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MICR222 > Plant Microbiome Interactions > Flashcards

Plant Microbiome Interactions Flashcards

1
Q

Features of plant microbiomes

A

Every part of a plant has a different microbiome eg leaves, stem, roots, soil surrounding them
Many mcrobes benefit host by boosting immunity, helping absorption of nutrients or protection against drought conditions/ other abiotic features

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2
Q

Plant roots as microbial habitats- different terms to know

A

Rhizosphere- zone of root influence, extends less than 5mm from root
Rhizoplane- root surface
Endorhizosphere- microbes inbetween the cells in the root
Root hair- incr SA, appendages from epidermal cells
Root cap and plant mucilage- polysaccharide secreted to cover
Sloughed root cap cell- cells the root gets rid off- way of exporting nutrients
Epidermis
Endodermis
Cortex
Bacterial mucilage
Root colonisation- bacteria grow as microcolonies over ~5% of root

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3
Q

Features of the rhizosphere

A

Influences microbial growth around the root with chemicals and nutrients
Stimulates growth and creates a highly competitive environment

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4
Q

How are the rhizosphere and endophytic compartment inflenced

A

By soil type and to a lesser degree by host genotype
Studied to control plant growth and susceptibility to pathogens in sustainable agricultural regimes

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5
Q

Communication between plant and bacteria

A

Plant adds molecules in the rhizosphere to communicate to bacteria who can add molecules to it aswell in response back to the plant

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6
Q

Composition of microbes in roots

A

Alpha and beta proteobacteria are most predominant
There is a slight difference in composition between different plant species

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7
Q

What is the rhizosphere effect

A

Populations around plant roots usually 20-100 x surrounding soil

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8
Q

Plants feeding microbes in the rhizosphere?

A

~15% of carbon and energy the plant makes is exported to the rhizosphere for bacteria

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9
Q

How does bacterial colonisation of plant roots work

A

Chemotaxis of microbes towards the root where they can then attach
Primary attachment (reversible) where electrostatic forces or hydrophobic interactions cause a loose attachemnt, adhesion of single cells strengthened by flagella, pili, fimbriae and surface adhesins
Secondary attachment (irreversible) where cellulose binds to bacteria and species specific factors strengthen attachment, polysaccharides caused microcolonies to form at adhesion site and then develop into mature biofilms

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10
Q

Where do microcolonies of pseudomonas fluorescens form

A

Slong junction between epidermal cells= make biofilms
The epidermal cells remain sterile and not colonised

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11
Q

One example of pathogenic fungus growth by rhizosphere

A

Containing microbes which can diffuse antibiotics against the fungus

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12
Q

Different interactions in the rhizosphere

A

Beneficial microbes- stimulated by plant but can also be inhibited by plant, bring benefits to the plant
Pathogenic microbes- damage the plant through infection or production of cytotoxic compounds
Commensal- no direct effect on pathogen or plant, can compete with pathogens
Interactions between these different microbes occurs

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13
Q

Ways beneficial microbes can benefit plants

A

Supply plants with beneficial nutrients like nitrogen and phosphorus
Enhance root growth= good start and physical support
Protect plants from disease and repel pests
Help plants tolerate conditions like heat, flooding and drought

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14
Q

Harmful effects of rhizosphere microbes on plant growth

A

Pathogenic fungi, oomycetes, nematodes and bacteria eg root rot fungi
Major and chronic threat to food production and ecosystem stability- pathogen resistance to the applied agents, envornmental impacts of pesticides, cost of pesticides, consumer demand for pesticide-free food

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15
Q

Beneficial effects of rhizosphere microbes on plant growth

A

Direct effects= facilitate uptake of nutrients from environment or synthesise compounds that effect plant growth
Indirect effects= lessen or prevent effects of plant pathogens

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16
Q

How can rhizosphere facilitate uptake of nutrients from environment

A

Phosphate solubilisation= make available for the plants
Associative nitrogen fixation
Siderophore production to allow for iron uptake

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17
Q

How can rhizosphere sythesise compounds that effect plant growth

A

Plant growth regulators such as IAA- produce ACC deaminase that inactivates precursor of stress-hormone ethylene which slows plant growth
Removing the precursor= plant growth isnt slowed

