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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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Scheme ofPBL1(At3g55450) gene structure and mutations in thecce5alleles. Genomic DNA was prepared from the five cce5 mutants and the PBL1 gene amplified by PCR and sequenced. The detected single nucleotide polymorphisms (SNPs) and the resulting amino acid exchanges are indicated above the exons. The gene model At3g55450.1 encoding a protein of 389 amino acids is used to designate the position of the amino acid exchanges. Locations of key kinase domains (such as ATP binding site and the kinase active center), relative to the corresponding mutations, are marked.
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Fig4: Scheme ofPBL1(At3g55450) gene structure and mutations in thecce5alleles. Genomic DNA was prepared from the five cce5 mutants and the PBL1 gene amplified by PCR and sequenced. The detected single nucleotide polymorphisms (SNPs) and the resulting amino acid exchanges are indicated above the exons. The gene model At3g55450.1 encoding a protein of 389 amino acids is used to designate the position of the amino acid exchanges. Locations of key kinase domains (such as ATP binding site and the kinase active center), relative to the corresponding mutations, are marked.

Mentions: To identify the CCE5 gene, an F2 population was generated by crossing cce5-1 with the Arabidopsis accession Ler-0. Segregation analysis with 36 F2 plants indicated that CCE5 is linked to the aequorin transgene, and located on chromosome 3 between the INDEL markers CER460928 (1 recombinant) and 473892 (1 recombinant) [30]. The map positions of CER460928 and 473892 are 17.243303 and 21.186345 Mbp (based on TAIR 10). This interval comprises 1107 gene loci, including the PBS1-like 1 gene (PBL1, At3g55450) that encodes a receptor-like cytoplasmic kinase (RLCK). Sequencing of the PBL1 gene of the cce5 mutants revealed single nucleotide polymorphisms (SNP) in all five cce5 alleles, but not in the PBL1 sequences from two other cce mutants, cce7 and cce8 [26]. These SNPs lead to two premature stops (cce5-2/R110- and cce5-4/Q272-) and three amino acid exchanges (cce5-1/G70D, cce5-3/A97V and cce5-5/R172Q) in the PBL1 sequence (Figure 4). Two gene models are predicted for PBL1 transcripts in the TAIR database, but since we could not detect transcripts for the predicted alternatively spliced gene model At3g55450.2 (data not shown), we used the 389 amino acid long PBL1 protein (predicted by the gene model At3g55450.1) to designate the positions of the amino acid exchanges in the cce5 mutated proteins.Figure 4


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)

Scheme ofPBL1(At3g55450) gene structure and mutations in thecce5alleles. Genomic DNA was prepared from the five cce5 mutants and the PBL1 gene amplified by PCR and sequenced. The detected single nucleotide polymorphisms (SNPs) and the resulting amino acid exchanges are indicated above the exons. The gene model At3g55450.1 encoding a protein of 389 amino acids is used to designate the position of the amino acid exchanges. Locations of key kinase domains (such as ATP binding site and the kinase active center), relative to the corresponding mutations, are marked.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Scheme ofPBL1(At3g55450) gene structure and mutations in thecce5alleles. Genomic DNA was prepared from the five cce5 mutants and the PBL1 gene amplified by PCR and sequenced. The detected single nucleotide polymorphisms (SNPs) and the resulting amino acid exchanges are indicated above the exons. The gene model At3g55450.1 encoding a protein of 389 amino acids is used to designate the position of the amino acid exchanges. Locations of key kinase domains (such as ATP binding site and the kinase active center), relative to the corresponding mutations, are marked.
Mentions: To identify the CCE5 gene, an F2 population was generated by crossing cce5-1 with the Arabidopsis accession Ler-0. Segregation analysis with 36 F2 plants indicated that CCE5 is linked to the aequorin transgene, and located on chromosome 3 between the INDEL markers CER460928 (1 recombinant) and 473892 (1 recombinant) [30]. The map positions of CER460928 and 473892 are 17.243303 and 21.186345 Mbp (based on TAIR 10). This interval comprises 1107 gene loci, including the PBS1-like 1 gene (PBL1, At3g55450) that encodes a receptor-like cytoplasmic kinase (RLCK). Sequencing of the PBL1 gene of the cce5 mutants revealed single nucleotide polymorphisms (SNP) in all five cce5 alleles, but not in the PBL1 sequences from two other cce mutants, cce7 and cce8 [26]. These SNPs lead to two premature stops (cce5-2/R110- and cce5-4/Q272-) and three amino acid exchanges (cce5-1/G70D, cce5-3/A97V and cce5-5/R172Q) in the PBL1 sequence (Figure 4). Two gene models are predicted for PBL1 transcripts in the TAIR database, but since we could not detect transcripts for the predicted alternatively spliced gene model At3g55450.2 (data not shown), we used the 389 amino acid long PBL1 protein (predicted by the gene model At3g55450.1) to designate the positions of the amino acid exchanges in the cce5 mutated proteins.Figure 4

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