High cell surface expression and peptide binding affinity of HLA-DQA1*05:03, a susceptible allele of neuromyelitis optica spectrum disorders (NMOSD)

Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing autoimmune disease characterized by the presence of pathogenic autoantibodies, anti-aquaporin 4 (AQP4) antibodies. Recently, HLA-DQA1*05:03 was shown to be significantly associated with NMOSD in a Japanese patient cohort. However, the specific mechanism by which HLA-DQA1*05:03 is associated with the development of NMOSD has yet to be elucidated. In the current study, we revealed that HLA-DQA1*05:03 exhibited significantly higher cell surface expression levels compared to other various DQA1 alleles, and that its expression strongly depended on the amino acid sequence of the α1 domain, with a preference for leucine at position 75. Moreover, in silico analysis indicated that the HLA-DQ encoded by HLA-DQA1*05:03 preferentially presents immunodominant AQP4 peptides, and that the peptide major histocompatibility complexes (pMHCs) are more energetically stable in the presence of HLA-DQA1*05:03 than other HLA-DQA1 alleles. In silico 3D structural models were also applied to investigate the validity of the energetic stability of pMHCs. Taken together, our findings indicate that HLA-DQA1*05:03 possesses a distinct property to play a pathogenic role in the development of NMOSD.

GhTBL34 Is Associated with Verticillium Wilt Resistance in Cotton

Verticillium wilt (VW) is a typical fungal disease affecting the yield and quality of cotton. The Trichome Birefringence-Like protein (TBL) is an acetyltransferase involved in the acetylation process of cell wall polysaccharides. Up to now, there are no reports on whether the TBL gene is related to disease resistance in cotton. In this study, we cloned a cotton TBL34 gene located in the confidence interval of a major VW resistance quantitative trait loci and demonstrated its relationship with VW resistance in cotton. Analyzing the sequence variations in resistant and susceptible accessions detected two elite alleles GhTBL34-2 and GhTBL34-3, mainly presented in resistant cotton lines whose disease index was significantly lower than that of susceptible lines carrying the allele GhTBL34-1. Comparing the TBL34 protein sequences showed that two amino acid differences in the TBL (PMR5N) domain changed the susceptible allele GhTBL34-1 into the resistant allele GhTBL34-2 (GhTBL34-3). Expression analysis showed that the TBL34 was obviously up-regulated by infection of Verticillium dahliae and exogenous treatment of ethylene (ET), and salicylic acid (SA) and jasmonate (JA) in cotton. VIGS experiments demonstrated that silencing of TBL34 reduced VW resistance in cotton. We deduced that the TBL34 gene mediating acetylation of cell wall polysaccharides might be involved in the regulation of resistance to VW in cotton.

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.

T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/Du) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/Du and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vβ13a T cell receptor β chain, completely prevents diabetes. Using the RT1B/Du-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 β chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vβ13a TCR and its class II RT1u ligand suggests a mechanism by which a germline TCR β chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vβ13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity.

Development of a New DNA Marker for Fusarium Yellows Resistance in Brassica rapa Vegetables

In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.

Genetic Changes in Experimental Populations of a Hybrid in the Cryptococcus neoformans Species Complex

Hybrids between Cryptococcus neoformans and Cryptococcus deneoformans are commonly found in patients and the environment. However, the genetic stability of these hybrids remains largely unknown. Here, we established mutation accumulation lines of a diploid C. neoformans × C. deneoformans laboratory hybrid and analyzed the genotypes at 33 markers distributed across all 14 chromosomes. Our analyses found that under standard culture conditions, heterozygosity at most loci was maintained over 800 mitotic generations, with an estimated 6.44 × 10−5 loss-of-heterozygosity (LoH) event per mitotic division. However, under fluconazole stress, the observed LoH frequency increased by > 50 folds for the two markers on Chromosome 1, all due to the loss of the fluconazole susceptible allele on this chromosome. Flow cytometry analyses showed that after the 40th transfer (120 days), 19 of the 20 lines maintained the original ploidy level (2N), while one line was between 2N and 3N. The combined flow cytometry, genotyping at 33 markers, and quantitative PCR analyses showed the allelic loss was compensated for by amplification of the resistant ERG11 allele in eight of the ten fluconazole-stress lines. Our results suggest that hybrids in C. neoformans species complex are generally stable but that they can undergo rapid adaptation to environmental stresses through LoH and gene duplication.

