Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. Significance Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey.

Summary: The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot‐inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non‐host rhizobia, lipo‐chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40–1 peptide or different flavonoids. The results show that clover‐nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot‐inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O‐acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot‐inoculation, but only after delivery by microtargeting. Non‐O‐acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40–1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

▪ Abstract Rhizobia are soil bacteria that can engage in a symbiosis with leguminous plants that produces nitrogen-fixing root nodules. This symbiosis is based on specific recognition of signal molecules, which are produced by both the bacterial and plant partners. In this review, recognition factors from the bacterial endosymbionts are discussed, with particular attention to secreted and cell surface glycans. Glycans that are discussed include the Nod factors, the extracellular polysaccharides, the lipopolysaccharides, the K-antigens, and the cyclic glucans. Recent advances in the understanding of the biosynthesis, secretion, and regulation of production of these glycans are reviewed, and their functions are compared with glycans produced by other bacteria, such as plant pathogens.