Extraction and molecular detection of viral dsRNA from different infected plants

Extraction of viral double stranded RNA (dsRNA) from infected plants is helpful in identification of the viruses involved in infection. To date, there have been several methods developed to isolate dsRNA; however, type of the plant and virus is determinative in extraction efficiency. In this study we extracted dsRNA from different woody and herbaceous plants through a modified method which reduces the costs and time of extraction procedure. This method is based on different affinity of nucleic acids for the cellulose CF-11 in1X STE (Sodium chloride Tris EDTA) buffer containing 16 % ethanol. There is no phenol treatment or mini columns used in the isolation procedure. Extracted dsRNAs were identified by ribonuclease treatment and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). We have applied the procedure on five different hosts representing Amaranthaceae, Vitaceae, Fabaceae and Rosaceae infected with four different viruses representing Secoviridae and Bromoviridae.&nbsp

Molecular detection of Grapevine fanleaf Virus by the isolation of ssRNA and dsRNA from Xiphinema index

Xiphinema index is an important grapevine pathogen nematode which also vectors Grapevine fanleaf virus. The viral genes involved in transmission by the vector nematode are mapped to the C-terminal residues of RNA2-encoded polyprotein. To recognize viruliferous nematodes, there are some serological and molecular methods. In this study, we extract RNA and dsRNA of the virus, then Reverse transcription-polymerase Chain Reaction was done with virus specific primers to detect virus in its vector. The virus was detected by visualizing the desired 350 and 750 bp gene fragments in electrophoresis. This study reduces the virus detection time to only couple of hours with least imposed charges, and could be employed in transmission experiments as well.&nbsp

Molecular Characterization of Snowdrop Lectin (GNA) and its Comparison with Reported Lectin Sequences of Amaryllidaceae

WOS: 000389669800002Plant lectins have become efficient sources of insect resistance in crops. The present study was conducted to identify, amplify, clone and characterize the plant lectin gene GNA. The lectin, present in Galanthus nivalis (snowdrop), is an agglutinin toxic to hemiptera. The attempt was made to elucidate the relationship of the lectin gene trGNA (GNA isolated and characterized from Turkey) with other previously cloned lectins having insecticidal activity and to ensure the presence of the conserved mannose-binding region/site in the gene sequence. The full-length cDNA of trGNA was 477 bp that contained a 333 bp open reading frame encoding 157 amino acid proteins with 23 amino acids of signal peptide. BLAST results showed that trGNA has 89-97% similarity with previously reported GNA sequences while it has 84-96% similarity with earlier reported GNA protein sequences. No intron was detected within the region of genomic sequence corresponding to trGNA full-length cDNA. According to the search results from the NCBI (National Centrer for Biotechnology Information database), trGNA from Galanthus nivalis is most similar to the previously reported lectin sequences of Narcissus tazetta with a similarity percentage of 87%. The obtained results are useful for engineering of plants with enhanced insecticidal activity against chewing and sucking insects, causing crop pests. In addition, medical application of lectins may also be considered.Scientific and Technological Research Council of Turkey (TUBITAK) - BIDEB [2215]The authors are grateful to The Scientific and Technological Research Council of Turkey (TUBITAK) - BIDEB for granting PhD. fellowship (2215) to Mr. S.D. Khabbazi

Development of insect-resistant cotton lines with targeted expression of insecticidal gene

In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T0 progeny were further raised under greenhouse conditions to obtain progeny (T1). The introduced gene was properly inherited and expressed in T1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized

DEVELOPMENT OF INSECT-RESISTANT COTTON LINES WITH TARGETED EXPRESSION OF INSECTICIDAL GENE

WOS: 000389771500008In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T-0 progeny were further raised under greenhouse conditions to obtain progeny (T-1). The introduced gene was properly inherited and expressed in T-1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized.TUBITAK (The Scientific and Technological Research Council of Turkey) [2216]The corresponding author is grateful to TUBITAK (The Scientific and Technological Research Council of Turkey) for providing postdoctoral fellowship Code: 2216) to carry out the research activities at University of Ankara. The authors acknowledge Dr. Umut Toprak, Associate Professor, Department of Plant Protection, Faculty of Agriculture, University of Ankara for providing Helicoverpa armigera for our lab bioassays experimen