Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.     

Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.     

Fine structure of the eggs of blowflies Aldrichina grahami and Chrysomya pacifica (Diptera: Calliphoridae)

We report here the fine structure of the eggs of blowflies Aldrichina grahami (Aldrich) and Chrysomya pacifica Kurahashi. For A. grahami, the plastron is wide and extends to almost the entire length of the eggs. The plastron near the micropyle is truncated. The polygonal patterns of chorionic sculpture bear a distinct swollen boundary. Regarding C. pacifica, the plastron is narrow and extends to almost the entire length of the eggs. The plastron near the micropyle bifurcates to a Y-shape, but the arms of the 'Y' are short. Information presented herein allows some distinctive features to differentiate among eggs of blowfly species.     

Independent translation of ORFs in dicistronic operons,synthetic building blocks for polycistronic chloroplast gene expression

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ultrastructure of male accessory glands of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae)

The ultrastructure of the male accessory glands of the blow fly, Chrysomya megacephala (Fabricius), was presented using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). A pair of accessory glands was separated at opposite sites. Morphometric results using LM yield evidenced no significant difference in the median of either length or width of the left and right glands. A significant increment in both length and width was seen to plateau between three to six days. SEM observation showed that the surface of the glands revealed a faint irregular groove pattern throughout, and it was occasionally penetrated by tracheoles. Each gland was a slender, elongated sac‐like tubule having apical rounded ends, with a slight constriction at the sub‐apical part of the gland being observed occasionally. TEM analyses of three‐day‐old males showed that the glands consisted of external capsular cells with a basement membrane underneath, glandular cells, and gland lumen. The capsular cell was flat and contained a nucleus with electron dense material in the nuclear envelope. The glandular cell, appearing as columnar, consisted of a vacuolated component that contained a large oval nucleus centrally or sub‐basally located, with dense mitochondria, numerous rough endoplasmic reticulum, and secretory vesicles containing electron‐lucent materials. In the gland lumen, the cross‐section through the middle portion revealed dense secretory materials, characterized by electron‐dense materials. Some sections revealed a large lumen where secretion accumulates within the delicate sac. The seven‐day‐old glands exhibited a remarkable change in the lumen, where the whole space contained a large amount of secretory materials, with the electron‐dense materials being characterized as similar to those observed in three‐day‐old glands. About four prominent types of secretions were observed on the basis of difference in electron‐density.     

Ethiprole resistance in Nilaparvata lugens (Hemiptera: Delphacidae): possible mechanisms and cross-resistance

This article reports the current status of ethiprole resistance in Nilaparvata lugens Stål in the central region of Thailand, together with the associated resistance mechanisms. A resistance survey found that a field population had developed 308.5-fold resistance to ethiprole. Further selection with ethiprole for nine generations in the laboratory led to 453.1-fold ethiprole resistance. However, following this selection procedure, the resistance of N. lugens to other insecticides decreased to about one-third of its original resistance. This result implies that there is no cross-resistance between ethiprole and other kinds of insecticides in this pest. In an in vivo study of synergisms, triphenyl phosphate (TPP) exhibited a strong synergism (SR 4.2) with ethiprole in the resistant hoppers, piperonyl butoxide (PBO) also showed significant synergistic effects with ethiprole (1.6), but diethyl maleate (DEM) did not show any obvious synergism with ethiprole (1.2). An in vitro biochemical study indicated that esterase activity increased with ethiprole resistance in N. lugens, that P450 monooxygenase activity also increased significantly with high resistance, but that glutathione S-transferase activity did not. These results reveal that increases in esterase activity and P450 monooxygenase activity cause the ethiprole resistance observed in the field populations of N. lugens. Whether the mechanisms for ethiprole resistance involve target-site sensitivity is not yet known; further molecular analysis is required. However, an analysis of insecticide cross-resistance and the insecticide application history of the resistant populations indicated that target resistance was present and that rotation between insecticides with different modes of action will provide a key countermeasure to maintain the efficacy of ethiprole.     

A guide to choosing vectors for transformation of the plastid genome of higher plants

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3'-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3'-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.     

Observations on first and second-instar larvae of Megaselia scalaris (Loew) (Diptera: Phoridae).

The ultrastructure of the first and second-instar larvae of Megaselia scalaris (Loew) was studied using scanning electron microscopy (SEM). Significant changes in morphological features were observed in the anterior and posterior spiracles, but only minimal changes in the labium and mouthhooks were seen. The ultrastructure of M. scalaris larvae not only provides chronological transformation of their larval instars, but it can also be used to explain their feeding behavior and mode of respiration. In addition, morphological structures useful for specific identification of first or second-instar larvae collected from human corpses may be used in forensic investigations.