Lecture 10: GM and virus resistance: Opportunity or environmental disaster?

Question 1

example of plant virus:

Answer 1

Tulip break virus, 17th Century

Question 2

viruses are devastating ___ of many crop species

Answer 2

Pathogens

Question 3

treatments of viral plant diseases: pre or post infection?

Answer 3

treatments post infection are generally ineffective, therefore focus on prevention of infection

Question 4

controls used to prevent infection of viral diseases:

Answer 4

• spray insecticides to control aphid vectors
• delay planting to avoid insect vectors
• destroy infected crops
• maintain good field hygiene
• certified virus-free seed
• –all these are easier in developed countries

Question 5

example of insect vectors for viral diseases:

Answer 5

• Aphids
• Whitefly
• Hoppers
• Thrips

Question 6

examples of virus damage and their viral causes:

Answer 6

• Fruit distortion on eggplant fruit caused by Tomato bushy stunt virus
• yellow vein-banding on grapevine caused by Grapevine fan leaf virus

Question 7

simplified typical virus lifecycle:

Answer 7

viruses inject genetic material (DNA or RNA ) into the plant cell which reprogrammed the cell machinery to produce new viral particles

Question 8

simplified typical virus lifecycle: the nucleic acids injected (DNA/RNA) ENCODES:

Answer 8

• enzymes necessary to replicate the viral genome
• proteins to allow transmission from cell to cell
• coat proteins which encapsulate the viral genome

Question 9

A strategy for making virus resistant plants (concept:A good idea, but incorrect)

Answer 9

• Engineer plants so that they produce coat protein inside the cell
• as nucleic acid is injected into the cell it becomes encapsulated (transcapsidation) within the coat protein and cannot be transcribed/translated
• -> the experiment works (plant became virus resistant) but mechanism was wrong, resistance wasn’t due to encapsulation

Question 10

virus resistant plants: how the mechanism was wrong

Answer 10

plants which expressed coat protein were virus resistant (supporting concept) BUT
-further experiments showed:
–a complete coat protein was not required, part of one would still produce resistance
-framshift mutations which still allowed RNA production but which produced no protein produces resistance
-coding regions inserted in reverse produced resistance
-gene fragments as short as 25bp produced resistance
EXPERIMENT WORKED BUT CONCEPT WAS WRONG

Question 11

virus-induced Gene silencing (VIGS)

Answer 11

• infected plants display sever localised symptoms
• recovered tissues have low virus load and no symptoms
• recovered tissues are resistant to subsequent infection by the virus and other related viruses

Question 12

Post -transcriptional gene silencing (PTGS) what is it?

Answer 12

-expression of high levels of foreign genes (viruses or transgenes) can lead to repression of gene expression

Question 13

Post -transcriptional gene silencing (PTGS How does it work?

Answer 13

• If double stranded RNA’s (mRNA) are produced they are ‘diced’ (by Dicer) into small 25 nucleotide RNAs which activate a sequence specific nuclease.
• resulting in degradation of mRNA

Question 14

__ gene silencing mechanisms exists

Answer 14

MANY

-in terms of viral resistance all make RNA that the plant can recognise as foreign

Question 15

commercial application of virus resistance plants:

Answer 15

• a portion of the viral genome is introduced into the crop using a construct which will generate dsRNA molecules
• any proportion of the viral genome can be targeted
• Viruses naturally produce proteins which suppress PTGS so resistance is not guaranteed (antagonistic to the dicer)

Question 16

field resistance against viruses in squash (Upjohn)

Answer 16

transgenic grew larger and produced larger & non infected fruit

Question 17

concerns regarding transgenic virus-resistant squash

Answer 17

• squash plant may become a weed
• viruses resistance genes may be transferred to weedy species improving their fitness
• recombination of viral genes in plants with other viruses may create new viruses with different host ranges (demonstrated in lab experiments)

Question 18

increased possibility of recombination due to use of virus resistant plants

Answer 18

• when a virus infects a cell it quickly proliferates and destroys the cell
• the probability that two separate viruses infect a cell at the same time & recombine is VERY low

Question 19

with transgenic plants the probability of viral recombination is ___

Answer 19

GREATLY INCREASED

• all transgenic plants contain part of the viral genome
• any virus infection the plant cell has the possibility of recombining
• new viruses with different host ranges may be produced

Question 20

papaya & papaya ring spot virus:

Answer 20

• Papaya is an important crop in Hawaii & SE Asia
• it is rich in vitamins A & C
• susceptible to Papaya Ring Spot virus spread by APHIDS

Question 21

papaya ring spot virus and hawaii islands

Answer 21

• PRSV discovered in Oahu in 1940’s
• papaya production destroyed by 1950’s –>wasn’t economically sustainable & virus
• papaya production moved to Puna district of Hawaii island in 1960’s
• PRSV found in Puna district in 1992
• attempts to limit spread by felling affected trees were unsuccessful

Question 22

transgenic papaya resistant to PRSV

Answer 22

transgenic papaya produced using biolistics containing part of the coat protein of PRSV

Question 23

transgenic plants successful in field trials:

Answer 23

Following incolutation with PRSV, non-transgenic plants are killed

• deregulated by USDA in 1997
• commerical use in 1998
• now being introduced in SE asia where Papaya is a staple but criticised as a way of getting GM crops accepted

Question 24

General PRSV resistant transgenic plant production

Answer 24

• isolate PRSV coat protein from PRSV itself
• used transformation
• -> high speed particle bombardment method

Question 25

transgenic papaya & Allergens?!

Answer 25

• the coat protein used to make PRSV-resistant papaya contains amino acid sequences which MIGHT be allergenic
• -6 amino acids shared with allergen from roundworms Ascaris lumbricoides
• -this doesnt mean it IT an allergen
• defninitve proof requires blood testing

Question 26

How id PRSV spread

Answer 26

by aphids

Question 27

decision made with papaya and allergenic qualities

Answer 27

USDA (1997) gave an exemption to the requirement that the levels of coat protein in the papaya are shown to be safe.
• Allergies kill people. Peanut allergies are common and dangerous – so why don’t we ban peanuts? 150-200 people die each year in the US, 10 in the UK
Transgenes which are allergens have been used but have always (so far) been identified at the safety testing stage.

Question 28

transgenic virus resistant plants: containment is a __

Answer 28

PROBLEM
-In Thailand GM papaya resistant to PRSV has been found outside of the field trial areas.
– It is thought that the field station distributed GM seeds to local farmers (in error).
-In Hawaii, organic papaya growers have found that their trees contain GM transgenes.
–It is thought contaminating pollen from GM varieties is responsible.
-In India one third of all cotton grown contains Bt.
– 80% of this (3 million acres) is unlicensed and has been produced by
local seed merchants without regulation.