GTPP

->is found in Bacillus thuringiensis (Bt)

->are pore-forming proteins and toxic to some insects

->very specific to their respective hosts

->used in biological insecticides

->alkaline digestive tract denature the insoluble crystals, making them soluble and available to being cut with proteases found in the insect gut

->insect stops eating and starves to death

->genetically modified crops (e.g. maize, cotton) have been developed expressing insect tolerance through genes from Bt

-> contain one or more genes from a different species that have been introduced through genetic engineering

can be produced by:

-> cloning of the gene construct

-> introduction (transformation) into the plant cell

-> selection of transformed lines

mutants = plants with changes in their DNA

can be:

->induced naturally,

->by radiation,

->by chemicals and

->genetic engineering

For every (resistance) gene in the host that determines the reaction of the host there is a corresponding gene in the pathogen, which determines the pathogenicity of the pathogen.

->The basis of vertical resistance

->Vertical resistance is a race-specific resistance

Ti-plasmids are very large -> binary vectors have been developed -> transfer-plasmid

plant transformation systsem: A. tumefaciens und E. coli

binary vector (transfer-plasmid) contains the T-DNA as well as “ori“ (origin of replication) for A. tumefaciens und E. coli

it has a marker gene for selection, restriction sites or polylinker for cloning the DNA

Alkaloids: widely distributed in plants, volatile poisons, plant defense against herbivores because of toxic properties

->e.g. nicotine, morphine

Phenolic compounds: Phenolic acids, Flavonoids, Tannins

-> e.g. coffein, anthocyanidins, lignans

Terpenes: scents (smells) and chemical signalling between plants and between plants and vertebrates

->e.g. saponins, isoprene, limonene

Main methods:

->Meganucleases,

->Zinc-finger nucleases (ZFNs),

->TALENs,

->CRISPR/Cas system

The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation) into the DNA.

->Enzyme Cas9 —> ‘molecular scissors’ that can cut both strands of DNA at a specific location so that bits of DNA can then be added or removed

->guide RNA (gRNA) —> consists of pre-designed RNA sequence

The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA

—>DNA is damaged —> repair is needed

PR proteins ("pathogenesis related") are part of biochemical defense mechanisms

induced as part of systemic acquired resistance (SAR)

->glucanases and chitinases

->inhibitors of polygalacturonases

->inhibitors of proteinases

DNA is a double-stranded molecule, uses deoxyribose, thymine, read by polymerase, is read 3´-5´

—>transcription in mRNA, tRNA, rRNA

RNA is single-stranded, uses ribose, uracil, read by ribosomes, is read 5´-3´, can have multiple structures, e.g. mRNA, tRNA, rRNA, is copied from DNA

—> mRNA, tRNA, rRNA ->translation->protein

Elicitors are foreign molecules often associated with plant pathogens -> part of PTI (pattern-triggered immunity)

Effectors are part of ETI (effector triggered immunity)

Exogenous elicitors: e.g. oligosaccharides (glucan, chitin, LPS), lipids (arachidonic acid, ergosterol)

endogenous elicitors: e.g. oligogalacturonic acid

Abiotic elicitors: e.g. Ozone, copper chloride

loss of susceptibility:

->Blocking key pathogen strategies

->Losing interaction with key host targets

->Host reprogramming

Specific members of the Mildew Locus O gene family act as powdery mildew susceptibility factors. Their inactivation is associated with a peculiar form of resistance, referred to as mlo resistance

Reporter genes are used to identify if the desired gene is transformed into the plantvells

-> the marker has to be used to find the transformant

gus (uidA)

GUS reporter gene:

Using a colorless substrat -> where color appears the transformation was successful

Base

Sugar

Hydrogen Bonds

phosphate

etc.

Vertical resistance (qualitative resistance, race resistance):

->is targeted against one or a few pathotypes of the pathogen

->based on gene-for-gene interactions and is inherited monogenically or oligogenicall

->usually manifests itself as a hypersensitive reaction (HR) of the host plant

Horizontal resistance (quantitative resistance):

->is directed against all individuals of a pathogen species

->inherited polygenically

->manifests itself in a delayed development of the pathogen

->T-DNA mutagenesis (integration of the T-DNA of Agrobacterium tumefaciens into the genome of a plant),

->TILLING (targeting induced local lesions in genomes) and

-> transposon mutagenesis

-> cutting enzymes, recognize one or a few target sequences and cut DNA

Type II

-> cuts DNA at defined positions within the recognition sequence

-> for each restriction enzyme there is a corresponding methylase to protect the own DNA ->high practical importance

->these restriction enzymes cut too often and mutations could only be induced at a restriction sequence

Type IIS

-> Cut DNA at a defined position close to the recognition site

-> Important for certain cloning techniques

infects the plant through its Ti (tumour-inducing) plasmid

Ti plasmid integrates a segment of its DNA (T-DNA) into the chromosomal DNA of its host plant cells

leads to the synthesis of important nutrients for A. tumefaciens

—> plants provide nutrients to the colonizing bacteria

Agrobacterium´s plasmid T-DNA is naturally transferred to a host plant and thus can be used as a vehicle for genetic engineering.

This is done by cloning a desired gene sequence into T-DNA binary vectors that will be used to deliver a sequence of interest into plant cells.