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18
Q

How can rhizosphere lessen or prevent effects of plant pathogens

A

Pseudomonas fluorescens can control root rots caused by fungal pathogens
Make an antibiotic against them

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19
Q

Soils suppressive to take-all

A

Of wheat caused by Ggt, leads to conductive growth (diseased)
In many cases where wheat has grown in the same soil over years, natural suppression of the fungus occurs= take all decline (TAD)
Correlates with development of specific fluorescent pseudomonas population in rhizosphere
Bacteria produce antibiotics that kill the fungus (2,4-diacetylphloroglucinol)

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20
Q

Study of disease suppressive soils integrated approach

A

Use metataxonome, metagenome, metatranscriptome, metaproteome and metabolome to find key microbial species and traits causing this (by comarison in conductive and suppressive soils)
Identify bacterial genera and particular traits
Test microbes to confer disease suppressiveness by direct application in seed/seedling/soil and augmentation of indigenous microbial populations and/or their activities in situ

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21
Q

Test microbes to confer disease suppressiveness by direct application in seed/seedling/soil

A

Single isolates
Synthetic communities, consortia
Transplantation of microbial communities

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22
Q

Test microbes to confer disease suppressiveness by augmentation of indigenous microbial populations and/or their activities in situ

A

Soil microbiome engineering towards disease suppressiveness eg by soil amendments
Plant mediated microbiome engineering eg by exudation

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23
Q

Ways plants shape their microbiome/ biological controls in rhizosphere- how microbes can provide resistance to pathogens

A

Antibiosis- bacterium colonises growing root system and delivers antibiotic molecules around root harming pathogens that approach
Induced systematic resistance (IRS)- local root colonisation= IRS, induces systemic signalling in most cases= protection of the whole plant from detection in one area
Competition for nutrients and niches- biocontrol bacteria acting, exceed in fast chemotactic movement along growing root in efficient hunt for root exudate components= outcompeting pathogen and occupying niches

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24
Q

What is the phyllosphere as a microbial habitat

A

Phylloplane is the leaf surface
Harsh environment- rapid moisture fluctuations, rapid temperature fluctuations, UV radiation
Microbial populations fluctuate rapidly if the conditions are right
Phylloplane inhabitants= epiphytic bacteria (grows on surface where nutrients are leaked), gram negative eg pseudomonas, erwinia, xanthomonas

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25
Q

Bacterial habitat modifications in the phyllosphere

A

Syringomycin= affects release of nutrients from plant cells and bacterial cell dispersal. Is a phytotoxin and surfactant (breaks-down hydrophobic nature or disperses across)
Auxin secretion= release of saccharides from plant cell wall
EPS in bacterial aggregates= protection from environmental stresses such as tolerance to UV and dessication

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26
Q

Bacterial traits involved in adaptation to the phyllosphere

A

Antibiotic and biosurfactant (enzyme) release- controlled by quorum sensing signalling molecules released
Pigment release for UV and radiation protection
Auxin for protection

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27
Q

Pseudomonas syringae and frost damage

A

Ice-nucleation active (INA+)
Function as nuclei for the formation of ice crystals that can spread into plant tissues
Prevent supercoiling of leaves so frost damage is initiated at -3deg instead of -8deg= agricultural problem
Dependent on production of INA outer-membrane protein

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28
Q

INA+ and snow making

A

Used in snow making machines on mountains
Kill bacteria, proteins are on surface so still able to be used
Put bacteria into water= produce ice at warmer temperatures of -3deg on mountains for snow sport

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29
Q

INA+ and rain/snow/hail making

A

Swept into atmosphere by wind, adaptations allow their survival by making rain and snow, can also be found in the middle of hail
Come back down to earth
Allows spreading to different plants

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30
Q

Example of competition exclusion from bacteria causing disease in phyllosphere

A

Caused by erwinia amylovora of pear and apples from flowers (bees get it from flowers and bring to fruit trees)
Spray inoculation with suspension of P.fluorescens and rapid multiplication of the antagonist occurs
Then when infection arrives by bees, no disease occurs as colonisation by the other bacteria is so high

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31
Q

What is a legume

A

Bear seeds in pods eg soybeans, clover, beans, peas, alfalfa, gorse

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32
Q

What is rhizobium-legume symbiosis

A

Infection of legume roots by rhizobia leads to root nodule formation and nitrogen fixation (conversion of N gas to combined N in form of ammonia)
Host specificity high- one rhizobium nodulates one legume type- 2 compatible partners need to recognise each other
Symbiosis adds over 200 million tonnes of N to soils worldwide every year

