2 siRNA Based Techniques

Transcript

Hello, welcome back to the second talk of the third week and that is si based RNA based detection technology . RNAi is a basic phenomenon that exist in all living beings, including plant system, where, a foreign RNA is degraded by the inherited small nucleotide of RNAs strengths and there by this mechanism plant defense itself from certain pathogen that invades the plant tissue system, and this particular technology has now been utilized commercially for basically develop resistance in the plants again certain plant pathogens. So what is RNAi silencing or RNA interference – It is a cytoplasmic cell surveillance system to recognize double stranded, RNA and specifically destroys single and double stranded RNA molecules, homologous to the end user using small interfering RNAs normally known as siRNA as a guide. Viruses are both inducers and targets of RNAi that constitutes a fundamental antiviral defense mechanism in eukaryotic organisms. It is particularly important in plants that use RNAi to recover from virus diseases. The use of high throughput sequencing of small RNAs (sRNA) from plants can successfully identify the viruses infecting them, including previously unknown viruses, even in extremely low titre symptoms infectious. So plants high throughput sequencing helped in identification of those viruses particularly of unknown origin because of this high throughput sequencing of the small RNA fragments that is get sequence along with the plant RNAs. RNA silencing constitutes a fundamental antiviral defense mechanism in plants in which host enzyme cut viral RNA into pieces of 20-24 nucleotides in size. When isolated and sequence and en-mass properly assembled, this virus derives small RNAs sequences can reconstitute genomic sequence, information of the virus being targeted in the plant. So once these high throughput sequencing is done followed by its assembly is done, then it takes out the genomic sequences which is differing from the plant origin, and that is why it is able to now differentiate the genomic sequence of the unknown pathogen particularly the viruses which infect the plants and causes disease.

This approach is independent of the ability to culture or purify the virus and does not require any specific amplification and our enrichment of viral nucleic acids. Using this technique known and novel DNA RNA viruses as well as virus as well as viroids have been identified at sensitivity levels comparable to PCR. So, even presence off a low amount of RNA or DNA genomes of the viruses this technique can be useful to detect of this unknown viruses. In short, the double stranded RNA is broken down or cut into piece smaller pieces of twenty to twenty four nucleotides sizes by a plant enzyme known as dicer, then one strand of the small double stranded RNA is then incorporated into the RISC complex and then, along with the RISC camp, complex this guide RNA goes for surveillance in the plant system and whenever it finds a complimentary sequence or particularly mRNA of a particular gene. Then it goes and bind with that particular side and then, with the help of these, RISC complex, this mRNA is then digested and then further fragmented into smaller pieces. So this is how the RNAi technology works, and if it is the originated from a virus , then this is a technology how this virus can be negated in the plant system. So here the same mechanism is depicted where double stranded RNA virus is then cut down into smaller fragments and then one fragment which serves as a guide RNA it incorporates with the RISC complex and then it goes and find the new template of the RNA which has complementary sides and then it cuts down into smaller fragments. So this small fragments of the cut virus RNA can be subjected to high throughput sequencing, followed by assembly into contigs and search for similarity in database using the blast can help us to identify the virus off which these fragments belong to or if it doesn’t belong to any of the known sequences in the database, it can be identified as a new virus. RNA silencing recognize double stranded, RNA and eliminates RNAs homologues to the inducer RNA, by cleavage using RNase III endonucleases called dicers. Plants encodes several Dicer-like enzymes that recognize and cleave long double stranded RNA (dsRNA) which is off 21-,22-, upto 24-base: pairs, fragments and acts as siRNAs. siRNAs then binds to ribonuclease H-like proteins in the RNA induced silencing complex which is in short known as (RISC) and are used to detect homologous single-stranded RNA (ssRNA) molecules for cleavage, producing more siRNAs. In plants, RNAi becomes amplified when the cleaved RNA recruits and RNA-directed RNA polymerase to generate more double stranded (dsRNA), which is again cleaved by a dicer protein to produce secondary siRNAs, that are once again able to detect and cleave homologous RNA in a type of ‘degradative PCR’ cycle. This leads to the accumulation of large amounts of siRNAs with homologous to the invading virus. So, this is the basic mechanism how the siRNAs are generated in the plant system which can be even enhanced its copy number with the technology using RNA-directed RNA polymerase and then further accumulation of siRNAs which we call it as secondary siRNAs and this accumulate siRNAs are being now sequenced for identification of the new virus. Application of Next Generation Sequencing (NGS) for siRNA detection. NGS platforms such as illumina (Solexa) and SOLid generates massive amounts but rather short reads of nucleotide sequences. With them a set of bioinformatics tools are used to de novo assembly of such short reads and deep sequencing of siRNAs could thus lead to detection and diagnosis of plant viruses. So, in this particular case for detection of viral organism which is noble in nature:

The NGS platforms Illumina and SOLiD is being proved to be very helpful. NGS platforms have been used to detect- Reverse Transcribing Viruses, Known Viruses, New Viruses and even mixed infections or Defective RNA/DNAs as well as Contamination. So, NGS platforms have been used for various purposes for correctly identifying and detecting the casual agent of plant symptoms. So, with this we have come to an end of the siRNAs based diagnostic technology. In brief, it deals with particularly the NGS technologies are used for sequencing of siRNAs and this nucleotide sequenced siRNAs can be assembled and then we can get an idea of the entire genome of virus that is causing the plant symptom and if it is subjected to similar sequences in the NCBI database we can identify the existing virus that is being infecting plants and different plant species. Or if the sequences are entirely different from the sequences data being deposited in NCBI database, then it categorized to be a new virus that is causing the plant disease.

So with this we have come to an end of second talk of this week that is siRNAs based diagnosis and in the next topic will be talking about genomic based diagnosis. Till then….

Thank you very much.