(a) isolation of the genes of interest from the source organism

(b) development of a functional transgenic construct including the gene of interest

(c) insertion of the transgene into the Ti-plasmid

(d) introduction of the T-DNA-containing-plasmid into Agrobacterium

(e) mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into plant chromosome

(f) regeneration of the transformed cells into genetically modified (GM) plants

The overall advantages of using Agrobacterium-mediated transformation methods are: reduction in transgene copy number, intact and stable integration of the transgene into the plant genome.

tms (auxin formation)

tmr (cytokinin formation)

nos (nopaline synthesis)

Reporter genes are marker genes whose gene products are easily detectable by simple biochemical, histochemical, microscopic or photometric methods

e.g.

ß-D-glucuronidase (GUC)

luciferase (LUC)

green fluorescent protein (GFP)

replacement vector:

-> cloning vector that replaces a portion of the vector DNA with the desired insert DNA

insertion vector:

-> vector in which the cloned sequence is inserted into a single restriction enzyme site

-> the DNA that is to be cloned is inserted in the polylinker.

Step 1: Denaturation (> 90 °C)

-> two strands in the DNA double helix separate

Step 2: Annealing (55-65 °C)

-> Primers bind to the target DNA sequences and initiate polymerisation

Step 3: Extension

-> New strands of DNA are made using the original strands as templates

-> DNA polymerase (often Taq polymerase) enzyme joins free DNA nucleotides together

Ingredients

the DNA segment of interest

specific primers

heat-resistant DNA polymerase enzyme

the four different types of DNA nucleotides

buffer

quadratic check

When the plant is attacked, substances are released

->pathogen-associated molecular pattern/PAMP-> plant recognises the presence of a pathogen

e.g. flagellin, chitin, glucans, proteins

molecular patterns associated with non-pathogens and the plant are called MAMP´s/microbe-associated-molecular pattern

e.g. oligoglucan, oligochitin, oligochitosan, oligogalacturonic acid

PAMP´s and MAMP´s are also called elicitors and are recognised by receptors on the cell surface

pattern triggered immunity (PTI):

-> triggered by general elicitors (e.g. glucan, chitin) those can attach to special receptor proteins located on plant cell membranes

These receptors are able to recognize the molecular pattern of elicitors and trigger intracellular defense signaling

effector-triggered immunity (ETI):

-> triggered by specific elicitors (e.g. oligogalacturonic acid) which interact with cytoplasmic receptor molecules

Jasmonic acid (JA) mediates systemic resistance after wounding

->Protease inhibitors can inhibit the activity of proteases through binding at the active site (Animals need proteases for the digestion of their food)

Defense response:

herbivore induced plant volatiles (HIPVs),

secondary metabolites (flavonoids, tannins, anthocyanins, glucosinolates)

Glucosinolates are organic compounds and part of a variety of preformed biochemical defense substances

Myrosinase seperates glucose from glucosinolates

Glucosinolates and myrosinase are stored in different cells

Chewing insects destroy the cells and myrosinase comes into contact with glucosinolates -> removal of glucose leads to unstable molecules and the production of toxic metabolites

SAR (Systemic Acquired Resistance) is activated trough biotic and abiotic elicitors

-> dependent on the phytohormone salicylic acid (SA) and associated with the accumulation of pathogenesis-related (PR) proteins (npr1)

-> plants infected with one pathogen become more resistant to subsequent infections (same/other pathogens)

ISR (Induced Systemic Resistance) is a resistance mechanism activated through beneficial microorganisms (non-pathogenic rhizobacteria)

-> increasing physical or chemical barriers of the host plant

-> signal transduction pathways activated by jasmonic acid and ethylene

-> jasmonic acid activates genes that encode protease inhibitors, which protects the plant from insect attacks, or genes associated with the formation of phytoalexins

-> ethylene induces certain PR proteins and precursors of phytoalexins and lignin

-> it is responsible for regulation of gene expression as well as post-transcriptional gene silencing

-> a cellular mechanism that degrades unwanted/foreign RNAs (e.g. viral RNA) in the cytoplasm but not in the nucleus

-> in molecular biology it is used to block expression of genes and generate phenotypes that can deliver clues about the function of the gene

-> it has a great potential in plant protection as GM-free exogenous RNA spray application

when the avirulence (AVR) gene of the pathogen and the resistance (R) gene of the host plant "fit", i.e. their respective gene products interact with each other, recognition occurs and defense can be triggered

This requires the dominant expression of R and AVR genes.

effector target necessary for R protein function and, in the absence of its cognate/related R protein, has a function in host defense or susceptibility

Guardee effector target is required for R protein function and function in host defense

Alteration of the guardee results in enhanced pathogen fitness in plants that lack the R protein and triggers innate immunity in plants that carry the R protein.

Effector target required for R protein function but with no function in host defense or susceptibility in the absence of its cognate R protein

Alteration of the decoy does not result in enhanced pathogen fitness in plants that lack the R protein and doesn´t trigger innate immunity in plants that carry the R protein

Effectors are elicitors that become active in the cytoplasm by binding to receptor molecules, and either trigger or inactivate defense mechanisms

Effectors with elicitor function trigger Effector-triggered immunity (ETI)

phytoalexins (isoflavonoids & phenolics):

Secondary metabolites which are only expressed after induction following pathogen infection

Engeneering resistant plants through production of phytoalexins

example: resveratrol (polyphenolic)

phytoanticipins (terpenoids)

Secondary metabolites which are consistently expressed

Glyphosate occupies the PEP-binding site of the shikimate-3-phosphate complex of the enzyme-> inhibiting EPSP synthase

Glyphosate-insensitive EPSPS from A. tumefaciens (CP4):

-> CP4-EPSPS combines a high affinity for PEP coupled with a very high tolerance for glyphosate

-> The result is an ability to ‘bypass’ the endogenous EPSPS system with the CP4-EPSPS insertion that allows the shikimate pathway to function normally