Capsid protein-mediated recruitment of host DnaJ-like proteins is required for Potato virus Y infection in tobacco plants

Abstract

The capsid protein (CP) of potyviruses is required for various steps during plant infection, such as virion assembly, cell-to-cell movement, and long-distance transport. This suggests a series of compatible interactions with putative host factors which, however, are largely unknown. By using the yeast two-hybrid system the CP from Potato virus Y (PVY) was found to interact with a novel subset of DnaJ-like proteins from tobacco, designated NtCPIPs. Mutational analysis identified the CP core region, previously shown to be essential for virion formation and plasmodesmal trafficking, as the interacting domain. The ability of NtCPIP1 and NtCPIP2a to associate with PVY CP could be confirmed in vitro and was additionally verified in planta by bimolecular fluorescence complementation. The biological significance of the interaction was assayed by PVY infection of agroinfiltrated leaves and transgenic tobacco plants that expressed either full-length or J-domain-deficient variants of NtCPIPs. Transient expression of truncated dominant-interfering NtCPIP2a but not of the functional protein resulted in strongly reduced accumulation of PVY in the inoculated leaf. Consistently, stable overexpression of J-domain-deficient variants of NtCPIP1 and NtCPIP2a dramatically increased the virus resistance of various transgenic lines, indicating a critical role of functional NtCPIPs during PVY infection. The negative effect of impaired NtCPIP function on viral pathogenicity seemed to be the consequence of delayed cell-to-cell movement, as visualized by microprojectile bombardment with green fluorescent protein-tagged PVY. Therefore, we propose that NtCPIPs act as important susceptibility factors during PVY infection, possibly by recruiting heat shock protein 70 chaperones for viral assembly and/or cellular spread.

FIG. 1.

Isolation of NtCPIPs from N. tabacum that interact with PVY CP. (A) Specific interaction between PVY CP and NtCPIP1 in the yeast two-hybrid system. Yeast cells transformed with bait and prey vectors were plated on Trp⁻/Leu⁻ medium to test for double transformation and on Trp⁻/Leu⁻/His⁻ medium for protein interaction. As a second reporter of the interaction, lacZ activity was tested using a β-galactosidase filter assay (β-Gal). Reporter gene activation was observed only for colonies cotransformed with BD-PVY CP and AD-NtCPIP1 (1) or with BD-SNF1 and BD-SNF4 representing a positive control (6). No interaction was detectable for any of the other transformations. Combinations of transformed plasmid: 1, BD-PVY CP/AD-NtCPIP1; 2, pGBT9 vector/AD-NtCPIP1; 3, BD-p53/AD-NtCPIP1; 4, BD-SNF1/AD-NtCPIP1; 5, BD-PVY CP/AD-SNF4; 6, BD-SNF1/AD-SNF4. (B) Alignment of deduced amino acid sequences of NtCPIPs with a DnaJ-like protein from N. tabacum (NtM541) using the CLUSTAL W program (DNASTAR, Madison, WI). NtCPIP2a and NtCPIP2b, isolated by cDNA library screening by using NtCPIP1 as a probe, were both shown to specifically interact with PVY CP in the yeast two-hybrid system (data not shown; see Fig. 1C), whereas NtM541 was previously identified to bind to the TSWV NSm movement protein (66). Regions of identity are shaded in black and gaps introduced for alignment are indicated by dashes. The predicted J domain is marked by a gray horizontal bar, and two conserved motifs (K-X-X-X-K-E/K) indicative for a lysine-enriched domain are boxed. (C) Interaction ability of PVY CP and TSWV NSm with NtCPIPs or NtM541.Yeast cells expressing combinations of the indicated viral bait and DnaJ-like prey proteins were grown on Trp⁻/Leu⁻/His⁻ medium and analyzed qualitatively for β-galactosidase activity.

FIG. 2.

Identification of the PVY CP interaction domain in the yeast two-hybrid system. PVY CP deletion and various single and double amino acid substitution mutants were individually cotransformed with NtCPIP1 or NtCPIP2a into yeast cells and qualitatively assayed for β-galactosidase activity. Mutations introduced into the PVY CP gene are indicated. Amino acids (S125, R157, and D201) targeted in the substitution mutants refer to highly conserved residues in the core domain of potyviral CPs (22).

FIG. 3.

PVY CP interacts with NtCPIPs in vitro and in planta. (A and B) In vitro binding assay between PVY CP and NtCPIP1 (A) or NtCPIP2a (B). MBP alone (lanes 1 and 4) or in fusion with PVY CP (lanes 2 and 5) was expressed in E. coli, coupled to an amylose matrix, and incubated with 25 μg of affinity-purified His₆-tagged NtCPIP1 or NtCPIP2a protein. Aliquots of the eluates (75% of total amount) were separated by SDS-PAGE and tested for the presence of NtCPIPs (arrowheads) by Coomassie blue staining or Western blot analysis with anti-His antibodies. Then, 2 μg of His₆-NtCPIP1 (lane 3) or His₆-NtCPIP2a (lane 6) was loaded onto the respective gels as input controls. (C to H) BiFC analysis of PVYCP/NtCPIP interaction in plant cells. The coding regions of NtCPIPs and PVY CP were fused with the N-terminal (YFP^N, in pSPYNE-35S) or C-terminal (YFP^C, in SPYCE-35S) region of YFP, respectively. Plasmids were Agrobacterium infiltrated in N. benthamiana leaves, and the reconstructed YFP signal was detected in the epidermal cell layer by confocal microscopy. Coexpression of empty vectors pSPYNE/pSPYCE (C), PVY CP:YFP^C/pSPYNE (D), pSPYCE/NtCPIP1:YFP^N (E), pSPYCE/NtCPIP2a:YFP^N (F) PVY CP:YFP^C/NtCPIP1:YFP^N (G), or PVY CP:YFP^C/NtCPIP2a:YFP^N (H) reveals specific YFP complementation only by PVY CP/NtCPIP interactions. YFP-derived fluorescence signals (in green) of single confocal sections (left) and the transmission mode (middle) were superimposed in the merged image (right). Bars, 50 μm.

