Identification and Validation of High LD Hotspot Genomic Regions Harboring Stem Rust Resistant Genes on 1B, 2A (Sr38), and 7B Chromosomes in Wheat

Stem rust caused by Puccinia graminis f. sp. tritici Eriks. is an important disease of common wheat globally. The production and cultivation of genetically resistant cultivars are one of the most successful and environmentally friendly ways to protect wheat against fungal pathogens. Seedling screening and genome-wide association study (GWAS) were used to determine the genetic diversity of wheat genotypes obtained on stem rust resistance loci. At the seedling stage, the reaction of the common stem rust race QFCSC in Nebraska was measured in a set of 212 genotypes from F3:6 lines. The results indicated that 184 genotypes (86.8%) had different degrees of resistance to this common race. While 28 genotypes (13.2%) were susceptible to stem rust. A set of 11,911 single-nucleotide polymorphism (SNP) markers was used to perform GWAS which detected 84 significant marker-trait associations (MTAs) with SNPs located on chromosomes 1B, 2A, 2B, 7B and an unknown chromosome. Promising high linkage disequilibrium (LD) genomic regions were found in all chromosomes except 2B which suggested they include candidate genes controlling stem rust resistance. Highly significant LD was found among these 59 significant SNPs on chromosome 2A and 12 significant SNPs with an unknown chromosomal position. The LD analysis between SNPs located on 2A and Sr38 gene reveal high significant LD genomic regions which was previously reported. To select the most promising stem rust resistant genotypes, a new approach was suggested based on four criteria including, phenotypic selection, number of resistant allele(s), the genetic distance among the selected parents, and number of the different resistant allele(s) in the candidate crosses. As a result, 23 genotypes were considered as the most suitable parents for crossing to produce highly resistant stem rust genotypes against the QFCSC.

Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae

The evolution of resistance to insecticides threatens the sustainable control of many of the world’s most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

Current Effector and Gene-Drive Developments to Engineer Arbovirus-Resistant Aedes aegypti (Diptera: Culicidae) for a Sustainable Population Replacement Strategy in the Field

Arthropod-borne viruses (arboviruses) such as dengue, Zika, and chikungunya viruses cause morbidity and mortality among human populations living in the tropical regions of the world. Conventional mosquito control efforts based on insecticide treatments and/or the use of bednets and window curtains are currently insufficient to reduce arbovirus prevalence in affected regions. Novel, genetic strategies that are being developed involve the genetic manipulation of mosquitoes for population reduction and population replacement purposes. Population replacement aims at replacing arbovirus-susceptible wild-type mosquitoes in a target region with those that carry a laboratory-engineered antiviral effector to interrupt arboviral transmission in the field. The strategy has been primarily developed for Aedes aegypti (L.), the most important urban arbovirus vector. Antiviral effectors based on long dsRNAs, miRNAs, or ribozymes destroy viral RNA genomes and need to be linked to a robust gene drive to ensure their fixation in the target population. Synthetic gene-drive concepts are based on toxin/antidote, genetic incompatibility, and selfish genetic element principles. The CRISPR/Cas9 gene editing system can be configurated as a homing endonuclease gene (HEG) and HEG-based drives became the preferred choice for mosquitoes. HEGs are highly allele and nucleotide sequence-specific and therefore sensitive to single-nucleotide polymorphisms/resistant allele formation. Current research efforts test new HEG-based gene-drive designs that promise to be less sensitive to resistant allele formation. Safety aspects in conjunction with gene drives are being addressed by developing procedures that would allow a recall or overwriting of gene-drive transgenes once they have been released.

Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.

Surveillance of a kdr Resistance Mutation in Culex pipiens (Diptera: Culicidae) and Culex quinquefasciatus in California

Culex pipiens Linnaeus and Culex quinquefasciatus Say are the primary vectors of West Nile and St. Louis encephalitis viruses in California. Pyrethrins and pyrethroids (synthetic pyrethrins) are the most widely used insecticides to control adult stage mosquitoes to prevent disease transmission. The most abundant and widespread mutation associated with pyrethroid resistance is the L1014F mutation of the voltage-sensitive sodium channel gene. Statewide, based on the testing of almost 2,000 mosquitoes from 14 counties, the resistant allele frequency was 71%. Although the L1014F mutation was found in all counties assessed, the resistance allele profiles differed between regions of California. The highest resistant allele frequency occurred in the Central region and lowest frequencies were from the Northern and Southern regions. Resistance allele frequencies observed in 2014–2016 are nearly 1.5 times higher than those from pre-2012, indicating that resistance profiles can change over time. Regular monitoring of the L1014F kdr mutation will help aid in operational decisions.

Genetic Resistance Against Scrapie Disease Related to Lamb Growth Performance Traits

The objective of the study was to identify the impact of selection on genetic resistance against scrapie disease related to lamb growth performance attributes for Suffolk, Kent, Charollais, and Texel lambs.The allelic genotypes were grouped according to the presence of scrapie resistant allele (ARR): ARR homozygotes, ARR heterozygotes, no presence of ARR allele. The influence of these groups on lamb live weight (LW), musculus longissimus lumborum et thoracis depth (MLLT), and back-fat thickness at 100 days of age was investigated using SAS software. No significant differences for Suffolk and Charollais breeds were detected. Significantly highest LW (34.41 kg) and MLLT (27.80 mm) were noticed for Kent ARR homozygotes lambs, while significantly lowest values were estimated at lambs with absent ARR allele (LW = 33.42 kg, MLLT = 26.68 mm). Significantly lower muscle depth (–0.69 mm) was detected for Texel ARR homozygote compared to ARR heterozygote lambs. As a result, we were unable to detect a consistent evidence for reduced growth performance traits in relation to genetic resistance against scrapie disease. However, the number of animals in some groups caused as a limiting factor. This can be a potential reason of opposed trends in Texel and Kent lambs.

