1
Q
What are 4 different methods of sequencing?
A
- Sanger sequencing
- incorporation of chain terminating DNTPs that tagged with 4 different colours - Oxford Nanopore
- DNA is passed through a small whole with a voltage detector to analyse the base type - Illumina (shotgun)
- generate fragments, sequence and then assemble into complete genome - RNA-seq
- converts RNA to cDNA and then uses illumina to sequence
2
Q
Describe the structure of cyanobacteria
A
- Contains PSI and PSII
- Flexible chemistry, links photosynthesis with respiration due to shared ATP synthase NADH etc.
- Found in oligotrophic conditions (deficiency of plant nutrients that is usually accompanied by an abundance of dissolved oxygen.)
- Contains carboxysomes that help concentrate CO2 for Rubisco
3
Q
What are archaeplastids and what are the 3 subgroups?
A
Archaeplastids - contain a primary plastid (chloroplast) due to primary endosymbiosis of a cyanobacterium
Glaucophytes - rare, chloroplasts contain peptidoglycan
Red algae - extermophiles, contains phycobilisomes
Green algae - from which land plants evolved from (embryophytes)
4
Q
What are the requirements for VitB12 by algae?
A
Algae commonly require VitB12 for methionine synthesis (an essential amino acid)
- Many algae are unable to produce their own VitB12, instead form symbiotic relationships with bacteria
- Some red algae e.g. nori, green algae and some species of diatoms, rely on EXTERNAL sources
- Potentially due to environment and availability that has led to evolution of VitB12 independent pathways, e.g. in the southern ocean of the Antarctic
5
Q
What is secondary endosymbiosis?
A
When a eukaryote engulfs another eukaryote, leading to plastids having more than 2 membranes surrounding them
6
Q
What species have secondary chloroplasts? (5)
A
- Diatoms
- Dinoflagellates
- Cryptomonads
- Haptopyhtes
- Apicomplexans
7
Q
Describe the structure of cryptomonads
A
- fresh and marine water algae
- Retains nucleomorph (reduced nucleus of red algae endosymbiont)
- Retains phycobilisomes
8
Q
Describe the structure of diatoms
A
- Large, live inside a silicon box
- DOMINATE nutrient rich envs, well adapted to Fe stress (due to siderophores, ferritin, and HA transporters)
- e.g. well adapted to upwellings
9
Q
Describe the structure of haptophytes
A
- Calcium shells (coccoliths) that form chalk and limestone
- Form algal blooms visible from space
- Constrained by viral infections
B.bigelowii evolved a nitrogen fixing organelle
10
Q
Describe the structure of dinoflagellates
A
- Photosynthetic and heterotrophic
- Chromosomes are constantly condensed, but not by histone proteins, instead by viral proteins
- Undergone serial endosymbiosis so contain chloroplasts of green and red algal originD
11
Q
Describe the structure of apicomplexans
A
- vestigial plastid that is no longer able to carry out photosynthesis (apicoplast)
- retains genome
e.g. Plasmodium falciparum
12
Q
What is the chromalveolate hypothesis? What are the issues with it?
A
- Suggests that there is a single common ancestor for secondary red algal endosymbiosis
- Many are no longer photosynthetic, oomycetes show no real evidence of ancient chloroplast in genome
- Cryptomonads more closely related to archaeplastids, which are distinct from the secondary red algae proving that there isn’t a common secondary red algal chloroplast conserved
13
Q
What may be the reason behind the green signals found in secondary red algal symbionts?
A
- Horizontal gene transfer
- Serial endosymbiosis
Signals present in diatoms, found red chloroplasts supported by proteins of green origin
14
Q
What is a mosaic plastid?
A
Plastid that has genes from a mix of sources as a result of multiple endosymbiosis events.
15
Q
Describe the structure of the bacterial genome
A
- Around 3000 genes
- Packaged into a nucleiod
- Operons that are co-transcribed by RNA pol
- Presence of TATAAT promoters and sigma factors
- Contain plasmids
16
Q
Describe the structure of chloroplast genomes
A
- Fewer genes min 12 in dino and max 250
- Contains operons like in bacteria
- Encode PS, large subunit of Rubisco and RNA Pol
17
Q
What are some examples of gene gain by horizontal gene transfer in algae?
A
- Rubisco in red algae, derived from proteobacteria has a greater specificity
- Dinoflagellates replaced their Rubisco with one from proteobacteria that has faster catalytic activity but lower specificity. And gained toxin production from cyanobacteria
18
Q
What are the similarities and differences between the chloroplast and bacterial genome?
