Identification of multiple salicylic acid-binding proteins using two high throughput screens
Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA i...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-01-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2014.00777/full |
| _version_ | 1818911413900410880 |
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| author | Daniel F. Klessig |
| author_facet | Daniel F. Klessig |
| author_sort | Daniel F. Klessig |
| collection | DOAJ |
| description | Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. |
| first_indexed | 2024-12-19T22:58:19Z |
| format | Article |
| id | doaj.art-f7cc2a07285e4066931c1d53a8170c89 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-19T22:58:19Z |
| publishDate | 2015-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-f7cc2a07285e4066931c1d53a8170c892022-12-21T20:02:35ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2015-01-01510.3389/fpls.2014.00777122736Identification of multiple salicylic acid-binding proteins using two high throughput screensDaniel F. Klessig0Boyce Thompson Institute for Plant ResearchSalicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays.http://journal.frontiersin.org/Journal/10.3389/fpls.2014.00777/fullDisease ResistancePlant ImmunitySalicylic AcidSalicylic acid signalingsalicylic acid-binding proteins |
| spellingShingle | Daniel F. Klessig Identification of multiple salicylic acid-binding proteins using two high throughput screens Frontiers in Plant Science Disease Resistance Plant Immunity Salicylic Acid Salicylic acid signaling salicylic acid-binding proteins |
| title | Identification of multiple salicylic acid-binding proteins using two high throughput screens |
| title_full | Identification of multiple salicylic acid-binding proteins using two high throughput screens |
| title_fullStr | Identification of multiple salicylic acid-binding proteins using two high throughput screens |
| title_full_unstemmed | Identification of multiple salicylic acid-binding proteins using two high throughput screens |
| title_short | Identification of multiple salicylic acid-binding proteins using two high throughput screens |
| title_sort | identification of multiple salicylic acid binding proteins using two high throughput screens |
| topic | Disease Resistance Plant Immunity Salicylic Acid Salicylic acid signaling salicylic acid-binding proteins |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2014.00777/full |
| work_keys_str_mv | AT danielfklessig identificationofmultiplesalicylicacidbindingproteinsusingtwohighthroughputscreens |