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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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In vivophosphorylation of PBL1 or BIK1 after flg22 treatment. Protoplasts were transfected with plasmids expressing HA-tagged BIK1 (A), PBL1 (B), or the indicated variants. After overnight expression of the proteins, the protoplasts were treated with 100 nM flg22 (10 min), harvested and subjected to western blotting with anti-HA. In vivo phosphorylation is implicated by a reduced mobility of the protein (highlighted with black arrowheads). Amido black staining of the nitrocellulose membranes was used to estimate equal loading. In (C), autophosphorylation of the immunoprecipitated kinases was used to determine if the kinase activities have been compromised by the mutations. The experiment was performed three times with similar outcome. Note that the autophosphorylation of the wild type (WT) kinases in the untreated protoplasts is variable and typically low but a weak band can be seen (indicated by asterisks). Autophosphorylation of the G70D, A97V and R172Q variants were always not visible (or lower than the wild type kinases).
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Fig8: In vivophosphorylation of PBL1 or BIK1 after flg22 treatment. Protoplasts were transfected with plasmids expressing HA-tagged BIK1 (A), PBL1 (B), or the indicated variants. After overnight expression of the proteins, the protoplasts were treated with 100 nM flg22 (10 min), harvested and subjected to western blotting with anti-HA. In vivo phosphorylation is implicated by a reduced mobility of the protein (highlighted with black arrowheads). Amido black staining of the nitrocellulose membranes was used to estimate equal loading. In (C), autophosphorylation of the immunoprecipitated kinases was used to determine if the kinase activities have been compromised by the mutations. The experiment was performed three times with similar outcome. Note that the autophosphorylation of the wild type (WT) kinases in the untreated protoplasts is variable and typically low but a weak band can be seen (indicated by asterisks). Autophosphorylation of the G70D, A97V and R172Q variants were always not visible (or lower than the wild type kinases).

Mentions: After MAMP stimulation of plants, a reduced mobility of PBL1 and BIK1 protein bands in polyacrylamide gels (i.e. a “mobility shift”), indicative of in vivo phosphorylation of the kinases, has been reported [34,35]. Since three of the cce5 alleles are predicted to encode PBL1 proteins with a single amino acid exchange (Figure 4), we tested these mutated PBL1 proteins as well as BIK1 with regard to the gel mobility shift. As a negative control, we mutated the presumed myristoylation site (G2A) of PBL1 and BIK1, which is expected to prevent the proteins from targeting to the plasma membrane. All these constructs were tagged with a C-terminal HA epitope for western blot detection and transiently expressed in Arabidopsis mesophyll protoplasts. A mobility shift could be seen for wild type PBL1 and BIK1 after flg22 treatment of the protoplasts, but not for the G2A myristoylation site variants and the G70D, A97V and R172Q PBL1 variants (Figure 8A, B). This indicates that there is no in vivo phosphorylation of the mutated protein variants after flg22 treatment.Figure 8


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)

In vivophosphorylation of PBL1 or BIK1 after flg22 treatment. Protoplasts were transfected with plasmids expressing HA-tagged BIK1 (A), PBL1 (B), or the indicated variants. After overnight expression of the proteins, the protoplasts were treated with 100 nM flg22 (10 min), harvested and subjected to western blotting with anti-HA. In vivo phosphorylation is implicated by a reduced mobility of the protein (highlighted with black arrowheads). Amido black staining of the nitrocellulose membranes was used to estimate equal loading. In (C), autophosphorylation of the immunoprecipitated kinases was used to determine if the kinase activities have been compromised by the mutations. The experiment was performed three times with similar outcome. Note that the autophosphorylation of the wild type (WT) kinases in the untreated protoplasts is variable and typically low but a weak band can be seen (indicated by asterisks). Autophosphorylation of the G70D, A97V and R172Q variants were always not visible (or lower than the wild type kinases).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: In vivophosphorylation of PBL1 or BIK1 after flg22 treatment. Protoplasts were transfected with plasmids expressing HA-tagged BIK1 (A), PBL1 (B), or the indicated variants. After overnight expression of the proteins, the protoplasts were treated with 100 nM flg22 (10 min), harvested and subjected to western blotting with anti-HA. In vivo phosphorylation is implicated by a reduced mobility of the protein (highlighted with black arrowheads). Amido black staining of the nitrocellulose membranes was used to estimate equal loading. In (C), autophosphorylation of the immunoprecipitated kinases was used to determine if the kinase activities have been compromised by the mutations. The experiment was performed three times with similar outcome. Note that the autophosphorylation of the wild type (WT) kinases in the untreated protoplasts is variable and typically low but a weak band can be seen (indicated by asterisks). Autophosphorylation of the G70D, A97V and R172Q variants were always not visible (or lower than the wild type kinases).
Mentions: After MAMP stimulation of plants, a reduced mobility of PBL1 and BIK1 protein bands in polyacrylamide gels (i.e. a “mobility shift”), indicative of in vivo phosphorylation of the kinases, has been reported [34,35]. Since three of the cce5 alleles are predicted to encode PBL1 proteins with a single amino acid exchange (Figure 4), we tested these mutated PBL1 proteins as well as BIK1 with regard to the gel mobility shift. As a negative control, we mutated the presumed myristoylation site (G2A) of PBL1 and BIK1, which is expected to prevent the proteins from targeting to the plasma membrane. All these constructs were tagged with a C-terminal HA epitope for western blot detection and transiently expressed in Arabidopsis mesophyll protoplasts. A mobility shift could be seen for wild type PBL1 and BIK1 after flg22 treatment of the protoplasts, but not for the G2A myristoylation site variants and the G70D, A97V and R172Q PBL1 variants (Figure 8A, B). This indicates that there is no in vivo phosphorylation of the mutated protein variants after flg22 treatment.Figure 8

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