The northern corn leaf blight resistance gene Ht1 encodes an nucleotide-binding,leucine-rich repeat immune receptor

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E. turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E. turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3.     

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

     

A Novel PRPF31 Mutation in a Large Chinese Family with Autosomal Dominant Retinitis Pigmentosa and Macular Degeneration

Purpose

This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration.

Methods

A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons'' sequencing was executed to identify mutations.

Results

A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain).

Conclusion

This report is the first to associate PRPF31 gene''s nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.     

Abscisic acid-triggered guard cell l-cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to heme oxygenase-modulated stomatal closure

Recent studies have demonstrated that hydrogen sulfide (H₂S) produced through the activity of l -cysteine desulfhydrase (DES1) is an important gaseous signaling molecule in plants that could participate in abscisic acid (ABA)-induced stomatal closure. However, the coupling of the DES1/H₂S signaling pathways to guard cell movement has not been thoroughly elucidated. The results presented here provide genetic evidence for a physiologically relevant signaling pathway that governs guard cell in situ DES1/H₂S function in stomatal closure. We discovered that ABA-activated DES1 produces H₂S in guard cells. The impaired guard cell ABA phenotype of the des1 mutant can be fully complemented when DES1/H₂S function has been specifically rescued in guard cells and epidermal cells, but not mesophyll cells. This research further characterized DES1/H₂S function in the regulation of LONG HYPOCOTYL1 (HY1, a member of the heme oxygenase family) signaling. ABA-induced DES1 expression and H₂S production are hyper-activated in the hy1 mutant, both of which can be fully abolished by the addition of H₂S scavenger. Impaired guard cell ABA phenotype of des1/hy1 can be restored by H₂S donors. Taken together, this research indicated that guard cell in situ DES1 function is involved in ABA-induced stomatal closure, which also acts as a pivotal hub in regulating HY1 signaling.     

Impaired modulation of GABAergic transmission by muscarinic receptors in a mouse transgenic model of Alzheimer's disease

It has long been recognized that muscarinic acetylcholine receptors (mAChRs) are crucial for the control of cognitive processes, and drugs that activate mAChRs are helpful in ameliorating cognitive deficits of Alzheimer's disease (AD). On the other hand, GABAergic transmission in prefrontal cortex (PFC) plays a key role in "working memory" via controlling the timing of neuronal activity during cognitive operations. To test whether the muscarinic and gamma-aminobutyric acid (GABA) system are interconnected in normal cognition and dementia, we examined the muscarinic regulation of GABAergic transmission in PFC of an animal model of AD. Transgenic mice overexpressing a mutant gene for beta-amyloid precursor protein (APP) show behavioral and histopathological abnormalities resembling AD and, therefore, were used as an AD model. Application of the mAChR agonist carbachol significantly increased the spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude in PFC pyramidal neurons from wild-type animals. In contrast, carbachol failed to increase the sIPSC amplitude in APP transgenic mice, whereas the carbachol-induced increase of the sIPSC frequency was not significantly changed in these mutants. Similar results were obtained in rat PFC slices pretreated with the beta-amyloid peptide (Abeta). Inhibiting protein kinase C (PKC) blocked the carbachol enhancement of sIPSC amplitudes, implicating the PKC dependence of this mAChR effect. In APP transgenic mice, carbachol failed to activate PKC despite the apparently normal expression of mAChRs. These results show that the muscarinic regulation of GABA transmission is impaired in the AD model, probably due to the Abeta-mediated interference of mAChR activation of PKC.     

Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+/calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons

We have studied the regulation of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT(1A) receptor agonists and blocked by 5-HT(1A) antagonists, indicating the mediation by 5-HT(1A) receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT(1A) agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT(1A) receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice.     

Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.     

ABCB6 mutations cause ocular coloboma

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.