Mark K. Nakhla

Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex.

Summary.Tomato yellow leaf curl disease (TYLCD) is caused by a group of geminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complex and are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causes great yield losses in many countries throughout the Mediterranean region and the Middle East. In this study, the efficacy of post-transcriptional gene silencing (PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curl virus – mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curl Malaga virus, and tomato yellow leaf curl Sardinia virus – Spain [2] were selected and used to design a construct that can trigger broad resistance against different viruses that cause tomato yellow leaf curl disease. The silencing construct was cloned into an Agrobacterium-binary vector in sense and antisense orientation and used in transient assay to infiltrate tomato and Nicotiana benthamiana plants. A high level of resistance was obtained when plants were agro-infiltrated with an infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or challenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16–20 days post infiltration with the silencing construct. Results of the polymerase chain reaction showed that the resistance was effective against all three viruses. Furthermore, dot blot hybridization and PCR failed to detect viral DNA in symptomless, silenced plants. A positive correlation between resistance and the accumulation of TYLCV-specific siRNAs was observed in silenced plants. Together, these data provide compelling evidence that PTGS can be used to engineer geminivirus-resistant plants.

Multiplex Detection of Potato Virus S, Potato Virus X, and Potato Virus Y by Non-radioactive Nucleic Acid Spot Hybridization in Potato Tissue Culture Plantlets

Potato plantlets initiated into tissue culture must be tested for numerous viruses prior to propagation for seed potato production. Ideally, one plantlet is tested for all pathogens of concern and, if found pathogen-free, this plantlet is propagated for production of seed potatoes. Commercially available ELISA kits are generally used for the pathogen tests, but the commercial kits have some limitations. For example, the protocols differ for different viruses, so multiple extractions must be completed, increasing the time and expense of testing. This is a significant problem with tissue culture plantlets, for which there is limited material available to test and an ever-increasing number of pathogens that must be tested for, including viruses in the potyvirus, carlavirus, potexvirus, luteovirus, pomovirus, tobravirus, tospovirus, alfamovirus, and tymovirus groups. We have optimized a non-radioactive nucleic acid hybridization (NASH) assay for the simultaneous detection of carlavirusPotato virus S (PVS), potexvirusPotato virus X (PVX) and potyvirusPotato virus Y (PVY) in potato tissue culture plantlets. This assay requires a single extraction from a small portion of a tissue culture plantlet for the detection of viruses from three different families.ResumenLas plántulas de papa provenientes de cultivo de tejidos se prueban contra varios virus antes de su propagación para semilla. Generalmente se prueban las plántulas para todos los patógenos y cuando se encuentra una libre, esta se propaga para producir papa semilla. Los kits comerciales ELISA se usan generalmente para la prueba de patógenos, pero estos tienen ciertas limitaciones. Por ejemplo, los protocolos son diferentes para virus diferentes, por lo tanto se tienen que hacer múltiples extracciones, incrementado así el tiempo y costo de la prueba. Esto es un problema significativo con plántulas de cultivo de tejidos, para las cuales hay material limitado para las pruebas y un gran número de virus contra los que hay que probar, incluyendo grupos de potyvirus, carlavirus, protexvirus, luteovirus, pomovirus, tobravirus, tospovirus, alfamovirus y tymovirus. Hemos optimizado una prueba de hibridación no radioactiva de ácido nucleico (NASH) para la detección simultánea de carlavirus Virus S de papa (PVS), protexvirus Virus X (PVX) y potyvirus Virus Y (PVY) en plántulas de cultivo de tejidos. Esta prueba requiere de una sola extracción de una pequeña porción de tejido para la detección de virus de tres diferentes familias.

Occurrence and distribution of Citrus tristeza virus (CTV) in the Jordan Valley

In a survey conducted in 2002 and 2003, Citrus tristeza virus (CTV) was detected in the Jordan Valley. The direct tissue blot immunoassay (DTBIA) indicated that 12.7 and 15.2% of samples tested in the central and northern Jordan Valley respectively were infected with CTV. Similar results showed that all citrus species grown in the Jordan Valley were susceptible to CTV. DAS-ELISA analysis of samples from a citrus orchard in the Dir Alla area with severe CTV symptoms indicated that 49% of samples were infected with CTV. Using a CTV specific primer pair (CTV1/CTV10), the coat protein gene of the virus was successfully amplified from leaf extracts obtained from CTVinfected trees by IC-RT-PCR. After cloning and sequencing the coat protein gene, the sequence of the amplified product was deposited in the GenBank.

Detection and molecular characterization of grapevine virus A in Jordan

Summary. In a study on grapevines in Jordan conducted between 2002 and 2003, grapevine virus A (GVA) was detected in all areas where grapevines were planted. DAS-ELISA analysis of samples from symptomatic trees found that 16.1% of samples were infected with GVA. Using a GVA- specific primer pair (H587/C995), a portion of the coat protein gene of the virus was amplified by IC-RT-PCR and RT-PCR, using leaf extracts and RNA extracted from infected grapevines respectively. After cloning and sequencing the coat protein gene of the Jordanian isolate of GVA (GVA-Jo), the sequence of the amplified product was compared with sequences of other GVA isolates from different countries.