Genome sequencing and assembly of Indian golden silkmoth, Antheraea assamensis Helfer (Saturniidae, Lepidoptera).

Muga silkworm (Antheraea assamensis), one of the economically important wild silkmoths, is unique among saturniid silkmoths. It is confined to the North-eastern part of India. Muga silk has the highest value among the other silks. Unlike other silkmoths, A. assamensis has a low chromosome number (n = 15), and ZZ/ZO sex chromosome system. Here, we report the first high-quality draft genome of A. assamensis, assembled by employing the Illumina and PacBio sequencing platforms. The assembled genome of A. assamensis is 501.18 Mb long, with 2697 scaffolds and an N50 of 683.23 Kb. The genome encompasses 18,385 protein-coding genes, 86.29% of which were functionally annotated. Phylogenetic analysis of A. assamensis revealed its divergence from other Antheraea species approximately 28.7 million years ago. Moreover, an investigation into detoxification-related gene families, CYP450, GST, and ABC-transporter, revealed a significant expansion in A. assamensis as compared to the Bombyx mori. This expansion is comparable to Spodoptera litura, suggesting adaptive responses linked to the polyphagous behavior observed in these insects. This study provides valuable insights into the molecular basis of evolutionary divergence and adaptations in muga silkmoth. The genome assembly reported in this study will significantly help in the functional genomics studies on A. assamensis and other Antheraea species along with comparative genomics analyses of Bombycoidea insects.

Intrinsically Disordered Kiwellin Protein-Like Effectors Target Plant Chloroplasts and are Extensively Present in Rust Fungi.

The effector proteins produced by plant pathogens are one of the essential components of host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal ß1/ß2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein-like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level.

Genetic characterisation of an Iflavirus associated with a vomiting disease in the Indian tropical tasar silkworm, Antheraea mylitta.

Antheraea mylitta, the Tropical tasar silkworm, is frequently affected by a vomiting disease called Virosis by sericulturists although not confirmed being of viral origin. Based on the symptoms and the disease pattern, the causal agent is however suspected to be a virus. The condition involves a series of characteristic and progressive symptoms that generally culminates in the death of the larva. The disease is common in autumn season (Sep- Oct), with widespread distribution causing severe damage to the tasar silk industry. The leads for this study were obtained from a transcript identified in the EST database in a different study, which matched the positive strand of Iflavirus, an RNA virus known to infect insects. In the present study the genome of this novel Iflavirus was characterised and the full length of the genome was found to be 9728 nucleotides long encoding for a single large open reading frame (ORF) with flanking NTR regions at 5' and 3' ends and a natural poly A tail at the 3' end. The ORF encoded structural proteins at the N-terminal end and non-structural proteins at the C-terminal end with a predicted 2967 amino acid long polyprotein. The structural proteins consisted of 4 proteins (VP1-VP4) and the non-structural proteins consisted of helicase, RNA-dependent RNA polymerase and 3C-protease. The virus is found in various tissues (midgut, fatbody, trachea, Malpighian tubules and silk gland) and also has a vertical route of transmission, i.e., from gravid females to the offspring. Based on the available data, the virus is a new member of Iflaviridae for which we propose the name Antheraea mylitta Iflavirus (AmIV).

Smut fungi as a stratagem to characterize rust effectors: opportunities and challenges.

The rust pathogens are one of the most complex fungi in the Basidiomycetes. The development of genomic resources for rust and other plant pathogens has opened the opportunities for functional genomics of fungal genes. Despite significant progress in the field of fungal genomics, functional characterization of the genome components has lacked, especially for the rust pathogens. Their obligate nature and lack of standard stable transformation protocol are the primary reasons for rusts to be one of the least explored genera despite its significance. In the recently sequenced rust genomes, a vast catalogue of predicted effectors and pathogenicity genes have been reported. However, most of these candidate genes remained unexplored due to the lack of suitable characterization methods. The heterologous expression of putative effectors in Nicotiana benthamiana and Arabidopsis thaliana has proved to be a rapid screening method for identifying the role of these effectors in virulence. However, no fungal system has been used for the functional validation of these candidate genes. The smuts, from the evolutionary point of view, are closely related to the rust pathogens. Moreover, they have been widely studied and hence could be a suitable model system for expressing rust fungal genes heterologously. The genetic manipulation methods for smuts are also well standardized. Complementation assays can be used for functional validation of the homologous genes present in rust and smut fungal pathogens, while the species-specific proteins can be expressed in the mutant strains of smut pathogens having reduced or no virulence for virulence analysis. We propose that smuts, especially Ustilago maydis, may prove to be a good model system to characterize rust effector proteins in the absence of methods to manipulate the rust genomes directly.

No association of TNFRSF1B variants with type 2 diabetes in Indians of Indo-European origin.

BACKGROUND: There has been no systematic evaluation of the association between genetic variants of type 2 receptor for TNFα (TNFR2) and type 2 diabetes, despite strong biological evidence for the role of this receptor in the pathogenesis of this complex disorder. In view of this, we performed a comprehensive association analysis of TNFRSF1B variants with type 2 diabetes in 4,200 Indo-European subjects from North India. METHODS: The initial phase evaluated association of seven SNPs viz. rs652625, rs496888, rs6697733, rs945439, rs235249, rs17883432 and rs17884213 with type 2 diabetes in 2,115 participants (1,073 type 2 diabetes patients and 1,042 control subjects). Further, we conducted replication analysis of three associated SNPs in 2,085 subjects (1,047 type 2 diabetes patients and 1,038 control subjects). RESULTS: We observed nominal association of rs945439, rs235249 and rs17884213 with type 2 diabetes (P < 0.05) in the initial phase. Haplotype CC of rs945439 and rs235249 conferred increased susceptibility for type 2 diabetes [OR = 1.19 (95%CI 1.03-1.37), P = 0.019/Pperm = 0.076] whereas, TG haplotype of rs235249 and rs17884213 provided protection against type 2 diabetes [OR = 0.83 (95%CI 0.72-0.95, P = 7.2 × 10-3/Pperm = 0.019]. We also observed suggestive association of rs496888 with plasma hsCRP levels [P = 0.042]. However, the association of rs945439, rs235249 and rs17884213 with type 2 diabetes was not replicated in the second study population. Meta-analysis of the two studies also failed to detect any association with type 2 diabetes. CONCLUSIONS: Our two-stage association analysis suggests that TNFRSF1B variants are not the determinants of genetic risk of type 2 diabetes in North Indians.