Bayram Çevik

Distribution and Characterization of Citrus tristeza virus in South Florida Following Establishment of Toxoptera citricida

The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.

In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus

SummaryExpression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84–99% to 81–100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

The RNA-dependent RNA polymerase of Citrus tristeza virus forms oligomers.

The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRpΔ1-166-HA, RdRpΔ1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV.

Detection of Citrus tristeza virus (CTV) from Satsuma Owari mandarins (Citris unshiu) by direct tissue blot immunoassay (DTBIA), DAS‐ELISA, and biological indexing

Abstract Three different citrus‐producing regions of Turkey—Edremit Gulf, Coastal Aegean, and eastern Black Sea—were surveyed in 2005 and 2006. A total of 119 samples were collected from Satsuma Owari mandarin (Citrus unshiu) trees grafted on Citrus tristeza virus (CTV)‐resistant Poncirus trifoliata rootstocks in commercial groves and home gardens. All samples were tested for the presence of CTV by direct tissue blot immunoassay (DTBIA) and double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) using young shoots. The samples that tested positive were indexed for biological properties. The results obtained from DTBIA tests showed that 20 of 119 (16.8%) tested trees were infected with CTV and 99 trees were virus free. All samples that tested positive in DTBIA were also positive in DAS‐ELISA. After biological indexing, none of these 20 isolates showed any symptoms on sour orange, grapefruit, and sweet orange plants, but all isolates induced vein‐clearing symptoms on Mexican lime (Citrus aurantifoli) in 9 months. Some isolates also caused leaf‐cupping and chlorosis in Mexican lime. However, 15 months after initial grafting, all isolates induced mild to moderate stem‐pitting on Mexican lime. In addition, young tissues, petioles, and leaf samples were collected periodically at monthly intervals for 1 year (2006) from a 25‐year‐old Satsuma Owari mandarin grafted on P. trifoliata in the Edremit Gulf. CTV was readily detected in tissue blots of young shoots and petioles from CTV‐infected plants during the autumn, winter, and spring seasons. Similarly, the highest ELISA values were obtained in April; the lowest values were noted in September. This study showed that DTBIA is a rapid, sensitive, and reliable procedure for detecting CTV under field conditions.

Expression analysis of WRKY genes from Poncirus trifoliata in response to pathogen infection

The majority of WRKY transcription factors (TFs) play a role in the regulation of defense response in plants. Three WRKY genes, PtrWRKY1, PtrWRKY2 and PtrWRKY3, were previously identified in Poncirus and their expressions were characterized in response to cold and drought in Poncirus and Citrus. In this study, expressions of these WRKY genes were studied in response to infection with two major pathogens of Citrus, Citrus tristeza virus (CTV) and Phytophthora citrophthora, in resistant Poncirus and susceptible pummelo (Citrus grandis) plants. Northern blot analysis showed that the expression of the PtrWRKY1 gene was induced earlier and stronger in Poncirus than in pummelo in response to CTV infection. On the other hand, the expression of the PtrWRKY1 gene was not altered in response to P. citrophthora infection neither in Poncirus nor in pummelo. When the expression of the PtrWRKY2 gene was analyzed, it was repressed by CTV inoculation in Poncirus plants, whereas the expression was not changed in response to CTV infection in pummelo or in response to P. citrophthora inoculation in Poncirus or pummelo. Similarly, the expression of the PtrWRKY3 gene was repressed in Poncirus, but not changed in pummelo by CTV inoculation; however, the expression of the PtrWRKY3 gene was induced in Poncirus, but it was repressed in pummelo in response to P. citrophthora inoculation. The expression analysis of three different WRKY genes revealed that they are differentially expressed in response to CTV and P. citrophthora infection in resistant Poncirus and susceptible pummelo suggesting that they may play a role in disease resistance in Poncirus.