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33
Q

What are rhizobia

A

Gram negative rod shaped soil bacteria
Can form nodules on atleast one particular legume
Symbiosis characterised by high degree of host specificity
Not all able to fix nitrogen
Diverse between alpha and beta proteobacteria, then after diversity formed, ability to do symbiosis, create nodules and fix nitrogen occurred from HGT

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34
Q

First steps in nodule formation

A

Recognition of correct plant partner by rhizobia and attachment to root hairs
Root hair curling, rhizobia trapped in the curl, leads to cortical cell division of once terminally differential cells (due to signal)
Invasion of root hair by formation of infected thread (tube produced by the plant cell wall and membrane as a response)

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35
Q

Second steps in nodule formation

A

Rhizobia travel to root cortex via infection thread
Bacterial release from infection thread into plant cytoplasm by endocytosis (surrounded by a membrane)
Formation of misshapen bacterial cells (bacteriods- become terminally differential) within plant root nodule cells and development of a nitrogen fixing state
Plant provides carbon for energy to fix nitrogen, bacteria produce nitrogen as ammonia for the plant- transport through surrounding vascular tissues

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36
Q

What is a determinant nodule

A

Cell division stops after a short period of time and the cell expands to further grow= finite division

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37
Q

How does the two-way signalling occur between host legume and rhizobium cell

A

Legume produces flavonoid (many types with different chemical groups in certain areas)
Flavonoid is recognised by particular bacterium which turns on the expression of nod genes from accessory genetic element
Nod leads to creation of a nod factor which is a lipochito-oligosaccharide of N-acetyl glucosamine, fatty acid attached and differences in bound chemical groups depend on the rhizobium type to make it unique for their legume= host specificity
Recognised be receptors in legume, allows induction of N fixation

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38
Q

Features of nod genes

A

Borne on accessory genetic elements eg plasmid OR IGE or symbiosis island on chromosome (not on core chromosome)
Sym plasmids in Rhizobium and Sinorhizobium and islands in mesorhizobium loti

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39
Q

Features of mesorhizobium loti

A

Nodulates legumes of genus lotus (incl L.corniculatus and L.japonicus)
Studies contribute to understanding bacterial and plant components of the symbiosis
Symbiosis genes are on the chormosome

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40
Q

Discovery of the symbiosis island

A

Site lacked indigenous rhizobia capable of nodulating plant and uninoculated seedlings die from nitrogen deficiency within a year
Sampling 7 years later found diverse strains in nodules containing chromosomal symbiotic DNA identical to that of R7A= original nodulation had transferred to others

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41
Q

Features of the R7A symbiosis island

A

Integrative and conjugative element
~10% of genome
Converts non-symbiotic into Lotus symbionts in environment and lab
Integrates into a phe-tRNA gene in a process mediated by a phage-type integrase
Converts by a one off transfer event- excise from chromosome and transfer by conjugation and insert into chromosome recipient

42
Q

Two types of nod genes

A

Regulatory= nodD gene product turns on other nod genes in response to flavonoid signal from plant
Structural= responsible for synthesis of a lipochito-oligosaccaride signalling molecule Nod factor

43
Q

How are nod genes turned on

A

Host legume secretes signal molecule i root exudate
Different factor for each legume/rhizobium pair (mostly flavonoids= three ring compounds)
Different NodD’s recognise different flavonoids= host-specificity

44
Q

Plant responses to nod factors

A

Recognises nod factor via receptor
Influx of calcium at root hair tip
Calcium spiking in root hair nucleus
Deformation (curling) of root hair
Initiation of cortical cell division
Nod factors are like plant hormones as they share similar characteristics= changes in plant growth and can induce a new organ

45
Q

How are nod factors perceived by a plant

A

Nfr1 and Nfr5 are receptor for Nod factor
Extracellular LysM domains bind Nod factors= symbiotic signals through intracellular kinase domain of Nfr1
Leads to downstream signalling cascades= rhizobial infection and nodulation
Cascade involves several additional genes and includes activation of Ca-calmodulin kinase through calcium spiking
Multiple steps leads to infection thread formation and organogenesis= infected nodule

