Lent Flashcards
What was the traditional basis for grouping micro-organisms, and how is phylogeny determined nowadays?
Traditionally, organisms were grouped based on cell shape, features, and gram stain. Nowadays, phylogeny is determined based on molecular data, such as 16S rRNA for prokaryotes and 18S rRNA for eukaryotes.
How does the gram stain work, and what colors do Gram-positive and Gram-negative bacteria appear?
The gram stain involves using crystal violet dye.
Dye gets stuck in thick layer of peptidoglycan wall
Gram-positive bacteria = purple
Gram-negative bacteria = pink.
What is the unique feature of Gram-negative bacteria’s outer membrane, and why are they less susceptible to antibiotics?
Gram-negative bacteria have an outer membrane made of lipopolysaccharides. This membrane controls molecule movement and provides resistance to antibiotics.
How are Gram-positive bacteria classified, and what is an example of a genus with low genomic GC content?
Gram-positive bacteria are classified into Firmicutes (low genomic GC content, found in the gut)
Actinobacteria (high genomic GC content, found in soils).
What are the key differences between bacteria and archaea?
- Bacteria have peptidoglycan in walls V archaea lack peptidoglycan.
- Most bacteria have an outer cell membrane, while archaea commonly don’t.
- Archaeal and bacterial flagella are constructed differently
- Archaea reproduce by fission while some bacteria produce spores, or by binary fission
- Bacterial cell membrane is bilayer linked with ester bonds, Archaeal is single layer linked with ether bonds
How are fungi classified, and what is an example of a fungus with septate hyphae?
Ascomycota (septate hyphae, e.g., Saccharomyces)
Basidiomycota (club fungi, e.g., common mushrooms).
Glomeromycota- AM fungi, non septate, obligate biotrophs
What is the primary contribution of algae to the environment, and what are the pigments responsible for their diversity?
Algae contribute to 50% of global carbon fixation. Pigments like chlorophyll, phycoerythrin, and phycobilins provide diversity in different environments.
What is metagenomics, and which sequencing techniques are commonly used?
Metagenomics uses Next-Gen sequencing techniques like Illumina to identify and classify microorganisms without the need for culturing.
What factors influence fungal richness, and what are predictors for different fungal groups?
Fungal richness is influenced by factors like vegetation age, precipitation, and soil pH.
AM fungi = age of vegetation
Saprotrophs = mean annual precipitation
Yeasts = soil pH
Describe the seasonal changes in the Bermuda Atlantic Time-series study.
Winter = oceans are turbulent
Spring = phytoplankton bloom
Summer = prokaryotic plankton dominates, with distinct communities in the upper euphotic zone, deep chlorophyll maximum, and upper mesopelagic zone.
What does syntrophy/cross-feeding refer to in microbial interactions?
Syntrophy or cross-feeding involves one organism utilizing the waste product of another, indicating a potentially mutualistic relationship.
Name the three nutritional types of microorganisms and provide examples for each.
Photo(auto)troph: Cyanobacteria, Purple and Green Bacteria
Litho(auto)troph: Few bacteria, many archaea, methanotrophs
Heterotroph/Chemotroph: Most bacteria, some Archaea
How do fungi with saprotrophic lifestyles obtain nutrients from dead organic matter, and what enzymes are involved?
- Fungi secrete digestive enzymes from hyphal tips
- Break down substances like starch (via amylase), sucrose (via invertase), and cellulose (via endocellulase, exocellulase, β-glucosidase, and β-glucanase).
- Take up nutrients in the sub-apical region through H+ couple symport.
What are methanogens, and where are they commonly found?
Methanogens are archaea that produce methane. They are commonly found in anoxic habitats and the stomachs of ruminants.
Describe the phases of methanogenesis, and what is the significance of syntrophy in this process?
CO2 is reduced to a formyl which is attached to methanofuran (MF), occurring by ferredoxin
Transfer to methanopterin
Reduction by F420 to methyl group attached to methanopterin
Then reduction to methane, generating a H+ pmf
Syntrophy- due to requirement of H2 as source for e-, derived from fermentation
Explain the complex microbial community involved in the digestion of wood by termites.
- Termites chew wood, releasing endoglucanase.
- Protists take up fragments, using endo and exo glucanases to form glucose and ATP.
- Methanogens convert H2 to methane.
- Ectosymbiotic acetogens convert H2 to acetate, usable by termites.
What is the role of methanotrophs, and what enzyme is crucial for their function?
Methanotrophs convert methane to CO2 and use the enzyme Methane Monooxygenase (MMO) for this process.
How is cellulose broken down to glucose?
Using exo and endo cellulases to form cellubiose
β- Glucosidase- convert Cellobiose to β-glucose
β- glucanase- converts β-glucose to glucose
How is lignin broke down?
Only carried out by white rot fungi, due to the presence of ether bonds which doesn’t make it hydrolysable
1. Lignin peroxidases- strong oxidant, Haem containing and Trp171 for attachment
2. Manganese peroxidase- strong oxidant Haem containing, no Trp so allows for diffusion of Mn(III) bound to oxalate to diffuse in, for internal degradation
What are extremophiles, and what types of extreme conditions can they adapt to?
Extremophiles are organisms adapted to extreme conditions such as high or low temperatures, pressures, and pH.
They can thrive in environments that might be stressful to other organisms.
Explain the difference between acclimation and adaptation in extremophiles.
Acclimation is the ability to change in response to imposed stresses within a certain range
Adaptation involves changes caused by evolution occurring over a long period of time.
Describe the heat shock responses in mesophiles, taking Escherichia coli as an example.
In E. coli, a shift from 30-42°C induces a heat shock response activating the Heat Shock Regulon (HSR)
Activates: FeS proteins, chaperone proteins, and DNA repair enzymes.
The response is controlled by the σ32 factor.
How is the activity of the σ32 factor controlled and regulated during heat shock responses?(4)
- Translation regulation through exposure of the Shine-Dalgarno (SD) sequence and AUG region in the stem loop
- Expression of chaperones
- Unfolded Protein Titration (UPT) Model
- Short half-life (<1min) with degradation by FtsH.
How does the sigma 32 factor work?
- rpoH mRNA encodes σ32 factor
- High temperatures cause stem loops to melt and exposure of SD sequence
- At 30°C σ32 is bound to chaperone proteins (DnaK/J and chaperonins GroEL/ES)
- Higher temperatures other proteins interact with DnaK/J
- Release σ32 so that they can interact with HSR promoters
What are the key differences in the adaptation of thermophiles, specifically in the phospholipid structure, between bacteria and archaea?
Archaea often have a phospholipid monolayer with ether-linked bonds, branched isoprene side chains
Bacteria have a phospholipid bilayer with ester-linked bonds.
Explain the differences in proteins, specifically Cytochrome C, between Pseudomonas aeruginosa and Hyperthermus thermophilus.
Hyperthermus thermophilus = 87.5°C
P. aeruginosa = 47.3°C
Extremophile has more aromatic compounds and is more compact
How do thermophilic proteins stabilize themselves, and what structural features contribute to this stability?
Ion pairs (S-S bonding)
Increased abundance of charged residues
Decreased flexibility with cyclopentene rings
Existence as oligomers.
What strategies do extremophiles employ to enhance resilience to stresses in their environment?
- Increase genome copy for redundancy
- Large number of DNA repair proteins
- Have a highly compacted nucleoid
In halophiles, the presence of glycerol methylphosphate contributes to low membrane permeability under high salt concentration.