The Inhibition of Gck Affect MAPK Cascade in Multiple Myeloma

Introduction: Germinal Center Kinase (GCK) is an essential regulator of stress-activated MAPK core signaling pathways. Recent studies revealing the role of GCK in tumorigenesis and cancer progression have generated increasing interest in validating GCK as a therapeutic target in cancer. We previously showed that GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation (Li et al, Blood 2017 130:1795). Our further research established GCK as a critical kinase in MM and as an attractive target for treatment of MM. Methods and Results: The over-expression of GCK in primary MM compared to normal cells was confirmed by immunohistochemistry staining of primary MM patient bone marrow biopsies. By quantitatively comparing the staining scores of GCK of primary MM patient bone marrows (n=26) and normal donor bone marrows (n=26), we found a significantly higher GCK expression (p<0.001) in MM patient BM samples (98%) compared to healthy donor BM samples (47%) (Figure 1). GCK protein expression level was induced by several cytokines that are critically involved in MM cell survival and proliferation including IL-6, TNF-a, LPS and CD40L in MM cells after treatment for 1h. The role of GCK expression in MM signaling transduction and tumor progression was addressed by inducible GCK-knockdown in MM cells. By generating Tet-on-shGCK-MM cell lines, we found that doxycycline-induced GCK silencing blocks IL-6 induced phosphorylation of MKK4, MKK7, JNK and ERK in MM.1S cells. Moreover, knockdown of GCK causes downregulation of IKZF1/3, c-MYC and BCL-6 in MM cells. To exclude the off-target effects of the GCK-shRNA and confirm the specific and critical role of GCK, we further generated recombinant GCK-WT and GCK-shRNA resistant allele construct with C-terminal c-Myc tag, and introduced them into the GCK inducible KD cells to determine if shRNA-resistant GCK can rescue the growth inhibition induced by the shRNA. Tet-on-shGCK-RPMI-8266 cells transduced with empty vector (EV) or GCK wild-type with c-Myc tag (WT) or GCK shRNA resistant mutation with c-myc tag (MT) were generated by routine lentiviral infection. Doxycycline-induced tet-on-shGCK expression resulted in significant decrease of GCK protein. Both endogenous GCK and wild-type GCK-c-Myc expressions were silenced. In contrast, expression of GCK shRNA resistant allele with c-Myc tag (MT) was not affected. The rescue effects of this allele on MM cells growth inhibition are undergoing. Bay 61-3606 is a small molecule that selectively inhibits GCK activity and shows promising anti-MM effects in our previous in vitro and in vivo studies. To further confirm the mechanism of GCK inhibition, we examined its effects on the MAPK cascade and found that pharmacologic blockade of GCK by Bay 61-3606 dose-dependently downregulated ERK, p38 and JNK phosphorylation in MM cells. In MM, IMiDs treatment leads to the selective ubiquitination and degradation of IKZF1 and IKZF3 by the CRBN-CRL4 ubiquitin ligase. Suppression of CRBN in MM cell lines induces IMiDs resistance. To our surprise, in MM cells with CRBN-knockdown, Bay 61-3606 retained the ability to decrease IKZF1 protein level and inhibit MM cell growth, indicating that GCK-induced IKZF1 downregulation is independent of CRBN. Thus, this study further validates the therapeutic potential of GCK inhibitors in IMiDs-resistant MM. Conclusion: Our findings show that GCK is overexpressed in MM and an attractive target for multiple myeloma therapy. The inhibition of GCK blocks the phosphorylation and activation of MAPK cascade, and induces the degradation of the substrate proteins IKZF1, IKZF3 and c-Myc, (Figure 2.). These studies further confirm the critical role of GCK in MM tumorigenesis. GCK inhibitors represent a novel anti-MM therapy especially in relapsed and IMiDs-resistant MM patients. Disclosures Lentzsch: Caelum Biosciences: Consultancy, Other: Dr. Lentzsch recused herself as an investigator from the Phase 1a/b trial testing CAEL-101 in 11/2017., Patents & Royalties: Shareholder for Caelum Biosiences; Janssen: Consultancy; Bayer: Consultancy; BMS: Consultancy.

Chicken Mx gene polymorphisms in Indian native chicken breeds and White Leghorn by real time multiplex allele specific PCR

Chicken Mx protein confers resistance against viral infections including avian influenza virus. Mx protein is one of the strong antiviral proteins induced by interferon system. Different alleles of chicken Mx gene have been shown to vary in their antiviral activity against influenza virus. A non-synonymous mutation at position 2032 of chicken Mx gene responsible for variation of amino acid (Ser to Asn) in Mx protein is responsible for negative to positive antiviral activity of Mx protein. In the present study it was analyzed that SNP at G2032A in chicken Mx gene in two Indian native chicken breeds, Aseel, Kadaknath and compared with White Leghorn layer. A total of 90 samples, including 50 of White Leghorn and 20 each from native chicken breed were screened for SNP at position 2032 of Mx gene by using real time multiplex allele specific PCR. The resistant allele (A) frequency is higher than susceptible allele in all three chicken breeds investigated. Among the breeds, the resistant allele frequency (A) is higher in White Leghorn (0.95) followed by Aseel (0.75) and Kadaknath (0.625). Homozygous to resistant allele (AA) is the major genotype in White Leghorn whereas heterozygous to both alleles is the major genotype in Kadaknath. Variations in the gene frequency might have emerged from varying degree of selection pressure the different breeds had undergone. In future, the resistant genotype could be selected in modern breeding programs to improve the genetic status for disease resistance against viral infections