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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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Early responses are reduced in thechanged calcium elevation 5(cce5) mutants compared to the parental HVA1 line. Seedlings (~8 days old) were elicited with 1 μM of flg22 (A) or 1 μM elf18 (B) and the calcium levels measured. Relative calcium levels are depicted as L/Lmax ratio (where L/Lmax = luminescence counts per sec/total luminescence counts remaining). Error bars represent standard deviation (n > 12 seedlings). MAPK activation is shown by immunoblotting (α-pTEpY) for the phosphorylated MAPKs after 1 μM elf18 elicitation (C). Amido black staining of the nitrocellulose membrane was used to estimate equal loading. For MAPK activation after flg22 treatment, see Additional file 1: Figure S1.
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Fig1: Early responses are reduced in thechanged calcium elevation 5(cce5) mutants compared to the parental HVA1 line. Seedlings (~8 days old) were elicited with 1 μM of flg22 (A) or 1 μM elf18 (B) and the calcium levels measured. Relative calcium levels are depicted as L/Lmax ratio (where L/Lmax = luminescence counts per sec/total luminescence counts remaining). Error bars represent standard deviation (n > 12 seedlings). MAPK activation is shown by immunoblotting (α-pTEpY) for the phosphorylated MAPKs after 1 μM elf18 elicitation (C). Amido black staining of the nitrocellulose membrane was used to estimate equal loading. For MAPK activation after flg22 treatment, see Additional file 1: Figure S1.

Mentions: Four other independently isolated changed calcium elevation (cce) mutants did not restore a “normal” calcium response to flg22 in the F1 generation when crossed to the previously described cce5 mutant [26] (data not shown). The lack of complementation suggests that these five cce mutants are allelic and thus designated as cce5-1 to cce5-5. All five cce5 mutant lines show a reduced flg22- and elf18-induced calcium rise compared to the parental HVA1 line; however, the reduction in the elf18-induced calcium levels appears to be stronger than with flg22 (Figure 1A, B). Correspondingly, elf18-induced MAPK activation was partially reduced and delayed (Figure 1C). Surprisingly, the reduction in flg22-induced MAPK activation was not as obvious as for elf18. It was only visible if a lower concentration (e.g. 10 nM) of the flg22 peptide was used; at higher concentrations, no difference in comparison to HVA1 was discernible (Additional file 1: Figure S1). Thus, CCE5 may have different signaling role(s) for these two MAMPs. Similarly, other rapid responses such as reactive oxygen species (ROS) accumulation was also reduced for the cce5 alleles, when treated with flg22 or elf18 (Additional file 1: Figure S2). Since MAPK activation and ROS accumulation occur within minutes upon elicitation, cce5 is “mutated” in some early signaling component(s).Figure 1


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)

Early responses are reduced in thechanged calcium elevation 5(cce5) mutants compared to the parental HVA1 line. Seedlings (~8 days old) were elicited with 1 μM of flg22 (A) or 1 μM elf18 (B) and the calcium levels measured. Relative calcium levels are depicted as L/Lmax ratio (where L/Lmax = luminescence counts per sec/total luminescence counts remaining). Error bars represent standard deviation (n > 12 seedlings). MAPK activation is shown by immunoblotting (α-pTEpY) for the phosphorylated MAPKs after 1 μM elf18 elicitation (C). Amido black staining of the nitrocellulose membrane was used to estimate equal loading. For MAPK activation after flg22 treatment, see Additional file 1: Figure S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Early responses are reduced in thechanged calcium elevation 5(cce5) mutants compared to the parental HVA1 line. Seedlings (~8 days old) were elicited with 1 μM of flg22 (A) or 1 μM elf18 (B) and the calcium levels measured. Relative calcium levels are depicted as L/Lmax ratio (where L/Lmax = luminescence counts per sec/total luminescence counts remaining). Error bars represent standard deviation (n > 12 seedlings). MAPK activation is shown by immunoblotting (α-pTEpY) for the phosphorylated MAPKs after 1 μM elf18 elicitation (C). Amido black staining of the nitrocellulose membrane was used to estimate equal loading. For MAPK activation after flg22 treatment, see Additional file 1: Figure S1.
Mentions: Four other independently isolated changed calcium elevation (cce) mutants did not restore a “normal” calcium response to flg22 in the F1 generation when crossed to the previously described cce5 mutant [26] (data not shown). The lack of complementation suggests that these five cce mutants are allelic and thus designated as cce5-1 to cce5-5. All five cce5 mutant lines show a reduced flg22- and elf18-induced calcium rise compared to the parental HVA1 line; however, the reduction in the elf18-induced calcium levels appears to be stronger than with flg22 (Figure 1A, B). Correspondingly, elf18-induced MAPK activation was partially reduced and delayed (Figure 1C). Surprisingly, the reduction in flg22-induced MAPK activation was not as obvious as for elf18. It was only visible if a lower concentration (e.g. 10 nM) of the flg22 peptide was used; at higher concentrations, no difference in comparison to HVA1 was discernible (Additional file 1: Figure S1). Thus, CCE5 may have different signaling role(s) for these two MAMPs. Similarly, other rapid responses such as reactive oxygen species (ROS) accumulation was also reduced for the cce5 alleles, when treated with flg22 or elf18 (Additional file 1: Figure S2). Since MAPK activation and ROS accumulation occur within minutes upon elicitation, cce5 is “mutated” in some early signaling component(s).Figure 1

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