Epstein-Barr virus reactivation in sepsis due to community-acquired pneumonia is associated with increased morbidity and an immunosuppressed host transcriptomic endotype.

Epstein-Barr virus (EBV) reactivation is common in sepsis patients but the extent and nature of this remains unresolved. We sought to determine the incidence and correlates of EBV-positivity in a large sepsis cohort. We also hypothesised that EBV reactivation would be increased in patients in whom relative immunosuppression was the major feature of their sepsis response. To identify such patients we aimed to use knowledge of sepsis response subphenotypes based on transcriptomic studies of circulating leukocytes, specifically patients with a Sepsis Response Signature endotype (SRS1) that we have previously shown to be associated with increased mortality and features of immunosuppression. We assayed EBV from the plasma of intensive care unit (ICU) patients with sepsis due to community-acquired pneumonia. In total 730 patients were evaluated by targeted metagenomics (n = 573 patients), digital droplet PCR (n = 565), or both (n = 408). We had previously analysed gene expression in peripheral blood leukocytes for a subset of individuals (n = 390). We observed a 37% incidence of EBV-positivity. EBV reactivation was associated with longer ICU stay (12.9 vs 9.2 days; p = 0.004) and increased organ failure (day 1 SOFA score 6.9 vs 5.9; p = 0.00011). EBV reactivation was associated with the relatively immunosuppressed SRS1 endotype (p = 0.014) and differential expression of a small number of biologically relevant genes. These findings are consistent with the hypothesis that viral reactivation in sepsis is a consequence of immune compromise and is associated with increasing severity of illness although further mechanistic studies are required to definitively illustrate cause and effect.

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence.

BACKGROUND: NAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function. RESULTS: We identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence. CONCLUSIONS: We combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.