A
Similarities
- Nucleoid structure
- Small
- Presence of operons
- Encoding RNA pol
Differences
- Nucleoid in chloroplast condensed by cp-proteins
- Chloroplast genome is smaller
19
Q
Where are chloroplast proteomes derived from?
A
- Nucleus and chloroplast genome
In land plants RNA pol is nuclear encoded
PS and associated proteins are encoded in the chloroplast genome
20
Q
How can chloroplast proteomes be identified?
A
- Mass spec - expensive, and sometimes hard to isolate only the chloroplast
- Fluorescence labelling - GFP tagging proteins and seeing them localised in the chloroplast
- N-terminal sequencing - analysis of N terminal to predict where the protein will locate to
21
Q
How can proteins be imported into the chloroplast?
A
- Hydrophilic N -terminal transit peptide
- Protein unfolds
- Moves through the TIC/TOC complex across the double membrane
- Refolded
- Transit peptide cleaved
22
Q
How are peptides moved across membranes of secondary chloroplasts?
A
- Requires movement across 4 membranes
1. N terminus recognised
2. Moved across the outermost membranes by sec61, then by SELMA (symbiotic-specific ERAD-Like machinery)
3. Then moved through the other two membranes by the TIC/TOC complex (Translocon on inner/outer chloroplast membrane) = innermost/original plastid membrane
23
Q
Why is there transfer of plastid genome to the nuclear genome?
A
- Protection from mutations
- Sexual recombination bias when found in the chloroplast
- Regulation of expression is better controlle in the nucleus
24
Q
Why does transfer of plastid genome to the nucleus stop?
A
- Gene transfer incomplete- perhaps it does all move to the nucleus, however, species that are unable to photosynthesis have also lost plastid genome so may be reason for the stop
- Proteins can’t be imported - some proteins if encoded in the nucleus may not be able to be imported due to characteristics
- Requirement for a rapid response - more rapid change can be carried out if proteins are encoded in the organelle instead of having the signal transduced and sent to the nucleus e.g. ROS stress in the chloroplast
- Co-location of Redox Response (CoRR), requirement for genes in redox reactions to be controlled, specific and rapidb
25
What evidence suggests that UCYN-A endosymbiosis has evolved into an organelle?
1. Combination with cell architecture - surrounded by mitochondria matrix providing energy
2. Synchronous in replication and division with the whole cell
3. Import of nuclear encoded proteins - shows integration e.g. HemeO required for heme catabolism for Fe acquisition, and flavodoxin a e- donor for nitrogenase (along with ferredoxin which is encoded in UCYN-A). 1/3 of UCYN-A proteome is nuclear encoded
Bacteria and Haptophyte
Coale et al., 2024
26
Describe the structure of the world oceans, including nutrients and stratifications
1. Nutrients are commonly found at the ocean floor, where dead organisms are found
2. Nutrient rich env are more commonly at the poles due to less stratification and greater mixing due to storm events
3. Greater stratification and lack of mixing is found in the tropics due to heating of the surface and it being less dense and remaining at the top
4. Fe- distribution and content is low in open oceans, and higher near the coast of desert areas
27
Describe the different stratification/ populations formed in the tropic and the arctic
Tropic - 2 populations, surface level where photic levels are high and the Deep Chlorophyll Maximum (DCM), where nutrient levels are higher but light levels lower
Arctic - less stratification of nutrient levels so not found in separate populations
28
Describe the distribution of microbial species in the oceans
Tropic - cyanobacteria, able to survive in low nutrient conditions
Poles - Eukaryotes (Haptophytes, dinos, diatoms)
- Spring more nutrient rich, and then decrease into autumn due to ice melt and dilution
29
Why is it difficult to sample and culture microbes found in open oceans?
- Bad weather and inaccessibility
- Used to low nutrient environments, and lab culture media has too much so 99% don't survive
30
What are different types of metagenomics?
1. Metabarcoding - amplify short regions of V9 region of 18SrRNA to identify species. However, abundance can be inaccurate
2. Transcriptomics - study of all RNA molecules
3. Single cell amplified genes
4. Metagenome assembled genomes - by binning look at similar gene abundance and group them due to co-ocurrence
31
Describe some outcomes of the Tara Oceans expedition
1. De vargas (2015) by MetB found that dinos are the most abundant. Aimed at analysing diversity in the photic zone
2. Ibarbalz (2019) found that the tropics show greatest biodiversity
3. Lima-Mendez (2015) showed that most, except diatoms, formed symbiotic relationships, and that there is a top-down biotic interaction between parasites/ phages and microbes
4. Ustick (2021) looked at what nutrients limited growth of algae by looking at changes in the genome of Prochlorococcus a phytoplankton that can rapidly adapt to different nutrient environments.
