| Claim area | Key findings (with numbers) | Evidence type | Source (short citation with year) | URL | Publication date |
|---|---|---|---|---|---|
| Biochemical activity | PpMPK4a is a bona fide MAP kinase activated by phosphorylation on the TEY motif after elicitation; the MPK4a band corresponds to ~42.8 kD, and an MPK4a-GFP fusion appears at ~70 kD. Immunoprecipitated MPK4a-GFP phosphorylated myelin basic protein (MBP) in vitro after chitin treatment. No endogenous substrate was identified in the retrieved sources; MBP was used as a generic kinase substrate (pqac-00000000, pqac-00000001, pqac-00000006) | KI GFP line, anti-pTEpY immunoblot, in-gel kinase assay, GFP-trap immunoprecipitation kinase assay | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Activation stimuli | Two moss MPKs including MPK4a were rapidly activated by PAMPs including chitin/chitosan and peptidoglycan; activation was detectable within 1 min, and chitin assays commonly used 100 µg/mL chitin. MPK4a-GFP was also activated after Botrytis cinerea spore treatment, though more weakly than with soluble chitin (pqac-00000005, pqac-00000004, pqac-00000001) | Time-course elicitation, immunoblot, in-gel kinase assay, pathogen inoculation | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Negative stimuli / specificity | MPK4a was not activated by 500 mM NaCl, 800 mM mannitol, or 10 mM ABA in the reported assays. In the same study, osmotic stress instead activated SnRK2-class kinases around ~40 kD, supporting pathway specificity distinct from MPK4a (pqac-00000001, pqac-00000003) | Stress treatments, comparative kinase assays, anti-pTEpY immunoblot | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Substrates | The retrieved experimental work supports kinase activity toward the artificial substrate MBP; in-gel kinase assays also used peptide substrates including P3 in pathway-discrimination experiments. No physiological in vivo substrate of PpMPK4a was identified in the retrieved sources, so substrate specificity remains incompletely resolved experimentally for this protein (pqac-00000000, pqac-00000001, pqac-00000002) | In-gel kinase assay, immunoprecipitation kinase assay | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Localization | MPK4a-GFP localized to both cytoplasm and nucleus, with strongest signal in apical caulonemal cells and rhizoids / newly formed apical tip cells. The localization pattern did not change substantially after chitin treatment (pqac-00000000, pqac-00000001, pqac-00000007) | Knock-in GFP fusion, confocal microscopy | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Genetic phenotypes | Δmpk4a knockout lines were morphologically close to wild type under normal growth, unlike Arabidopsis mpk4 developmental mutants. However, Δmpk4a plants showed reduced chitin-induced cell wall depositions, reduced induction of defense genes, greater Evans blue staining after B. cinerea infection, and higher Alternaria brassicicola spore production than wild type, indicating impaired immunity (pqac-00000004, pqac-00000001, pqac-00000007) | Targeted knockout by homologous recombination, pathogen assays, staining, qRT-PCR | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Pathway placement | PpMPK4a functions in a canonical PAMP-triggered immunity MAPK cascade downstream of chitin perception and upstream of defense outputs. The study concludes that MPK4a primarily functions in pattern-triggered immunity rather than ABA/osmotic signaling; MPK4b likely provides partial redundancy. The pathway was described as requiring a chitin receptor plus upstream MEKK(s) and MKK(s) (pqac-00000005, pqac-00000003, pqac-00000004) | Genetic analysis, elicitor-response biochemistry, pathway inference from mutant/KI data | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Quantitative data | MPK4a transcript rose within 15 min after chitin, peaked at about 8-fold by 2 h, and returned near baseline by 8 h. MPK4b transcript abundance was reported to be ~20-fold lower than MPK4a in untreated plants. Pathogen assays quantified increased cell death at 2 days after inoculation with 2×10^5 B. cinerea spores/mL and increased A. brassicicola sporulation 4 days after inoculation with ~2,500 spores per plant; significance was reported at p<0.01 to p<0.001 for susceptibility phenotypes (pqac-00000000, pqac-00000001, pqac-00000004, pqac-00000007) | qRT-PCR, pathogen quantification, Evans blue assay, spore count assay | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |
| Methods | Key methods included targeted knockout/knock-in by homologous recombination; PEG-mediated moss protoplast transformation with 30 µg linearized DNA; selection with 50 µg/mL G418 or 30 µg/mL hygromycin B; in-gel kinase assays using 40 µg protein on 13% SDS-PAGE containing 14 µM MBP or peptide substrate; GFP-trap immunoprecipitation from extracts adjusted to 1 mg/mL; kinase reactions at 30°C for 30 min in 40 µL buffer with 12.5 µM ATP, 5 µg MBP, and 10 µCi γ-32P-ATP; anti-p42/p44-ERK used at 1:2000 and anti-GFP at 1:1000 (pqac-00000002, pqac-00000003) | Detailed biochemical and cell-biological protocols | Bressendorff et al., 2016 | https://doi.org/10.1105/tpc.15.00774 | June 2016 |


*Table: This table summarizes the experimentally supported functional annotation of Physcomitrium patens MPK4a (UniProt A9T142) from the retrieved literature. It highlights what is directly shown for kinase activity, activation context, localization, immune function, and the main quantitative methods and results.*