; VIRAL disease
Documents
Resources
Learning Center
Upload
Plans & pricing Sign in
Sign Out
Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

VIRAL disease

VIEWS: 114 PAGES: 43

  • pg 1
									Reducing the effects
 of VIRAL disease



     RT 20104 Agriculture Biotechnology
      Viral infections of plants
• Cause:
  – Cell necrosis
  – Hypoplasia – retarded cell division & growth
  – Hyperplasia – excessive cell division/ cell
    expansion
• Symptoms:
  – Growth retardation
  – Distortion
  – Mosaic patterning of leaves
  – Yellowing
  – Wilting
              RT 20104 Agriculture Biotechnology
         Types of plant viruses
• Virus – composed                  of a nucleic acid
  genome: 5 types
  1.   ssRNA, +ve sense
  2.   ssRNA, -ve sense
  3.   dsRNA
  4.   ssDNA
  5.   dsDNA
• Genome – surrounded by the capsid/
  protein coat – nucleocapsid

              RT 20104 Agriculture Biotechnology
Genome structure of plant viruses
Genome structure      Families and genera                        Notes
ssRNA, +ve sense      Families: Bromoviridae, Comoviridae,       70% of known plant viruses.
(act as mRNA          Potyviridae, Sequiviridae &                Both non-segmented & segmented (two or more RNAs in
directly)             Tombusviridae                              different virus particles)
                      Unassigned genera: luteo-, potex-,
                      tobamo- & tobraviruses
ssRNA, -ve sence      Family: Bunyaviridae, genus:               Bunyaviridae possess a lipidic envelope in addition to their
(needs to be copied   topspovirus                                nucleocapsid
before can act as     Family: Rhabdoviridae, genus:
mRNA)                 rhabdovirus
                      Unassigned genera: tenuivirus
dsRNA                 Family: Reoviridae, genera: fijivirus,     The plant membranes of the Reoviridae family have a
                      phytoreovirus, oryzavirus                  genome consisting of 10-12 segments of RNA. Each has 1
                      Family: Partitiviridae, genera: alpha- &   ORF which produces a protein
                      betacryptovirus
dsDNA                 Unassigned genera: caulimovirus (e.g.      The only plant viruses of this group are the caulimoviruses.
                      cauliflower mosaic caulimovirus) &         Their genome consists of one ds-circular DNA molecule
                      badnavirus                                 with specific as discontinuities in both strands. It codes for
                                                                 6 or 8 ORFs located on one strand only. DNA replication
                                                                 occurs by a process of reverse transcription (i.e. via an
                                                                 RNA intermediate) similar to that of animal retroviruses
ssDNA                 Family: Geminiviridae, includes            3 groups, the only plant viruses possessing either one or
                      bigeminiviruses                            two molecules of ss-genomic DNA. DNA replication occurs
                                                                 via ds-DNA intermediates. ORFs located on both the viral
                                                                 strand and its complement.
                                  RT 20104 Agriculture Biotechnology
• Plant viruses – morphologies
   – From rod shapes to isometric particles
   – Some have membranes envelopes around
     the nucleocapsid
• Virus genomes code for similar ‘core’ proteins:
   – The capsid/ coat protein(s)
   – Replication related proteins (e.g. polymerases
     & helicases)
   – Capping enzymes/ proteins
   – Movement proteins (facilitate the spread of
     the viruses)
• Some viruses have extra, small RNA segments
  – satellite RNA
   – Alter the pathology of the virus infection
              RT 20104 Agriculture Biotechnology
• 2 members of the most common group, +ve-
  sense RNA viruses:
   – Tobacco mosaic tobamovirus (TMV)
   – Comoviruses (CPMV)




           RT 20104 Agriculture Biotechnology
Tobacco mosaic tobamovirus (TMV)
Genome
• Composed of
  – One, ss linear RNA molecule – ~6400 bases
  – Has a tRNA-like structure at the 3’-end
• Code for 5 translated ORFs
  1. 183-kDa protein
  2. 126-kDa protein
  3. 30-kDa protein
  4. 54-kDa protein
  5. 17-kDa protein            symptoms of tmv in tobacco
                RT 20104 Agriculture Biotechnology
                             5 translated ORFs of TMV
                          183-kDa protein • Involved in the replicase complex
Non-structural proteins




                                          • A read-through product of the 126-
                                            kDa protein
                                          • Has a polymerase function
                          126-kDa protein • Involved in the replicase complex
                                          • Has a methyltransferase activity –
                                            involved in RNA capping
                                          • Has a helicase function

                          30-kDa protein   • A movement protein (MP)
                          54-kDa protein   • Unknown function
                          17-kDa protein   • Capsid/ coat protein (CP)
                                     RT 20104 Agriculture Biotechnology
                                 Sub-genomic promoters
                              I1 Leaky stop I2        CP
                                 codon
Genomic RNA                                                         tRNA like structure
       5’                                                                 3’
        Translation                                    CP            Sub-genomic RNA
  126-kDa
                                                                          CP sgRNA
  183-kDa
            Replication associated proteins
                                              I2                    17-kDa coat protein

                                                                            I2 sgRNA

                            I1
                 Transcription                               30-kDa Movement protein

  Translation                                                               I1 sgRNA
  of the TMV
     ORFs                                          54-kDa protein
                        RT 20104 Agriculture Biotechnology
Comoviruses (CPMV)
• Cowpea mosaic virus
• Genome: Segmented
  – RNA 1
     • Core polymerase
     • Protease                     symptoms of CPMV

     • VPg (genome-linked protein) protein
     • Attached to the 5’-end of the molecule
     • Fulfils a cap function
  – RNA 2
     • 2 coat-protein subunits
     • Movement protein
• Entry into the cell, the genomes act as
  messages & can be translated directly
              RT 20104 Agriculture Biotechnology
       Translation of the CPMV genome
     RNA1                                    RNA2
     VPg
   RNA                        AAAAA(A)n        RNA                  AAAAA(A)n

                                                           Translation
                     Translation

                                            Polyprotein             105-kDa
Polyprotein                   200-kDa
                                                                    95-kDa

                     Proteolytic cleavage                  Proteolytic cleavage
Protease cofactor
32-kDa                                        58-kDa
                                                           CP L   CP S
                                               48-kDa
     58-kDa VPg Protease Core polymerase                   37-kDa 23-kDa
                                             Transport
                32-kDa 87-kDa
                      RT 20104 Agriculture Biotechnology
             Entry and Replication
             – points of inhibition

Virus infection:

     Virus

   Host cell

  Replicate



                   RT 20104 Agriculture Biotechnology
Initial entry into the cell:
• Specific mechanisms
• Transferred between plants by vectors
   e.g. – nematodes
          – various arthropods – aphids,         left
             hoppers, whiteflies
          – Fungi



            RT 20104 Agriculture Biotechnology
•   Systematic infection
    – Virus must move from one cell to cell via the
      plasmodesmata/ vascular tissues
    – Exclusion limit of the plasmodesmata pore is
      normally too small to allow passage of the virus
      particles, but
    – Viral movement proteins may function by increasing
      the molecular size
      a. TMV – 30-kDa MP
         Modifies the plasmodesmata to allow the passage
         of genomic RNA coated by the 30-kDa protein to
         pass from cell to cell
      b. CPMV – 58/48-kDa proteins
         Form       tubular     structures    through the
         plasmodesmata to allow the passage of intact
         virus particles from one cell to another

                RT 20104 Agriculture Biotechnology
• Different viruses, different modes of movement
  – Transferred as RNA, may be coated with
     capsid/ movement proteins
  – Transferred as virus particles




             RT 20104 Agriculture Biotechnology
      How has industry dealt with
              viruses?
• Chemical control
  – If virus transmitted by an insect/ another vector
  – Limited impact
• Cross-protection
  – Some plants infected with a virus were
    apparently protected against infection by a
    related virus
  – Success to combat disease caused by
      • Citrus tristeza closterovirus (CTV)
      • Papaya ringspot potyvirus (PRSV)
      • Zucchini yellow mosaic potyvirus (ZYMV)
               RT 20104 Agriculture Biotechnology
• Production of virus-free plant stocks
  – Combinations of heat therapy, meristem tip
    culture and antiviral chemicals
  – Can be used in field
  – DO     NOT     provide    protection against
    subsequent infection




            RT 20104 Agriculture Biotechnology
• To operate a quarantine system
   – To prevent the import of virus diseases into
     non-endemic regions
     e.g. sugar beet disease – rhizomania
          • Caused by beet necrotic yellow vein
            virus (BNYVV)
          • A +ve-sense RNA furovirus
          • Carried in soil from one plant to another
            by spores of the fungus Polymyxa betae
   – Soil bait test scheme
   – Molecular test for viral nucleic acids
• Development of crops with resistance gene

              RT 20104 Agriculture Biotechnology
Soil bait
  test
scheme
            RT 20104 Agriculture Biotechnology
Molecular test for viral nucleic acids

                 Plant material/soil
                     +/- Virus


 Total nucleic acid                      Virus nucleic acid




    Hybridization                          PCR

              RT 20104 Agriculture Biotechnology
  The transgenic approach - PDR
• PDR       – pathogen-derived resistance
            – parasite-derived resistance
• Pathogen genomic sequences are deliberately
  engineered into the host plant’s genome
• During the infection cycle, the sequence may be
  expressed
   – at an inappropriate time              Induce
   – in an inappropriate amounts           some form
                                           resistance
   – in an inappropriate form during the
                                           in plant
             RT 20104 Agriculture Biotechnology
• Potential types of interaction between
  transgenic sequences and virus life cycles:
  2 groups
   1.Those involving the synthesis of viral
     proteins
   2.Those involving viral RNA




            RT 20104 Agriculture Biotechnology
 Interaction involving viral proteins
• Transgenic expression of the coat protein (CP)
  coding sequence
• Some variations in the level of resistance to
  virus infection, related to
   – Transcriptional gene silencing
   – Transgene protein effects
   – Relationship between the coding sequence &
     target virus


             RT 20104 Agriculture Biotechnology
Examples of CP-mediated resistance
Plant        Source of coat protein gene                    Virus resistance exhibited to
Alfalfa      Alfalfa mosaic alfamovirus (AIMV)              AIMV
Citrus       Citrus tristeza closterovirus (CTV)            CTV
Papaya       Papaya ringspot potyvirus (PRSV)               PRSV
Peanut       TSWV (N-gene)                                  TSWV
Potato       Potato X potexvirus (PVX)                      PVX, PVY
             Potato Y potexvirus PVY                        PVY
             Potato leafroll luteovirus (PLRV)              PLRV
Rice         Rice tungro spherical waikavirus (RTSV)        RTSV
             Rice stripe tenuivirus (RSV (N gene))          RSV
             Rice hoja blanca tenuivirus (RHBV (N gene))    RHBV
             Rice yellow mottle sobemovirus (RYMV)          RYMV
Squash       Cucumber mosaic cucumovirus (CMV)              CMV
             Watermelon mosaic 2 potyvirus (WMV2)           WMV2
             Zucchini yellow mosaic potyvirus (ZYMV)        ZYMV
Sugar beet   Beet necrotic yellow vein furovirus (BNYVV)    BNYVV
Tobacco      Tobacco mosaic tobamovirus (TMV)               TMV, PVX, CMV, AIMV
             Cucumber mosaic cucumovirus (CMV)              CMV
             Alfalfa mosaic alfamovirus (AIMV)              AIMV
Wheat        Wheat soil-borne mosaic furovirus (SBWMV)      SBWMV
             Barley yellow dwarf luteovirus (BYDV)          BYDV

                            RT 20104 Agriculture Biotechnology
• Important features of the Coat Protein (CP)
  strategy
   – There is some level of cross-protection
     against infections by related viruses
      • Resistance is not limited to viruses with the
        same nucleic acid sequence
   – TMV & several viruses from other groups, the
     level of resistance has been related to the
     level of transgenic coat protein produced
      • Several different resistance mechanisms
        may be at work


              RT 20104 Agriculture Biotechnology
               RNA effects
• Approaches developed include:
   1. Satellite RNAs
   2. Antisence RNA
   3. Ribozyme technologies
• The most important development in this area –
  gene silencing




             RT 20104 Agriculture Biotechnology
         Satellite sequences
• Produce transgenic plants containing satellite
  sequences
• Plant viral satellite RNAs
   – Small RNA molecules
   – Unable to multiply in host cells without the
     presence of a specific helper virus
   – Not required for virus replication
   – May affect disease symptoms
   – Presence of a satellite RNA may reduce the
     effects of the virus

             RT 20104 Agriculture Biotechnology
• Combinations of genes
  – Generate more durable resistance
  – BUT, severity of the disease can be increased
• Related approach
  – Inclusion of small defective interfering RNAs &
    DNAs (DI)
     • Small sequence fragments
     • Generated during the replication of some
       viruses
     • Presence
         –Redirects the replication machinery
          towards the DI
         –Reducing the amount of virus
     • Some relationship to co-suppression
              RT 20104 Agriculture Biotechnology
       Antisense & Ribozymes
• Antisense approach
  – To specifically interfere with virus replication

                Target virus RNA
        5’                                                 3’
                                                    AAAAA(A)n



               3’                       5’
                    Anti-sense
  Vpg or Cap

               RT 20104 Agriculture Biotechnology
• Ribozymes
  – Antisense catalytic RNA molecules
  – Capable of catalyzing the cleavage of the
    target sense RNA sequence
       5’                                             3’
                                             AAAAA(A)n
             3’               5’


                                                     ribozyme
                                        3’            5’

                  P

            OH                        AAAAA(A)n
             RT 20104 Agriculture Biotechnology
• Different
  – A short catalytic sequence is embedded within
     a specific target region
• Aim
  – To block replication by the formation of a ds
     RNA:RNA hybrid
  – To cut a key region of the virus genome (e.g.
     within a replicase complex genes) before it is
     able to replicate
• Both technologies
  – Based on sequence homology
  – Limited cross-protection value unless the
     antisense sequence is designed against a
     very conserved region
              RT 20104 Agriculture Biotechnology
• Antisense RNA
  – Provide protection
  – May be due to gene silencing
• Ribozyme
  – Generally not been very successful in plants
    as an antiviral tool




             RT 20104 Agriculture Biotechnology
Gene silencing/ co-repression – the
 explanation for the RNA effects
• Post-transcriptional gene silencing (PTGS)
   – Found in plants, fungi, nematode worms,
     humans
   – An important system for preventing invasion
     by viruses and transposon sequences
   – Having some regulatory functions in the cell
• Co-suppression: both the introduced gene & the
  endogenous genes were turned off


             RT 20104 Agriculture Biotechnology
• The cell has a mechanism – can monitor RNA
  levels
   – Scans for unusual amounts of ds nucleic
     acids (e.g. in a viral infection where +ve & -ve
     hybrids may form during the replication
     process)
   – Above a certain critical level the RNA is
     rapidly degraded



               RT 20104 Agriculture Biotechnology
  A model for the mechanism of PTGS
The ds RNA is recognized by the Dicer
                                               1 ds RNA forms in the plant cell
complex & is degraded to ss siRNA           Replication,
                                            antisense RNA,
                    ds RNA                  aberrant RNA      Virus genome
       Dicer
       RNase                                                             ss RNA

  2
                            helicase                               3   Invading virus
                                                                       is detected
          ds siRNA                         ss siRNA                    entering the
                                                                       cell and is 

                                RdRP

   ds RNA                    Alternatively, the        RISC-RNA-induced silencing
                        5
      (amplification)        RNA is part of an         complex   4 Degraded
                             amplification                          by the RISC
                                               Degraded RNA
                             complex                                complex
                              RT 20104 Agriculture Biotechnology
                     siRNAs
• Small interfering RNAs
• Also known as ‘guide RNAs’
• May act as systemic signals that are transported
  throughout the plant thereby inducing systemic
  gene silencing




             RT 20104 Agriculture Biotechnology
 If plants contain effective defense
             mechanisms
 How do viruses manage to cause
             an infection
• Virus proteins – can act as suppresser to gene
  silencing
• E.g. 1 Potyviruses, the HcPro protein
          – Blocks the maintenance of already
            established gene silencing
       2 CMV, the 26 protein

          – Suppresses the initiation of gene
            silencing
             RT 20104 Agriculture Biotechnology
 What has been commercialized in
            the West
• Yellow squash & zucchini
  – Independence II, Liberator III, Freedom III &
    Destiny III
  – Resistance to 3 important viral diseases
      • Zucchini yellow mosaic potyvirus
      • Watermelon mosaic 2 potyvirus
      • Cucumber mosaic cucumovirus
  – Curcubits, causing leaf mottling, yellowing,
    dwarfing & deformed fruit

             RT 20104 Agriculture Biotechnology
• Papaya
  – SunUp & Rainbow
  – Resistance to papaya ringspot potyvirus
• Potato
  – Newleaf potato
  – Resistance to
     • BT
     • Potato leafroll polerovirus
     • Potato Y potyvirus

             RT 20104 Agriculture Biotechnology
                         Risk
• Producing a new virus
• Transcapsidation & Recombinant:
  RNA recombination




• Heterologous encapsidation, transencapsidation
  or genome masking
             RT 20104 Agriculture Biotechnology
• The CP protein produced from the transgene
  could form chimeric particles with invading
  viruses




            RT 20104 Agriculture Biotechnology
• Or package the nucleic acid of compatible
  viruses




• Coat proteins may affect a range of virus
  properties, e.g.
   – Protection of the nucleic acid
   – Vector transmission and specificity
   – Systemic invasion
   – Symptom expression
           RT 20104 Agriculture Biotechnology
• The frequency of transcapsidation is likely to be
  very low, and effects, such as changes of
  vectors, will be transitory
• Strategies: to reduce the likelihood of
  transcapsidation
   – CP sequences have been mutated, either to
     remove sequences that may be involved in
     transfer or to truncate the sequence to
     minimum size required for protection
   – PTGS, fully precludes protein expression;
     constructs are being generated that do not
     allow any protein expression, but rely on gene
     silencing to produce resistance

              RT 20104 Agriculture Biotechnology

								
To top