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Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1.

Ranf S, Eschen-Lippold L, Fröhlich K, Westphal L, Scheel D, Lee J - BMC Plant Biol. (2014)

Bottom Line: They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers.Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling.

View Article: PubMed Central - PubMed

Affiliation: Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany. ranf@wzw.tum.de.

ABSTRACT

Background: Plant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.

Results: We characterized here the "changed calcium elevation 5" (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.

Conclusions: Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction - perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components.

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MAPK activation profile in the T-DNA insertionpbl1,bik1orpbl1bik1double mutants. 14-day-old liquid-grown seedlings were equilibrated in 1.5 ml of fresh MS medium for ~24 h, and elicited by adding 0.5 ml of media containing a 4-fold concentrated stock of the indicated MAMP/DAMPs. Samples were collected at the indicated time points (min) after treatment and proteins were extracted for immunoblotting to detect phosphorylated (i.e. activated) forms of the MAPKs. Amido black staining of the nitrocellulose membranes was used to estimate equal loading.
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Fig7: MAPK activation profile in the T-DNA insertionpbl1,bik1orpbl1bik1double mutants. 14-day-old liquid-grown seedlings were equilibrated in 1.5 ml of fresh MS medium for ~24 h, and elicited by adding 0.5 ml of media containing a 4-fold concentrated stock of the indicated MAMP/DAMPs. Samples were collected at the indicated time points (min) after treatment and proteins were extracted for immunoblotting to detect phosphorylated (i.e. activated) forms of the MAPKs. Amido black staining of the nitrocellulose membranes was used to estimate equal loading.

Mentions: Since calcium acts upstream of MAPK activation [9,22], we analyzed MAPK activation in the T-DNA mutants of PBL1, BIK1 and the double mutant. However, there was no reduction in MAPK activation by flg22, elf18 and Pep1 in pbl1, bik1 and pbl1bik1 compared to their Col-0 (pMAQ2) background line (Figure 7). This is in contrast to the reduced elf18-induced MAPK activation (Figure 1C) and the dose dependent reduction in flg22-induced MAPK activation (Additional file 1: Figure S1) in the cce5 mutants. Since the reduction of MAPK activation could be seen in multiple cce5 lines, the difference is unlikely to be due to secondary mutations in the ems-mutagenized lines.Figure 7


Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1.

Ranf S, Eschen-Lippold L, Fröhlich K, Westphal L, Scheel D, Lee J - BMC Plant Biol. (2014)

MAPK activation profile in the T-DNA insertionpbl1,bik1orpbl1bik1double mutants. 14-day-old liquid-grown seedlings were equilibrated in 1.5 ml of fresh MS medium for ~24 h, and elicited by adding 0.5 ml of media containing a 4-fold concentrated stock of the indicated MAMP/DAMPs. Samples were collected at the indicated time points (min) after treatment and proteins were extracted for immunoblotting to detect phosphorylated (i.e. activated) forms of the MAPKs. Amido black staining of the nitrocellulose membranes was used to estimate equal loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279983&req=5

Fig7: MAPK activation profile in the T-DNA insertionpbl1,bik1orpbl1bik1double mutants. 14-day-old liquid-grown seedlings were equilibrated in 1.5 ml of fresh MS medium for ~24 h, and elicited by adding 0.5 ml of media containing a 4-fold concentrated stock of the indicated MAMP/DAMPs. Samples were collected at the indicated time points (min) after treatment and proteins were extracted for immunoblotting to detect phosphorylated (i.e. activated) forms of the MAPKs. Amido black staining of the nitrocellulose membranes was used to estimate equal loading.
Mentions: Since calcium acts upstream of MAPK activation [9,22], we analyzed MAPK activation in the T-DNA mutants of PBL1, BIK1 and the double mutant. However, there was no reduction in MAPK activation by flg22, elf18 and Pep1 in pbl1, bik1 and pbl1bik1 compared to their Col-0 (pMAQ2) background line (Figure 7). This is in contrast to the reduced elf18-induced MAPK activation (Figure 1C) and the dose dependent reduction in flg22-induced MAPK activation (Additional file 1: Figure S1) in the cce5 mutants. Since the reduction of MAPK activation could be seen in multiple cce5 lines, the difference is unlikely to be due to secondary mutations in the ems-mutagenized lines.Figure 7

Bottom Line: They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers.Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling.

View Article: PubMed Central - PubMed

Affiliation: Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle/Saale, D-06120, Germany. ranf@wzw.tum.de.

ABSTRACT

Background: Plant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.

Results: We characterized here the "changed calcium elevation 5" (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.

Conclusions: Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction - perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components.

Show MeSH
Related in: MedlinePlus