Lecture 2 - engineering resistance of crops to pathogens and insects

Question 1

Give examples of diseases and organisms that cause plant disease and affect yield

Answer 1

Diseases: Powdery mildrew, spotted wilt, anthracnose, root knot nematode, crown gall disease, fusarium wilt, tomato mosaic virus, bacterial speck

Oranisms: insects, bacteria, viruses, nematodes, fungus

Question 2

What are non host plants?

Answer 2

Incompaitble interaction/no interaction

No disease

Question 3

What makes a plant a host plant to a microoganisms leading to disease?

Answer 3

1. Compatible interaction (disease)
   
   • suceptible plant
   • virulent pathogem
2. Incompatible interaction (no disease)
   
   • resistant plant
   • avirulent pathogen

Question 4

Define a biotrophic organism

Answer 4

Certain pathogens that are dependent on living tissue e.g. the biotrophic fungus Cladosprium fulvum

Question 5

Define a necrotrophic organism

Answer 5

Organism that attains its nutrients from dead cells and tissues of its host organism

e.g. necrotrophic fungus otrytis cinerea

Question 6

What are the two types of preformed plant defenses?

Answer 6

• physical (constantly present)
• biochemical

differs from plant to plant

Question 7

Give some examples of preformed physical barriers to plant infection in the leaves

Answer 7

• cuticle (epidermal cells)
• Wax (wax prjections, wax layer, wax lamelliae)
• Cutin
• Thick cell wall (cellulose lamellae, pectin lamellae, cellulose layer, plasma membrane, cytoplasm)

Pathogens cannot easily enter, although may have enzymes that degrade these layers

e.g. fungus with proteinase/pectinase activity to degrade cell wall and enter into plant tissue

Question 8

Give some examples of preformed biochemical barriers to plant infection

Answer 8

Preformed anti-microbial copounds

• saponins
• glucosinolates

Question 9

Outline saponins as preformed biochemical barriers to plant infection

Answer 9

• Avenacin A-1
• produced by oaks
• toxic activity on certain fungi that attempt to enter the wood system
• certain strains of fungi act against saponins to inactivate

Question 10

Outline glucosinolates as preformed biochemical barriers to infection of plants

Answer 10

Glucosinolates

• Preformed glucosunolates, wounding activity increases myrosinase activity, formation of an unstable aglycone intermediate - > e.g. Isothiocyanate, Nitrile, Thiocyanolate
• toxic compounds that can be anti-fungal

Question 11

What inducible compounds do plants produce to prevent infection?

Answer 11

• Anti-microbial peptides (e.g. defensins)
• Inactivation of microbial toxins (e.g. HC toxin inactivation by maize toxni reductase

Question 12

How was it discovered that the plant defesin peptide alfAFP protects potato against fungal pathogens (inducible defence example 1)?

Answer 12

• screen for natural defences and identify the corresponding genes
• once the gene has been isolated from alfalfa and expressed in potato (vector control transferred by agrobacterium for transformation)
• control was less tolerent than the transgenic plant to fungal attack
• Plants that grew best in the presence of the fungus showed most expression

Question 13

What disadvantages is there to transforming a plant with a single gene conferring resistance?

Answer 13

Often don’t get a long lasting effect

Side effects if a gene is simply overexpressed continuously

May adversely affect optimal yield

Question 14

How do plants respond to viral/biotrophic infection?

Answer 14

The hypersensitive response (HR)

Inducible response example 2

e.g. HR induced by tobacco mosaic virus on resistant tobacco

Question 15

How does the hypersensitive response kill the spread of a viral infection?

Answer 15

Kills plants cells surrounding those infected, viruses need living cells to survive and spread

Question 16

How does the HR response protect plants against inefction with Phytophtorn?

Answer 16

Phytophtorn

In the susceptible plant, the fungus enters the tissue and grows between cells eventually the plant is severly affected by the disease

HR of the plant triggers programmed cell death of the cells in response to the presence of the fungi - fungi is isolated and cannot grow further

Question 17

How does the speed of the HR response affect yeild?

Answer 17

The quicker the HR response occurs, the less of the plant has to be sacrificed, yeild penalty is lower the quicker the response

Question 18

Give an example of the HR response at a single cell level

Answer 18

HR on barely induced by powdery mildew

Single spore of the fungus lands on plant tissue. Fungal hyphae grow out appressorium forms at the cell surface and hyphae try to enter the cell

HR is induced and under UV light it has been shown that particular conpounds accumulate in the cells, indicating the initiation of programmed cell death

Question 19

Give an example of the HR response on nematode

Answer 19

HR on soybean induced by nemtaodes

Nematodes feed on root cells. Nematodes need living tissue. Following HR induction, the growth of nematodes is slowed. More suceptible plants nematodes grow faster.

Question 20

How is programmed cell death triggered in plants?

Answer 20

Triggered by the plant by a recognition system involving a gene-for-gene interaction

Certain genes in pathogen produce compounds e.g. proteins, enzymes, which are recognised by proteins in the host plant. If there is a successful recognition a response will be triggered.

A viral inspectors interact with resistance proteins to prevent disease.