What are porins?
Porins are protein channels present in the outer membrane of bacteria.
They form trimeric complexes with an 18-strand antiparallel beta barrel structure.
What are the pros and cons of porins?
Pros: Provide protection, restricted access to hydrophobic molecules.
Cons: All nutrients have to cross the membrane, no electrochemical gradient.
Name examples of non-selective porins and their characteristics.
OmpC (smaller 1.1nm, present at high osmolarity)
OmpF (slightly larger 1.2nm, more expressed at low osmolarity) allow passive diffusion of molecules <700Da.
How is maltose transported into bacteria?
LamB, a specific porin, allows maltose to enter. MalE binds maltose, transporting it across the periplasmic space to the inner membrane, where ABC transporters move it against the concentration gradient using ATP.
Name different types of bacterial secretion systems.
Sec translocation (unfolded proteins)
Tat translocation (folded proteins)
Type I (ABC transporters)
Type II (secretin protein)
Type III (needle-like injectisome)
Type IV (DNA and protein injection).
What is quorum sensing?
Quorum sensing is a bacterial communication system.
In Vibrio fischeri, LuxI synthesizes AHL, which, when reaching a threshold, binds LuxR, activating the expression of LuxI and LuxAB, leading to bioluminescence.
What are the roles of quorum sensing in bacteria?
Quorum sensing regulates bioluminescence, secretion of virulence factors, conjugation, and biofilm formation.
Describe the stages of biofilm formation.
Initial reversible attachment
Irreversible attachment
Maturation I and II
Dispersal (active or passive).
P. aeruginosa biofilm formation involves PelA and extracellular polysaccharide (EPS)
How do bacteria and plants interfere with quorum sensing?
Bacteria can secrete AiiA to degrade AHL, Plants (e.g., legumes) can secrete L-canavanine to inhibit AHL-mediated communication.
What are the characteristics of viruses?
Viruses are obligate intracellular parasites that hijack host cell machinery, replicate, assemble, and can capture host membranes to form envelopes.
What components make up the structure of a virus?
Viruses consist of a capsid that encloses their genomes, which can be either DNA or RNA. The genomes are very small.
How do viruses exhibit diversity in their genetic makeup?
Viruses show diversity through overlapping reading frames, ambisense RNA (coding both ways), and splicing mechanisms.
What are some methods used to provide evidence for the existence of viruses?
- Visualization through electron microscopes
- Metagenomics
- Koch’s postulates by isolating and reinfecting a healthy organism.
What is the role of symbiotic polydnavirus in parasitoid wasps?
Symbiotic polydnavirus inhibits the host immune response against wasp eggs, being integrated into the wasp’s DNA and transmitted vertically.
How do retrovirus elements contribute to placental vertebrates?
Retroviral elements, for example the env gene code for syncytin proteins, are essential for placental development in vertebrates, and about 8% of the human genome consists of retroviral elements.
The LTR enhancer is also important for positive feedback and release of corticotropin RH, during parturition
What is the significance of mycovirus-infected fungal endophytes in relationships and plant health?
Virus-infected fungi play a role in forming relationships that impact plant health positively.
Without vius infection, the fungi doesn’t aid plant health
How do viruses contribute to biogeochemical cycles?
Viral shunt
1. Mass lysis of biomass (phytoplankton)
2. Dissolved organic matter (DOM) recycling carbon
3. Used by marine bacteria, releasing CO2 into the air for photosynthesis.
- without it photosynthesis would not have sustained for so long
What are effector proteins?
Proteins produced by pathogens that directly manipulate host cell functions.
Able to modulate signalling, immune responses and cellular structures
Why is the quantification of the effect of disease challenging?
Imperfect and sparse data
Some countries may provide inaccurate information
Difficult to quantify with different types of pathogens and crops
What are the consequences of plant diseases?
Plant death
- 20-30% yield loss, sporadic yield loss that is hard to prepare against,
- Quality loss leading to issues like kidney disease due to mycotoxins.
What are the three strategies employed by pathogens, and how do they differ?
Necrotrophs kill cells and consume contents with a broad host range, Hemibiotrophs have an intermediate host range
Biotrophs keep plant cells alive with a narrow host range.
Describe the infection mechanism filamentous biotrophs like fungi and oomycetes.
They form an appressorium, puncture the cell wall, create a haustorium, and increase surface area for better nutrient exchange.
What is unique about nematode biotrophs?
They invade the host, cause plant cells to differentiate and divide, and become linked together, leading to the death of both if one dies.
What is the effector concept in the context of plant diseases and give an example?
Pathogens produce effectors, often proteins, that alter host defense to benefit the pathogen for example increase the transcription of susceptibility genes
~
TAL effector, secreted by Xanthomonas, enters the nucleus, binds to DNA upstream of the promoter, and upregulates plant gene expression. It is used in early gene editing for directing enzyme cutting.
HopZ1 from P.syringae, suppresses plant immune responses by inhibiting MAP kinase cascades
What are susceptible genes in the context of plant diseases?
Susceptible genes are plant genes required by pathogens to cause disease, such as SWEET sucrose transporters. Mutations in these genes lead to reduced sucrose and less bacterial colonization.
Define passive and active resistance in plants.
Passive resistance - occurs when plants lack a susceptibility gene, making them less susceptible to disease.
Active resistance - action by the plant to resist pathogens and involves genetically dominant resistance factors/genes. The two sub-groups are Constitutive (active all the time) and Induced (deployed when infected).
Name some physical and chemical barriers that plants use against pathogens.
Physical barriers: cell wall with cross-linked glycans, lignin, and suberin, as well as the cuticle.
Chemical barriers: antimicrobial metabolites and substances like nicotine and caffeine.
Describe how fungi and oomycetes penetrate physical barriers in plants.
Fungi and oomycetes, such as the Rice blast fungus, form an appressorium that penetrates the cuticle and cell wall, facilitated by increased turgor pressure.
How do pathogenic bacteria overcome physical barriers in plants?
Pathogenic bacteria secrete proteins, DNA, hydrolases, effector proteins, cellulase, and lipases through various secretion mechanisms.
Explain how nematodes penetrate plant cells.
Nematodes use needle-like structures called stylets, which puncture the cell wall using muscle force, and secrete enzymes and effectors.
What is the mode of entry for viruses into plant cells?
Viruses rely on damage to host defense or transmission via invertebrates like nematodes, insects, or fungi. Once inside, they move through plasmodesmata and the phloem.
Provide an example of a chemical barrier and its role.
Avenacin is a constitutive, preformed antimicrobial barrier. Some wheat G. graminis species secrete avenicinase to overcome its function.
What is flg22, and how does it trigger plant immunity?
- flg22 is a peptide in flagellin recognized as a pathogen-associated molecular pattern (PAMP).
- Detection by the receptor-like protein kinase FLS2 triggers PAMP-triggered immunity (PTI) through a kinase signaling cascade.
- Formation of a complex with FLS2 and BAK1
What are the characteristics of PAMPs?
- Highly conserved
- Essential to the pathogen’s life, and include substances like chitin, heptaglucan, and pheromones.
How do pathogens avoid PTI, and provide examples?
- Blocking signaling (e.g., AvrPTO)
- Preventing PAMP detection (e.g. LysM in fungi, greater affinity for PAMP)
- Inhibiting PAMP creation (e.g., AVR4 preventing breakdown of chitin).
