A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

The IMD pathway regulates lysozyme-like proteins (LLPs) in the silkmoth Antheraea mylitta.

Lysozyme-like proteins (LLPs) are members of the glycoside hydrolase family 22 (CAZY GH22). Unlike conventional c-type lysozymes (EC 3.2.1.17), LLPs lack specific catalytic amino acid residues essential for muramidase activity. Previous reports indicated upregulation of LLPs upon bacterial infection in the wild silkworm, Antheraea mylitta as well as in the domesticated silkworm, Bombyx mori. In the present work, we studied the signaling pathways mediating the production of LLPs using RNA interference-mediated knockdown of Spätzle, Relish and STAT, the key regulators of Toll, IMD (Immune deficiency) and JAK/STAT pathways, respectively. We observed that knockdown of the Relish variant RD1 resulted in reduced expression levels of the ALLP1. We also showed that recombinant LLP has antiviral activity. We infer that LLPs showing both antibacterial and antiviral activity are regulated by the conventional IMD pathway in the silkmoths.

Genetic analysis of reciprocal differences in the inheritance of in vitro characters in pearl millet.

Reciprocal differences persist in nature because of the unequal contribution of cytoplasmic determinants from male and female gametes to the zygote. The inheritance of genetic differences is an important factor that influences various traits, including somatic embryogenesis and regeneration in vitro. In this report, we estimate the cytoplasmic and maternal effects in pearl millet and their adequacy in describing the observed reciprocal differences based on an in depth study of the parents, F2s and reciprocal backcross progenies needed for fitting genetical models. Our study revealed that of the two characters examined, embryogenic callus quantity and regeneration frequency, the former showed a greater proportion of cytoplasmic nuclear interaction whereas the latter showed a greater role of nuclear factors. Additive-maternal effects influenced total callus quantity and dominance-maternal effects influenced total callus quantity, embryogenic callus quantity and regeneration frequency. Dwarfing was associated with the production of large quantities of embryogenic callus that had visually recognizable characteristics. The phenotypic nature of dwarf parents (green dwarf with long narrow leaves) with a genetic basis for a given character controlled by nuclear and cytoplasmic determinants can be exploited for other breeding programs.

Noduler an immune protein augments infection-induced cell proliferation through cross-talking with p38 MAPK.

Noduler, an immune protein that mediates nodule formation by binding to specific bacteria and hemocytes was previously reported in the wild tasar silkworm, Antheraea mylitta. However, the molecular mechanism underlying nodulation in lepidopterans remains unclear. The present study is performed to investigate the functional connection between Noduler with various signalling pathways. It was observed that Noduler is an upstream factor in the phenoloxidase cascade and its knockdown has no direct effect on Toll/Imd pathway inducible genes. Additionally, Noduler was shown to stimulate cell proliferation via activation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 in the infected hemocytes cultured in vitro resulted in reduced cell proliferation and melanization. These results suggest that Noduler mediates nodulation via p38/MAPK signalling. This is the first report implicating the p38 MAPK signalling pathway in the nodulation response of insects.

Nodulation: an unexplored cellular defense mechanism in insects.

Nodulation is a highly conserved process that involves aggregation of cells around microorganisms, leading to their entrapment with the help of cellular milieu. In insects upon infection, the humoral and cellular arms of the innate immune system orchestrate recognition of pathogens facilitating effector responses through various signaling pathways. Existing data suggests a wide range of immune functions for multiple pattern recognition molecules but their role in nodulation is not known. Hence, an in-depth knowledge of components implicated in the signaling pathways across diverse species is crucial for understanding their evolutionary conservation. Here, we attempted to consolidate available information on the nodulation response in insects and made an analogy with other known systems.

Engineering silkworms for resistance to baculovirus through multigene RNA interference.

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.