46
Q

Nitrogen fixation in root nodules

A

Rhizobia genes needed are nif and fix
Requires high energy and low O2 (O2 inactivates nitrogenase enzyme, inhibit transcription of nif and fix)

47
Q

Ways plants avoind having O2 too high for N fixation (even though it is required for respiration to make more ATP)

A

Bacteroids have high O2 affinity ETC= can have low O2 to be just as efficient
Plant compound leghaemoglobin facilitates diffusion of O2 to bacteroids at high flux but low concentration
Nitrogen fixation only expressed in absence of intracellular oxygen regulated by FixLJ

48
Q

How does FixLJ work

A

FixL= Membrane spanning with majority in the cytoplasm
Contains a heme domain in the cytoplasm area, when O2 is present, FixL phsophatase dephosphorylates FixJ and target genes arent expressed
When no intracellular O2, autophosphorylation of his in FixL occurs= kinase leading to phosphorylation of FixJ= activates target genes involved in nitrogen fixation (P from His transferred to Asp on FixJ)

49
Q

Metabolic reactions and nutrient exchange in nitrogen fixing nodules

A

Host supplies bacteroids with C4 dicarboxylic acids as energy into CAC to make electrons- enters through 2 transporters one in symbiosome membrane and one in bacteroid membrane
Bacteria reduce N2 to NH3= translocated to host and bacteria make amino acids for itself with NH3
Plant has adaptations for O2, C and N metabolism

50
Q

Ammonium assimilation

A

NH3 enters host cytosol by passive diffusion (not used by bacteria)
NH3 assimilated through actions of glutamine synthetase and glutamate synthetase= glutamine and glutamate
Used to synthesise amides or ureides which are transported to the rest of the plant via the xylem

51
Q

Another example of plant mutalistic association and some features- fungi being good

A

Mycorrhixa fungi= root fungus
Integrated into physical structure of roots
Widespread occurrence: >90% of land plants (350,000 species), and >50,000 taxa of fungi
Little species specificity

52
Q

What are the different types of mycorrhizae

A

Ectomycorrhizae
Endomycorrhizae- orchid, ericoid, arbuscular

53
Q

What does the mycorrhizal fungus get from the plant

A

Carbohydrates and fatty acids (FAs needed as they are FA auxotrophs)

54
Q

Why is the mycorrhizal relationships so widespread (thought to be)

A

Essential for the movement of plants from the ocean to land
Crucial role in land colonisation

55
Q

Features of ectomycorrhizae

A

Form a sheath around root with little intercellular penentration and no intracellular penetration (can go around/ between cells a little bit, doesnt go in)
Hyphae spread out from the sheath and can form networks with others in other plants= nutrient exchange through sheath
Found mostly in temperate forest trees eg conifers, beech, oak
Root system is long and short
Short roots are colonised by micorrhizae which prevents pathogen attach
Can form fruity bodies
Mycelium in roots, rhizomorphs (where exchange of carbs to outer and stuff for plant occurs), fungal mantle/ sheath, hartig net (fungus/ plant interface)

56
Q

Features of orchid endomycorrhizae

A

Restricted to orchids
Non-pathogenic penetration of root cortex by septate fungal hyphae= intracellular coils
Orchids need the fungus (obligatory mycorrhizal)
Fungus mildly pathogenic- balance by plant to control growth using orchanol (so doesnt become parasitic)

57
Q

Features of ericoid endomycorrhizae

A

Restricted by ericaceae eg rhododendron, blueberry)
Non-pathogenic penetration of root cortex by septate fungal hyphae that form intracellular coils (like orchid)
The plants here are also obligatory mycorrhizal

58
Q

Features of arbuscular endomycorrhizae

A

Most common type- in most crops
Can be grown in culture by addition of FA to medium (discovered in 2019)
Makes a hyphopodium which allows the fungus to put pressure on the plant cell and enter
Inter and intracellular: non-septate (long with multiple nuclei rather than many divisions between) hyphae in cortex, directly linked to external mycelia that spread into soil and form loose netowrk around the root= high SA between (arbuscule)

