Knocking Out the Wall: Protocols for Gene Targeting in Physcomitrella patens

Roberts, Alison W.; Dimos, Christos S.; Goss, Chessa A.; Lai, Virginia

doi:10.1007/978-1-61779-008-9_19

Cited by 35 publications

(70 citation statements)

References 35 publications

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“…The CESA8KO vector was cut with EcoRI and NsiI for transformation into wild type P. patens. The CESA3KO, CESA4KO, CESA6/7KO, and CESA10KO vectors were constructed using Gateway Multisite Pro cloning (Invitrogen) as described previously (Roberts et al, 2011). Flanking sequences 59 and 39 of the coding regions were amplified with appropriate primer pairs (Supplemental Table S1) using Phusion DNA polymerase (New England Biolabs) and cloned into pDONR 221 P1-P4 and pDONR 221 P3-P2, respectively, using BP Clonase II (Invitrogen).…”

Section: Vector Constructionmentioning

confidence: 99%

“…All entry clones were sequence verified. For vectors conferring hygromycin resistance, entry clones with flanking sequences in pDONR 221 P1-P4 and pDONR 221 P3-P2 were inserted into BHSNRG (Roberts et al, 2011). For vectors conferring G418 resistance, entry clones with flanking sequences in pDONR 221 P1-P4 and pDONR 221 P3-P2 were linked with the entry clone containing the nph selection cassette and inserted into pGEM-gate (Vidali et al, 2009) using LR Clonase II Plus (Invitrogen).…”

Section: Vector Constructionmentioning

confidence: 99%

“…Wild type and transformed P. patens lines were cultured on basal medium supplemented with ammonium tartrate (BCDAT) as described previously (Roberts et al, 2011). Protoplasts were prepared and transformed as described previously (Roberts et al, 2011).…”

Section: Culture and Transformation Of P Patensmentioning

confidence: 99%

See 2 more Smart Citations

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Norris

Huang

et al. 2017

Plant Physiol.

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Section: Vector Constructionmentioning

confidence: 99%

Section: Vector Constructionmentioning

confidence: 99%

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Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Norris

Huang

et al. 2017

Plant Physiol.

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“…Tissue Culture of P. patens and Generation of pdk1 Knockout and PpPDK1-6His Constructs Wild-type P. patens cv Gransden (Ashton and Cove, 1977) was routinely grown on BCD plates (Roberts et al, 2011) overlaid with cellophane discs (AA Packaging) at 25°C in continuous light following standard protocols (Roberts et al, 2011). Seven days before transformation, wild-type P. patens was passaged to BCD plates containing 5 mM diammonium tartrate.…”

Section: Protein-lipid Overlaysmentioning

confidence: 99%

“…To transform P. patens, 50 mg of each construct was linearized by simultaneous digestion with 80 units of HindIII and SpeI for pdk1 knockout generation or XbaI and SpeI for the PpPDK1 gene replacement. Linearized DNA was introduced into wild-type P. patens cv Gransden protoplasts using a previously described polyethylene glycol transformation protocol (Roberts et al, 2011). Transformants were initially selected by 7 d of growth on plates containing 20 mg mL 21 hygromycin or 25 mg mL 21 G418 followed by 10 d of growth on nonselective plates and an additional 7 d of growth on hygromycin or G418 plates.…”

Section: Protein-lipid Overlaysmentioning

confidence: 99%

Characterization of a PDK1 Homologue from the Moss Physcomitrella patens

Dittrich

Devarenne

2011

Plant Physiology

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The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases, PDK1 controls many basic cellular processes, from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms, it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants, we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1), a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However, unlike PDK1 from other plants, the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain, which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site, indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.

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Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms

et al. 2021

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Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes.Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

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Knocking Out the Wall: Protocols for Gene Targeting in Physcomitrella patens

Cited by 35 publications

References 35 publications

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Characterization of a PDK1 Homologue from the Moss Physcomitrella patens

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms

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