What is Effector Triggered Immunity (ETI)?
Caused by effector proteins secreted into host plant cells by pathogens.
It serves as a second layer of protection which is stronger and more effective compared to pathogen triggered immunity (PTI), the combination of these two types of immunity creates a zig zag scheme
How does ETI differ from PTI (PAMP Triggered Immunity)?
- ETI is a larger response compared to PTI
- Only triggered when intracellular receptors, such as NLRs, bind and detect effectors, indicating the pathogen has breached physical defenses.
What are NBS-LRRs (NLRs), and how do they differ from PTI receptors like RLKs?
NBS-LRRs, or NLRs, are intracellular receptors
Have additional domains
Both have Leucine-rich repeats (LRR), making them variable for protection against different effectors.
Explain convergent evolution in the context of plant and animal immune responses.
= Unrelated organisms, indepently evolve similar mechanisms in response to similar environments
1. Both plants and animals detect PAMPs and effectors entering the cell
2. Recognized by NLRs with similar structures,
3. Activating the resistosome in plants and inflammasome in humans.
What is race-specific resistance, and what is its significance?
Race-specific resistance is coded by a dominant resistance gene, controlling susceptibility and resistance against pathogens.
The shape of the receptor is crucial, especially in direct recognition.
Differentiate between direct and indirect recognition in ETI.
Direct recognition involves detecting and binding to the effector protein, requiring a wide variety of receptors.
Indirect recognition involves binding to the guard model, which is then bound to the effector protein, providing a more efficient but less specific response.
What is the role of the guard model in indirect recognition?
Host protein acts as a guardee, changes to the guardee trigger the R protein.
Providing resistance against a greater variety of effector proteins.
Explain negative regulation and cooperation in the context of sensor and helper proteins.
Negative regulation involves a bound sensor and helper proteins, with inhibition removed upon binding of effector
Cooperation involves the binding and execution of the response upon sensing.
List some responses activated after the recognition of pathogens in ETI.
- antimicrobial molecules
- activation of defense protein genes,
- alterations to the cell wall
- reactive oxygen species
- hypersensitive response (HR)
- systemic acquired resistance (SAR).
What is Systemic Acquired Resistance (SAR), and how is it signaled?
heightened triggered immunity in non-inoculated leaves, signaled by Salicylic acid (SA).
Describe the role of Pathogenesis Related Proteins (PR) in 1° and 2° leaves.
1° leaves initially infected have a greater concentration of PR proteins.
2° leaves, non-inoculated, also increase in PR proteins, but the response is more delayed.
Showing SAR
What is the role of WRKY transcription factors in defense gene expression?
WRKY transcription factors, activated by MAPK, increase resistance to stress by amplifying the signal and activating various defense genes.
What is the mechanism of function of NPR1?
NPR1 is a master regulator of defense gene expression.
- SA signalling for SAR
1. Oligomer, broken to monomer
2. activates TAG TF
3. Moves to nucleus increases R gene transcription
What is active and passive resistance?
Active resistance- plant produces a defence e.g cell wall or pesticide encoded in an R gene
Passive resistance- when plant lacks a susceptibility gene
What are the positives and negatives of conventional plant breeding?
Positives- Doesn’t require knowing exact location, conventional
Negatives- Dependent on having resistance crop variety, difficult if multiple genes are involved, requires resistance testing + Linkage drag (bring along other genes)
What is marker assisted selection?
- Use a DNA marker that is polymorphic
- Located close to resistance gene, unlikely to be split apart
- Reliable, simpler than phenotypic screening
What is conventional breeding?
- Desired characteristics x resistance plant
- F1 is heterozygous
- Backcrossed with desired characteristics
What are transgenics and cisgenics?
Cisgenesis- transfer between hybridisable species
Transgenesis- transfer between non-hybridisable species
How can R genes be identified?
Renseq
1. Extract DNA and form a library
2. Find baits that code for NLRs
3. Introduce the oligonucleotides from the NLRs
4. R genes in the library will hybridize with some mismatch
5. Use markers on NLRs to quantify
How can overexpression lead to plant immunity?
Overexpression of resistance genes leads to genetic priming
- Primes immune response so that it’s more rapid
e.g. NPR1 response activated by SA signalling
What are the two main categories for gene editing to improve resistance?
Susceptibility knock out
- Reduces susceptibility to disease
Negative regulators of immunity knock out
- Remove the negative regulators that down-regulate the immune response
How is genetic editing commonly achieved?
CRISPR/Cas9
- target dsDNA
- cause a break in the DNA
- insert new sequence or just knockout gene
Describe the example of gene editing of TAL
Natural mutation
- Mutation of the TAL promoter binding area
- Transfer of TAL binding element to R gene instead of S gene
Gene editing
- Move TAL promoters to code for R genes
- Use TAL to transcribe small interfering RNA which inhibits S gene activation
- Insert a TAL to interfere with another TAL encoding an S gene
How does varying durability of disease develop?
Low durability- single dominant R gene production, which pathogen can easily develop resistance against
High durability- recessive mutation e.g. mutation in the TAL promoter preventing binding of TAL
How can defence priming be generated by the use of chemicals?
Spray chemicals onto plants, priming their defence
- Defence can also be passed on, however it doesn’t last forever
How is ETI signalled? Give examples of ETI
Signal
- SA for SAR
- ROS production
- MAPK cascade
Examples
- Detect AvrPto by Pto gene in tomato
- Detecte AvrB by RPM1 gene in A.thaliana
How is RNA silencing achieved?
By use of siRNAs
1. Single stranded viral RNA
2. Converted to a dsRNA
3. Diced up by dicer into small interfering RNAs
4. siRNAs binds to argonaute
5. Double stranded segments dissociates and one is degraded.
6. Other siRNA guides argonaute to cleave viral RNA
What are the components of the argonaute slicer?
- sRNA binding domain, to bind the siRNA
- RNAase H domain that cleaves viral genome
What evidence is there for RNA silencing in transgenic plants?
- Integration of transgene with GFP as a marker
- Green flourescence seen under UV
- When transgene is detected as foreign then silencing occurs
- Gene no longer expressed GFP, so fluoresces red
- Also with grafting one plant to another that doesn’t showing spread of RNAi
What are the positives of RNA silencing?
- Sequences specific, derived from viral RNA
- Amplifiable, dicing leads to many catalytic siRNAs
- Mobile, able to move in the plant and stay ahead of virus
How can the virus evade from RNA silencing?
- Rapid movement
- Produces suppressor proteins that block Argonaute
e.g. P19 from the Carnation Italian Ringspot virus, which prevents binding to dsRNA
What are miRNAs?
MicroRNAs that bind to RNA and cause the suppression of certain proteins, such as NLRs.
- Can be used to generate parasite resistance
What is an example of miRNA suppression in NLRs?
Suppression of NLRs e.g. Phytophthora infestans
1. Pathogen infect and suppress miRNA, which is suppressing NLR
2. This leads to NLR being activated instead of suppressed
How can RNA silencing be implemented in nematodes?
Introducing RNA silencing genes into plants that are taken up by the nematode when they attack
e.g. targeting HYP effector that suppresses the plant defence mechanism
- However not effective as resistance can be generated
How can RNA silencing occur in response to fungi and how can this be overcome?
RNA silencing of Avr10 effector
Response
- Insertion of transposed elements that bind to argonautes preventing RNA silencing
What are the positives and negatives of using RNA as a pesticide?
