Project description:In order to analyze the production of small RNA (sRNA) by Potato spindle tuber viroid- RG1 strain (PSTVd-RG1) upon infecting the plants, the tomato plants (Lycopersicum esculentum cv. Rutgers) were inoculated with the PSTVd-RG1. After 21-days of post inoculation, total RNA was extracted and subjected for deep-sequencing using Illumina MiSeq platform. The primers were trimmed and the 21- to 24-nt long small RNA species were filtered after quality check of the raw data.
Project description:5′ RNA ligase mediated rapid amplification of cDNA ends (5′ RLM-RACE) is the widely used tool for confirming the direct cleavage of target mRNA by a miRNA. Recently, 5′ RLM-RACE combined with high-throughput sequencing such as parallel analysis of RNA ends (PARE) are gaining importance as these techniques allows to assess the large number of sample in a single experiment. In order to validate the cleavage of predicted a target mRNA in potato spindle tuber viroid-RG1 (PSTVd-RG1) infected tomato plants, the leaf samples collected at 21-days post inoculation were subjected for RNA extraction. A custom RNA adaptor containing MmeI restriction endonuclease was ligated to the free 5'-phosphate end of an uncapped mRNA using T4 RNA ligase, followed by reverse transcription (RT) and second strand synthesis using poly (T) primers. Obtained product was slightly amplified, cleaved with MmeI restriction endonuclease and then, a dsDNA adaptor was added to the 3’-end of the MmeI restriction endonuclease cleavage site. Resulted products were sequence in an Illumina Mi-Seq machine. Retrieved data was then used to verify the 5’-termini of predicted target mRNA.
Project description:In order to verify the production of viroid specific small RNAs (vd-sRNA) by viroids upon infecting plants, the tomato plants (Lycopersicum esculentum cv. Rutgers) were inoculated with Potato spindle tuber viroid (PSTVd) variants. After 21-days of post inoculation, total RNA was extracted and subjected for deep-sequencing using Illumina platform. Obtained data was analyzed for the presence of PSTVd specific small RNAs.
Project description:In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Lycopersicum esculentum cv. Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of post inoculation, total RNA was extracted and subjected for deep-sequencing using Illumina platform. The primers were trimmed and only 21- to 24-nt long small RNAs were filtered after quality check of the raw data. The filtered 21- to 24-nt was mapped against the genomic and antigenomic strands of the respective PSTVd variants using standard pattern matching algorithm. The profiling of viroid derived sRNA (vd-sRNA) revealed that the viroids are susceptible to host RNA silencing mechanism. Evaluation of the vd-sRNA production in PSTVd infected tomato plants by high-throughput sequencing of small RNAs.
Project description:In order to analyze the production of small RNA (sRNA) by Potato spindle tuber viroid-intermediate strain (PSTVd-I) upon infecting the plants, the tomato plants (Lycopersicum esculentum cv. Rutgers) were inoculated with the PSTVd-I. After 21-days of post inoculation, total RNA was extracted and subjected for deep-sequencing using Illumina MiSeq platform. The primers were trimmed and the 21- to 24-nt long small RNA species were filtered after quality check of the raw data.
Project description:The tomato SlWRKY3 transcription factor was overexpressed in cultivated tomato (Solanum lycopersicum)and transgenic plants transcriptome was compared to that of wild-type plants.