Insect resistance

Sometimes insects go into crops, so insecticides are very difficult to use

waarom kan je niet altijd insecticides gebruiken?

produce proteins that are toxic (to larvae of) certain insects.

Wat doet Bt?

Bacillus thuringiensis

afkorting Bt

Many Bt proteins form crystals: Cry-proteins

Wat is bijzonder aan de Bt proteins?

highly specific, rapid biodegradation, used as a bio-pesticide since 1936

voordelen Bt

There are so many because of an arms race between bacteria and insects (evolution of toxins vs insect resistance)

Waarom zijn er zoveel Bt genes?

Uptake of Bt crystal and spore by insect

Dissolution of crystal in alkaline digestive system of insects

Activation from 120 kDa pro-toxin > 55-65 kDa toxin by proteases

Binding to enterocytes

Different Bt proteins target different receptors or transporters

Pore formed in cell membrane > cell death

Spore germinates and bacteria grow

Bt-mechanism

plant makes the protein itself

Strategie voor Bt-plants

Bacterial Bt gene was very poorly expressed

~ codon optimized gene

origineel probleem en oplossing bij de first Bt-plants

35S promoter for high constitutive expression

promotor bij de first Bt-plants

Bt176 replaced by MON810

twee belangrijke events voor Bt maize

MON810 has higher expression of the Bt-protein and an event of better quality, since Bt176 still has the vector backbone (ampicillin gene)

waarom is MON810 beter dan Bt176?

ZmHSP70 intron in 5’ UTR enhances expression of p35S

wat zorgde bij MON810 voor betere expressie?

Pollen from a Bt corn (Cry1Ab, Bt11 event) was found toxic to larvae of the Monarch butterfly in lab experiments

Decrease in population due to:

●Loss of host plants due to herbicide use.

●Mortality during migration.

●Climate change

controversy en weerlegging rond Bt corn

even higher expression of Bt

Refuges

Gene pyramiding

Hoe resistentie tegen Bt tegengaan?

A refuge allows part of the insect population to escape selection.

These Bt-sensitive insects (SS) mate with resistant survivors (red) in Bt fields and produce Bt-sensitive (heterozygous) offspring.

principe Refuge

Strategy in which seeds of common (non-Bt) crops and seeds of Bt crops are pre-mixed in one bag. This mixture automatically creates a refuge in the field

principe refuge-in-the-bag

Bt plants → less insect damage → fewer fungal infections → lower mycotoxin levels

indirect benefit Bt-plants

aflatoxins: linked to liver cancer deaths, most of these occurring in low- and middle-income countries where corn and peanuts are dietary staples

bekende, schadelijke mcyotoxins

Reduced pesticide use/cost

Biodiversity (through selectivity)

Yield

Corn: less mycotoxins

voordelen Bt-plants

Resistance

Cost of seeds

nadelen Bt-plants

using RNAi’s that target essential genes for the insects

hoe kan je RNAi gebruiken tegen insecten?

Plant lectins are proteins that specifically recognize and bind to carbohydrate structures, but do not possess enzymatic activity

plant lectins

Plant lectins zijn eiwitten die specifiek binden aan carbohydrate structures
ze hebben zelf geen enzymatische activiteit

In insecten kunnen ze binden aan glycoproteïnen in de darmwand of aan darmreceptoren

Daardoor verstoren ze de darmfunctie
bv. minder goede voedingopname, schade aan darmcellen of celdood

Voorbeeld: ASAL uit knoflook
geeft resistentie tegen belangrijke sapzuigende insecten

Principe:
Lectines werken als natuurlijke plantverdediging doordat ze aan suikergroepen in de insectendarm binden en zo de darmwerking van herbivoren verstoren.

rol plant lectins tegen insecten

PIs alter enzyme conformation, reducing catalytic capacity.

Insect gut proteases (trypsin, chymotrypsin) impacted by PIs.

PIs are not lethal, but inhibit insect growth, reproduction, and development

toepassing proteinase inhibitors

1. Defense pathways verhogen
bv. jasmonate signalling
jasmonate is een plantenhormoon dat belangrijk is bij verdediging tegen herbivoren

2. Anti-insect eiwitten verhogen
bv. proteinase inhibitors en lectins
deze kunnen insectengroei, vertering of overleving verstoren

3. Specialized metabolism aanpassen
meer of andere metabolieten met anti-insect activiteit

4. Natural enemies aantrekken
bv. meer VOCs produceren
vluchtige stoffen die herbivoren afstoten of predatoren aantrekken

Principe:
CRISPR maakt planten niet rechtstreeks “toxisch”, maar kan bestaande verdedigingsroutes versterken of herprogrammeren tegen insectenvraat.

CRISPR approaches voor insect defense

Boosting volatile organic compounds (VOCs) that repel herbivores or can attract their natural enemies

principe attracting natural enemies

CRISPR library screens

hoe vind je welke genen je moet targetten?

CRISPR library: a collection of sgRNAs, that all target a different gen / region

principe CRISPR library