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, 83 (19), 9720-30

Interaction of the Tobacco Mosaic Virus Replicase Protein With a NAC Domain Transcription Factor Is Associated With the Suppression of Systemic Host Defenses

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Interaction of the Tobacco Mosaic Virus Replicase Protein With a NAC Domain Transcription Factor Is Associated With the Suppression of Systemic Host Defenses

Xiao Wang et al. J Virol.

Abstract

An interaction between the helicase domain of the Tobacco mosaic virus (TMV) 126-/183-kDa replicase protein(s) and the Arabidopsis thaliana NAC domain transcription factor ATAF2 was identified via yeast two-hybrid and in planta immunoprecipitation assays. ATAF2 is transcriptionally induced in response to TMV infection, and its overexpression significantly reduces virus accumulation. Proteasome inhibition studies suggest that ATAF2 is targeted for degradation during virus infection. The transcriptional activity of known defense-associated marker genes PR1, PR2, and PDF1.2 significantly increase within transgenic plants overexpressing ATAF2. In contrast, these marker genes have reduced transcript levels in ATAF2 knockout or repressor plant lines. Thus, ATAF2 appears to function in the regulation of host basal defense responses. In response to TMV infections, ATAF2 and PR1 display increased transcript accumulations in inoculated tissues but not in systemically infected tissues. ATAF2 and PR1 transcript levels also increase in response to salicylic acid treatment. However, the salicylic acid treatment of systemically infected tissues did not produce a similar increase in either ATAF2 or PR1 transcripts, suggesting that host defense responses are attenuated during systemic virus invasion. Similarly, noninfected ATAF2 knockout or ATAF2 repressor lines display reduced levels of PR1 transcripts when treated with salicylic acid. Taken together, these findings suggest that the replicase-ATAF2 interaction suppresses basal host defenses as a means to promote systemic virus accumulation.