= Found more about abundance and diversity as well as overall distribution of organisms, used to predict future responses to climate change
32
Describe the interaction between the mitochondria and the chloroplast
- Mitochondria adapt following endosymbiosis to support the metabolism of the endosymbiont
- Evidence for this includes experiments in anoxic conditions where the algae where then exposed to dark, which led to hibernation and when exposed to light. Potentially due to the imbalances in ATP, which could be shared between chloroplast and mitochondria to fuel reactions
33
How might targeting of proteins to the mitochondria and chloroplast evolved?
- Potentially from antimicrobial peptides that commonly target microbes, and are a form of plant defence
- Potentially the endosymbiont was targeted initially and now adapted to use the mechanism to insert proteins
- Amphipathic α helices present to mediate insertion of the protein
- Modifications to the peptides showed that replacing lysine with arginine improves targeting. Arginine commonly found in N-terminal targeting peptides, while lys is found in AMPs (Caspari et al., 2023)
34
What are the differences between fungi and oomycete?
- Oomycetes are protists
- Oomycete cell wall component = beta glucans, and cellulose. While fungi have chitin
- Number and location of flagella differ, oomycete has 2 anterior
35
What are the 5 major phyla in fungi?
Chytridiomycota
Zygomycota
Mucuromycota
Ascomycota (septate + dimorphic)
Basidiomycota (septate + dimorphic)
36
What are the two different life cycles of fungi?
Asexual - form mitospores from hyphae
Sexual - form meiospores
37
What is plasmogamy and karyogamy?
Plasmogamy - fusion of cytoplasms
Karyogamy - fusion of nuclei
38
What does heterothallic and homothallic mean?
Heterothallic - self-incompatible, promotes outcrossing of compatible MAT
Homothallic - male and female reproductive structures on the same individual. self-compatible
39
Describe the life cycle of basidiomycota
Prolonged dikaryote phase, plasmogamy without karyogamy
- Karyogamy induced by environment
40
Describe the life cycle of ascomycota
Rapid proliferation and mitospore formation (conidiospores)
- Extra round of mitotic division leads to 8 meiospores instead of 4
- Turgor pressure generated to eject ascospores
41
Describe mating type switching in S.cerevisiae
Two mating types a and α
Hidden MAT left (HML) = α copy
Hidden MAT right (HMR) = a copy
1. Binding and unequal distribution of ASH1 results in
2. Repression of HO endonuclease transcription
3. In the other cell, HO cleaves DNA at the MAT locus
4. Repair of break utilises either HMR or HML (regulated by the recombination enhancer)
5. Resulting in the cell with low ASH1, undergoing MAT type switching
42
What experiments suggest that asexual fungi can reproduce sexually?
Aspergillus fumigatus
- Genome sequencing detected sex-related genes
- In vitro crossing of these fungi, was successful
43
Describe the two forms in which fungi exist?
Hyphae - Ancestral form, grow into a mycelium
Yeast - found in dikarya, Pseudohyphal growth in nutrient poor conditions. Divide by fission
44
How does hyphal growth occur?
Apical extension
1. Fusion of vesicles to the membrane allowing for change in hyphal shape
2. Requires a degree of turgor for directed change
- no evidence of change in CW thickness that contributes to polar growth, in some areas the tip was thicker. Chevalier et al., 2023
3. Increase in ROS prevent hyphal branching
Fungal wound healing *hyphae growth goes on- Gellin proteins, mechanosensitive Gii domain, in bursting hyphae, unfolds the domain (active once burst) binds/ holds the membranes together to form a mesh Nguyen et al.,
45
How is hyphal polarity generated?
Spitzenkorper - an area with high density of vesicles (dense reservoir)
- chitinases and glucanases to loosen cell wall, and secretory vesicles for digestion
46
Describe the modes of transport at the hyphal tip
Microtubules - Vesicle transport from ER and golgi towards the tip
F-actin - moves vesicles to the membrane
47
How do fungi obtain nutrients?
- External digestion, by secretion of enzymes
- Uptake of the nutrients at the subapical region
- Also release antibiotics subapically to prevent other microbes from accessing the nutrients
48
How do fungal enzymes demonstrate the remarkable diversity of fungi?
- White rot fungi with peroxidase enzymes, capable of breaking down lignin
- CAzymes for breaking down carbohydrates
49
Why do you think brown rot fungi lost the ability to degrade lignin while white rot fungi retained it?