Question 21

What is the gene-for-gene interaction in plant disease resistance?

Answer 21

Pathogen genotype is recognised by specific host plant genotype.

If recognised -> no disease, plant and pathogen are incompatible.

If not recognised -> disease -> plant and pathogen are compatible

If the resistance protein is not there/has a different structure -> can’t interact with Avr then defense cannot be triggered

Question 22

Why would the pathogen produce factors that would make it reognisable to the plant?

Answer 22

Many genes are produced by pathogens, avirulence genes are often polypeptides/metabolites

Question 23

How do resistance genes exhibit dominance?

Answer 23

If have one copy which can interact with Avr/avr then the disease is prevented, but if the Avr is mutated then you get no interaction and disease

Question 24

What compounds are avirulence genes?

Answer 24

• polypeptides
• metabolites

Question 25

What is syringolide?

Answer 25

Metabolite

race-specific elicitor (trigger defense response)

produced by pseudomonus syringae

aviurlence gene AvrD

Question 26

What are the two types of disease resistant proteins?

Answer 26

RACE-specific R proteins: confers resistance to one pathogen strain e.g. Xa21 with a kinase domain. Recognition of perturbed self proteins.

RACE non-specific R proteins: Recognition of conserved pathogen associated molecular patterns (PAMP). Confers a broad spectrum of resistance to more than one pathogen strain, potentially more stable than RACE specific protein (bacterial strains evolve, those which are dominant one year may not be dominant the next) e.g. flagella (PAMP) conserved to many bacteria

Question 27

What types of structures are R proteins?

Answer 27

Many have been identified, genes and proteins of different types. Can be located in the plant cells, transmembrane receptors, or consist solely of kinase domain. Proteins consisting solely of a kinase domain interact directly with the Avr product to trigger a response, e.g. by initiating downstream signalling. Different cultivars may/may not recognise different pathogen strains.

Transmembrane proteins may recognise a number of different pathogens. Can use these tools to transfer broad resistance between plants.

Question 28

What are the proposed mechanisms of R gene action?

Answer 28

• R proteins interact with proteins that are common targets of pathogen effectors
• Modification of the target protein by an effector triggers a conformational change in the R protein
• Two mechanisms: plant is challenged by a pathogen, e.g. pseudomonus syringae bacterial secretion system injects Avr vomponent whih has in cell targets
  
  • a phosphorylation of the target is triggered. modification of target is recognised by an R gene product e.g. RPF1, and this interaction leads to an activation of the R protein beginning a signal transduction cascade e.g. initiating HR (effector triggered immunity)
  • or a cleavage of the target, this is recognised by the R gene product, resulting in a conformational change in the R protein so that it is active, to initiate effector triggered immunity

Question 29

What does a pattern recognition receptor do?

Answer 29

PRR

recognition of conserved pathogen associated molecular patterns (PAMP)

RACE non specific R proteins

Question 30

How was it shown that the manners by which plants protect themselves can be transferred into other plants?

Answer 30

1. The arabidopsis EFR (recognises a translational elongation factor) gene was isolated and overexpressed in tomato (cultivar money maker)
2. Got healthy plants when challenged by bacteria of several different species (broad-spectrum resistance)
3. Non transformed plant had low resistance
4. Used another tomato plant that was related to one where a resistance gene is active. Achieved similar level of resistance with a broad spectrum than with a RACE-specific R protein.

But: what about long term effects? Is it possible in other crops?

Question 31

Why do plants have inducible resistance systems?

Answer 31

programmed cell death should only happen when it is needed or else it could problematic - if began the process prematurely it compromises the plant as there are lesions where there shouldn’t be

Question 32

What is sytematic acquired resistance?

Answer 32

SAR: inducible resistance system

If an older tissue is attacked, younger leaf becomes more resistant.

Requires signalling involving siaylic acid (for aquired resistance)

modified versions of sialyic acid can be volatile and move to different parts of the plants

Question 33

How was it demonstrated that SA is required for systematic aquired resistance?

Answer 33

1. Introduced gene encoding an enzyme that degrade sialyic acid, NahG.
2. Studied many transgenic lines with differential expression of the enzyme (if create transgenic plants for expression there is always a variety of expression levels, depends on the integration location in the chromosome. Also sometimes get gene silencing, where the gene is recognised as ‘foreign’ and expression is switched off)
3. Innoculated older leaves first, then upon infection of younger leaves tissue and lesion size was evaluated
4. High NahG low SA bigger lesions so the HR response came later, low NahG high SA good response, lesions were so small couldn’t be seen

Question 34

What is benzothiadiazole?

Answer 34

An analogue of sialyic acid, more stable.

Has been tested on inducing SA by spraying on fields but wasn’t v effective at providing protection to plants.