Positives
- Easy application
- Specific, targets certain organisms
- Low toxicity to humans
Negatives
- Difficult to control uptake
- RNA prone to degradation by sunlight
- Expensive
What is the phyllosphere and Rhizosphere?
Phyllosphere- aerial part of the plant
Rhizosphere - area around the root
What nutrients are in rhizosphere?
Photosynthate - 40%
Amino acids
Species richness 30,000
What are the effects of microbes on plants, focus on symbiosis?
Symbiosis: intimate living relationship between different species, can be described as a continuum
Parasitism - One partner benefits at the detriment to another
Commensalism - one partner benefits at no cost to the other
Mutualism - both individuals benefit
What are the different exchanges in mutualistic symbiosis? and give examples
1 .Exchange of services
- Shelter (acacia) for alkaloid (ants)
2. Resources for services
- Pollination
3. Resources for resources
- AM symbiosis
Give two examples of exchange of resources for service
- Pollination
- Plant provides food
- Insect disperses pollen - Endosymbiotic fungi (Epichloe festucae)
- Plant provides food
- Fungi produces alkaloids (protect from insects)
Give two examples for resource- resource exchange
- AM interactions
- Plant shelter and food
- Fungi inorganics (phosphate) - Rhizobia interaction
- Plant shelter and food
- Bacteria provides ammonium
How does the Epichloe festucae (fungus) show a continuum from mutualism to parasitism?
Beneficial (Asexual)
- Grows intercellularly and at the same rate as the plant
- Generates drought tolerance for the plant
- Secretes insect and mammalian toxins, alkaloids against herbivory
Parasitic (Sexual)
- Asynchronous growth with the plant
- Inflorescence (flowering) is suppressed, Grass choke disease, where the fungi form spores in inflorescence
- Secretes alkaloids that attract insects to aid its own spread
What are fungal factors for mutualism?
- Single gene alteration can lead to relationship turning parasitic
- MAP kinase SakA responsible for ROS production, which is for communication
- Mutation leads to rapid proliferation
How has the development of an infection thread by a rhizobia evolved?
- Used to weave inbetween cells to reach primordia layer
- Then carried out crack entry, which includes breaking the plant cell barrier
- Developed an infection thread which is generated by a plant but hijacked by the bacteria
Nodulation symbiosis evolved 90-Mya
What is the dose response relationship for symbiotic reactions?
Mutualism occurs when the benefit outweighs the cost
Parasitism occurs when the costs outweigh the benefits
What are the taxa of bacteria that carry out N symbiosis and what plants do they target?
Fabales- Rhizobia
Frankia
- Fagales
- Cucurbitales
- Rosales
What are the signalling steps in Rhizobia symbiosis?
- Plant secretes flavonoids when N deficient
- Rhizobia perceives the flavonoids and secretes NOD factors (MAMPs) that are lipo-chitooligosaccharides
- Plant detection of NOD factors leads to increase in Ca and noduel formation
Describe the structure of the nodule formed by Frankia symbiosis
Vasculature is found in the central
Because intracellullar infections occurs when a pre-nodule has already formed
Describe the different nodule structures formed by Rhizobia, comparing between determinate and indeterminate
Vasculature is found peripherally
Determinate - no meristem retained
Indeterminate - meristem is maintained
Describe the steps in root hair formation around rhizobia
- Plant secretes lectin
- Root hair grows an apoplastic envelope and entraps the bacteria
- Cytoplasmic bridge forms moving bacteria to the base of the root hair
- Root hair structure begins to curl around
How has forward genetics been used to find genes regulating nodule formation?
Legume models Medicago truncatula and Lotus japonicus
1. Mutate the genes by radiation or transposon insertion
2. Analyse response to N deficiency
= Results
- Mutation in NFR1 and NFR5 which are NOD-factor receptors, leads to impaired symbiosis
- nin (nodule inception) encodes TF for cell division, arrests at rhizobia recognition
- rpg (rhizobium- directed polar growth) encodes coiled protein for protein-protein interaction, arrests in rhizobia recognition
How does the nodule primordia form?
- Requires LHK1 (lotus histidine kinase receptor) which binds to cytokinin
- Binding causes Ca release and increase in signalling
- Causes a local increase in cytokinin
= Mutant of lhk1, forms infection thread but no primordia
What systemic regulation is there for nodule symbiosis?
Positive
- CEP1 peptide produced when N is low
– Detected by CRA2 receptors in the shoot
— Increases engagement with rhizobia
Negative
- CLE13 peptide released when N is available
– SUNN receptor in the shoot
— Suppresses nodulation by secreting micro RNAs
Describe the evidence for different theories of nodulation symbiosis evolution
Multiple origin hypothesis
- Mutation occurred in a clade that rendered it pre-disposed to nodulation
Evidence against
- Phylogenomics suggests there has been a loss of symbiosis ability not gain
- No clear gene that shows pre-disposition
- Potentially used to to be more common however has been lost due to its high cost
How old is the AM symbiosis relationship and what percentage of plants form these relationships?
470Mya in the mid-Ordovician
80% of terrestrial plant species form these relationships
- Occurred when roots hadn’t developed yet due to transition from water to land
- 23-37% yield increase in a variety of plants onion, parsley, cress etc. (Ortas et al.,2019)
What evidence is there for the evolution of AM symbiosis?
- Fossil records
- Found in Rhynie chert - DNA sequencing
What are the characteristics of glomeromycotina AM symbiosis?
- No host specificity
- Obligate biotrophs (cannot be grown solely in lab)
- Asexual life cycle
- Aseptate
What are the stages of AM symbiosis?
- Pre-symbiotic talk
- Root penetration
- Arbuscule formation
- Vesicle and spore formation
What occurs in pre-symbiotic cross talk?
- Plant secretes strigolactone, with a steep gradient being formed
- Fungi detect signal and secretes chitin based signals (e.g. lipo-chitooligosaccharides)
- Plant sense chitin based signals by LysM receptors
- Cause calcium spike in root epidermal cells
Evidence: a membrane was introduced which only allowed strigolactone through which allowed hyphal branching
What occurs in arbuscule formation?
- Highly branched tree-like structure
- Fill up most of the plant cell
- Don’t penetrate the cell membrane
= Able to recover to initial structure quickly
How is vesicle and spore formation triggered?
- Movement of FA triggers their formation
Evidence: mutants in FA transporters or RAM1 (TF in FA biosynthesis) - Leads to smaller arbuscular structures
What is forward genetics?
Begins with phenotype and aim to identify the genotype changes
- Phenotypes have to be unambiguous
e.g. mutant screens
What is reverse genetics?
Begins with genotype and see different phenotypes
- May require CRISPR-cas 9, or doing gene knockouts
How has transcriptomics been used to identify normal plant response to pathogens?
Transcriptosome- includes the whole RNA set expressed
- Monitor expression of certain RNA and secreted response proteins
e.g.
1. Entry causes release of Jasmonic acid isoleucine (JA-Ile)
2. JA-Ile sensed by COI1
3. COI1 binds to JAZ6 and ubiquitinates it
4. JAZ6 is broken down and no longer inhibiting MYC2 (a pathogen response protein)
Laccaria bicolor, produces MiSSP7 that inhibits JAZ6 so not broken down and allows infection
What is phylogenomics?
Analysis of evolutionary relations using genomic data analysis, for example Next-Gen sequencing
What is the common signalling pathway in symbiosis interactions?