- Energy intensive to produce ROS and maintain the enzymes
- Colonisation of different environments, nutrient rich (brown) and nutrient poor (white rot)
- Genomic streamlining, resulting in loss of gene
50
What are the diverse lifestyles of fungi on plants?
Biotrophy - requires a living host
Hemibiotroph - starts of biotrophically, then switches to necrotroph
Necrotroph - Kills the plant, to then feed off it
Saprotroph - feeds off already dead material
51
Describe the relationship of C.carbonum with plants
C.carbonum (ascomycote) produces HC-toxin
- This interferes with chromatin unbinding and growth of maize
Some maize plants have adapted Hm gene, that encodes a HC-toxin reductase
52
How have fungi affected ecosystems?
- Saprotroph playing key roles as decomposers as part of the carbon cycle
- White rot fungi, being able to breakdown lignin led to a change in the amount of lignin-generated carbon
- Important for EVOLUTION plant nutrition and development of roots (transition from water to land)
- As pathogens, regulating and changing the size of populations
53
What are the role of some effectors? Give some examples (5)
1. Suppress plant immunity
- MoAvr-Pita reduced COX expression on mitochondria reducing ROS production
- MoAvr-Pik targets HMA proteins (such as NLRs)
- P.sojae releases PsAVh52 that relocates an acetyltransferase to the nucleus where is acetylates a histone, upregulating genes that result in increased susceptibility (Li et al., 2018)
- PiAvr3a binds CMPG1 (E3 ligase required for cell death)
- PiAvrblb2 binds and prevents C14 protease release
2. Host nutrient reallocation
- ToxA initiating programmed cell death
- Psyringae HopM1 disrupts vesicle trafficking causing cell leakage
3. Modulate host gene expression and signalling
4. Alters host metabolism
- ACE1 from Magnaporthe orzyae alters lipid metabolism in rice to favour fungus
5. Promotes symbiosis
- MiSSP7 by ectomycorrhizal fungi
54
What promotes mobile pathogenicity chromosome spread in fungi?
Hyphal anastomosis
- As a form of horizontal gene transfer
e.g. of chr14 between Fusarium species
55
Describe the genomic phylostratigraphy of symbiosis-induced genes
74% of ectomycorrhizal-induced genes predated the symbiotic lifestyle
- Suggesting genes were co-opted for this function (Exaptation)
22% of novel genes evolved after symbiosis
15% of genes are shared by all species
31% are species-specific genes involved in symbiosis
56
What is an example of a gene that has been co-opted for symbiosis with fungi?
1. VAPYRIN
Symbiosis = vesicle trafficking to support development of arbuscules
Previous = endosomal, vesicular trafficking
2. PT11
Symbiosis = specific gene upregulated in rice, or MtPT4
Previous = PT transporters involved in direct uptake
3. Cell wall degrading enzymes - for symbiosis toned down to not cause too much damage but promote entry
4. Gene duplication of HYDROPHOBIN, repurposed for adhesion of appressorium in rice blast fungus
57
What's unique about the Glomeromycotina/ AMF?
- Fatty acid auxotrophs, due to the lack of fatty acid synthesis gene
- Requires association with plants to complete their life cycle
58
What do AMF usually use for nutrient storage? Give an exceptional case
- Commonly use vesicles to stores excess nutrients
- except for gigaspora, which is incapable of forming vesicles for hibernation and short term survival when the plant is dead
59
Describe the structure of the AMF genome
- Larger than average fungal genome
- No evidence for gene duplication, but high number of transposable elements
- Reductive evolution = loss of fatty acid synthase
60
AMF are largely seen as asexual fungi, however give an example of recombination that has occurred
Collection of R.irregularis isolates from a largely undististurbed field in Switzerland
- Found that some strains had undergone recombination and lived in a dikaryon state (two nuclei)
- Certain genes associated with a MAT type had been switched around, suggesting recombination
61
Describe the dynamics of nutrients exchange at the peri-arbuscular membrane
- Plant provides up to 20% of photosynthates, while fungi supplies 70% of Pi
- Temporal analysis shows that STR1/2 is expressed earlier in development, while PT11 is in a mature arbuscule.
- Then STR1/2 expression reduces before PT11
62
Describe the translation process of viral positive ssRNA
1. VPg (viral protein genome-linked) binds to 5' terminus mimicking the cap to recruit the ribosome
2. CITE (cap-independent translation element) RNA element in 5'UTR, bypasses need for VPg, helps binding directly to host proteins
3. Interaction of the strand with PABP (PolyA binding protein), resulting in circulisation
63
What is a gene cluster and how can they evolve?
