Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1.

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1ΔKL308-309), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.

Creatine transporter deficiency, an underdiagnosed cause of male intellectual disability.

X-linked creatine transporter deficiency is caused by the deficiency of the creatine transporter encoded by the SLC6A8 gene on Xq28. We here report a 3-year-old boy with global developmental delay, autism and epilepsy. He had a normal MRI of the brain. Brain magnetic resonance spectroscopy (MRS) subsequently showed an abnormally small creatine peak. His high urine creatine/creatinine ratio further suggested the diagnosis, later confirmed by hemizygous mutation detected in the SLC6A8 gene. His mother was also heterozygous for the same mutation. Supplementation with creatine monohydrate, arginine, and glycine (precursors of creatine) and supportive therapies, resulted in modest clinical improvement after 12 months. This case highlights the importance of doing MRS for boys with global delay/intellectual disability, autism and epilepsy even with a normal MRI of the brain, to pick up a potentially treatable cause.

Infantile spasms: Etiology, lead time and treatment response in a resource limited setting.

This study explores the etiology and lead time to treatment for infantile spasm (IS) patients and their effect on treatment responsiveness, in a limited resource setting. Patients with IS onset age ≤12 months', seen over 3 years were recruited retrospectively. Clinical information, neuroimaging and genetic results retrieved. Patients categorized into three primary etiological groups: Structural (including Structural Genetic), Genetic, and Unknown. The effect of etiology and lead time from IS onset to initiating appropriate treatment on spasm resolution, evaluated. Total 113 patients were eligible. Mean IS onset age was 6.86(±4.25) months (M: F 3.3:1). Patients were grouped into: Structural 85, Genetic 11 and Unknown 17. Etiology was ascertained in 94/113 (83.1%) with neonatal hypoglycemic brain injury (NHBI) being the most common (40/113, 36%). A genetic etiology identified in 17 (including 6 Structural Genetic, of which five had Tuberous Sclerosis). Structural group was less likely to be treatment resistant (p = 0.013, OR 0.30 [0.12-0.76]). Median treatment lead time - 60 days. Longer lead time to treatment was significantly associated with resistant spasms (χ2 for trend = 10.0, p = 0.0015). NHBI was the commonest underlying cause of IS. There was significant time lag to initiating appropriate treatment, affecting treatment responsiveness.

Correction to: small RNA discovery in the interaction between barley and the powdery mildew pathogen.

Following the publication of the original article [1], the authors noted several typesetting errors which are noted in this Correction article.

Small RNA discovery in the interaction between barley and the powdery mildew pathogen.

BACKGROUND: Plants encounter pathogenic and non-pathogenic microorganisms on a nearly constant basis. Small RNAs such as siRNAs and miRNAs/milRNAs influence pathogen virulence and host defense responses. We exploited the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare) to explore fungal and plant sRNAs expressed during Bgh infection of barley leaf epidermal cells. RESULTS: RNA was isolated from four fast-neutron immune-signaling mutants and their progenitor over a time course representing key stages of Bgh infection, including appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. The Cereal Introduction (CI) 16151 progenitor carries the resistance allele Mla6, while Bgh isolate 5874 harbors the AVRa6 avirulence effector, resulting in an incompatible interaction. Parallel Analysis of RNA Ends (PARE) was used to verify sRNAs with likely transcript targets in both barley and Bgh. Bgh sRNAs are predicted to regulate effectors, metabolic genes, and translation-related genes. Barley sRNAs are predicted to influence the accumulation of transcripts that encode auxin response factors, NAC transcription factors, homeodomain transcription factors, and several splicing factors. We also identified phasing small interfering RNAs (phasiRNAs) in barley that overlap transcripts that encode receptor-like kinases (RLKs) and nucleotide-binding, leucine-rich domain proteins (NLRs). CONCLUSIONS: These data suggest that Bgh sRNAs regulate gene expression in metabolism, translation-related, and pathogen effectors. PARE-validated targets of predicted Bgh milRNAs include both EKA (effectors homologous to AVRk1 and AVRa10) and CSEP (candidate secreted effector protein) families. We also identified barley phasiRNAs and miRNAs in response to Bgh infection. These include phasiRNA loci that overlap with a significant proportion of receptor-like kinases, suggesting an additional sRNA control mechanism may be active in barley leaves as opposed to predominant R-gene phasiRNA overlap in many eudicots. In addition, we identified conserved miRNAs, novel miRNA candidates, and barley genome mapped sRNAs that have PARE validated transcript targets in barley. The miRNA target transcripts are enriched in transcription factors, signaling-related proteins, and photosynthesis-related proteins. Together these results suggest both barley and Bgh control metabolism and infection-related responses via the specific accumulation and targeting of genes via sRNAs.

A prospective randomized controlled double-blind trial to assess the effects of dexmedetomidine during cleft palate surgery.

