Frank Shotkoski

Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti: Conservation of the gene and resistance associated mutation with Drosophila

In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a γ‐aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non‐Drosophilids is discussed.

Field trial of Xanthomonas wilt disease-resistant bananas in East Africa

volume 32 NumBeR 9 SePTemBeR 2014 nature biotechnology to intensified production of active oxygen species and activation of the hypersensitive response when plants are challenged with bacterial pathogens4. Overexpression of Pflp has been shown to provide resistance against various bacterial pathogens, such as Erwinia, Pseudomonas, Ralstonia and Xanthomonas spp., in transgenic tobacco4, tomato5, orchids6, calla lily7 and rice8. However, none of these transgenic plants was tested for disease resistance under field conditions. In previous work, transgenic plants of two banana cultivars ‘Sukali ndiizi’ (apple banana; AAB group) and ‘Nakinyika’ (EAHBs; AAA group) were generated by constitutively expressing the Hrap9 or Pflp10 gene. Here we evaluated the best 65 resistant lines (40 lines expressing Hrap gene and 25 lines with Pflp gene) in a confined field trial at National Agriculture Research Laboratories, Kawanda, Uganda, against X. campestris pv. musacearum for two successive crop cycles (see Supplementary Methods) (Fig. 1). To the Editor: Banana is a major staple crop in East Africa produced mostly by smallholder subsistence farmers. More bananas are produced and consumed in East Africa than in any region of the world. Uganda is the world’s second foremost grower with a total annual production of about 10.5 million tons. The average daily per capita consumption in Uganda ranges from 0.61 to over 1.6 kg, one of the highest in the world. In this Correspondence, we report preliminary results from a confined field trial in Uganda of transgenic bananas resistant to the deadly banana Xanthomonas wilt (BXW) disease. BXW caused by Xanthomonas campestris pv. musacearum is threatening banana production, the livelihoods of the smallholder growers in East and Central Africa1, and the stability of food security in the region. The disease has caused estimated economic losses of about

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

2–8 billion over the past decade and substantial reductions in production have resulted in major price increases1. BXW was originally reported1 in Ethiopia and first identified in Uganda in 2001 and subsequently in the Democratic Republic of Congo, Rwanda, Kenya, Tanzania and Burundi. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas (EAHBs) and exotic dessert and beer bananas. The economic impact of the disease is potentially disastrous because it destroys whole plants leading to complete yield loss. Once these pathogens have become established in smallholder plantations, disease control is very difficult. As diseases continue to spread, demand grows for new improved varieties. There are currently no commercial pesticides, biocontrol agents or resistant cultivars available to control BXW1. Although BXW can be managed by following phytosanitary practices, the adoption of such practices has been inconsistent as they are labor intensive1. Given the rapid spread and devastation of BXW across Africa, the lack of known genetic resistance in banana against X. campestris pv. musacearum, and the difficulties associated with conventional breeding of this highly sterile crop, genetic transformation through the use of modern biotech tools offers an effective and viable way to develop resistant varieties. Two potential transgenes for controlling BXW are those encoding hypersensitive response-assisting protein (Hrap) and plant ferredoxin-like protein (Pflp) from sweet pepper (Capsicum annuum). Both have been proven effective against related bacterial pathogens, such as Erwinia, Pseudomonas, Ralstonia and Xanthomonas spp., in other crops2–8. Hrap is one of the important hypersensitive cell death–associated genes that could be used to protect plants from bacterial pathogen attack2. HRAP has been reported to intensify the hypersensitive response mediated by harpinPSS (harpin derived from Pseudomonas syringae pv. syringae), by dissociating multimeric forms of the hairpin into dimers and monomers that trigger stronger hypersensitive cell death necrosis. Constitutive expression of Hrap genes in transgenic tobacco2 and Arabidopsis3 has been shown to confer enhanced resistance against virulent pathogens under laboratory and glasshouse conditions. Resistance resulting from overexpression of the Pflp gene in transgenic plants is due Field trial of Xanthomonas wilt disease-resistant bananas in East Africa

Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15 days in the presence of selective medium containing 1 microM methotrexate. The transformed clones contained an estimated 100-500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.

Stable expression of insect GABA receptors in insect cell lines promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines

Two welldescribed elements of the Immune response of insects include encapsulation of metazoan parasites (blood‐cell‐mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co‐regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitold Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal‐related immunity factor (Dil), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.

Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ‐aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non‐Drosophilid insects.

Functional analysis of a mosquito γ-aminobutyric acid receptor gene promoter

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homo‐muitimeric expression of a γ‐aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus‐infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100‐fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.

An amplified mosquito dihydrofolate reductase gene: ampiicon size and chromosomal distribution

Abstract A single point mutation in the insect γ-aminobutyric acid receptor (GABAR)-encoding gene ( Rdl ) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the Rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti . Promoter activity is contained within a 2.53-kb fragment immediately upstream from the Rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals a number of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis -acting control elements underlying ion-channel gene regulation are discussed.

Sequence of a mosquito ribonucleotide reductase cDNA and evidence for gene amplification in hydroxyurea-resistant cells

The dihydrofolate reductase ampiicon in metho‐trexate‐resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate‐resistant cells. Subsequent to amplification, an additional polymorphism at a ten base‐pair XmrA recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed‐field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase ampiicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx‐5011‐128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.

The Drosophila Cytochrome P450 Gene Cyp6a2: Structure, Localization, Heterologous Expression, and Induction by Phenobarbital

Ribonucleotide reductase is essential for production of the deoxyribonucleotide substrates required for DNA synthesis in all eukaryotic cells. The full‐length cDNA encoding a mosquito ribonucleotide reductase R2 subunit was obtained from Aedes albopictus cells using a polymerase chain reaction (PCR)‐based approach. The cDNA contained 1197 nucleotides, and encoded a 398 amino acid R2 protein. Overall, mosquito R2 shared approximately 70% amino acid sequence identity with R2 proteins from vertebrates, but conservation at the N‐terminus of the protein was relatively low. The sequence of R2 cDNA was identical in hydroxyurea‐resistant mosquito cells and in wild‐type cells, while the R2 gene copy number was increased ten to twentyfold in resistant cells.