport; oomycete effector; plant innate immunity.

Figure 1

The C-terminal 58 amino acids of HaRxL106 are sufficient and required for active nuclear import.(a) Confocal images of RFP and the indicated RFP–HaRxL106 fusion constructs in epidermal cells of N. benthamiana. The images were taken 48 h after infiltration with A. tumefaciens. Upper panels show RFP channel, lower panels show RFP channel overlaid on bright field images. Scale bars 50 μm.(b) Western blot of soluble proteins extracts for the RFP fusions used in (a). Samples were harvested 48 h post infiltration with A. tumefaciens and probed with α-RFP antibody. NS = non-specific signal of the α-RFP antibody. Coomassie stain shows RubisCO band as loading control.

Figure 2

HaRxL106 and MOS6 form a stable complex in vitro with a K_d in the low micro-molar range.(a) Elution volumes of His6-tagged HaRxL106, HaRxL106ΔC and ΔIBBMOS6 on a Superdex HR 200 30/10 size exclusion chromatography column determined by absorption at 280 nm. The upper two panels show elution profiles of the three proteins alone. The lower two panels show elution profiles of mixtures of ΔIBBMOS6 with either HaRxL106 or HaRxL106ΔC at a molar ratio of 1^ΔIBBMOS6:2^{HaRxL106(ΔC)}.(b) SDS-PAGE of fractions of ΔIBBMOS6/HaRxL106 and the ΔIBBMOS6/HaRxL106ΔC control eluting from the column.(c) ITC binding isotherms and associated fits for the interactions between His6–ΔIBBMOS6 and His6–HaRxL106, His6–HaRxL106ΔC or His6–SAP11. K_d values are representative of two ITC experiments.

Figure 3

The armadillo repeat domain of MOS6 has the canonical importin-α fold.(a) Crystal structure of the ΔIBBMOS6 protein in cartoon representation and superposition of the armadillo repeat domains of MOS6 (green) and rice importin-α1a (light blue, PDB 4B8O) (Chang et al., 2012).(b) Superposition of ΔIBBMOS6 (green) and the ΔIBB variant of rice importin-α1a (light blue, PDB 4B8O) in complex with an SV40NLS (orange) bound at the major NLS-binding site. Residues of rice importin-α1a that contribute to the NLS-binding site and the corresponding MOS6 amino acids are shown in stick representation.(c) Superposition of ΔIBBMOS6 (green) and the ΔIBB variant of rice importin-α1a (light blue, PDB 2YNS) in complex with the B54NLS (orange) bound at the minor NLS-binding site. Residues of rice importin-α1a that contribute to the NLS-binding site and the corresponding MOS6 amino acids are shown in stick representation. Residue labels in (b) and (c) correspond to the MOS6 sequence.

Figure 4

Conservation of the NLS-binding sites of importin-α proteins expressed in Arabidopsis rosette leaves.(a) Sequencing reads of the nine Arabidopsis importin-αs detected by RNA-Seq in Col-0 rosette leaves (Asai et al., 2014). Error bars show standard deviation (SD) of three biological replicates.(b) Conservation of residues contributing to the MOS6 NLS-binding sites in Arabidopsis importin-α1, -α2, -α4 and -α6. The figure shows the MOS6 armadillo repeat domain and amino acids contributing to the inner concave site of the protein are shown in surface representation. Residues coloured in yellow are conserved in importin-α1, -α2, -α4 and -α6. Orange colour indicates amino acids that diverge from MOS6 in at least one of the other importin-αs. For a sequence alignment of all Arabidopsis importin-α protein sequences, see Wirthmueller et al. (2013).(c) Conservation of residues contributing to the MOS6 NLS-binding sites in Arabidopsis importin-α9. Representation as in (b).(d) GFP fusion proteins of importin-α1, -α2, -α4, -α9, MOS6 and free GFP were transiently co-expressed with StrepII-3xHA (HS)-tagged HaRxL106 in N. benthamiana. At 48 h post infiltration GFP-tagged importin-αs were IP-ed and co-purifying HS–HaRxL106 was detected by an α-HA western blot. Coomassie stains show RubisCO band in total protein extracts and IP-ed importin-αs in the IP blot. Similar results were obtained in two independent experiments.

Figure 5

The HaRxL106 NLS mediates stronger complex formation with importin-αs than the SV40NLS in plant cells.(a) BiFC between MOS6–YFP^C and the indicated YFP^N-tagged NLS-cargo proteins in nuclei of N. benthamiana 48 h post infiltration. Images are representative of at least 10 nuclei analysed. Scale bars 5 μm.(b) MOS6–GFP was transiently co-expressed with the indicated StrepII-3xHA (HS)-tagged NLS-cargo proteins in N. benthamiana. At 48 h post infiltration MOS6–GFP was IP-ed and co-purifying HS-tagged proteins were detected by an α-HA western blot. Coomassie stains show RubisCO band in total protein extracts and IP-ed importin-αs in the IP blot. Similar results were obtained in two independent experiments.

Figure 6

Competition for importin-α binding in plant cells is not only mediated by the IBB domain.Importin-α2–YFP or the corresponding ΔIBB constructs were transiently co-expressed with the indicated HS-tagged NLS-cargo proteins in N. benthamiana. At 48 h post infiltration YFP-tagged importin-αs were IP-ed and co-purifying StrepII-3xHA (HS)-tagged proteins were detected by an α-HA western blot. Coomassie stains show RubisCO band in total protein extracts and IP-ed importin-αs in the IP blot. Similar results were obtained in two independent experiments.