BACKGROUND: We investigated whether the intraoperative administration of dexmedetomidine would attenuate the profound sympathoadrenal response associated with cleft palate (CP) surgery. METHODS: Sixty children aged 6 months to 12 years undergoing CP surgery under general anesthesia were randomly assigned to the control (C) or dexmedetomidine (D) groups. Group C received benzodiazepine (0.05 mg/kg midazolam followed by infusion of normal saline) fentanyl isoflurane anesthesia, and Group D received dexmedetomidine (loading 1 µg/kg followed by infusion of 0.5 µg/kg/h) fentanyl isoflurane anesthesia. Heart rate (HR), mean blood pressure (MBP), intraoperative fentanyl and isoflurane requirements, recovery scores, emergence agitation, pain scores, time and requirement of rescue analgesic, and surgeon satisfaction were noted. RESULTS: Intraoperative HR and MBP in Group D were significantly lower than the corresponding values in Group C (P < 0.001). HR decreased up to 16% in Group D. By contrast, HR increased up to 20% in Group C. Group D had comparable MBP to its baseline, whereas Group C had higher MBP until extubation (P = 0.015). Two children in Group D developed bradycardia and hypotension, which was successfully treated. The fentanyl and isoflurane requirements decreased by 43% and 30%, respectively, in Group D patients compared to those in Group C (P < 0.001). Group D had lower pain scores and less emergence agitation (P < 0.001). Time until requirement of first rescue analgesic was longer in Group D than that in Group C (P < 0.001). Surgeon satisfaction was higher in Group D than that in Group C. CONCLUSIONS: Intravenous dexmedetomidine during CP surgery attenuated hemodynamic responses with excellent surgeon satisfaction. Close monitoring of hemodynamics is recommended.

Interchromosomal Transfer of Immune Regulation During Infection of Barley with the Powdery Mildew Pathogen.

Powdery mildew pathogens colonize over 9500 plant species, causing critical yield loss. The Ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh), causes powdery mildew disease in barley (Hordeum vulgare L.). Successful infection begins with penetration of host epidermal cells, culminating in haustorial feeding structures, facilitating delivery of fungal effectors to the plant and exchange of nutrients from host to pathogen. We used expression Quantitative Trait Locus (eQTL) analysis to dissect the temporal control of immunity-associated gene expression in a doubled haploid barley population challenged with Bgh Two highly significant regions possessing trans eQTL were identified near the telomeric ends of chromosomes (Chr) 2HL and 1HS. Within these regions reside diverse resistance loci derived from barley landrace H. laevigatum (MlLa) and H. vulgare cv. Algerian (Mla1), which associate with the altered expression of 961 and 3296 genes during fungal penetration of the host and haustorial development, respectively. Regulatory control of transcript levels for 299 of the 961 genes is reprioritized from MlLa on 2HL to Mla1 on 1HS as infection progresses, with 292 of the 299 alternating the allele responsible for higher expression, including Adaptin Protein-2 subunit µ AP2M and Vesicle Associated Membrane Protein VAMP72 subfamily members VAMP721/722. AP2M mediates effector-triggered immunity (ETI) via endocytosis of plasma membrane receptor components. VAMP721/722 and SNAP33 form a Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor (SNARE) complex with SYP121 (PEN1), which is engaged in pathogen associated molecular pattern (PAMP)-triggered immunity via exocytosis. We postulate that genes regulated by alternate chromosomal positions are repurposed as part of a conserved immune complex to respond to different pathogen attack scenarios.

Outcome of four pretreatment regimes on hemodynamics during electroconvulsive therapy: A double-blind randomized controlled crossover trial.

CONTEXT: Electroconvulsive therapy (ECT) is associated with tachycardia and hypertension. AIMS: The aim of this study was to compare two doses of dexmedetomidine, esmolol, and lignocaine with respect to hemodynamics, seizure duration, emergence agitation (EA), and recovery profile. METHODOLOGY: Thirty patients undergoing ECT were assigned to each of the following pretreatment regimes over the course of five ECT sessions in a randomized crossover design: Group D1 (dexmedetomidine 1 µg/kg), Group D0.5 (dexmedetomidine0.5 µg/kg), Group E (esmolol 1 mg/kg), Group L (lignocaine 1 mg/kg), and Group C (saline as placebo) before induction. Heart rate (HR), mean arterial pressure (MAP), seizure duration, EA, and time to discharge were evaluated. RESULTS: Groups D1, D0.5, and esmolol had significantly reduced response of HR, MAP compared to lignocaine and control groups at 1, 3, 5 min after ECT (P < 0.05). Motor seizure duration was comparable in all groups except Group L (P = 0.000). Peak HR was significantly decreased in all groups compared to control. Total propofol requirement was reduced in D1 (P = 0.000) and D0.5 (P = 0.001) when compared to control. Time to spontaneous breathing was comparable in all the groups (P > 0.05). Time to eye opening and time to discharge were comparable in all groups (P > 0.05) except Group D1 (P = 0.001). EA score was least in Group D1 (P = 0.000). CONCLUSION: Dexmedetomidine 1 µg/kg, 0.5 µg/kg, and esmolol produced significant amelioration of cardiovascular response to ECT without affecting seizure duration, results being best with dexmedetomidine 1 µg/kg. However, the latter has the shortcoming of delayed recovery.

The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions.

Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of basal defense mechanisms. In the current report, we show that BLN1 and BLN2 can both be secreted to the apoplast and Barley stripe mosaic virus (BSMV)-mediated overexpression of Bln2 increases susceptibility of barley to Bgh. Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 can interact with each other, and with calmodulin. We then used BSMV-induced gene silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional host genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin α1-b and Sec61 γ subunits. Further functional analysis of these two affected genes showed that when silenced, they also reduced susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of disease-related host proteins or effectors, influencing the establishment of Bgh compatibility on its barley host.

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.).

The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to ß-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni.

The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.