Plants viruses as biological vectors

Transcription

1 Plants viruses as biological vectors

2 Virus very small infectious particles composed of a protein coat and a nucleic acid core. Most viruses have at least 3 genes: One (or more) concerned with replication of the nucleic acid, One (or more) concerned with cell-to-cell movement of the virus and One (or more) encoding a structural protein that is assembled into the virus particle (usually called the "coat" or "capsid" protein). There may also be additional genes that have a regulatory function or which are required for transmission between plants.

3 Viruses provide natural examples of genetic engineering since viral infection provide addition of new genetic material to the target cell Advantage of viral infection: High efficiency gene transfer: as amplification of genes occurs via viral genome replication cross protection a plant infected by one virus usually can t be superinfected by a second strain of a related virus- infection by mild strain or non-infectious strain provide protection against infection by more severe isolates this has encouraged the use of viruses for genetic transformation of plants

4 Virus as vectors Viruses are able to adsorb to and introduce their nucleic acid into intact plant cells However for many viruses, naked DNA or RNA are also infectious, allowing recombinant vectors to be directly introduced into the plants by methods such as leaf rubbing Recombinant viral vectors have potential for high level of transgene expression since infected plant cells yield large amount of virus Viral infection being systemic result in expression of transgene in all cells Viral infection are rapid and therefore large amounts of recombinant proteins can be produced in a few weeks Viral vectors are non integrative episomal vectors, therefore they have high copy number per cell and they are not subjected to the position effect. The gene product is very rapidly accumulated. Viral genome sequences are excellent source of promoters, enhancers and other components useful for designing gene vectors

5 Characteristics of viral vector Broad Host range Virulence Ease of mechanical transmission Rate of seed transmission Virus whose capsid is filamentous or rod shaped or viruses with multi-partite genome or helper or satellite component offer potential for carrying extra nucleic acid Genetic material must be able to be manipulated and be infectious

6 Most notable plant virus based vectors Caulimovirus based vectors Gemini virus based vectors TMV based vectors

7 Caulimovirus Unique plant viruses in having genome made up of double stranded DNAmakes it more compatible for manupulations Caulimovirus is the collective name for a group of tightly related viral species usually transmitted by aphids. Symptoms occur two to three weeks after infection and can be recognized by the mosaic-like lesions of infected leaves. The virus spreads systemically, its secondary symptoms are similar to those of the primary infection. Leaves that were infected during their development display deformed leaf blades. Caulimoviruses have, however, been important in genetic engineering as the source of highly active promoters that work in all plants and that are used to obtain expression of genes introduced by Ti plasmid cloning or direct gene transfer. The prototype of a plant virus with double-stranded DNA is the cauliflower mosaic-virus (CaMV).

8 Cauliflower mosaic virus (CaMV) dsdna virus of size 8kb. Mechanical and aphid mediated transmission Virion DNA alone or cloned CaMV DNA is infectious when simply rubbed on leaves High copy number (Up to 10 6 copies per cell). Rapid spread within 3-4 weeks for systemic infection through plant. The promoters (particularly 35S) and terminator for both transcripts have been utilized in plant expression vectors.

9 Genome properties of CaMV Linear, open circular and twisted or knotted form Unusual structure characterized by the presence of three discontinuities in the duplex genome Eight tightly packed genes expressed as two major transcripts : 35s RNA and 19s RNA 35s RNA: represent entire genome 19s RNA: encodes for gene VI

10 Genome properties of CaMV (contd..) ORF I - Movement Protein ORF II - Insect Transmission Factor ORF III - Structural Protein, DNA- Binding Capabilities ORF IV - Capsid Protein ORF V - Protease, Reverse Transcriptase and RNaseH ORF VI - Translational Activator, Inclusion Body Formation/Trafficking; Possibly more functions ORF VII - Unknown (Appears to not be required for infection)

11 CaMV activity in plant cell Gene I uncoating nucleus Inclusion body (gene VI) Gene III/IV transcription assembly 35S RNA 19S RNA Reverse transcription Gene V Gene IV translation

12 Genome properties of CaMV (contd..) Two regions of the CaMV genome- ORF II and VII do not seem to be essential for infection, as both can be either deleted or expanded by small inserts for foreign DNA Since CaMV has an icosahedral capsid, the size of the genome can t be increased greatly without affecting the efficiency of packaging The maximum insert size is of less than 1kb after deletion of all non-essential genes

13 Recent research has shown that it might be possible to circumvent this problem by adopting a helper virus strategy In this strategy, the cloning vector is a cauliflower mosaic virus (CaMV) genome that lacks several of the essential genes, which means that it carry a large DNA insert but cannot by itself direct infection. Plants are inoculated with the vector DNA along with a normal CaMV genome. The normal viral genome provides the genes needed for the cloning vector to be packaged into virus proteins and spread through the plant.