An abiotic stress-responsive WRKY gene is transiently induced in response to cold and drought stresses in Poncirus trifoliata

Abstract A partial cDNA sequence sharing significant homology with WRKY transcription factors (TFs) was previously amplified from Poncirus trifoliata by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers. In this study, the full-length cDNA sequence of this cDNA, PtrWRKY3, was obtained by RACE method from P. trifoliata. The length of PtrWRKY3 cDNA was 1908 bp and contained open reading frame coding for a 512-amino-acid polypeptide. The ortholog of this gene, CpWRKY3, was also cloned and sequenced from Citrus paradisi (grapefruit). The gene consists of 1550 bp cDNA encoding a protein of 516 amino acids. Both genes have double WRKY domains with Cys2His2 signature motif and showed 98% amino acid sequence identity with each other. They were tightly clustered together and showed a close phylogenetic relation with putative WRKY TFs from different woody perennial plants. The expression of the WRKY3 gene was analyzed quantitatively by northern blot and real-time RT-PCR in response to cold and drought stresses in both cold-hardy Poncirus and one of the cold-sensitive Citrus species, C. paradisi. While the expression of the WRKY3 gene was initially induced transiently in response to cold in cold-hardy Poncirus, no significant changes in the expression of this gene were observed in cold-sensitive grapefruit plants. On the other hand, the expression of the WRKY3 gene was transiently induced by drought stress in both Poncirus and grapefruit plants. These results indicated that the WRKY3 gene from Poncirus and Citrus is conserved and its response to drought stress was similar in both species. However, its expression in response to cold was different in cold-hardy and cold-sensitive species indicating that it is transiently induced early in cold acclimation and may be involved in cold tolerance in Poncirus.

Biochemical and molecular characterizations of cypermethrin resistance in laboratory-selected cypermethrin-resistant strains of Tetranychus urticae Koch. (Acari: Tetranychidae)

ABSTRACT Pyrethroids are commonly used pesticides for controlling agricultural pests. A pyrethroid resistant strain of T. urticae was obtained after laboratory selection with cypermethrin for determining mechanisms of cypermethrin resistance. TaqMan diagnostic assay as well as sequencing of voltage gated sodium channel (VGSC) gene were performed in order to detect previously known mutations in German susceptible strain (GSS) confirming that it does not carry any of the known VGSC mutations. Therefore, GSS strain was selected with cypermethrin and the resistant C6 strain was generated that is free of VGSC mutations. Toxicity assay data revealed relatively high resistance levels of the C6 strain (54.24-folds) in comparison to GSS. All inhibitors (piperonyl butoxide (PBO), esterases, P450; S-benzyl-O, O-diisopropylphosphorothioate (IBP), esterases; and diethyl maleate (DEM), glutathione S-transferase (GST)) were used in combined toxicity assays against the major detoxification enzymes, synergized resistance by 1.41-, 1.61-, and 2.27-fold, respectively. In the C6 strain, activities of P450, GST, and esterase showed elevated activity in comparison to GSS. By application of TaqMan diagnostic assay, we confirmed that both GSS and C6 T. urticae strains are free of VGSC mutations, indicating that detoxification-based cypermethrin resistance mechanisms are involved in resistance and have significant effect on the phenotype.

Identification of drought-induced genes from the leaves of Rangpur lime (Citrus limon (L) Osbeck)

ABSTRACT Water stress is one of the limiting factors for citrus production and Citrus species show great variation in their response to drought stress. Although the majority of Citrus rootstocks are sensitive to water stress, Rangpur lime (Citrus limon (L) Osbeck) (RL) shows a high degree of drought tolerance. Therefore, it has been used as a rootstock in drought-prone environments, but mechanisms of drought tolerance are not yet known. In this study, to explore the mechanisms of drought adaptation and tolerance, a subtractive cDNA library was constructed from the leaves of 14-day drought-stressed and non-stressed RL for identification of drought-induced genes. 285 cDNA sequences were obtained from randomly selected clones from the subtracted library containing the drought-induced genes. The expression analyses of 200 cDNAs in 14-day drought treated and untreated RL by macroarray hybridisation revealed that the expression of 56 cDNAs increased two to 11-fold. 30 non-redundant drought-induced genes were identified from these cDNAs and drought induction of eight selected genes was confirmed by a real-time RT-PCR assay suggesting that expressions of these genes were regulated by drought-stress. Genes identified in this study were mostly related with cell rescue and defense pathways involved in drought adaptation and tolerance of RL.