Lecture 2 - engineering resistance of crops to pathogens and insects Flashcards

Question

What is syringolide?

Answer 1

Metabolite race-specific elicitor (trigger defense response) produced by pseudomonus syringae aviurlence gene AvrD

Answer 2

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

Answer 3

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.

Answer 4

* 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

Answer 5

PRR recognition of conserved pathogen associated molecular patterns (PAMP) RACE non specific R proteins

Answer 6

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?

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

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.

Answer 11

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.

Answer 12

NPR3 and NPR4 directly bind SA, have differential affinity to SA NPR3 low affinity NPR4 high affinity

Answer 13

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.

Answer 14

Each gene may have a small contributive effect.

Answer 15

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.

Answer 16

**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.

Answer 17

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.

Answer 18

**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.

Answer 19

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.

Answer 20

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?