14 Example I: Expression of bacterial gene in plants No functional genes have yet been introduced into these ORFs. Here we report the replacement of CaMV ORF II by the R67 plasmid-encoded dihydrofolate reductase (DHFR) gene; this gene (dhfr) confers resistance to methotrexate in Escherichia coli. The chimeric viral DNA can be stably propagated in turnip plants and the dhfr gene is expressed, producing a functional enzyme

15 Example II: Mammalian metallothionein Functions in Plants A recombinant cauliflower mosaic virus (Ca-MTII) was constructed by inserting a cdna clone of Chinese hamster metallothionein II into the ORF II of the cloned virus pca- BB1. Systemically-infected Brassica campestris tissue contained metallothionein at a level of 0.5% of the soluble leaf protein. This efficient expression conferred 4X the Cdbinding capacity when compared with Ca-BB1 infected leaves. Ca-MTII-infected leaves exposed to 1 mm CdCl2 bound all the free Cd whereas uninfected leaves possessed 43.8 nmol free Cd per milligram of protein. This may be responsible for Cd resistance in the Ca-MTII plant cells. Metallothionein is the first mammalian gene product shown to be functional in plants.

16 Challenges with CaMV vector Small insertions (10-30 bp) in various sites abolished infectivity. Only gene II could tolerate insertion of significant size and could be entirely removed But the largest insert tolerated so far is bp. Complicated polycistronic design (ATG of cloned DNA must not interfere with the termination of gene I). CaMV derived vectors are restricted to members of cruciferae and some species of solanceae only CaMV DNA has multiple cleavages sites which also limits its usefulness Due to these limitations, CaMV vectors have not be widely used.

17 Gemini Viruses Characterized by twin (geminate) virions comprising two partially fused icosahedral capsids Small single stranded DNA in circular form represents the genome Genome in some species divided into two segments namely DNA A and DNA B Insects (greenhouse whitefly, grasshoppers, and others) help usually in spreading Gemini-viruses in nature.

18 Monopartite C1 WDV V1 V2: capsid protein V1: systemic spread C1 and C2: replication C2 V2 Bipartite AC1 TGMV DNA A AV1 BV1 TGMV DNA B BV1 AC1: replication AV1: Coat protein AC2: transactivation of sense genes AC3: delayed or attenuated symptoms BC1 and BV1: spread AC2 AC3

19 [The EMBO Journal (2000) 19, doi: /emboj/ ]

20 So far two genera of gemini viruses namely Begomovirus and Mastrevirus have been developed as vectors Begomovirus: with bipartitite genomes Transmitted by whitefly Bemisia tabaci Infects dicots Examples of the vectors: African cassava mosaic virus (ACMV) and Tomato golden mosaic virus (TGMV) Mastrevirus: monopartite genome Transmitted by leaf hoppers Mostly infect monocots Vectors developed so far: Maize streak virus (MSV) and Wheat dwarf virus (WDV) Mastreviruses are not mechanically transmissible Solved this problem by Grimsley et al, 1987 using the principle of Agroinfection Agroinfection: a plasmid containing tandem dimer of the MSV genome inserted into binary vector at T-DNA region Usually replacement of coat protein gene used as strategy for construction of expression vectors

21 Gemini virus vectors have their coat protein coding sequence replaced with a reporter gene or gene of interest Expression vectors based on these virus have been found to successfully deliver, amplify and thus express the gene of interest into the protplasts, cultured cells and the intact plants as well Able to accommodate gene inserts of size up to 3 kb

22 Advantages of Gemini virus as expression vectors The ssdna genome replicate via a double stranded intermediate and this makes the in vivo manipulation more convenient Due to presence of bipartite genome it is possbile to delete or replace the viral coat protien encoding genes with the gene of interest without interfering the replication of viral genome; thus increases the efficiency of gene expression These are particularly interesting because their natural hosts include plants such as maize and wheat, and they could therefore be potential vectors for these and others monocots.

23 But Gemini difficulties; Constraints viruses have presented their own set of one problem being that during the infection cycle the genomes of some Gemini viruses undergo rearrangements and deletions, such that the target gene insert might get excised, which would scramble up any additional DNA that has been inserted, an obvious disadvantage for a cloning vector. not readily transferred by mechanical means from plants to plants The size of gene of interest that can be inserted still very small

24 RNA viruses as expression vectors in Plants Most plant RNA viruses have filamentous morphology and this sort of packaging don t present the constraints regarding the limitation of the size of insert

25 Tobacco mosaic virus (TMV) One of the most extensively studied plant viruses Has monopartite RNA genome of size 6.5 kb Movement protein and coat protein encoded by the subgenomic RNA Replacement of coat protein gene with the cat gene showed high level CAT activity at the site of infection. But the expression was found to be confined at the point of viral entry because the coat protein is required for systemic infection

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27 Assignment Elaborate about the uses of viruses as vectors in generation of transgenic plants along with their advantages and constraints. [7.5]