- Detection of NOD or Myc factor
- CASTOR/ POLLUX proteins cause an influx of calcium
- CYCLOPS (Rhizobia only) - TF that decodes the Ca2+ spike causing downstream signalling
- NSP2 is downstream of CYCLOPS and reprogramms plant cell for symbiosis establishment
- nin, and rgp for nodulation or GRAS TF complex for arbuscules
What is the evidence for the pathways being similar in both symbiotic pathways?
Mutation in Common Symbiosis Signalling Pathway ( CSSP) altered ability
- e.g. SYMRK and CCaMK (Ca/ Calmodulin dependent protein kinase) and CYCLOPS
What are the similarities and differences between nodule and AM symbiosis?
Similarities
- Both release lipo-chitooligosaccharides (LCOs)
- Signal is perceived by LysM, containing RLK
- Activation of SYMRK (symbiosis receptor-like kinases)
- Activation of the MAPK cascade
Differences
- Nodule formation forms a whole other organ
- Nodule for fixing nitrogen, arbuscule mainly for uptake and more so phosphate than nitrate
- Bacteria in nodule, while a fungi for arbuscule
What impact does the discovery of the symbiosis pathway have on science?
- Impact of phosphate uptake
- Increase in symbiosis by mutating SMAX1 that can be ubiquitinated by a complex of D14L and Fbox - Impact on nitrate uptake
- Research into the development of nodule formation and symbiosis in crop plants also
= Reduce the use of fertilisers
What are the 3 conditions for an epidemic? (Epidemic triangle)
- Large number of susceptible hosts
- Suitably virulent pathogen
- Favourable environment
What are the 3 susceptibility factors?
- Host factors
- resistance/ susceptibility genes
- Genetic uniformity - Pathogen factors
- Level of virulence
- Amount and position of inoculum
- Life cycle and timing
- Mode of spread - Environmental factors
- Temp and moisture
- Other microorganisms (competition)
- Effects of human activity
What are the different types of cycles in epidemiology?
Life cycle- stages of pathogen development
Epidemiological cycle - stages that are relevant to infection and spread of the pathogen
Infection cycle- Stages of invasion, incubation, replication, symptom development and transmission
What is the reproductive number?
Parameter that quantifies the transmission potential of a pathogen
Representing how many secondary infections are generated from a single infectious agent
What is an example of roguing/ culling to control the spread of a pathogen?
Citrus Canker caused by Xanthomonas axonopodis (bacterium)
- occurred in Florida
Doesn’t kill trees so would eventually lead to all trees becoming infected, so roguing to kill
- Culling not effective as radii changed to drastically and many farmers didn’t agree (from 38m to 580m)
What case study involves Rhizomania and its infection of sugar beet?
- Causes Beet necrotic yellow vein virus
- Can be picked up by machinery
- Caused 80% yield loss in the UK
- Cause cryptic infections, can pass virus without showing symptoms
Treatment - Stewardship scheme - Farmers able to sell off crops if they think it’s infected
- Annual surveys
What is the sudden oak death case study?
Caused by oomycete Phythophthora ramorum
- Different effects on different species, oak causes death within years, Bay laurel survives indefinitely
- Host distribution, Environmental drivers (water runoff), Pathogen dispersal (estimated with Markov Chain Monte Carlo (MCMC))
- Control is difficult and expensive
How is plant development different to animal development?
- Determinate/ Indeterminate
- Plants have a mix of both, determinate (flowers), indeterminate (how many branches) - No unique parts in plants
- Due to sessile nature, no central point of control - Plant development is continuous
- Occurs throughout life - Sensitive to the environment
- No cell rearrangement
What is the structure of the shoot apical meristem (SAM)?
Central zone- small group of slowly diving cell
Peripheral zone - give rise to cells that contribute to the organs of the plant
Rib zone - give rise to a file of cells, that later become ground meristem
L1 = differentiates into the epidermis
L2 = differentiates into mesophyll
L3 = differentiates into the vasculature
What factors maintain stem cell identity?
Wuschel (wus)
- TF expressed at the centre of the SAM
Clavata (clv)
- Peptides that suppress the activity of wus
- clv1 and clv2 encode receptor kinases, clv3 is a mobile peptide
How can the movement of Wus be detected?
By GFP tagging
- Block the plasmodesmata by increasing callose production
- This leads to GFP being concentrated and so evidence for transport via plasmodesmata
How does CLV3 move in plants?
Movement is apoplastic
- Labeling clv3 shows location outside in-between cells
What is the role of shoot meristemless (STM) on WUS and CLV3?
TF that prevents cells from gaining organ identity
- similar to WUS but effects a larger area
- Activates CLV3 (negative regulator of stem cell proliferation)
Evidence: stm mutant can’t maintain a meristem
What is phyllotaxis and what is Hofmeister’s model?
Arrangement of organs around the meristem
Hofmeister’s model
- new organs are inhibited from forming due to existing organs, however organs are able to develop further away due to weaker inhibition
What is the role of auxin?
Lack of auxin leads to lack of auxin depletion
- Movement is via PIN proteins, mutants lead to the development of a pin shape
1. Pin movement re-inforces the maxima
2. Spacing is usually with a golden angle of 137°
How is floral formation co-ordinated?
Different combinations and suppression of different TFs cause different structures
1. A class TF (APETALA1/P1)
- Expressed in first whorls, promoting formation of septals and petals
2. B class TF (APETALA3/ AP3)
- Expressed in second and third whorl, required for stamen
3. C class TF (AGAMOUS)
- Expressed in fourth whorl, differentiates into carpel
= Triplet mutant forms leaves
Describe the primary root structure
Root cap- protective layer that is responsible for sensing gravity
Root apical meristem (RAM)- generates slowly dividing cells
Division zone- division occurs more rapidly
Elongation zone - Cells elongate and stop dividing
Maturation/ differentiation - cells acquire different characteristics, lateral roots begin to form
Describe the structure of the root apical meristem (RAM)
Quiescent zone- in the middle of the RAM, where cells divide slowly an maintain stem cell population
Transition zone - located above the meristematic zone, division occurs rapidly
Elongation zone- elongate rapidly
Maturation zone- at the proximal end of the root, become specialised
How is the identity of the cells in the root controlled?
Dependent on the transcription factors Short Root (SHR) and Scarecrow (SCR)
1. Movement of SHR from the stele (vasculature) to the endodermis or cortex via the plasmodesmata
2. In the endodermis SHR forms a complex with SCR
3. Activate transcription of genes, controlling differentiation of cells
e.g. development of Casparian strip
What controls the continuous root production?
Auxin- maintains meristem
Cytokinin - initiates cell differentiation
How is the movement of auxin controlled?
- Auxin freely enters in via AUX1
- Deprotonated so cannot freely exit
- Moved out by PIN proteins
What is the reverse fountain model for auxin movement?
PIN1 localised on the basal membrane, promotes downward movement of auxin
PIN4 and PIN7 for lateral movement and regulate vascular development
PIN2 moves upwards and regulates elongation and differentiation
How is root length controlled?
Cytokinin promotes cell differentiation = shorter root
Auxin opposite = longer root
1. Ck upregulates expression of short hypocotyl 2 (SHY2)
2. SHY2 disrupts PIN proteins
3. Preventing auxin maxima formation
4. Prevents elongation
Evidence: application of cytokinin leads to smaller root, as well as mutation in CK receptor ahk3-3
How is lateral root formation regulated?
