EDS1 complexes are not required for PRR responses and execute TNL‐ETI from the nucleus in Nicotiana benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown

Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing

Abstract Purpose The study aimed to compare the effect of mincing bovine articular cartilage with different shaver blades on chondrocyte viability. Methods Bovine articular cartilage was harvested either with a scalpel or with three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage harvested with a scalpel was then minced into fragments smaller than 1 mm3 with a scalpel. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, the following measurements were performed: metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1 and ACAN) and catabolic genes (MMP1 and MMP13), live/dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes. Results Mincing the cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p < 0.001). Gene expression of anabolic genes (COL2A1 and ACAN) was reduced, while catabolic genes (MMP1 and MMP13) were increased after day 7 in all shaver conditions. Confocal microscopy showed a thin line of dead cells at the lesion side with viable cells beneath for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p < 0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Conclusion Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing immediately after harvest and after seven days in culture. This suggests that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Level of evidence Level II therapeutic study

Differential requirement for the EDS1 catalytic triad in A. thaliana and N. benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Biochemical activities underlying these mechanistic frameworks remain unknown. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, mutations within the catalytic triad of Solanaceae EDS1 can abolish or enhance TNL immunity in N. benthamiana. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Dependency of Solanaceae but not A. thaliana EDS1 on catalytic triad residues raises the possibility that a TNL-derived small molecule binds to the Solanaceae EDS1 lipase-like domain, and that EDS1 lipase-like domain pocket contributions to TNL immune responses vary between lineages. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown

Differential requirement for the EDS1 catalytic triad in A. thaliana and N. benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Biochemical activities underlying these mechanistic frameworks remain unknown. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, mutations within the catalytic triad of Solanaceae EDS1 can abolish or enhance TNL immunity in N. benthamiana. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Dependency of Solanaceae but not A. thaliana EDS1 on catalytic triad residues raises the possibility that a TNL-derived small molecule binds to the Solanaceae EDS1 lipase-like domain, and that EDS1 lipase-like domain pocket contributions to TNL immune responses vary between lineages. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown

EDS1 complexes are not required for PRR responses and execute TNL‐ETI from the nucleus in Nicotiana benthamiana

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown