Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the Myzus persicae

Plant-mediated RNAi has been developed as a powerful weapon in the fight against agricultural insect pests. The gap gene hunchback (hb) is of crucial importance in insect axial patterning and knockdown of hb is deforming and lethal to the next generation. The peach potato aphid, Myzus persicae (Sulzer), has many host plants and can be found throughout the world. To investigate the effect of plant-mediated RNAi on control of this insect, the hb gene in M. persicae was cloned, plant RNAi vector was constructed, and transgenic tobacco expressing Mphb dsRNA was developed. Transgenic tobacco had a different integration pattern of the transgene. Bioassays were performed by applying neonate aphids to homozygous transgenic plants in the T2 generation. Results revealed that continuous feeding of transgenic diet reduced Mphb mRNA level in the fed aphids and inhibited insect reproduction, indicating successful knockdown of the target gene in M. persicae by plant-mediated RNAi.     

Co-occupancy of two Pumilio molecules on a single hunchback NRE

Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3′ untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8–10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3′ Box B site than the 5′ Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.     

Expression of <Emphasis Type="Italic">hunchback</Emphasis> during oogenesis and embryogenesis in <Emphasis Type="Italic">Locusta migratoria manilensis</Emphasis> (Meyen)

hb (hunchback) is a contributing factor in anteroposterior axial patterning of insects. Although the hb function in Locusta migratoria manilensis has been investigated, its expression pattern remains unknown. Here, the mouse polyclonal antibody was produced against Hb fusion protein, and then its expression pattern during oogenesis and embryogenesis of L. migratoria manilensis was examined by immunohistochemical staining. Hb protein was detected in the oocyte nucleus which was positioned centrally within the developing oocyte. The oocyte nucleus gradually moved to the posterior end of the egg along with the oocyte maturing. In freshly laid eggs, Hb formed gradient at the posterior end of the egg, and then hb was expressed as a band in the middle of the blastodisc. As the blastodisc differentiated into the head and trunk, the expression region became wide, which would develop into spatial gnathal and thoracic segments. With abdominal segmentation, the expression domain in the gnathal and thoracic region became faint and eventually faded out, while the Hb expression domain appeared at the posterior growth zone in a discontinuous expression manner. The hb expression pattern of L. migratoria manilensis is greatly similar to that of other locusts, such as Schistocerca americana and another L. migratoria. Compared with other insects, hb expression is conserved in the gnathal and thoracic domains, while divergent in oogenesis and abdomen.     

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.     

Molecular characterization of a new synthetic cry2ab gene in Nicotiana tabacum

A newly-synthesized cry2Ab gene was characterized in Nicotiana tabacum, before its further transformation in cotton. Synthetic cry2Ab gene was cloned in pGreen0029 and its expression was transiently analyzed at mRNA level through agroinfiltration in tobacco. The mRNA of cry2Ab was detected after 72 h agroinfiltration through PCR using total plant RNA. This construct was then transformed into N. tabacum through Agrobacterium. Insect bioassays were conducted on detached leaves using first instar Spodoptera exigua larvae; after 96 h significant insect mortality was recorded. This newly synthesized gene was effective in controlling S. exigua first instar larvae. It can be used in combinations with other Bt genes like cry1Ac for developing resistance against major insect pests of cotton and further widening the insect control spectrum.     

Segmentation gene expression in the mothmidge Clogmia albipunctata (Diptera, Psychodidae) and other primitive dipterans

 To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, we studied the expression of two gap genes hunchback (hb) and Krüppel (Kr) as well as the pair-rule gene even-skipped (eve) in the dipteran Clogmia albipunctata (Nematocera, Psychodidae). This species has features of both short- and long-germ mode of embryogenesis. In Clogmia hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region. These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis. Krüppel expression, on the other hand, was found to be rather similar to the Drosophila expression, both at early and late stages. eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension. Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans. We therefore also studied another nematoceran, Coboldia, to address this question and found that some segmental stripes form by intercalation as in Drosophila, although belatedly. Our results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects. Received: 24 August 1998 / Accepted: 29 October 1998     

The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation. Received: 28 May 1999 / Accepted: 21 December 1999     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

