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Science 338 (6107): 659-662

Copyright © 2012 by the American Association for the Advancement of Science

Tricking the Guard: Exploiting Plant Defense for Disease Susceptibility

J. Lorang1, T. Kidarsa1,*, C. S. Bradford1,{dagger}, B. Gilbert1, M. Curtis1, S.-C. Tzeng2, C. S. Maier2, and T. J. Wolpert1,{ddagger}

1 Department of Botany and Plant Pathology and Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA.
2 Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
* Present address: Horticultural Crops Research Lab, USDA-ARS, Corvallis, OR 97331, USA.
{dagger} Present address: Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA.


Figure 1
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Fig. 1. Victorin binds to TRX-h5. (A and B) Immunoblot of wild-type and mutant, myc-tagged TRX-h5 proteins from Arabidopsis leaves hours after treatment (hpi) with victorin (A) or biotinylated victorin (B). Proteins were detected with antibody to myc (A) or immunoprecipitated with antibody to myc and detected with antibodies to biotin and myc (B). Victorin binding is seen as a ~1-kD increase in the apparent mass of TRX-h5. (C) Purified, His-tagged TRX-h5 incubated in vitro with 1 mM dithiothreitol (DTT) and 10 μM native victorin or victorin in which the aldehyde moiety has been reduced to a primary alcohol (Vic-OH). Protein detected by silver staining. (D) Purified, His-tagged TRX-h5 incubated in vitro with or without 10 μM victorin, 1 mM DTT, and 1 mM N-ethylmaleimide and detected by Coomassie staining.

 

Figure 2
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Fig. 2. Victorin inhibits thioredoxin activity. (A) Left: TRX-h5–catalyzed reduction of insulin measured by change in optical density at 650 nm ({Delta}OD650) of insulin (0.1% w/v) in the presence of 1 mM DTT (solid circles) or 1 mM DTT and 1 μM (solid squares), 5 μM (triangles), or 10 μM (open circles) victorin. Open squares denote insulin reduction in the presence of 1 mM DTT without TRX-h5. Right: Relative specific activity of TRX-h5 in the presence of 0, 1, 5, or 10 μM victorin. Error bars represent SE from triplicate assays. (B) Quantitative reverse transcription polymerase chain reaction analysis of PR1 from leaves of 3-week-old Arabidopsis (lov1, lov1) grown in hydroponic solution and transferred to water or victorin (20 μg/ml) 48 hours before spraying with water or 1 mM salicylic acid (SA). RNA was isolated 24 hours after SA treatment. SE bars represent N = 8 (4 biological x 2 technical replicates). Data were reproducible in separate experiments. (C) Colony-forming units of Pseudomonas syringae pv. maculicola (Psm) from 3-week-old Arabidopsis (lov1, lov1) leaves 2 days after treatment with Psm, 0.1 OD600. Leaves were infiltrated with water or victorin (100 μg/ml) 2 hours before treatment with Psm. SE bars represent N = 6 biological replicates containing three leaf discs each.

 

Figure 3
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Fig. 3. LOV1 guards TRX-h5. (A) Transient expression of LOV1 (left), TRX-h5 (center), or LOV1 and TRX-h5 (right) in N. benthamiana infiltrated with water or victorin (1 μg/ml). (B) Yeast two-hybrid assay of LOV1, TRX-h5, and control proteins on SD/-Trp/-His medium (left) and SD/-Leu/-Trp/-His medium (right). (C) Confocal fluorescence microscopy of N. benthamiana leaves transiently expressing proteins tagged with yellow fluorescent protein (YFP) or split-YFP* (BiFC). From left to right: top row, 35S:LOV1-YFP; 35S:TRX-h5-YFP; 35S:YFP-RPS5; 35S:cYFP-LOV1+35S:nYFP-GST; bottom row, 35S:cYFP-LOV1+35S:nYFP-TRX-h5; 35S:cYFP-GST+35S:nYFP-TRX-h5; 35S:cYFP-RPS5+35S:nYFP-TRX-h5; 35S:cYFP+35S:nYFP-GST. Red, chlorophyll autofluorescence; yellow, YFP fluorescence. Scale bars, 10 μm. (D) Immunoblot of 10 μg of soluble (SOL), other membrane (OM), or plasma membrane (PM) protein fractions from two-phase extraction of N. benthamiana transiently expressing LOV1 and myc-TRX-h5. H+ATPase is used as a plasma membrane marker; Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) is used as a marker for soluble proteins.

 


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