Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes.

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica.

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants.

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain.

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory. The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee. The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes. The level of expression varies within all three neuropiles.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Distribution of dopamine receptors and dopamine receptor homologs in the brain of the honey bee, Apis mellifera L.

In the brain of the honey bee, Apis mellifera, the radioligands [3H]-SCH23390 and [3H]-spiperone recognise D1- and D2-like receptors, respectively. In addition to being pharmacologically distinct and exhibiting significantly different expression profiles during the lifetime of the bee, [3H]-SCH23390- and [3H]-spiperone-binding sites differ markedly in their distribution within the brain. Estimates of [3H]-SCH23390-binding site density are highest in the somatal rind, whereas [3H]-spiperone-binding sites are most concentrated in the beta lobe neuropil of the mushroom bodies. Molecular cloning techniques have been used to identify two honey bee genes encoding dopamine receptor homologs. The first is the honey bee counterpart of a Drosophila D1-like dopamine receptor and is expressed in the mushroom bodies of both workers and drones. The second is related to D2-like dopamine receptors from vertebrates and is expressed in the brain of the bee, but the precise distribution of expression is not yet known.

Isolation of seven unique biogenic amine receptor clones from the honey bee by library scanning.

The biogenic amine receptor genes constitute an ancient and highly divergent family within the larger superfamily of G-protein-coupled receptors. These receptors play a central role in modulating nerve cell activity and thus behaviour. Because the honey bee offers numerous advantages for behavioural studies we endeavoured to isolate as many members of this gene family as possible from the bee. We compared numerous approaches to gene isolation and found that PCR amplification from small subfractions of cDNA or genomic DNA libraries enabled us to isolate clones that are otherwise undetectable. In total we isolated seven biogenic amine receptor clones and identified five additional related sequences by low-stringency Southern hybridization. Two clones, AmBAR4 and AmBAR6, are 84% and 72% identical to the Drosophila 5-HT2 and D1b receptors, respectively, and probably represent orthologous genes. Phylogenetic analysis indicates that AmBAR5 clusters loosely with a variety of tyramine and octopamine receptors with which it shares <66% identity. The other four clones, AmBAR1, AmBAR2, AmBAR3 and AmBAR7, are weakly to moderately related (28-45% identical) to Drosophila dopaminergic or mammalian adrenergic receptors and probably represent receptors of these classes whose orthologues have not previously been isolated from any insect. The honey bee clones expand the size of the known insect biogenic amine receptor gene family to sixteen members. Therefore the size of the biogenic amine receptor gene family of insects approaches that of vertebrates. This is true despite the reduced behavioural and genetic complexity of the insects relative to vertebrate animals.

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum.

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S2 clone, with 1.6 kb of 5' sequence, nor the S6 clone, with 2.8 kb of 5' sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S2 clone was not detected, however the S6 clone was expressed (at low levels) in three out of four transgenic plants. An S6-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.

Primary sequence, copy number, and distribution of mariner transposons in the honey bee.

A single honey bee mariner transposon (TnM1a) was sequenced, revealing a transpositionally non-autonomous element of 937 bp delimited by 30 bp perfect inverted terminal repeats. The element is flanked by the TA duplication typical of mariner elements in general. There are approximately 435 copies of TnM1a homologous elements per haploid genome. These elements appear, by Southern blot analysis, to be dispersed throughout the genome. Thirteen individual genomic clones with an average size of 15 kb, were found to contain only a single element each, which also suggests that the elements are not tightly clustered. Finally, mariner elements are neither inactivated by methylation nor sequestered into a methylated fraction of the genome.

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum.

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.

Transformation and regeneration of the self-incompatible species Nicotiana alata Link & Otto.

A transformation and regeneration system has been developed for Nicotiana alata, a plant which is being intensively studied as a model of gametophytic self-incompatibility. Plantlets can be regenerated efficiently from seedling hypocotyls. Kanamycin-resistant, transformed plants have been obtained by cocultivation of regenerating hypocotyls with Agrobacterium tumefaciens strain LBA4404 containing a binary vector. The transformation frequency was low with less than 1% of tissue explants regenerating transformed plants. The transformed plants contained from one to three copies of the introduced DNA. In most cases, the kanamycin resistance phenotype was transmitted to the offspring as a normal Mendelian factor. In one unusual case, none of the offspring inherited the kanamycin resistance of the transformed maternal parent. This plant may have been chimeric or the kanamycin resistance gene may have been inactivated.

Style self-incompatibility gene products of Nicotiana alata are ribonucleases.

Self-incompatibility in flowering plants is often controlled by a single nuclear gene (the S-gene) having several alleles. This gene prevents fertilization by self-pollen or by pollen bearing either of the two S-alleles expressed in the style. Sequence analysis shows that three alleles of the S gene of Nicotiana alata encode style glycoproteins with regions of defined homology. Two of the homologous regions also show precise homology with ribonucleases T2 (ref. 4) and Rh (ref. 5). We report here that glycoproteins corresponding to the S1, S2, S3, S6 and S7 alleles isolated from style extracts of N. alata are ribonucleases. These style S-gene-encoded glycoproteins account for most of the ribonuclease activity recovered from style extracts. The ribonuclease specific activity of style extracts of the self-incompatible species N. alata is 100-1,000-fold higher than that of the related self-compatible species N. tabacum. These observations implicate ribonuclease activity in the mechanism of gametophytic self-incompatibility.

Recent developments in the molecular genetics and biology of self-incompatibility.

A summary of recent work on molecular aspects of self-incompatibility in Nicotiana alata is presented. The amino acid sequences of style proteins corresponding to different S-alleles of N. alata have a high level of homology in some regions and are variable in other regions. The regions of homology include N-terminal sequences as well as most of the glycosylation sites and cysteine residues. The glycosyl substituents may consist of a number of 'glycoforms'. The isolated style S-glycoproteins inhibit in vitro growth of pollen tubes. The S-glycoproteins tested inhibited the growth of pollen of several S-genotypes, and there was some specificity in the interaction. Heat treatment of the isolated S-glycoproteins dramatically increased their activity as inhibitors of pollen tube growth, although the specificity in the interaction was lost. The nature of the S-allele products in pollen is not yet established.

Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays.

We studied the fine structure of the nopaline synthase (nos) promoter, which is active constitutively in a wide range of plant tissues, by both transient and stable transformation expression analyses. 3' and 5' deletion fragments were linked to form a set of internal deletion and duplication mutants that scanned the nos promoter. These mutated promoters were linked to the gene for the marker chloramphenicol acetyltransferase (CATase) as a means to readily assay promoter strength. The stable transformation analysis revealed the functional importance of an extended CCAAT box region (-97 to -63). Deletion of an upstream region (-112 to -101) containing an octameric repeated element resulted in a reduction in promoter strength by a factor of 30. A further deletion (-119 to -101) disrupted a potential Z-DNA-forming element as well, totally eliminating promoter function. Thus, a 19-base deletion across a repeated octamer and a potential Z-DNA-forming element identifies an essential upstream activator in the nos promoter. Duplication of the upstream element tripled promoter activity. Electroporation-mediated transient analysis was unable to distinguish downstream promoter elements. However, the upstream element behaved similarly in both assays in that deletion of the entire upstream element resulted in no promoter activity and that duplication of the element significantly enhanced the promoter strength.

A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci.

The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25-kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements.