Parasitic Plants

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Parasitic Plants

• Video Private Life of Plants
• Ep. Living Together (3432 4814)
• Leafy mistletoes and dodders and Rafflesia
• Slideshow and Specimens
• Dodder
• Leafy Mistletoe
• Dwarf Mistletoe

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Dodder (Cuscuta sp.)
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• Dodder (C. cuspidata) of ragweed (Ambrosia spp.)
• Very common in Oklahomas prairies
• Specific to ragweed, will not infect alfalfa

Science article on volatile cues
http//www.sciencemag.org/content/vol313/issue5795
/images/data/1964/DC1/1131371_S1.mov
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Dwarf mistletoe of Douglas Fir (Arceuthobium
douglasii)
?
forcible ejection of seeds
?
?
Witches brooms
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Virus Disease Symptoms

• PLP 3343

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• Typical mosaic symptom Bean mosaic virus

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• Bean yellow mosaic virus
• Typical
• mosaic
• symptom

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• Leaf shoestringing
• symptom
• Cucumber mosaic virus
• In tomato

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eriophyid mite
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Tomato spotted wilt virus
Fruit distortion and chlorotic spotting
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Pear stony pit virus, internal fruit symptoms
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Wheat
Barley yellow dwarf leaf symptoms red and yellow
discoloration
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Barley Yellow Dwarf Virus Infection foci in
field
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Net blotch
Ringspot/mosaic
Range of symptoms with Rose Mosaic Virus
Mottle
Oakleaf mosaic
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Tomato Spotted Wilt Virus Broad host range and
symptoms
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Tulip breaking virus
Ornamental Viruses
Albutilon Mosaic Virus
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Fasciation Flattening of the stem and distortion
of meristems Can be caused by viruses,
phytoplasmas, somatic mutations, or physiological
injuries.
Normal flower
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Fanleaf of grape Cause?
2,4 D herbicide damage
Fanleaf and galls of grape
2,4 D herbicide damage and phylloxera
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Hackberry viral symptoms
ringspot
mottle
Cause of both unidentified viruses of Celtis
spp.
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Today in Lab

• Diagnosis of Viral Diseases
• Rub-inoculate tobacco plants
• Prepare sap from healthy and infected leaves
• Rub each with celite on tobacco leaves
• Resistant cv. Xanthi NN
• Susceptible cv. Xanthi nn
• Use immunostrip to detect TMV
• Understand ELISA and PCR diagnosis of viruses
• Molecular Plant Virology study viral movement

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Abrasive powders used to inoculate plant viruses
Carborundum (silicon carbide, SiC) Celite
(diatomaceous earth)
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Experimental Modes of Transmission
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TMV and N-gene resistance
Susceptible cv. Xanthi nn No symptoms 2-3d after
inoculation with with TMV Symptoms 1st appear in
apical leaves, 2-3 wks later
N-gene product recognizes TMV replicase,
triggering HR.
sap

• TMV-infected Nicotiana tabacum cv. Xanthi nn
• susceptible
• lower case n means recessive

Resistant cv. Xanthi NN Forms HR spots 2-3d after
inoculation with with TMV
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Virus DiagnosisDifferential Hosts

• Indicator host plants
• Systemic hosts
• Local lesion hosts
• Typical panel
• Tobacco (N. tabacum)
• Nicotiana benthiamiana
• Chenopodium quinoa
• C. amaranticolor
• Gomphrena globosum
• Cucurbits
• Datura sp.
• Pepper
• Tomato

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Virus Diagnosis

• Immunochemical (Serological)
• Antibodies and antiserum detect viral proteins
• Polyclonal vs. Monoclonal
• Methods
• Ouchterlony immunodiffusion assay
• Advantage Simple
• Disadvantage Large amounts of antibodies
  required, insensitive, nonspecific cross
  reactions
• Enzyme-Linked ImmunoSorbant Assay (ELISA)
• Advantage Sensitive, conserves antibodies,
  quantitative - viral titer
• Disadvantages nonspecific cross reactions
  possible, expensive equipment
• Immunostrips
• Advantages sensitive, simple, fast,
  field-testing possible
• Disadvantages nonspecific cross reaction
  possible,

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Ab



Ab
Immunodiffusion Assay
ELISA
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Agdia ImmunostripsFast antibody-based method
-

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Virus DiagnosisPCR now most commonly used

• Polymerase Chain Reaction (PCR)
• RNA viruses (Reverse Transcriptase (RT)-step)
• DNA viruses amplify directly
• Primers are extremely specific
• Sometimes too specific
• Most sensitive detection technique
• Disadvantage
• PCR Thermocyclers are expensive (4,000)

RT-PCR
Taq
RT
ssRNA ? cDNA ? PCR
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PCR
RT
cDNA template ? viral ssRNA

• Taq DNA polymerase
• DNA-dependent
• Thermo-tolerant

94C
35 cycles 2N 235 34 billion molecules
55C
72C

• PRIMERS
• ssDNA oligos
• Complementary to specific sequence
• Must use pairs to amplify DNA

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Studying plant virus movement

• Difficult and tedious using destructive assays by
  PCR or ELISA.
• Nondestructive live-imaging of a visible reporter
  gene is ideal.

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Engineering a plant virus to fluorescePotato
virus X (PVX) expressing green fluorescent
protein (GFP)

wild type PVX
PVX-GFP
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The Nobel Prize in Chemistry 2008
"for the discovery and development of the green
fluorescent protein, GFP"
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What is fluorescence?

• Shorter, higher energy wavelength of light
  excites a fluorophore which emits a longer, lower
  energy wavelength of light
• excite blue ? emit green
• absorb blue ? fluoresce green

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Studying plant virus movement with GFP

• OBSERVE
• PVX-GFP-inoculated Nicotiana benthamiana
  (universal suscept)
• uninoculated normal N. benthamiana
• transgenic (GMO) N. benthamiana engineer to
  express GFP
• Long wave ultraviolet (UV) light required to
  excite GFP
• GFP fluoresces GREEN wherever the virus PVX is
  replicating
• healthy green tissues fluoresce RED due to
  chlorophyll excited by UV
• Permits direct, naked-eye observations of viral
  movement in planta

chlorophyll b
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• Cauliflower Mosaic Virus
• Helper protein P2GFP
• Binds to tip of aphids stylet