Formed from the pericycle, forming a lateral root primordium
@low N - NRT1.1 transports auxin away
@high N- NRT1.1 transports nitrate, auxin accumulates and forms lateral root
How is gravitropism detected in roots?
- Movement of statoliths (amyloplasts) in the root cap
- Sediment due to gravity act as a signal
- Causes polarisation
- PIN3 receptors relocalise and move auxin
How does lateral root gravitropism occur?
- Grow at a set angle of 30°
- Gravity is still sensed by statocytes
- Caused by relocalisation of auxin, mutants don’t grow lateral roots
What occurs to the roots in different Pi environments?
Low pi
- many lateral roots form
- meristem is not maintained
- short primary root
High Pi
- long primary root
- meristem is maintained
What is the role of IAA (auxin)?
- IAA binds to TIR protein
- Inhibits a TF and triggers ubiquitination
- Degradation leaves ARF free to transcribe genome
How is shoot branching controlled?
Depends on the axillary bud, however apical dominance is present to control that
1. Auxin is transported from the newly developed leaves to the roots
2. Establishes a stream that bypasses the axillary bud
Evidence: removal of the tip - lack of auxin stream can cause branching, purposeful application of auxin
What is the role of strigolactone in lateral shoot formation?
- Causes PIN1 depletion
- Reduced auxin flow, meaning less auxin in the axillary buds so less branching
Evidence: mutants in strigolactone synthesis show high degree of branching
What are the three key structured on the leaf epidermis?
- Pavement cells
- forming impermeable barrier - Stomata
- control gas exchange and water loss - Trichomes
- protect against herbivores, especially important in early life
What are the steps in the development of stomata? (5)
- Protedermal
- Meristemoid mother cells (MMCs)
- Meristemoid
- Guard mother cell
- Guard cell
What are the 3 main regulators of stomata development?
- Speechless
- causes conversion from MMCs to meristemoid
Mutant= lack of stomata, synchronous division - Mute
- causes conversion from meristemoid to GMCs
Mutant = asynchronous division by no stomata - FAMA
- causes conversion from GMCs to GCs, ensuring only one division
Mutant = many divisions occur, no clear stomata in the guard cell
What controls speechless (SPCH)?
YODA, downregulates speechless
- Important in regulating stomata density
Mutant = many stomata, small green plant
What other factors interact with SPCH, MUTE and FAMA?
SCRM1 and SCRM2
- Prevent excessive stomatal formation
- Partially redundant, knockout of one is still OK
Mutant = abnormal stomatal density and development
How is stomatal patterning generated?
BASL polarises the MMC during cell division
- BASL crescent generated on the opposite wall
- Causing asynchronous division
Where BASL is present, leads to stomata fate
Orientate in such a way that stomata are separated by at least one cell
Robinson et al., 2011
What is the role of diffusible peptide factors in the patterning of stomata?
Epidermal patterning factor 1/2, binding to ERECTA and TOO MANY MOUTHS (receptors)
EPF1
- Negatively regulates MUTE
- Mutant - leads to stomata closer together
- Present in late meristemoids and GMCs
EPF2
- Negatively regulates SPCH, inhibiting MMC generation
- Leads to less stomata
- Present in MMC and early meristemoid
MUTANT = lead to too many stomata
What is the role of stomagen?
Peptide hormone that promotes stomatal development
Triggers asymmetric divisions, by promoting SPCH
How do environmental factors regulate the formation of stomata?
- CO2
HIC (high carbon dioxide) leads to reduced stomatal density
- upregulates EPF2 which inhibits SPCH - Temperature
- High temp sensed by PIF4, which downregulates SPCH - Drought
- ABA released reducing expression of SPCH and MUTE
Describe the presence of stomatal diversity in monocots and dicots
Monocots
- Dumb-bell shaped guard cells
- Prominent subsidiary cells
Dicots
- Sausage shaped guard cells
Adaptations
- Some may have sunken stomata, or trichome protected stomata
How is stomatal patterning controlled in monocots?
- SPCH not as important
- Cell are organised in files (vertical lines), and some file are unable to develop into stomata
What industrial application does researching stomata have?
Crop improvement
- Developing more drought resistant crops by changing stomatal density and increasing water use efficiency
Also used to prevent flower and plants from wilting
What is the different in the characteristics of human selected plants and naturally selected plants?
Natural
- Maximise offspring
- Flexible growth habit
- Seed may be poisonous
- Seeds are dispersed
Human
- Maximise fruit/ desired part of the plant
- Less flexible growth habit
- Edible seeds
- Seeds retained
What is the difference between maize and teosinte?
Maize (undergone human selection)
- Larger and softer kernels
- More ears, giving rise to flowers
- Seeds mature more rapidly
Teosinte (undergone natural selection)
- Greater degree of branching, in an attempt to outshade neighbours
What TF controls branching in maize/ teosinte?
TB1 suppresses bud activation
In maize promoter sequence and expression is different, compared to teosinte
Mutant: looks like teosinte
What controls branching in Arabidopsis?
BRC1 and BRC2 expressed in the axillary meristem
- Reduced branching
- Upregulated when plants perceive crowding
What characteristic was promoted in the green revolution and what are the benefits?
Reduced height, by activating a dwarfing gene RHT
Advantages
- Reduced lodging
- More energy to seeds
- Less shading of each other and self
What hormone and proteins are involved in stem elongation?
Gibberellic acid (GA) promotes elongation
- Breaks down DELLA which is bound to a TF on an elongation gene
How is shade sensed and signalled?
Detect: greater ratio of far red light, compared to red light
Signal:
- Increase in GA signalling
- Upright growth (etiolation)
What is an example of a commercial increase in branching?
Citrus fruits
- More branches, more fruit
How?
- Increase in WUS, means axillary stems become branches instead of thorns
Give an example of an orphan crop and the characteristics targeted by human selection
Tef
- Feed 70% of people in Africa
- Long and seeds are small
- Drought and flood resistant
Target: Increase in seed size and decrease in height to reduce lodging
What mechanisms have been implemented to increase crop yield?
- Greater area available for growth
- Irrigation
- Pesticides
- Green revolution
- Fertilisers
What are first and second generation biofuels?
First - Produced from edible crops such
Second - produced from the waste, non-edible parts of the crops
What are difficulties associated with the use of biofuels?
- Efficiency of plant to convert light to biomass is low (overall 0.13%)
- Effect on food security
- Not enough space to be a good enough alternative
- Emissions are also released in transport, growth and other processes
What are other good alternative renewable energy sources?
- Solar
- Photovoltaic solar panels with efficiency of 20% - Hydro
- Wind
What is radiative forcing?
Balance between incoming solar radiation and the outgoing IR radiation
What cells govern the movement of water and heat on the Earth?
Hadley cell - air rises at the equator and condenses at 30° latitude, so rain is present in between
Ferrel cell - transports warm air to higher latitudes
How have humans altered the water cycle?
- Aquifer exploitation
- e.g. Saudi Arabia, Mexico city, California for almonds - Land-use change
- more surface run-off and change in residency time or ability to replenish underground stores - Over-exploitation
- Aral sea turned Aralkum desert due to irrigation by using river water - Global warming
- indirect effect, causing greater rates of evapotranspiration
How has the concentration of CO2 in the atmosphere changed, and what are greenhouse gases?