59
Q

The arbuscule

A

Each fungal branch in a plant surrounded by plant-derived periarbuscular membrane (PAM)- continues with plant plasma membrane, excludes fungus from plant cytoplasm
When arbuscule goes into cell, it is surrounded by this PAM
Space between fungal cell wall and PAM is periarbuscular space made of fungal and plant cell wall material
Causes fungus to never be free in cytoplasm, prevents it from becoming pathogenic

60
Q

Communication betqween AM (arbuscular) fungi and root to form symbiosis

A

Plant lets out strigolactones, sensed by AM fungal spore, causes germination and mycelia growth which then expand
Expanding mycelia exude signalling molecules eg lipochitooligosaccharides (LCOs) and chitoologosaccharides (COs)
Molecules lead to reactions in the plant root: cytosol Ca increases= AM fungal induced gene expression= formation of pre-penetration apparatus
Reacting root secretes cutin monomers= fungi form hyphopodium and initiates arbuscular growth

61
Q

Fungal lipochitooligosaccharide and nod factor
(Relationship between arbuscular mycorrhiza and rhizobium)

A

Fungal lipochitooligosaccharide similar to a nod factor (same thing)
In fungus however, not all of them have lipid, can be chitoologosaccharide
Common symbiotic signalling pathway between Nod and Myc factors leading to nodulation and mycorrhization
Suggests AM symbiosis is ancient and rhizobium have taken over and modified the method to become symbiotic (evolved from AM symbiosis)
Possibly use the same receptor too

62
Q

Benefits of mycorrhizae

A

Essential for colonisation and growth of plants in nutrient-poor environments (key in nutrient and carbon cycle in forests)
Main effects from provision of P, N and other minerals and possibly water
Improved nutrient absorption by mycorrhizal plants due to greater SA provided by fungal micelia= enhanced resistance to drought stress from high water gathering ability due to same reason
Protection against some pathogens especially with ectomycorrhiza
Link together into communities= more resilient to stress and disturbance than single plants
Enable plant to plant communication
Less P needed to reach their maximum growth

63
Q

Examples of plant to plant exchange and communication in mycorrhizal networks

A

Eg molecules providing systemic resistance can be transferred
Carbon and nutrients can be transferred
Eg donor plant infested with aphids increases defence by emitting volatiles which are repellent to pea aphids and attract parasitic wasps that parasitise on aphids. Can share with other plants to increase their defenses (plants without system cant)

64
Q

Why are plant-pathogen interactions/ plant disease an exception rather than a rule

A

Most plant pathogens only attack one or limited no of plant species
Often find resistant varieties to a particular pathogen within a plant species
In susceptible plants, pathogen damage usually limited
Plants often able to recognise pathogen and mount co-ordinated defence against it
Once induced, plant defence response is usually effective

65
Q

Two plant immunity defence systems

A

Basal and gene-for-gene

66
Q

Features of basal defence

A

Induce upon infection by almost all microbes
Based on recognition of general elicitors- PAMPs by pattern recognition receptors (PRRs)
Rapid activation of defence but generally without ‘hyper-sensitive’ response
Similar in principle to innate immunity in mammals

67
Q

Features of gene-for-gene defence

A

Induced upon infection by specialised pathogens
Based on recognition of highly specific Avr-gene products- effectors, recognised by matching R-gene products
Rapid reaction usually including ‘hypersensitive response’

68
Q

The plant-pathogen arms race

A

Plant has PAMP recognition
Pathogen develops non-eliciting PAMPs that arent recognised by plant receptors= susceptible plant
Pathogen evolves effectors= susceptible plant (produces proteins that interfere with cellular signalling)
Plant evolves gene-for-gene defence

69
Q

Examples of PAMPs

A

Bacteria- flagellin, elongation factor EF-Tu, lipopolysaccharide, cold shock protein
Fungi- chitin, B-glucan, ergosterols

70
Q

Flagellin as a PAMP

A

Main building block of the flagellum
N-terminal and C-terminal domains highly conserved
22 aa sequence in N-terminal= flg22 which is recognised by plant FLS2
Leucine rich repeats with protein-protein interaction domains

71
Q

Recognition of flg22

A

In Arabidopsis, is a receptor-like kinase
Recognises= immediate changes (ion fluxes and generation of ROS)
Protein phosphorylation, mitogen activated phosphorylation (MAP) kinase activation= activation of transcription factors from them being phosphorylated-> gene expression of about 1000 genes (R-genes, RLKs, antimicrobials called phytoalexins)
Ethylene production
Callose deposition= strengthen cell wall (ROS earlier on can also strengthen cell wall)
Rapid oxidative cross-linking of hydroxy-proline rich proteins= cross link and strengthen

