Midterm 2 Flashcards
How do herbicides (in general) kill plants?
plants = autotrophs -> attack any pathways -> guaranteed knockout of any nutrients -> guaranteed death
Target any chloroplast pathways -> photodisruption
How does glyphosate kill plants?
Inhibition of EPSP Synthase activity, preventing amino acid synthesis or auxin growth hormones
How are crops GMO’d to survive glyphosate exposure
Transgenic EPSP genes
Transgenic EPSP mutant
provide detoxification pathway
Transgenic EPSP
counteract glyphosate inhibition by overexpressing EPSP
Mutated EPSP
provide CP4 gene (mutant)
Place under constitutive euk promoter (35S or NOS)
Agrobacterium delivery
glyphosate detoxification
method of providing glyphosate resistance by inserting transgenic glyphosate oxidases (sourced from soil bacterium)
GATs
glyphosate acetyltransferases - enzymes for glyposate detox by acetylation. Naturally occuring bacterial GATs are too weak to make plants HT to glyphosate -> required hybridization of several GAts to achieve a 200x-400x strength super GAT for crop use
What is ALS
acetolactate synthase - responsible for synthesis of branched AAs (eg isoleucine)
what can inhibit ALS
Suphonylureases
PPT
phosphinothricin (herbicide) - targets broadleaf plants. Inhibits glutamine synthase, leading to toxic NH3 accumulation
what detoxifies PPT
Can be neutralized phosphinothricin acetyltransferase (acetylation)
BT
B.thurigiensis endotoxin (pesticide) - encoded by “Cry” genes
Kills pests by binding to intestinal membranes -> gut breakdown -> septicemia
Transgenic BT
required heavy sequence modification of teh cry genes -> 21% base mods, 60% codons changed
Required chimeric/hybridized BT genes to achieve enough toxicity to kill pests
Importance of low pesticide GMO expression crops (a reservoir)
maintains a population of low resistance pests -> dilutes the overall pesticide tolerance in the pest population therefore preventing/slowing evolution of complete immunity to the pesticide
3 types of plant-bacteria interactions
necrotrophs -eat dead tissue
biotrophs - eat live tissue
hemitroph - eats both
2 types of disease resistances
host resistance - organism specific (a novel mutation)
non-host resistance - species wide resistance to the disease
Disease resistance by physical methods
cuticles/max to seal the exterior
bark (thick layers of dead cells)
Disease resistance by proteins/chems
antimicrobics (eg SN1 peptide)
defensins
Disease resistance by inducible pathways
usually protein synthesis in response to a disease
Pathogen -> plant cells die -> plant detects cell fragments -> cascade -> response protein synthesis (eg SN1 peptide antimicrobic)
MAPK disease response pathway
Antigen on the pathogen is detected –> binding to cell -> kinase activation -> MAPK phosphorylation -> MAPK cascade -> stromal closure (prevent further pathogen entry)
chitinases
recognize pathogen -> tagging the pathogen membrane
tagged membrane is targeted by lethal phenolic compounds –> kills the pathogen
Fungal/mould infections
usually oomycetes
eg A. flavus -> produces aflatoxin (carcinogen)
How is A.flavus infection countered
GMO a mutant of A.flavus with deactivated aflatoxin genes -> expose to plants -> occupy niches -> therefore teh natural (lethal) bacterium can’t infect that plants
Race specific responses
each pathogen is recognized based on a unique gene it carries (avirulence (avr) gene)
the plant carries a corresponding resistance gene (R gene) to match
if an avr is recognized by a present R gene -> defence response occurs
Systemic Acquired Response
Method of plant-plant comms for disease spread
pathogen -> detection -> plant excretes salicyclic acid -> ethylene + jasmonic acid production -> warning signal to other plants
what proportion of plant virusses are ssRNA
~70%
Tobacco mosaic virus (TMV)
used in biotech due to self-assembly capability
ssRNA genome is replicated into a (-) sense RNA -> (-) sense used as template for genome replication
subgenomic RNA (sgRNA) used to regulate viral spread
Cowpea mosaic virus (CPMV)
carries 2 ssRNAs
icosahedral capsid –> potential for nanoparticle delivery vesicle
Coat Proteins vs ArMV
Arabis mosaic virus (ArMV) resistance
coat proteins (CP) used for capsid synthesis -> overexpress CP = infecting virus reforms capsid -> therefore prevents machinery from expressing the viral genome
CP overexpression -> reform capsid -> can’t replicate virus
why is overexpression of CP genes bad?
overexpresison of CP rna –> PTGS effects –> negates the resistance
Pathogen derived resistance (PDR) using satellite virusses
satellite virus = virus that requires a helper virus to replicate
infection w/satellite forms dsRNA with helper virus -> RISC PTGS
how do virusses counter PDR?