Pre-industrial 280ppm, currently 421ppm
Greenhouse gases- gases that trap heat as IR and UV, and re-emit IR back to the Earth
What anthropogenic actions have affected the carbon cycle?
- Land use change
- Increase in livestock farming, less trees and more release of CH4 - Burning of fossil fuels
- Release long term C stores (fossils) into the atmosphere
KEELING CURVE - measured increase since 1958
List the evidence that organ initiation at the shoot apical meristem is triggered by local auxin accumulation (5)
- Response to exogenous auxin application
- Auxin gradients
- Auxin transport mutants (PIN)
- Analysis of temporal correlation between changing conc and organ formation
- Hofmeister’s model of lateral organ formation and auxin maxima
What evidence is there for the ancestral age of AM symbiosis?
- Fossils in Rhynie chert from early Devonian
- Phylogenomics - branching and formation of glomeromycota clade occurred early
- Ecological considerations
- Signalling pathways are highly conserved in many land plants (CYCLOPS and SYMRK)
How can microbial interactions between plants and microbes help plants respond to environmental stresses?
- Increase in drought tolerance, fungal hyphae can access water and generates a greater SA
- Endophytic bacteria and AMF secrete osmoprotectants that help plants develep stress response and control osmotic adjustment
- Interaction can lead to production of osmolytes and antioxidants in preparation for stress. Can help remove ROS and stabilise membranes
Describe an experimental design to identify plant genes required for both root nodulation and arbuscular mycorrhizal symbiosis
- Requires a model plant, such as Medicago truncatula
1. forward genetics where mutants are formed and their phenotypes analysed, to locate which genes regulate symbiotic interactions
-Mutations in LysM and NOD factor receptors, led to lack of signal perception and lack of symbiosis
2. Genetically map them to identify the mutated gene
3. Quantify mutants by looking at number or morphology of primordia formation or arbuscule intensity - e.g. discovery of CYCLOPS in M.truncatula by lack of formation of formation of nodules
4. Confirm genes by reverse genetics, causing knockouts by CRISPR/ Cas9, or complementation assays
What is the role of TB1?
- Teosinte branched 1 gene
- Mutant shows greater branching like teosinte
- Important as part of the green revolution
- TB1 also controls height of plant (Dixon et al,.2020)
What are the similarities and differences between NLRs and PRRs?
Similarities
- Both play a role in generating plant immunity
- Initiate defence responses, by triggering signalling cascades
Differences
- PRRs have a broader specificity, NLRs recognise more specific patterns. Can bind directly or indirectly
- PRRs are located on the cell surface or on endosomes, NLRs are cytoplasmic
- PRRs are constitutively active and rapidly activate, NLRs are inactive and activated when effector binds
- PRR occurs in early stage, more wide spread. NLRs more localised occurs later in infection
What is cryptic infection and how can it affect the control of disease?
-Cryptic infection is when a plant is infected, not showing symptoms but still able to transmit disease
- Makes controlling the disease harder because by the time control has been put in place the disease has already spread
- Makes culling and roguing less effective
- Requires integrated management such as sanitation, crop rotation and removal
Summarise the relative advantages and disadvantages of using SI and SIR models to analyse plant disease epidemics.
- SI and SIR are simple models
- Both useful to study the early stages of disease dynamics
- SI doesn’t consider recovery or death of plants
- SI assumes constant population size
-SIR includes recovery and the effect of control strategies - SIR assumes a constant homogeneous mix and constant transmission
- SIR is more complex than SI
What strategies can be implemented to better control the spread of diseases in plants? And what are their limitations
- Cull more trees (Greater radius)
- Look for infections more frequently
- More accurate detection of disease
- Decrease time between disease noticing and culling
Limitations - Ethically - public objection
- Money
- Increasing detection accuracy is difficult
Discuss with at least two examples the role of mobile factors in plant development
Auxin - promotes cell elongation, signals apical dominance, transported by PIN transporters. Involved in differentiation of the xylem and phloem
Gibberellins (GA)= regulates stem elongation, diffuses freely and act locally, promotes cell elongation and division
What are the similarities between rhizobia and AM symbiosis signalling?
- Initiation by plants AM = strigolactones, Rhizboia = flavonoids
- Secretion of LCO by microbes, AM = chitin, Rhizobia = Nod factors
- Detected by LysM receptors
- Ca2+ spike
- Increase expression of symbiosis genes
What is the stele?
Vascular tissue in the root
Contains the pericycle, phloem and xylem
What is the casparian strip?
Impermeable, hydrophobic material found in the cell walls of endodermal cells in plant roots.
- Made of suberin.
- Regulates the movement of water and solutes into the vascular system of plants
Describe an experimental design to identify plant genes required for both root nodulation and arbuscular mycorrhizal symbiosis.
- Mutagenesis screens - generating mutations and looking at the phenotype can be done by CRISPR (R)
- Use Medicago truncatula that can form root nodulation and AM
- RNA silencing - insert RNA constructs to see if certain proteins and RNA are important
- Also require phenotypic screens measuring root number, Arbuscule formation etc.
Describe three reporter genes commonly used in plant biology research.
- GFP
- Accurate, temporal resolution
- Can undergo photobleaching
- Seen using fluorescence microscope - GUS
- Easy to do but plant has to be dead
- Seen with light microscope - Luciferase
- Can be transiently expressed with diurnal cycles, high sensitivity
- Requires specific substrate luciferin
- Seen using a luminometer
Briefly describe, with evidence, how development of the Arabidopsis flower meristem terminates?
- Terminal flower 1 (TFL1) - prolongs meristem by suppressing flowering
- Mutant tfl1 flower early and lose the meristem quickly
- Cytokinin – promote meristem activity and delay flowering
- GA and Auxin – promote flowering
Give two examples of PTI
- Detection of flg22 by FLS2 which associates with BAK1
- Detection of chitin by CERK1 triggering kinase cascade, increase in chitinases
Give 3 examples of extremophiles
- Thermus aquaticus
- adapted to high temps
- DNA repair enzymes, Taq polymerase, heat stable proteins - Ferroplasma acidiphilum
- acidic conditions
- proton pumps, DNA repair enzymes - Photobacteria profundum
- deep sea, high pressure
- cavities in proteins to facilitate compression, more unsaturated FA to maintain membrane fluidity
Discuss with at least two examples the role of mobile factors in plant development.
Gibberellic acid (GA)
- Promotes growth, elongation and division
- Inhibits DELLA, which inhibits growth
Auxin
- Promotes growth
- Regulated by PIN proteins, and NRT1.1
- Important in apical dominance
- Response to gravitropism
When might roguing be an effective disease control strategy, and when might it fail?
Effective
- Localised area
- Clear pathology
- Specific to a species
Fail
- Cryptic infections
- Subtle symptoms
- Soil-borne pathogen
- Large area of land or transmission distance
How can RNA silencing be used to protect plants against pests and disease?
- Target essential genes in pathogens
e.g. Avr10 in fungi - Target specific pathogens
- As a spray or get plant to synthesise
- Common against viruses
Briefly describe three ways in which plants use proteins to help them cope with extremes of temperature.
- Heat shock proteins
- E.g. HSP70 and 90
- Chaperone proteins that help with folding
- activated by σ32 - Antifreeze proteins
- Bind to ice crystals prevent their growth - Osmoprotectants
- Compatible solutes such as mannitol and proline
What control the balance between mutualism and parasitism?