72
Q

Why cant most pathogens infect most plants

A

Because plant cells have multiple PRRs on their cell surfaces which each leads to an effect and prevents different diseases

73
Q

CERK1 and Nod factor

A

CERK1 is a PRR for chitin
Related to Nod factor receptors- both similar structure with 3 LysM domains
CERK1 homologous to legume Nod factor receptors NRF1 and NRF5 (mediates perception of lipo-chitin nod factors)
Likely Nod receptors evolved from CERK1

74
Q

What is compatible interaction

A

If a host is susceptible and a pathogen virulent, disease occurs

75
Q

What is incompatible interaction

A

If host is resistant and pathogen avirulent, no disease occurs

76
Q

What are races

A

Biotypes of pathogens that vary in their pattern of compatible or incompatible reactions on a set of host plant cultivars

77
Q

How does complementarity work for gene-for-gene

A

No disease if a plant had a dominant resistance gene AND pathogen had dominant avirulent gene
Resistance is able to recognise the avirulent gene and give resistance (as avirulent on its own can still have virulence factors, just not as well)
Plant may have several R genes directed against a pathogen, pathogen must lack all relevant avirulence genes to escape recognition
Leads to hypersensitive response

78
Q

What is the hypersensitive response

A

Rapid death of infected cells at point of infection (kills cells when R gene recognises avirulent)

79
Q

Another response by plant immunity when avirulent genes are recognised

A

Systemic acquired resistance
One recognises and spreads the resistance to the rest of the plant

80
Q

How do avr genes get into plants

A

Secreted into host cell cytoplasm by type III secretion systems
Called effector proteins (the avr genes)
Most can be mutated to restore pathogenicity
Most encoded hydrophilic proteins with no functional homologues in databases

81
Q

Avr genes and basal defence

A

In absence of R gene, avr effectors suppress basal defence

82
Q

Features of R genes

A

Most encode hydrophilic proteins with nucleotide-binding site and leucine rich repeat
Involved in protein-protein interactions
Others are protein kinases eg Pto
NB-LRR class further sub-divided based on N-terminal structure features

83
Q

Sub-dividing of NB-LRR R genes

A

TIR-NB-LRR= homology to intracellular signalling domains to Drosophila Toll and mammalian interleukin
CC-NB-LRR= contain coiled-coil domains

84
Q

What is the guard hypothesis

A

R-protein is linked to the host target protein of the effector
Senses the binding and is released or causes interactions= response to the infection

85
Q

Two possible methods of guard hypothesis

A

NB-LRR R gene is attached to host effector target and when bound to effector, R gene is released and causes effect
NB-LRR R gene is activated in response to effector target complex (R gene not bound to target)

86
Q

Pseudomonas syringae effectors; AvrB or AvrRpm1 AND AvrRpt2 (different processes of blocking innate immunity)

A

AvrB/ AvrRpm1 bind to RIN4 which is then P by RIP kinase= inactive= recognition by RPM1 resistance gene (R gene) leading to activation of the R protein and effector triggered immunity
AvrRpt2 is a protease, binds to RIN4 and cleaves it which makes it inactive= RPS2 resistance gene (R gene) recognises the cleavage and leads to R gene activation and effector triggered immunity

87
Q

Pseudomonas syringae effector AvrPto blocking innate immunity

A

Binds and blocks kinase domain of PRRs which prevents PAMP triggered immunity
R gene Pto mimics kinase domain= binds AvrPto, R gene Prf recognises this binding and leads to effector triggered immunity
Prf is a member of NB-LRR family

88
Q

Improved practices for breeding durable resistance by genomic strategies

A

Use next-gen seq to seq genomes of pathogens causing disease in local fields
Use bioinformatics to identify most successful core effectors in strains
Identify R genes activated by the effectors
Deploy multiple stacked R genes that recognise defined core effectors to reduce change pathogens overcome resistance
Identify and edit genome disease susceptibility genes to reduce pathogen growth and symptom development
Identify and deploy antipathogenic probiotic and/or antipathogenic microbial mixtures as seed coats

