Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

SUMMARYCoronavirus JHM contained six major proteins, four of which were glycosylated. The proteins were gpl70, gp98, gp65, p60, gp25 and p23. Sac(-) cells infected with JHM shut off host cell protein synthesis, and the synthesis of three major (150K, 60K and 23K) and three minor (65K, 30K and 14K) polypeptides was detected by pulse-labelling with aSS-methionine. Antiserum directed against purified virus proteins specifically immunoprecipitated the three major intracellular species and also the 65K minor species. During a chase period, species 150K and 23K were processed and three new immunoprecipitable species, 170K, 98K and 25K appeared. The intracellular species 170K, 98K, 65K, 60K, 25K and 23K co-electrophoresed with virion proteins.Two-dimensional gel electrophoresis of infected cell polypeptides showed that the 60K, 23K, 25K and 14K species were relatively basic polypeptides whilst the 98K and 170K were relatively acidic and heterogeneously charged polypeptides. Additionally, a charge-size modification of the 23K species during processing was detected, which was not apparent using one-dimensional gel analysis.

Optimal defense (OD) theory predicts that within a plant, tissues are defended in proportion to their fitness value and risk of predation. The fitness value of leaves varies greatly and leaves are protected by jasmonate (JA)-inducible defenses. Flowers are vehicles of Darwinian fitness in flowering plants and are attacked by herbivores and pathogens, but how they are defended is rarely investigated. We used Nicotiana attenuata, an ecological model plant with well-characterized herbivore interactions to characterize defense responses in flowers. Early floral stages constitutively accumulate greater amounts of two well-characterized defensive compounds, the volatile (E)-α-bergamotene and trypsin proteinase inhibitors (TPIs), which are also found in herbivore-induced leaves. Plants rendered deficient in JA biosynthesis or perception by RNA interference had significantly attenuated floral accumulations of defensive compounds known to be regulated by JA in leaves. By RNA-seq, we found a JAZ gene, NaJAZi, specifically expressed in early-stage floral tissues. Gene silencing revealed that NaJAZi functions as a flower-specific jasmonate repressor that regulates JAs, (E)-α-bergamotene, TPIs, and a defensin. Flowers silenced in NaJAZi are more resistant to tobacco budworm attack, a florivore. When the defensin was ectopically expressed in leaves, performance of Manduca sexta larvae, a folivore, decreased. NaJAZi physically interacts with a newly identified NINJA-like protein, but not the canonical NINJA. This NINJA-like recruits the corepressor TOPLESS that contributes to the suppressive function of NaJAZi on floral defenses. This study uncovers the defensive function of JA signaling in flowers, which includes components that tailor JA signaling to provide flower-specific defense

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000-and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecularweight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000-and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000-and 23,000-molecularweight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA. Coronaviruses cause a variety of diseases in both animals and humans. They are associated with respiratory and enteric diseases in humans, bronchitis in birds, transmissible gastroenteritis and encephalitis in pigs, and demyelinating encephalitis and hepatitis in rodents (11, 20; J. A.

SUMMARYCoronavirus JHM contains six major proteins, one of which, the 60000 rnol. wt. nucleocapsid protein pp60, is phosphorylated. In JHM-infected cells ip 60K, the intracellular precursor to pp60 is also phosphorylated. Associated with purified JHM virions is a protein kinase which will phosphorylate pp60 and a variety of exogenous substrates in vitro. The enzyme has the characteristics of a cyclic nucleotideindependent protein kinase. Both the in vivo reaction and the enzyme activity in vitro transferred the y-phosphate of ATP to serine residues on the nucleocapsid protein.