- Balance of signals
e.g. phytohormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene - Genetic studies have analysed the importance of genes in this network
- Infection with mutant strains influence stability
How do plants ensure that stomata are always spaced by at least one cell?
- Transcriptional factors
- SPCH, MUTE and FAMA - Patterning factors
- SCRM 1 and 2, promote stomata formation - Polarisation of cells
- BASL - EPF2 (inhibit SPCH) and EPF1 (inhibits MUTE)
Why is transpiration important for the global water cycle?
- Climate - important as release of water vapour (greenhouse gas)
- Generation of precipitation
- Plant growth
- Recycling and removal from soil
- Hydraulic conductance inside the plant (generates -ve water potential)
Why do pathogen genomes harbour avirulence genes?
- Co-evolution with host
- Improves pathogen fitness
- Help maintain diversity
- Different Avr allows infection of different hosts, ecological niches
- Non-virulent if the plant has a corresponding R gene
Describe an experimental design to identify plant genes required for both root nodulation and arbuscular mycorrhizal symbiosis
- Use M.truncatula or Lotus japonicus
- Use control, AM only and rhizobia treated and dual treated
- Grow in controlled conditions
- At certain times along development carry out RNA extraction and quantitative PCR
- Find what genes are being expressed, which ones vary
- Validate using targeted knockouts e.g. CRISPR or RNAi
Briefly describe, with evidence, how development of the Arabidopsis flower meristem terminates?
- Terminal flower 1 (TFL1) - lack of or mutation leads to flower formation
- Agamous (AG) - causes termination of FM, ag leads to loss of ability
- Requires high levels and central expression (Prunet et al., 2009) - Cytokinins upregulation and auxin downregulation
Give five examples of pathogen-derived effectors that block components of host plants’ defense systems.
- TAL effector - Xanthomonas, regulates transcription of R genes
- HopZ1 from P.syringae, inhibits MAP kinase cascades
- AvrPtoB, targets protein kinases involved in defense signaling pathways, such as FLS2
- Elicitins by Phytophthora species, alter lipid metabolism that alters the membrane
- ToxA – necrotic factor produced by some fungi
What are sub-genomic RNAs? Describe one mechanism by which they are generated.
- Early transcription termination strand
- Viral genes, overlapping reading frames
- DICER of viral genomes for siRNAs to be used as guides
Why is 70% ethanol added after X-Gluc staining?
To remove the chlorophyll
How can a dsRNA be formed for RNA silencing?
1) During viral replication dsRNA intermediate is made
2) Host dependent RNA pol
Describe the steps in the formation of the endodermis and the cortex
Longitudinal division
Short root (SHR) - TF for endodermis expression
Scarecrow (SCR) - maintains SHR to one side, so that other becomes the cortex
Describe the structure of SHR and SCR mutants
SHR - no endodermis is formed, only the cortex
SCR - mix of endodermis and cortex, due to lack of localisation of SHR by SCR
What is co-metabolism and why is it important in bioremediation?
- Bioremediation- remedying or reversing of pollutants in an area, by action of microorganisms
- Co-metabolism is the transformation of pollutants indirectly while using another growth substrate (primary sources for carbon).
- Production of by-products or enzymes, while using another carbon source, that then act to remove pollutants
- Bioremediation reduces toxicity of the land, so that other plants can grow on it
- Co-metabolism can be more efficient and cost-effective
Describe the similarities and differences between siRNAs and miRNAs
Similarities
- Short RNA strands (20-24bp)
- Complementary binding occurs to regulate gene expression
- Produced by DICER
- Associated with Argonaute
Differences
- Source of RNA strand (si exogenous template, mi endogenous template)
- siRNA more specific
- Many siRNAs arise from a precursor (diced)
How do fungi, bacteria, and viruses, influence or modify the environment?
- Carbon cycling by causing death of organsims
- Viral shunt in the oceans
- Mutualistic symbiosis, AM fungi and rhizobia bacteria
- Saprotrophic bacteria decompose dead matter
- Some extremophiles are bacteria, can survive in harsh conditions and help support life
What 2 genes are required for INFECTION THREAD formation?
nin - nodule inception
Mutant = recognition but no infection thread
rpg - rhizobium-directed polar growth
Mutant = defect in infection thread, encodes a coiled protein
What gene is required for NODULE formation?
hit1 (hyperinfected 1)
- histidine kinase receptor that responds to cytokinin to form nodule
Mutant = infection thread but no nodule
What are the benefits of using marker assisted selection?
- cheaper than growing fields
- can select for recessive phenotypes
- can be used to select multiple genes
- more efficient
Define latent and incubation period
Latent- period of time before becoming infectious
Incubation - period of time before becoming symptomatic
How do plants ensure proper spacing of stomata?
EPF1 and EPF2 regulate SPCH, MUTE and FAMA
- Act on receptors TMM and ERECTA
BASL - polarises cell, opposing end has stomatal fate, ensures 1 cell spacing
What are the various roles of cytokinin?
- Promotes shoot growth
- Delays senesence
- Promotes short root, by promoting differentiation
- Enhances drought tolerance
- Involved in signalling for nitrate and phosphate
- Signalling for rhizobia symbiosis, by detection by LHK1
How are extremophiles adapted to harsh environments?
- Heat
- Heat shock proteins E.coli
- DNA repair enzymes
- More copies of DNA
- Ether linked lipid membrane (archaea) - Pressure
- Cavities in proteins to allow compression (photobacteria profundum) - Acidic
- Transporters that move H+ out - Cold
- antifreeze proteins
How was NPR1 discovered?
- Overexpression in Arabidopsis and rice led to greater resistance
- BLAST sequencing showed evolutionary conservation - PR1 fused to GUS, mutated and analyse lack of GUS (reverse genetics) when infected
- Found a nonexpressor of PR1 (NPR1)
- Found that it was upstream and encoded PR1
- Cloned NPR1, introduced into mutant, regained SAR
Describe the degradation pathway for SMAX1
- Ligand binds to D14L
- Conformational change occurs
- D14L binds to Fbox and SMAX1
- Ubiquitinates SMAX1
- SMAX1 is degraded
How beneficial is AM symbiosis for P uptake in plants?
Around 70% of Pi is taken up by AM fungi e.g. in rice Yang et al., 2012
What evidence is there to reject the multiple origin hypothesis for FaFaCuRo and nodulation?
- Phylogenetic evidence - nodulating plants, more closely related than non-nodulating
- Comparative genomics- show a shared set of conserved genes encoding Nod factors
- Gene loss event- genomic studies into multiple gene loss events
- Molecular dating - divergence of FaFaCuRo and development of nodulation around the same time
What evidence is there for the common origin of AM and rhizobia symbiosis?
- Common symbiosis genes (SYMRK and CCaMK) Stracke et al., 2002
- Functional evidence (Ca2+ spike)
- Morphology studies
- Complementation (SYMRK from L.japonicus to M.truncatula)
Describe the difference between transcription in eukaryotes and prokaryotes (5)
- Location
E = Nucleus
P = cytoplasm - Enzymes
E = RNA pol I-III
P = single type of RNA pol - Promoter recognition
E = TATA box binding, TF bind to promoter
P = -10 and -35 elements in the promoter region - RNA processing
E = PolyA tail, 5- capping, splicing
P = Polycystronic (code multiple proteins) - Termination
E = cleavage and addition of PolyA tail
P = Rho in/dependent hairpin or Rho protein dissociation