89
Q

Agrobacterium tumefaciens and crown gall formation

A

Tumor growth on plants
Important in stone fruits, rose, grapes and apples
Only wounded cells can be transformed through freezing damage, grafting and mechanical injury
Majority of genes for crown gall induction are located on Ti plasmid, not part of core chromosome

90
Q

How crown gall formation occurs

A

Loose attachment via acidic capsular polysaccharide
Production of cellulose fibrils that enmesh large numbers of bacteria at wound surface
Vir genes expressed and T-DNA excised as single strand, transferred to plant nucleus via inter-kingdom sex/ conjugation
Integrated into plant cell DNA= expressed leading to tumor (from synthesis of cytokinin and auxin) and production of opines

91
Q

How does a bacterial cell recognise a plant which is wounded

A

Plant produces acetosyringone as part of its defence response
Activates virulence genes in the bacteria

92
Q

Parts of the Ti-plasmid

A

T-DNA and vir genes and opine catabolism genes
Only part of T-DNA required for transfer are borders- 25 bp sequences
Transfer occurs through action of expressed vir genes
T-DNA contains oncogenesis genes and opine synthesis genes

93
Q

Things naturally encoded in T-DNA

A

Enzymes for auxin and cytokinin synthesis= hormone imbalance= tumour formation
Opine synthesis genes eg octopine, nopaline
Opines are carbon and nitrogen sources for bacteria
Contains bacterial genes which have plant transcription sites on them to allow expression in the plants eg poly-A tails
Bacteria lack the appropriate set of transcription factors meaning these genes are only expressed in plants

94
Q

What is octopine

A

Condensation product of arginine and pyruvic acid

95
Q

What is nopaline

A

Condensation product of arginine and alpha-ketoglutaric acid

96
Q

How does expression of vir genes occur

A

VirA is part of a 2 component sensory system which resides in bacterial membrane
VirA detects phenolic compounds released from plant due to wound
Leads to autophosphorylation of virA
Phosphate is then transferred to virG which acts as a transcription factor activator for the expression of other vir genes

97
Q

How do vir genes excise T-DNA from Ti-plasmid

A

VirD2 nicks T-DNA at 5’/ right border, attaches to this end by covalent bonding to a Trp
Single strand of T-DNA unwinds, released from Ti by nicking at the left border
VirE single strand binding proteins bind to the excised T-DNA to stop it from coiling
Gap in the Ti-plasmid is repaired

98
Q

Transfer of T-DNA by conjugation bridge

A

Transferred through pilus encoded by virB operon
VirD4 also required
VirD2 acts as pilot protein and leads ss-T-DNA to the mating pore which has been formed by virB
Things secreted into bacterial cell by type IV secretion system (virF, virD2, virE2)
ATP-dependent process (type IV secretion) and allows transfer of effector proteins into cells

99
Q

Targeting of the plant nucleus for T-DNA integration

A

VirD2 and vieE2 contain plant-active nuclear localisation sequences (NLS)
VirE2 interacts with plant protein VIP1- transcription factor phosphorylated by MAP kinase from Agrobacterium infection (defence response)
Phosphorylation targets VIP1 to nucleus, T-DNA goes with it through interaction with virE2
VirD2 required for integration of T-DNA by protecting the 5’ end of the T-DNA
Integration occurs by illegitimate recombination at expressed loci (genes which are in loose chromatin from presence of RNA pol and are being synthesised)- leads to mutated DNA
Allows cell division and opine synthesis

100
Q

Responses to opines- things allowing for success of agrobacterium and opines

A

Each Agrobacterium strain catalyses only the opines synthesised by the tumours it induces- catabolic reactions encoded on Ti plasmid= ecological niche
Some opines induce conjugal transfer of Ti plasmid to other strains of Agrobacterium that may be present= way if ensuring plasmid is propagated

101
Q

Using Agrobacterium to genetically-engineer plants

A

Binary vector system
Clone DNA to be transferred between border sequences in a cloning vector
Transfer vector to disarmed/ defective Agrobacterium strain with mutated Ti plasmid containing vir genes but not T-DNA
Use strain to transfer cloned DNA to plant cells using either cells in petri dishes or by dipping flowers
Regenerate plant cells into whole plants or screen for transformed seeds

MICR222 (8 decks)

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