A novel intragenic marker targeting the ectodomain of bacterial leaf blight resistance gene Xa21 in rice

Background Among the diseases in rice (Oryza sativa L.), bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae results in devastating economic losses, both in terms of yield and quality. The Xa21 mapped to the rice chromosome 11, is known to convey resistance against BLB by its involvement in plant pathogen recognition and immunity responses. The closely-linked sequence-tagged site marker pTA248 is frequently used for marker-assisted selection (MAS) of Xa21. However, lower precision and linkage drag of linked-markers hinders the reliability and effectiveness in MAS compared to the use of flanking or intergenic markers. Results In the current study, a diagnostic intragenic marker was developed for MAS of Xa21 in rice. The new marker ABUOP0001 targets a 19-bp insertion/deletion (InDel) on the ectodomain of the Xa21, and amplifies a 200-bp amplicon from the rice line IRBB 62, which is known-to-carry the ‘resistance allele’, and a 181-bp amplicon from IRBB 7, which is known-to-carry the ‘susceptible allele’. The resistance allele was identified in ten IRBB lines, and five newly improved Sri Lankan rice accessions through ABUOP0001 marker assay. In a field study, 14 of these accessions conveyed highly resistant BLB disease responses, and one conveyed an intermediate response. While carrying the susceptible allele at Xa21, 30 accessions conveyed a resistance response to BLB, and it could be due to the contribution of other Xa resistance alleles in their genetic background. Further, the Xa21 resistance allele was identified in 1,675 rice accessions through an in silico analysis of genomic sequences available through the 3K Rice Genomes Project. The intragenic marker ABUOP0001 performs equally to that of the linked-marker pTA248 anchored 224 kbp upstream of Xa21, in detecting resistant and susceptible BLB phenotypes. The marker ABUOP0001 is compatible for high-throughput screening with high resolution melting (HRM) and can be multiplexed effectively with an intragenic marker of BLB resistance gene Xa4. Conclusion The marker ABUOP0001 is a diagnostic intragenic marker that can be recommended for MAS of Xa21 in rice. The marker can be assayed as a high-throughput marker using HRM and multiplexed PCR.

Development of InDel marker for rice blast resistance gene Pi9

Pi9 is one of the major blast resistance genes which encodes a nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain-containing protein. This gene was observed to show resistance against many pathotypes of the blast pathogen in Malaysia. Resistance allele Pi9 from rice variety 75-1-127 had previously been cloned using map-based cloning strategy. The gene sequence was used to design specific primers to amplify susceptible Pi9 allele from MR219 rice variety prior to cloning. The resistance and susceptible allele of Pi9 were 8.587kb and 8.785kb in length respectively. Allele mining was carried out by comparing between the susceptible and resistance allele of Pi9. One potential InDels polymorphism at position 590bp and 920bp was identified. Primer named as Pi9_InDel was designed targeting this region in such a way that the resistance and susceptible genotypes yielded 327 bp and 438 bp amplicon respectively.

Loss of premature stop codon in theWall-Associated Kinase 91(OsWAK91) gene confers sheath blight disease resistance in rice

The genetic arms race between pathogen and host plant is a tug of war that has been ongoing for millennia. The “battles” are those of disruption, restoration of signaling and information transmission on a subcellular level. One such battle occurs between rice an important crop that feeds 50% of the world population and the sheath blight disease (SB) caused by the fungusRhizoctonia solani. It results in 10□30% global yield loss annually and can reach 50% under severe outbreak. Many Receptor□like kinases (RLKs) are recruited as soldiers in these battles. Wall Associated Receptor Kinases (WAKs) a subfamily of receptor-like kinases have been shown to play a role in fungal defense. Here we show that rice geneOsWAK91, present in the major SB resistance QTL region on Chromosome□9 is a key component in defense against rice sheath blight. An SNP mutation C/T separates susceptible variety, Cocodrie (CCDR) from the resistant line MCR010277 (MCR). The resistant allele C results in the stop codon loss that results in 68 amino acids longer C□terminus carrying longer protein kinase domain and phosphorylation sites. Our genotype and phenotype analysis of the top 20 individuals of the double haploid SB population shows a strong correlation with the SNP. The susceptible allele appears as a recent introduction found in the japonica subspecies reference genome and a majority of the tropical and temperate japonica lines sequenced by the 3000 rice genome project. Multiple US commercial varieties with japonica background carry the susceptible allele and are known for SB susceptibility. This discovery opens the possibility of introducing resistance alleles into high yielding commercial varieties to reduce yield losses incurred by the disease.

Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, Anopheles funestus

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An. funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms. A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.