Figures

FIG. 1.
FIG. 1.
TMV replicase interacts with the NAC domain transcription factor ATAF2. (A) Schematic representation of TMV replicase and ATAF2 ORFs. Gray boxes represent the bait (126-/183-kDa replicase) and prey (ATAF2) interacting regions. MT, methyltransferase; HEL, helicase; POL, polymerase; TAR, transcriptional activation region. (B) Quantitative β-galactosidase assays for interaction between the helicase domain of the TMV replicase protein (nt 2508 to 3419) and the N-terminal 160-amino-acid NAC domain of ATAF2. (C) Coimmunoprecipitation (CO-IP) of ATAF2-GFP fusion protein and the TMV 126-kDa replicase protein within infected and noninfected tissues. α-Rep, anti-Rep antibody; α-GFP, anti-GFP antibody.
FIG. 2.
FIG. 2.
TMV-directed degradation of ATAF2. (A) Fluorescent micrographs of transient ATAF2-GFP or GFP expression in mock- or TMV-infected N. benthamiana epidermal cells. Fluorescent images were taken 14 to 16 h after bombardment. The numbers of cells expressing detectable ATAF2-GFP or GFP fluorescent signal in mock- or TMV-infected tissues are shown. Cell numbers were averaged from 10 independent bombardment experiments. (B) The upper panel shows the reduction of ATAF2-GFP levels in either mock- or TMV-infected 35S::ATAF2-GFP transgenic plants at 6 dpi. The middle panel shows the Coomassie blue detection of total proteins loaded in each lane (SDS-PAGE loading control). Results are averaged from three independent experiments ± the standard deviations (SD). The lower panel shows the real-time qRT-PCR analysis of transcript levels for the ATAF2-GFP transgene in mock- and TMV-infected tissues. (C) The upper panel shows that proteasome inhibition via treatment with MG132 restores ATAF2-GFP accumulation in TMV-infected tissues. In the lower panel, results are presented as averages ± SD from three independent experiments. N/T, not tested.
FIG. 3.
FIG. 3.
Effects of ATAF2 expression on virus accumulation. (A) 35S::ATAF2-GFP transgenic Shahdara plants (4 weeks old) show a moderate developmental phenotype (upper); ATAF2-GFP accumulation was confirmed by Western immunoblot (WB) analysis for the detection of GFP (middle); and the accumulation of TMV CP in 35S::ATAF2-GFP transgenic lines is reduced compared to that of nontransformed control (cont.) plants (lower). The means and standard deviations are averaged from three independent experiments using two different ATAF2 overexpression lines. α-GFP, anti-GFP antibody. (B) Six-week-old ATAF2 T-DNA knockout line Salk_136355 shows no obvious phenotype compared to that of nontransformed Columbia plants (upper); the knockout of ATAF2 was confirmed by RT-PCR analysis (middle); and TMV CP levels are not significantly different between nontransformed and ATAF2 T-DNA knockout lines (lower). Results represent the means ± standard deviations of three independent experiments.
FIG. 4.
FIG. 4.
Both ATAF2 and the defense gene PR1 are induced in locally but not systemically infected tissues. (A) The upper panel shows transgenic Shahdara lines carrying the ATAF2 promoter fused to a GUS reporter gene (PATAF2::GUS) that were inoculated with TMV and sampled for GUS activities. For the middle panel, after histochemical staining for GUS activity, individual leaves were analyzed for CP content by Western immunoblotting. The lower panel shows a tissue print immunoblotting method employed to monitor the pattern of virus accumulation in TMV-inoculated leaf tissues. (B) Induction of GUS activity was not observed in systemically infected tissues at 14 dpi. (C) Real-time qRT-PCR analysis monitoring the expression levels of ATAF2 and defense genes PDF1.2 and PR1 in both TMV-inoculated and systemically infected tissues of A. thaliana ecotype Shahdara plants. Total RNA samples were derived from three to five independent test plants. Changes (n-fold) in gene expression are presented as levels relative to those of the mock-inoculated tissue. Data represent averages ± standard deviations of three real-time qRT-PCR replicates.
FIG. 5.
FIG. 5.
Correlated induction of ATAF2 and defense-related marker genes. (A) ATAF2 overexpression stimulates the expression of host basal defense genes PDF1.2, PR1, and PR2. Real-time qRT-PCR analysis obtained from two representative 35S::ATAF2-GFP overexpression lines. (B) Expression of PDF1.2, PR1, and PR2 is reduced in ATAF2 knockout line SALK_136355. (C) Analyses of two representative 35S::ATAF2-SRDX repressor lines show reduced expression levels of PR1 and PR2 but not PDF1.2. Total RNA samples were derived from three to five independent test plants. Changes (n-fold) in gene expression are presented as levels relative to those of nontransformed plants. Data represent averages ± standard deviations of three real-time qRT-PCR replicates.
FIG. 6.
FIG. 6.
TMV infection inhibits SA activation of ATAF2 and the defense marker gene PR1. (A) SA inhibits TMV accumulation. TMV accumulation was monitored in 4-week-old A. thaliana ecotype Shahdara plants sprayed with water, 0.1 mM SA, or 0.5 mM SA 4 h prior to virus inoculation. Data are displayed as the means ± standard deviations of three replicates. (B) Systemic tissues from PATAF2::GUS transgenic plants, either mock or TMV inoculated (14 dpi), were sprayed with H2O (lanes 5 and 6) or 1 mM SA (lanes 1, 2, 3, and 4). Two and 4 h posttreatment the leaves were assayed for GUS activity (upper), and Western immunoblotting (WB) for the detection of the TMV CP was used to confirm systemic infection in the tested tissues (lower). (C) Real-time qRT-PC analysis showing the induction of ATAF2 and PR1 by SA (0.1 mM) in TMV-infected systemic tissue compared to induction for mock-infected tissue at 2 and 4 h posttreatment. Total RNA samples were derived from three to five independent test plants. Changes (n-fold) in gene expression are presented as levels relative to those of uninfected water-treated systemic tissues. Data represent averages ± standard deviations from three real-time qRT-PCR replicates.
FIG. 7.
FIG. 7.
ATAF2 plays a regulatory role in SA-mediated defense activation. Four-week-old mature leaves were sprayed with 0.1 mM SA, and leaf tissue was collected 4 h after SA treatment. Gene expression is presented relative to the levels observed in water-treated nontransformed plants. (A) ATAF2 knockout line SALK_136355 shows reduced PR1 induction after SA treatment compared to that of nontransformed A. thaliana ecotype Columbia control plants. (B) Two representative ATAF2-SRDX repressor lines show reduced PR1 levels after SA treatment. Total RNA was derived from three to five independent test plants. Data represent the averages ± standard deviations from three real-time qRT-PCR replicates.

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