Cluster = two or more genes located next to each other
Mechanisms for evolution
1. Gene duplication
2. HGT
3. Operon formation, cluster transcribed into single mRNA
4. Gene rearrangement
5. Mutations
64
Why do bacteria with complex lifestyles require a large genome?
- Diversity in metabolic activity
- Ability to switch to produce different enzymes
- Rhizobia - change lifestyle completely when in host than when free-living
65
What strategies are employed by viruses to increase the number of proteins transcribed + translated? (6)
1. Polyprotein that can then be cleaved
2. Transcriptional slippage, slips and misses or adds a base changing the mRNA seq
3. Translational read-through - leaky stop codon that can generate a small or larger protein
4. IRES - internal ribosome entry, start translating in the middle
5. Frameshift - shift in reading frame, generating more proteins
6. Multipartite - having multiple segments of the genome e.g. CMV in 3 segments
66
What is an ambisense genome? What are the positives and examples?
Both -ve and +ve sense can be translated into a protein
- Tospovirus tomato spotted wilt virus (TSWV) has multipartite genome and the small and medium strands are ambisense
67
What are pararetroviruses?
DNA viruses e.g. Cauliflower mosaic virus
- Uses reverse transcription, but doesn't integrate into the host genome
- Changes can occur due to lack of DNA repair machinery
68
What are satellite agents?
Satellite viruses
- Hyper-parasites that use a helper virus to replicate e.g. satellite tobacco mosaic virus.
- Presence of satellite and helper, leads to stronger phenotype
Satellite nucleic acids
- RNA and DNA satellites that require a virus for replication
69
What are viroids?
Small, circular single stranded RNA that DOESN'T encode proteins
- lack protein coat/capsid
- Use host RNA polymerase for transcription and no translation
- e.g. Potato Spindle Tuber viroid
70
What are the two main type of viroids?
1. Pospiviroidae
- replicate in the host nucleus
- rolling circle replication
2. Avsunviroidae
- replication in plastids
71
How do viruses move? What do they encode to facilitate this movement?
- Exploit natural pathways for movement (symplastic via plasmodesmata), following the movement of carbon to sinks also.
1. Directed movement
- TMV uses MP proteins that bind RNA to generate a ribonuclear protein (RNP) complex
- This complex is formed at the ER and associates with actin filaments that lead to plasmodesmata localisation
- Pi of the MP results in the release of RNA into the adjacent cell
- CMV requires interaction of the MP with the coat protein to strengthen the binding with RNA. 33amino acid deletion was able to generate strong interactions without CP (Kim et al., )
e.g. Cowpea mosaic virus - forms hairy protoplasts
72
What other proteins may be used to facilitate viral movement?
Potyvirus e.g. Turnip Mosaic Virus, uses P3N-PIPO that targets cylindrical inclusion (CI) to the PD
- Uses the actin filaments that are directed to the PD
- CI forms conical structures thought to help "tunnel" the virus through
(mutant P3N-PIPO unable to move between cells)
73
Name some symptoms of viral pathogen infection
1. Coat protein - accumulation e.g.. in the chloroplast can result in yellowing of the leaves as seen in certain strains of TM
2. Induction of cell division - Rep proteins interact with retinoblastoma proteins to regulate entry into the cell cycle e.g. geminivirus
3. Viral silencing suppression - disruption of ARGONAUTE proteins that are responsible for viral silencing e.g. CMV 2b protein disrupts the regulation by interfereing with miR159
74
What Arabidopsis mutant screens have been carried out to identify factors that affect virus infection?
1. Restricted Tobacco Movement (RTM)
- mutagenesis on plants that were infected with TEV containing resistance to herbicide
- plants that survive have mutated genes that prevent viral spread, and active genes that are required by the virus for protection
2. TOM1
- An essential membrane protein that interacts with TMV
- mutation in tom1 leads to reduced pathogenicity
75
How have yeast experiments been used to analyse factors that affect virus infection?
1. Replicon and viral replication plastid integration
- Generate yeast mutants (mutated in functions such as lipid metabolism), integrate 2 plasmids that promote antibiotic resistance
- Ability to survive means that those functions are required for viral replication and function
2. Changing the ratio of proteins controlling replication complex
- Brome Virus 1a and 2a are important for changing the cell membrane to generate an assembly factory
3. Identify Cell-Intrinsic Restriction Factors (CIRFs)
- Are antivrial host proteins, mutants and overexpression experiments have confirmed their functional role in yeast
76
Describe the function of the aphid stylet
SPIT (egestion)
- release from the salivary gland, containing effectors to dampen immune response
- may also release viral particles
SUCK (ingestion)
- take up from the cytoplasm, e.g. viral particles
77
What evidence is there that the aphid penetrates the cell membrane?
