Overexpression of OsMed16 Inhibits the Growth of Rice and Causes Spontaneous Cell Death

The Mediator complex transduces information from the DNA-bound transcription factors to the RNA polymerase II transcriptional machinery. Research on plant Mediator subunits has primarily been performed in Arabidopsis, while very few of them have been functionally characterized in rice. In this study, the rice Mediator subunit 16, OsMed16, was examined. OsMed16 encodes a putative protein of 1301 amino acids, which is longer than the version previously reported. It was expressed in various rice organs and localized to the nucleus. The knockout of OsMed16 resulted in rice seedling lethality. Its overexpression led to the retardation of rice growth, low yield, and spontaneous cell death in the leaf blade and sheath. RNA sequencing suggested that the overexpression of OsMed16 altered the expression of a large number of genes. Among them, the upregulation of some defense-related genes was verified. OsMed16 can regulate the expression of a wealth of genes, and alterations in its expression have a profound impact on plant growth, development, and defense responses in rice.

The TIR-NBS-LRR protein CSA1 is required for autoimmune cell death in Arabidopsis pattern recognition co-receptor bak1 and bir3 mutants

The BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed in bak1 knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutant bak1 bir3 genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocks bak1 and bak1 bir3-mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.

Dual Roles of GSNOR1 in Cell Death and Immunity in Tetraploid Nicotiana tabacum

S-nitrosoglutathione reductase 1 (GSNOR1) is the key enzyme that regulates cellular homeostasis of S-nitrosylation. Although extensively studied in Arabidopsis, the roles of GSNOR1 in tetraploid Nicotiana species have not been investigated previously. To study the function of NtGSNOR1, we knocked out two NtGSNOR1 genes simultaneously in Nicotiana tabacum using clustered regularly interspaced short palindromic repeats (CRISPR)/caspase 9 (Cas9) technology. To our surprise, spontaneous cell death occurred on the leaves of the CRISPR/Cas9 lines but not on those of the wild-type (WT) plants, suggesting that NtGSNOR1 negatively regulates cell death. The natural cell death on the CRISPR/Cas9 lines could be a result from interactions between overaccumulated nitric oxide (NO) and hydrogen peroxide (H2O2). This spontaneous cell death phenotype was not affected by knocking out two Enhanced disease susceptibility 1 genes (NtEDS11a/1b) and thus was independent of the salicylic acid (SA) pathway. Unexpectedly, we found that the NtGSNOR1a/1b knockout plants displayed a significantly (p < 0.001) enhanced resistance to paraquat-induced cell death compared to WT plants, suggesting that NtGSNOR1 functions as a positive regulator of the paraquat-induced cell death. The increased resistance to the paraquat-induced cell death of the NtGSNOR1a/1b knockout plants was correlated with the reduced level of H2O2 accumulation. Interestingly, whereas the N gene-mediated resistance to Tobacco mosaic virus (TMV) was significantly enhanced (p < 0.001), the resistance to Pseudomonas syringae pv. tomato DC3000 was significantly reduced (p < 0.01) in the NtGSNOR1a/1b knockout lines. In summary, our results indicate that NtGSNOR1 functions as both positive and negative regulator of cell death under different conditions and displays distinct effects on resistance against viral and bacterial pathogens.

Overexpression of OsMed16 inhibits rice growth and causes spontaneous cell death

Background The Mediator complex transduces information from the DNA-bound transcription factors to the RNA polymerase II transcriptional machinery. Research on plant Mediator subunits was mainly performed in Arabidopsis, while very few of them have been functionally characterized in rice. Results Here the rice Mediator subunit 16, OsMed16, was studied. OsMed16 encoded a putative protein of 1301 amino acids, which is longer than the reported version. It was expressed in various rice organs, and localized in nucleus. Knockout of OsMed16 caused rice seedling lethality. Its overexpression led to rice growth retardation, low yield, and spontaneous cell death in leaf blade and leaf sheath. RNA sequencing suggested that overexpression of OsMed16 altered the expression of a large number of genes. Among them, the up-regulation of some defense-related genes was verified. Conclusions Our results demonstrated that OsMed16 can regulate the expression of a wealth of genes, and alterations in its expression have profound impact on plant growth, development and defense response in rice.

Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.