High mortality caused by high dose of dsRNA in the green rice leafhopper Nephotettix cincticeps (Hemiptera: Cicadellidae)

RNA interference (RNAi) is a useful tool for gene functional studies in non-model insects and pest insects. Establishment of experimental conditions for RNAi, which differ from insect to insect, is important for evaluating the effect of dsRNA injection of relevant genes. When injecting dsRNA into the green rice leafhopper Nephotettix cincticeps (Uhler), high mortality was observed. Therefore, the adverse effects of injection of dsRNA on leafhopper development were examined to assess the suitable conditions for RNAi in this species. Injection manipulation by using a glass capillary did not affect leafhopper survival but delayed the molt of the corresponding instar. High mortality was observed when large amounts of dsRNA were administered. This adverse effect of dsRNA was examined in 2 genes, exogenous EGFP gene and endogenous peptidoglycan recognition protein gene (NcPGRP12). Injection of a high dose (60 ng/insect) caused high mortality in all stages tested: 4th instar, 5th instar, and female adult. A relatively low dose (6 ng/insect) did not cause high mortality, retaining a high potential for gene silencing. Since RNAi is highly effective in this species, the deleterious effect of large amounts of dsRNA could be avoided by administering a low dsRNA dose.     

A developmental genetic analysis of the gene Regulator of postbithorax in Drosophila melanogaster

We report the characterization of loss-of-function alleles of the homoeotic mutation Regulator of postbithorax (Rg-pbx) in Drosophila melanogaster. Rg-pbx is a dominant gain-of-function mutation which shows a transformation of posterior haltere to wing in the adult cuticle. This mutant phenotype mimics that of the bithorax complex lesion postbithorax (pbx). Loss-of-function alleles described here are lethal in the embryonic stage and affect the pattern of segmentation of the embryo. Examination of the terminal phenotype of null and hypomorphic alleles of Rg-pbx has shown that inactivation of the Rg-pbx gene leads to loss of the thoracic segments and the adjacent labial segment of the Drosophila embryo. An effect of the mutations is also seen in the seventh and eighth abdominal segments of embryos. The loss-of-function phenotype is similar to that described for the segmentation mutant hunchback (hb). Complementation tests show that Rg-pbx and hb are allelic. Temperature shift experiments using a temperature-sensitive loss-of-function allele show that the Rg-pbx gene product is required early in embryogenesis. We further report that the dominant Rg-pbx phenotype is sensitive to the gene dosage of another segmentation-controlling gene, fushi tarazu (ftz). Flies carrying a mutant copy of the ftz gene in trans to Rg-pbx show a dramatic enhancement of the penetrance of the homoeotic mutant phenotype. We were also able to demonstrate a suppression of the Rg-pbx phenotype by the addition of a duplication for the ftz⁺ gene to an Rg-pbx stock. Examination of the phenotype of ftz⁻, Rg-pbx⁻ double-mutant embryos did not reveal a clear pattern of epistasis between the genes nor was absolute additivity of phenotype seen. A possible formal relationship between Rg-pbx, ftz, and the postbithorax (pbx) locus is proposed.     

The effect of temperature upon Tipula iridescent virus infection in Tipula oleracea

The mean incubation period (time from inoculation with virus to first appearance of iridescence) was used as an indication of the rate of replication of Tipula iridescent virus (TIV) in Tipula oleracea larvae. The mean incubation period and survival time (time from inoculation with virus to death) were compared with the mean instar duration at a series of temperatures. In most stages of the insect the optimum temperature for the replication of TIV and the temperature for the shortest mean survival time coincided with the peak temperature (the temperature for the fastest development of the insect stage). The peak temperature for T. oleracea does not appear to be the same for each stage, and the optimum temperature for TIV replication appears to be closely linked to the peak temperature of the infected stage. The optimum temperature (the temperature at which most individuals survived from hatching to the adult stage) of the insect was 20°C. Tipula iridescent virus replicated in T. oleracea larvae and pupae at 3° and 27°C, which are near the temperature limits for the insect. Incubation periods and survival times in TIV-inoculated larvae incubated in the field were much longer in winter than in summer.     

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.