Ana M. Bailey

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase.

SummaryThe gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.

A cutinase-encoding gene from Phytophthora capsici isolated by differential-display RT-PCR

Abstract To detect and ultimately isolate genes of Phytophthora capsici the expression of which is induced during its interaction with pepper, a comparative analysis of gene expression in the wild-type pathogenic fungus with expression in a non-pathogenic (Nop) mutant reduced in cutinase and esterase activities was performed by the differential display of mRNAs. Discrimination of fungal genes induced in planta, from plant genes induced in response to the pathogen, was accomplished by exposure of the mycelium to bare-rooted seedlings of pepper (Capsicum annuum) in sterile water, to allow the initiation of infection, and then physical removal of the induced mycelium. With six sets of primer combinations, eight cDNA fragments (representing fungal genes) were present in planta only for the pathogenic strain. RNA-blot analysis showed that the transcripts detected accumulated to detectable levels only at early stages of the interaction. Sequence analysis and database searches revealed homology of one of the cDNA clones to fungal cutinases. The 218 amino-acid sequence predicted from sequencing a genomic clone of P. capsici suggested a protein of molecular weight of 23 980 Da with similarity to fungal cutinases previously characterized. These results indicated that differential-display analysis is sufficiently sensitive to be applied for the detection and isolation of fungal genes induced during a plant-pathogen interaction.

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and β-glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.

Transformation of four pathogenic Phytophthora spp by microprojectile bombardment on intact mycelia

SummaryPhytophthora capsici, P. citricola, P. cinnamomi and P. citrophthora were transformed without the removal of cell walls by particle acceleration with plasmids containing the β-glucuronidase gene and hygromycin B resistance. Transformants were detected by histochemical and fluorometric β-glucuronidase assays and confirmed by Southern-blot hybridization. It was found that the promoter of a plant virus is functional in Phytophthora. In addition, a method was designed to visually identify homogeneous transformed colonies, derived from zoospores of transformed multinucleated Phytophthora mycelia, based on blue color development on plates containing X-Gluc.

Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant viruses

PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan™, a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.

Transcriptome Analysis and Systemic RNAi Response in the African Sweetpotato Weevil (Cylas puncticollis, Coleoptera, Brentidae)

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

RNAi-based gene silencing through dsRNA injection or ingestion against the African sweet potato weevil Cylas puncticollis (Coleoptera: Brentidae)

BACKGROUND RNA interference (RNAi) technology can potentially serve as a suitable strategy to control the African sweet potato weevil Cylas puncticollis (SPW), which is a critical pest in sub-Saharan Africa. Important prerequisites are required to use RNAi in pest control, such as the presence of an efficient RNAi response and the identification of suitable target genes. RESULTS Here we evaluated the toxicity of dsRNAs targeting essential genes by injection and oral feeding in SPW. In injection assays, 12 of 24 dsRNAs were as toxic as the one targeting Snf7, a gene used commercially against Diabrotica virgifera virgifera. Three dsRNAs with high insecticidal activity were then chosen for oral feeding experiments. The data confirmed that oral delivery can elicit a significant toxicity, albeit lower compared with injection. Subsequently, ex vivo assays revealed that dsRNA is affected by degradation in the SPW digestive system, possibly explaining the lower RNAi effect by oral ingestion. CONCLUSION We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy.

Expression of the hygromycin B phosphotransferase gene confers tolerance to the herbicide glyphosate

Escherichia coli cells and tobacco (cv. Xanthi) plants transformed with the hygromycin B phosphotransferase gene were able to grow in culture medium containing glyphosate at 2.0 mM. The growth of tobacco calli in media containing increasing glyphosate concentrations was measured. The ID50 for glyphosate was 1.70±0.03 mM for hygromycin-B resistant plants, and 0.45±0.02 mM for control plants. Regenerated plants and progeny selected for resistance to hygromycin B were tested for glyphosate tolerance by spraying them with Faena herbicide (formulated glyphosate with surfactant) at a dose equal to 0.24 kg/ha. This was two times the dose required to kill 100 percent of the control plants. Phosphotransferase activity was measured in the extracts of the transformed leaves by the incorporation of 32P from [γ−32P]ATP and it was observed that hygromycin B phosphotransferase was able to recognize the molecule of glyphosate as substrate.

RNA interference : a promising biopesticide strategy against the African sweetpotato weevil Cylas brunneus

The African sweetpotato weevil Cylas brunneus is one of the most devastating pests affecting the production of sweetpotatoes, an important staple food in Sub-Saharan Africa. Current available control methods against this coleopteran pest are limited. In this study, we analyzed the potential of RNA interference as a novel crop protection strategy against this insect pest. First, the C. brunneus transcriptome was sequenced and RNAi functionality was confirmed by successfully silencing the laccase2 gene. Next, 24 potential target genes were chosen, based on their critical role in vital biological processes. A first screening via injection of gene-specific dsRNAs showed that the dsRNAs were highly toxic for C. brunneus. Injected doses of 200ng/mg body weight led to mortality rates of 90% or higher for 14 of the 24 tested genes after 14 days. The three best performing dsRNAs, targeting prosα2, rps13 and the homolog of Diabrotica virgifera snf7, were then used in further feeding trials to investigate RNAi by oral delivery. Different concentrations of dsRNAs mixed with artificial diet were tested and concentrations as low as 1 μg dsRNA/ mL diet led to significant mortality rates higher than 50%.These results proved that dsRNAs targeting essential genes show great potential to control C. brunneus.

Differentiation of "Citrus tristeza virus" (CTV) Isolates by Cleavase Fragment Lenght Polymorphism (CFLP) Analysis of the Major Coat Protein Gene

A panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/ RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequences of the variants composing the isolates. In artificial mixed infections of mild and severe isolates the patterns obtained were more closely related to the severe isolate. Thus the CFLP method was an accurate method for the identification, typing and clustering of CTV isolates. The usefulness of this technique as an alternative to SSCP analysis is suggested and discussed.