virus carries antisense RNA to hybridize with the satellite virus –> forms siRNA -> RISC complex -> PTGS of the satellite virus
non-PDR resistance against viral infection
introduce transgenic protein kinase to disable eIF2a (translation factor) –> disable viral protein translation
(Ideally place under wound-induced control)
benefit of non-PDR method?
disabled the eIF2a –> affects many virusses
broad-range resistance
Geminiviruses
DNA viruses
rolling replication relies on Ren + Rep + TrAP genes
transgenic Ren/Rep/TrAP antisense -> RISC -> PTGS therefore disabling geminivirus replication
Define water potential
the tendency for water to move from A to B
higher water potential = easier to move the water
env factors affecting water stress
T
[salts]
wind
soil porosity
how do plants control water flow?
water moves towards higher salt concentrations (osmosis) –> therefore concentrate salts in tissues with low water
water stress vs turgor pressure
more water = more turgor pressure
if turgor drops -> stroma closes to prevent water loss by transpiration
shell of hydration
proteins are surrounded by H2O –> prevents oxidation from O2 contact
therefore if SoH breaks -> proteins denatured
osmolytes/osmoprotectants
used to maintain the shell of hydration (may be natural or transgenic)
osmoprotectant examples (6 types) remember POSM
pinitol
ononitol
sorbitol
manitol
zwiterions
oligosaccharides
why is high salinity soil bad
due to osmosis, water will prefer to stay in the saline soil (reduced water potential)
halophyte adaptations
use sodium transports to intake salt -> cause osmosis into the plant
use sodium antiports to detox from the high salt intake
glycophytes
use high expression of osmolytes/osmoprotectants to maintain SoH
llimited salt tolerance due due to Na/Cl toxicity
they cope with the salt, not deal with it
ROS
oxidative stresses –> ROS –> free radicals –> damage to NA + proteins
types of counters to ROS
antioxidants
enzymatic free radical reduction
examples of antioxidants
glutathione
Beta carotene
vit C
Vit E
examples of enzymatic free radical reduction
superoxidase dismutase
catalase
peroxidase
Cold response genes (CDL acronym)
C-repeat element (CRT)
dehydration response element (DRE)
low T response element (LRTE)
transgenic cold response genes
transgenic cold genes + constitutive expression –> plants dont need to acclimatize to cold weather
Tomato ripening
ethylene -> ripening signal -> over expression of ripening leads to rot
ripening genes
pTOM5 = red pigment
pTOM6 = polygalacturonase (ripening) -> also affects pectin methylesterase
pTOM13 = ethylene synthesis
FlavrSavr Tomato GMO
goal: delay rotting to extend shelf life
method: antisense pTOM6 -> some PTGS of polygalacturonase (not full antisense expression, allow some PG)
less PG -> less pectin methylesterase -> less pectin degradation
effects of antisense other tomato ripening genes
antisense pTOM5 -> no red pigments –> yellow + impacted photosynthesis (dwarfism)
antisense pTOM13 -> no ethylene synth -> VERY slow rotting (2x shelf life)
Golden rice - VitA
rice normally = low vitA
golden rice -> modified with bacterial + daffodil enzymes -> able to synthesize vitA -> fix vitA deficiencies
Golden rice - treating diarrhea
transgenic lactoferrin -> antimicrobic + antiinflammatory -> decrease diarrhea for low hygeine regions
Golden rice - improving photosynthesis
attempted to replace photosynthetic rubisco enzyme with cyanobacterial analog (to get more efficient PS) -> not much better
-> concerns of HGT into weeds -> worsening weed growth/spread -> risk not worth the marginal PS increase
using plants for biosynthetics - Why are chloroplast transgenes so important
- chl genes are constitutively expressed
- chl genes are better conserved + less likely to be mutated (eg by infections)
- chl gene are maternally inherited –> can be used for tracking gene lines
carbohydrate biosynthetics - starch composition
20-30% linear amylose + 70-80% branched amylopectin
high starch barley GMO
want more linear amylose for berewing -> introduce ssRNA for amylopectin -> form dsRNA hairpin -> RISC -> PTGS of branched amylopectin. This forces teh barley to only produce the linear amylose
bioplastics synthesis
cellulose or starch -> nitrocellulose -> cellulose acetate -> cellulose acetate phthalate (bioplastic)
or
starch -> thermoplastic starch -> starch polycarbonate -> starch-modified cellulose -> starch vinyl alcohol
significance of PLA
it can be naturally biodegraded over 3-6 months into various byproducts -> useful as a packaging material
protein synthesis from GMOs
transgene expression of proteins -> Use see-specific promoters -> 36x more expression
chloroplast expression (more effective)
products: proteins, antibody domains, enzymes
plantibodies
plant-produced antibodies