1. Electrical Penetratio Graph
- attaching a wire to the aphid and clamping the leaf
2. Microscopy imaging
3. Fluorescent markers that show that the aphid stylet takes up some of the cytoplasm
78
Describe the different methods of viral retention in a vector
Non-circulate
- rapid and short retention time
- non-persistent and not vertically transmitted
- doesn't cross the vector membrane
Circulate
- slow process and spread
- can cross the membrane and may or may not replicate in the host (propagative nature dependent)
79
What is an example of a non-persistent transmission of a virus? How is the virus specially adapted?
Binding of potyvirus to the aphid stylet
1. via encoded Helper component proteinases (HC-pro)
2. Via coat protein binding to receptors
80
How can viruses directly and indirectly alter their spread by vectors?
Direct - altering the behaviour of their host vector/ insect e.g. changing male thrip feeding frequency when infected with Tobacco spotted wilt virus. Potato Leaf roll virus that promoted settling of aphids, which was reversed upon virus acquisition, Rajabaskar et al., 2014
Indirect - altering the smell of the plant to promote freeding e.g. production of volatile compounds, increase in terpenoids following infection with CMV promoting pollination by bees
- Y-satellite with CMV promotes ABCG4 that causes winged aphids
- Beta satellite with begomovirus suppresses MYC2 and JA production
81
Describe the different mechanisms attract & deter and attract & retain
A&D
- attract pollinators to the plant via volatiles produced. BUT make the plant distasteful
- leads to non-persistent infection that promotes viral spread to the next polinator
A&R
- attract and promotes extended feeding, if many aphids then congregate for example, this can lead them to develop wings
82
How can viral infections benefit the plant and increase their survival? and why does this occur?
- Improved drought and cold stress tolerance
e.g. CMV 2b protein acting on ABA pathway
REASON
- increase plant survival, and the virus being dependent is benefitted by this
- less pathogenic effect will lead to weaker PTI effect and greater chance of survival e.g. pay-back hypothesis
83
What modifications to viral genomes can be carried out to serve a purpose in research? (4)
1. Fusion to GFP to use as a marker
2. Viral induced gene silencing of target plant genes
3. Prime plants against pathogens, by modifying genomes
4. Production of vaccines and antibodies in plants
84
What is an example for which VIGS has been used?
Target Phytoene desaturase
- evidence for funtion as the protein decreases the amount of carotene in the leaf rendering it more prone to photobleaching which results in a white leaf
85
How can modification of viral genomes regulate gene expression?
- Using virus as a microRNA decoy
Targetting the virus RNA for degradation by ARGONAUTE and preventing degradation of target mRNA
86
Describe how TMV particles can be modified for use
1. Biosensor, or used as epitopes to generate an immune response (not using a fluorophore)
- Insert GFP or mCherry as an overcoat
- Insertion of the 2A protein from the foot and mouth disease means that the fluorophore and coat protein don't always bind, meaning the fluorophores are more spaced out and don't interfere with the structure
2. Modify to present RGD peptides that can bind to integrins in tumours to increase specificity of drug delivery
3. Encapsulate chemodrug cisplatin, preventing it from degradation, and increasing effectiveness
87
Describe potential uses for Cowpea Mosaic Virus in biotechnology
'Best developed spherical plant virus for nanotech'
1. β-barrel structure, that forms a spherical structure
- Can be used for drug delivery, by regulating the structure of the virus by disrupting ionic charges between the αα
- pH dependent swelling pH>6.5, non swollen form pH<6.5, generates openings Douglas + Young 1998
2. Cancer vaccine against S1009 gene that encodes a Ca2+ binding protein commonly expressed in cancerous cells
3. Biosensor to enhance MRI imaging
88
Describe potential uses for TMV in technology (biotech)
1. Cylindrical structure can be used as a vessel for drug delivery
e.g. anticancer drug cisplatin
- Modify the membrane to specifically target cells and prevent larger scale damage
= Useful as drug can be used in lower concentrations and cause less harm
2. Mineralised TMV rods to form nanowires, gold nanowires for nanocircuits
89
Name an example of a virus being used for pesticide delivery
Red Clover Necrotic Mosaic Virus (RCNMV)
- delivers Abamectin an anti-helminth drug
Virus targets the root and leads to targeted delivery, which is more effective and requires less drug
90
What is bio-pharming?