Question 35

What compound is a key regulator of SA signalling, how does it function and how was this identified

Answer 35

NPR1

1. Pathogen is recognised
2. SA levels are increased
3. SA interacts with NPR1
4. pathogen related products are produced (PR1)
5. Hypersensitive response triggered

Identified: Broad-spectrum disease resistance in arabidopsis by the overexpression of NPR1 an essential regulator of SAR, resistance against oomycete peronospora

1. Isolated mutant plants not able to use the PR protein
2. Identified NPR1 as a signalling mechanism in HR as an intermediate for inducing HR - if gene is lost no repsonse, if oxerexpressed, get better response
3. Analysed 3 different independent transgenic lines; one with high expression, intermediate and low
4. correlated perfectly with disease rating
5. also shown by the number spores on the leaves: more spores, higher disease rating (lowest NPR expression)
   6.

Question 36

What proetins interact with SA?

Answer 36

NPR3 and NPR4

directly bind SA, have differential affinity to SA

NPR3 low affinity

NPR4 high affinity

Question 37

How do NPR3 and NPR4 interact with SA and NPR1?

Answer 37

In order for NPR1 to work effectively and trigger programmed cell death it must be degraded. In the presence of high concentration of SA, NPR3 binds SA only at high concentrations and interacts with NPR1 to attract protein degradation machinery. Programmed cell death is triggered. If not degraded, NPR1 acts to prevent local cell death.

Away from the site of infection, where there is an intermediate concentraion of SA, NPR4 binds SA at high affinity. Does not bind NPR1, this stabilises NPR1. No cell death and the surrounding tissues are protected. Also involved in triggering SAR.

SA-bound NPR3 and unbound NPR4 degrade NPR1.

In the absence of SA, NPR4 leads to the degradation of NPR1.

Interfering with this process can have complex results. NPR1 level is highest at intermediate SA concentration. A simple overexpression of NPR1 can not be considered as an overall effective strategy for increasing SAR in crop plants.

Question 38

What is the premise of using QTLs for breeding?

Answer 38

Each gene may have a small contributive effect.

Question 39

How can classical breeding be considered as more effective than GM?

Answer 39

Trait may be controllled by multiple genes each having a small contributive effect. Classical breeding can be used - identify regions in the genome that are related to the desired trait and through classical breeeding these can be selected for.

e.g. Some wheat cultivars are resistant to two rust fungi, some eltive cultivars were susceptible. Aim: transfering these gene loci from one cultivar to another. QTL mapping identified QTL locus, then identified the allele that conferred resistance to the rust fungus. Identified subtle differences (few mutations) that resulted in resistant cultivars, not big changes like overexpression. Gene involved (LR34) identified (ABC transporter and mechanism). Once the specific allele was identified, transferred to other plants either quickly (gm) or via conventional breeding.

Question 40

Give an example of how QTL techniques have been used to generate new cultivars in Rice

Answer 40

Non-race specific durable resistance against rice blast

Crossed different cultivars, one tolerant to rice blast and one that wasn’t. Identified crossing over events with hybrids - however regions with resistance also showed bad grain quality. Identified the location of the gene that conferred bad grain quality was located very close to gene for resistance. Once these two loci were identified could identify crossing over events that resulted in a hybrid with resistance and good eating quality.

• loss of the function of a protein that slowed down plant defence
• segregation from a closely linked locus that reduces quality

This was better than introducing RACE-specific R proteins which can be easily overcome by new pathogen strains. Involved: QTL analysis, mapping and introgression benefitting from molecular marker techniques. Still classical breeding.

Question 41

How was resistance to root-knot nematodes engineered in tobacco?

Answer 41

GM tech. RNAi.

1. Introduce inverted RNA repeats
2. These form an RNA hairpin structure
3. Triggers the degradation of RNA and gene silencing
4. E.coli expressing RNAi constructs fed to nematodes, leading to a systematic knock down
5. Nematodes took up the expressed RNA hairpins adn triggered silencing
6. plants engineered to express RNAi constructs so when the nematodes fed on the plants -> gene silencing, led to nematode tolerance.

Also shown in arabidopsis. Benefit of using RNAs: can select multiple targets at the same time. Produce hairpins that target multiple pathogens. Transgenic arabidposis plants shown to be resistant to many species of root-knot nematode.

Question 42

How has RNA targeting been used to increase insect resistance?

Answer 42

Western corn root worm

Used RNA targeting a vacuolar ATPase essential in western corn root worm. some transgeneic lines weren’t resistant and some were. Enhanced resistance.

Fusarium

Targetted SME (sterol modifiying enzyme - target of a classical fungicide.) Tested transgenic barley, WT and empty vector controls under attack and the TP plants remained as healthy as the unattacked plants.

Question 43

How do plants communicate between tissues and other plants?

Answer 43

By sending out volatiles. Upon attack by chewing insects, Japonic acid and other volatiles are released -> younger parts of plants are ‘warned’ and produce/accumulate systematic wound response proteins and protinase inhibitors making the plant indigestible. Volatiles do not just stay on the plant, also sensed by the surrounding plants.

Question 44

Give an example of where plants produce different cocktails of volatiles at different times of the day and why

Answer 44

Different volatiles have different purposes.

In the daytime, tobacco plants attacked by catipillars release a cocktail of volatiles that attracts predators to feed on the caterpillars, and at night the plant releases a mix that repels moths that lay eggs on the plants. Ths signal benefits both the plants and the moths. Is this possible by genetic engineering? For other crops?