The Arabidopsis extracellular UNUSUAL SERINE PROTEASE INHIBITOR functions in resistance to necrotrophic fungi and insect herbivory
Version of Record online: 22 AUG 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 68, Issue 3, pages 480–494, November 2011
How to Cite
Laluk, K. and Mengiste, T. (2011), The Arabidopsis extracellular UNUSUAL SERINE PROTEASE INHIBITOR functions in resistance to necrotrophic fungi and insect herbivory. The Plant Journal, 68: 480–494. doi: 10.1111/j.1365-313X.2011.04702.x
- Issue online: 25 OCT 2011
- Version of Record online: 22 AUG 2011
- Accepted manuscript online: 12 JUL 2011 04:29PM EST
- Received 17 May 2011; revised 21 June 2011; accepted 4 July 2011.
Figure S1.UPI is not required for wound-induced resistance to B. cinerea. Disease symptoms of non-wounded (upper row) and forceps-wounded (bottom row) Arabidopsis leaves 3 days post-drop-inoculation with B. cinerea (5 µl, 3 × 105 spores/ml). Experiments were repeated at least three times with similar results. Wt, wild-type.
Figure S2.upi mutation alters induction of PR-3 and PR-4 but not PR-1 and PDF1.2. B. cinerea-induced expression of (a) PR-1, (b) PDF1.2, (c) PR-3, and (d) PR-4. Plants were spray-inoculated with a conidial-suspension (2.5 × 104 spores/ml). hpi, hours post-infection. Experiments were performed as described in the Methods and repeated at least three times with similar results. Wt, wild-type.
Figure S3. Genes co-expressed with UPI. Data generated by mutual rank (MR) based on weighted Pearson's correlation coefficients from publicly available microarrays (Obayashi et al., 2009). The highly co-expressed protease (At1g73260) was assayed for in vitro interaction with UPI.
Figure S4. RT-PCR analysis of UPI transcript. Primers were designed based on the sequences of the UPI cDNA clones S62951 (GenBank: BT010789) and U62959 (GenBank) including its 5′ untranslated region (UTR) (Yamada et al., 2003) and upstream genomic sequences. Arrows indicate locations of primers used for analysis. Lower panel shows RT-PCR results using the following primer combinations: Lane (L) 1: 1, R1; L2: 2, R1; L3: 3, R1; L4: 4, R2; L5: 5, R2; L6: 6, R2. The locations of primers 1 and R1 indicate the start and stop codons of the coding region, respectively whereas primer 2 denotes the start of the 5′ UTR.
Figure S5. Transient expression of UPI-GFP in N. benthamiana. GFP and UPI-GFP were transiently expressed in N. benthamiana leaf cells. Localization of proteins was examined using appropriate fluorescence, brightfield, and images merged to determine subcellular localization. Experiments were repeated at least three times with similar results.
Figure S6.UPI-HA expression restores upi phenotypes to wild-type. The restored (a) flowering, and resistance to (b) B. cinerea and (c) A. brassicicola of upi plants expressing 35S:UPI-HA. Experiments were performed as described in the Methods and repeated at least three times with similar results. Images (b) and (c) taken were taken 4 days post-inoculation. Wt, wild-type.
Figure S7. UPI-GST does not affect B. cinerea growth. Plates were supplemented with 5–7 µg UPI-GST or GST. Each plate was inoculated with a 2 mm punch of 10-day old B. cinerea growing on V8 media. Growth inhibition of protein supplemented plates was visually assessed daily relative to un-supplemented plates for each respective media type. Image taken 10 days post-inoculation. Experiments were repeated at least three times with similar results.
Figure S8.upi grafted to wild-type and reciprocal grafts do not affect flowering time compared to non-grafted controls. Labeling indicates which genotypes were grafted in which manner: labels above the line denote the shoot genotype that was grafted to the root genotype indicated below the line. Experiments were repeated at least three times with similar results. Wt, wild-type.
Figure S9. The upi mutant is not altered in the triple response. Seeds were surface-sterilized and plated on ACC- or un-supplemented MS media. Plates were kept in the dark and incubated 2 days at 4°C followed by 3 days at 23 ± 2°C. Experiments were repeated at least three times with similar results. Wt, wild-type.
Figure S10.upi displays wild-type germination responses to different phytohormones and abiotic stresses. Germination responses were assayed as described in the Methods. Image taken 7 days post-germination. Experiments were repeated at least three times with similar results. Wt, wild-type.
Figure S11.upi plants are not impaired in oxidative stress tolerance. Plants were inoculated with 5 µl of the indicated concentrations of methyl-viologen (MV) or water and kept in direct light for 2 days. Experiments were repeated at least three times with similar results. Wt, wild-type.
Table S1. Arabidopsis genotypes with altered flowering phenotypes and significantly reduced UPI expression.
Table S2. List of primers used in UPI study. Primer sequences read 5′–3′.
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