- Insert a gene encoding a protein from a pathogen (antigen) to generate a vaccine
- Commonly achieved by agrotransformatio into N.benthimiana
- Much more cost effective as plants are cheaper than using animals, and much faster
91
How can oomycetes be transformed?
1. Protoplast mediated transformation
- Produce protoplast, incubate with DNA and PEG to facilitate uptake
2. Ag-bac transformation
3. Electroporation
- electric pulses to generate pores for DNA to move in
92
What is the role of PEG in transformation?
Induces membrane permeability
Promotes aggregation of DNA to protoplast surface
93
Describe the characteristics of oomycetes
Protists that have 2 flagella
Lack a chloroplast
Live in a diploid state
Filamentous and coenocytic (no septa)
Cellulose and β-glucans in the cell wall
94
What are the two main lifestyles of oomycetes? What facilitates this?
Saprophytic - digest and take up dead matter
Pathogenic - live in a host and cause harm
Cause
- flexible metabolism
- genome encoding effector proteins for biotrophy or necrotrophy
95
Describe the two life cycles of oomycetes
Asexual - formation of biflagellate zoospore, rapid and mobile
Sexual - differentiate into oogonia and antheridia and mate
96
What are the differences between steroid based sex hormones and mating hormones?
Sex hormones - control the differentiation of the hyphae into antheridia and oogonia
Mating hormone - hormones for attracting opposite, compatible mating types
97
What is secondary homothallism?
When harsh environment is present and results in selfing, as unable to find compatible mating type
98
Describe the phytophthora infection cycle
1. Finding the host, chemo and electrotaxis
2. Entry into the host
3. Early plant response
4. Biotrophy and haustoria response
5. Switch to necrotophy
99
Describe the formation of a haustoria
Marker for biotrophy, doesn't penetrate the cell membrane
- Surface is known as the extrahaustorial membrane
Many haustoria can target one cell (unlike arbuscules)
- Secreting effectors
Callose deposition occurs at the neck
100
What are the two different types of effectors that can be released?
Apoplastic
Cytoplasmic - can diffuse or be taken up by transport mechanisms
101
Describe the structure of apoplastic effectors
- cysteine rich
- disulphide bridges that form increase stability
- contain a signal peptide in N-terminus for localisation
102
Describe the structure of cytoplasmic effectors
-RXLR motif for localisation (specific to haustoria, but not all)
- Over 550 candidate effectors in P.infestans
- CRINKLED, N-termini has motif for localisation
103
How can effector delivery be analysed?
1. Reporter fusion
- fusion of RXLR motif to GUS
2. Effector re-uptake (not commonly for oomycete)
- secretion of the effector from one cell and re-uptake by a neighbouring cell. Could be degraded or be retrograde transport from ER
3. Resistance protein reporter
- Monitor the immune responses that are triggered
4. Genomic sequencing
- identify potential targets by looking at similarities of known effectors
104
Describe important proteins secreted by plasmodium
Pf Erythrocyte Membrane Protein 1 (PfEMP1)
- adehere to erythrocytes to cause clotting
105
What similarities are shared between oomycetes and plasmodia?
- RXLR motif is similar to Plasmodium Export Element (PEXEL motif)
- Dependent on hosts for nutrients, pathogenic effect
- apical complex, similar to appressorium formed in oomycetes
- Oomycetes and Plasmodium both have two different mechanisms for secretion of proteins and other effectors
106
Describe the unique mechanism of PEXEL secretion
=Plasmodium Export Element
- Different protease to Sec, associated with it (Plasmepsin V protease)
- Provides a back-up for secretion
107
What is the role of Brefeldin A?