FIG. 4.

Identification of dominant-negative NtCPIP mutants and functional analysis in planta. (A) Interaction of N-terminal deletion mutants of NtCPIP1 and NtCPIP2a with PVY CP in the yeast two-hybrid system. Yeast cells cotransformed with the indicated bait and prey plasmids were grown on Trp⁻/Leu⁻/His⁻ medium and qualitatively assayed for β-galactosidase activity. (B) Transient expression of 3x-Myc epitope-tagged full-length and J-domain-deficient NtCPIP2a proteins in N. benthamiana leaves via agroinfiltration. Western blot analysis was performed with identical amounts of total protein extracts from leaves 0, 4, and 6 days after coinfiltration of NtCPIP2a-myc and NtCPIP2aΔN-myc with the p19 silencing suppressor, respectively. Infiltration of p19 alone served as a negative control. (C) Effect of transient expression of full-length and J-domain-deficient NtCPIP2a proteins on susceptibility to PVY infection in N. benthamiana. Leaves were infected with PVY 24 h after agroinfiltration and assayed for accumulation of viral coat protein 4 dpi by ELISA. Values represent means (n = 12) ± the standard error (SE) and are given as the percentage of the p19 control. The results indicate that expression of the dominant-negative mutant but not the full-length variant strongly interferes with the spread of infection.

FIG. 5.

Effect of stable expression of dominant-negative NtCPIP mutants on susceptibility to PVY infection. (A) Schematic representation of binary overexpression constructs used for transformation of N. tabacum. J-domain-deficient NtCPIP1Δ65N and NtCPIP2aΔ66N fragments shown to retain their interaction ability with PVY CP (Fig. 4A) were fused to the 5′-untranslated TMV U1 overdrive sequence (Ω) and placed between the CaMV 35S promoter and ocs terminator in the Bin19-derived vector. (B) Northern analysis of NtCPIP1Δ65N and NtCPIP2aΔ66N specific transcripts. Each lane contains 30 μg of total RNA isolated from WT plants and transgenic lines NtCPIP1Δ-9 and -17 and NtCPIP2aΔ-15, -16, -28, and -39. Northern blots were hybridized with NtCPIP1Δ65N or NtCPIP2aΔ66N cDNAs, respectively. (C) Immunoblot analysis of NtCPIP1Δ65N and NtCPIP2aΔ66N protein accumulation. Identical amounts of total protein extracted from leaf material of WT and transgenic lines were separated by SDS-PAGE and analyzed by Western blotting with rabbit-derived polyclonal anti-NtCPIP1 (dilution 1:3,000) or anti-NtCPIP2a (1:5,000) antibodies and goat-derived secondary antibody conjugated to horseradish peroxidase (dilution 1:100,000). (D) PVY titer in systemic leaves (five or six leaves above the inoculated leaf) of WT (n = 22) and transgenic ME-4 controls (ME, n = 24), as well as of the transgenic lines NtCPIP1ΔN-9 (n = 25) and -17 (n = 25) and NtCPIP2aΔN-15 (n = 23), -16 (n = 24), -28 (n = 24), and -39 (n = 24) at 6 dpi. Values represent means ± the SE and are given as the percentage of the WT level. Plants had developed six to eight leaves prior to PVY inoculation. (E) PVY coat protein levels in systemic leaves (seven or eight leaves above the inoculated leaf) of WT and transgenic lines at 13 dpi. Values represent means ± the SE and are given as the percentage of the WT level. (F) Development of virus-induced symptoms in PVY-infected transgenic lines compared to controls (WT, ME) at 13 dpi, indicating a dramatically increased virus resistance due to the expression of dominant-negative mutants of NtCPIPs.

FIG. 6.

Effect of dominant-negative NtCPIP mutants on cellular spread of PVY-gfp. PVY-gfp was cobombarded with a DsRed expression vector into leaves of WT (A) and transgenic (B) controls, as well as leaves of the transgenic lines NtCPIP1ΔN-9 (C) and NtCPIP2aΔN-30 (D). At 4 dpi, bombarded leaves were scanned for GFP- and DsRed-derived fluorescence by confocal microscopy. Representative lesions demonstrated that infections in transgenic lines NtCPIP1ΔN-9 and NtCPIP2aΔN-39 reached a considerably smaller area beyond the primarily bombarded cells (indicated in red) than in the control lines. Bars, 100 μm.