Inhibition of normal secretory pathway in oomycetes
- However RXLR are still secreted at haustoria, showing there are two methods of secretion
108
Describe the effector-target relationship
- One effector can target multiple different sites
- Many effectors targeting the same part of the pathway = redundancy
- Many targets target different aspects of the same pathway
109
Give some examples of effectors found in P.infestans (5)
1. AVRblb2
- interferes with C14 protease released at haustoria by plant which is part of MAPK
- Also inhibits flg22-induced Ca2+ influx by preventing dissocation between CaM and CNGC (by binding to CaM)
2. PexRD54 can bind to ATG8 and alter/prevent selective autophagy (which may be targeting microbial components)
3. Avr3a helps stabilise negative regulators of immunity (the E3 ligase CMPG1)
4. CRN8 (RXLR effector)
- promote cell death in P.infestans, localises to the nucleus and acts as a kinase
5. Extracellular Protease Inhibitor (EPICs)
- Prevent action of C14 in the apoplast
110
Describe how P.infestans interfere autophagy
Release of RXLR effector PexRD54
- PexRD54 can compete with Joka2 to bind to ATG8
- ATG8 is found on the autophagosome membrane and targets specific cargo for autophagy
- By binding of PexRD54 to ATG8, Joka2 can't bind and act as a mediator to bind cargo
- Joka2 can form part of immune response and bind cargo associated with microbes so PexRD54 effector acts to stop this
111
Give examples, and describe the function of effectors that acts as silencing inhibitors
PSR1/2 from P. sojae
PSR1 = binds to components of the RNA silencing machinery e.g. DRB proteins that are cofactors of DICER , inhibiting their function
PSR2 = interferes indirectly, by controlling the regulators that control RNA machinery
= Evidenced by GFP, mutated PSR had lower GFP as it was less effective at inhibiting silencing
PSR2 from P.capsica
- Prevents the formation of secondary siRNA which are commonly found in extracellular vesicles to target the pathogen. Suppression leads to greater infection (Hou et al., 2019)
112
What are the methods for identifying candidate effectors?
**1. Biochemical approach **
- purified and tested
**2. Functional genomics**
- CRISPR knockouts or RNA interference for silencing
**3. Genomic + Transcriptomic approach**
- look at sequence similarity using known/ conserved aspects e.g. RXLR motif
- RNA-seq to analyse gene expression during infection
113
What is the effect of jumping on effector evolution? Give an example
1. Gene loss, no longer beneficial/ functional
2. Effector redundancy
3. Promotion HGT
4. Co-evolution with new host
e.g. diversification of RXLR motif to avoid detection by the host
114
Describe the genome architecture of effectors
Speed associated
1. Two-speed genome
- Gene sparse - found spaced out as mutations and changes are common, and want to minimise effect on neighbouring genes. + Repeat -rich to promote adaptive evolution
- Gene dense - commonly for core genes, where there are less changes
2. One speed genome
- Evolution driven by copy-number variation
- characterised by an over-proliferation of TEs due to
the loss of genome defence mechanism and lack of compartmentalisation
3. Multi-compartment
- Accessory chromosomes, and compartments for core chromosomes.
- Conditionally Dispensable chromosomes – Chromosomes that encode virulence and effector genes, however, when lost doesn’t effect fungal viability.
Frantzeskakis et al., 2018
115
Describe the action of (BRI1 associated kinase) BAK1 as a coreceptor
1. Nlp20 detection
- nlp20 detected by RLP23, then SOBIR1 recruits BAK1, amplifying immunity signal
2. flg22 detection
- upong flg22 binding to FLS2, BAK1 is recruited
3. chitin detection
- upon chitin detection by CERK1, BAK1 coreceptor is recruited
4. BR detection
- BRI1 detection of brassinosteroids leads to complex formation with BAK1
116
What is the role of the effector PiAVR3a?
Interacts with and stabilises CMPG1 an E3 ligase involved in the hypersensitive response
- suppressing HR, which is commonly carried out to kill infected host cells and prevent spread
117
Describe an example of defence and counter defence system in P.sojae
1. Release of apoplastic effector = PsXEG1, which degrades the host cell wall
2. Plant responds by secreting GmGIP1
3. P.sojae releases PsXLP1 to mimic and act as a decoy
4. Receptor on the plant membrane REXG1 detects effector and elicits an immune response
5. P.sojae can release multiple RXLR effectors to suppress the immunity response
118
What intracellular receptors to effectors are found in plants?
Nucleotide-binding leucine-rich repeat (NLR)
- Direct (by binding) or indirect recognition (detect changes in guardee or decoy)
- e.g. RPM1 detecting AvrB from P.syringae
119
What is the role of ATR5, and what experiments have been carried out to analyse this?
ATR5 is an avirulence gene triggering ETI in a plant
- Resistance achieved by detection of ATR5 results in cell death to prevent spread by R gene (RPP5)
- Using a GUS reporter, lack of GUS expression due to cell death shows plant is resistant/ able to detect the effector
120
How can the presence/complementarity of an immune receptor be tested?
1. Agbac infiltration
- inoculation of the plant with a solution containing the desired T-DNA encoding the effector
- if this results in a large necrotic patch caused by the hypersensitive response, then this suggests the receptor is able to detect the specific effector
- can generate mutants lacking effectors to identify further
2. GUS assay
- hypersensitive response results in cell death and so GUS would not be expressed
121
How can the potato be saved from blight?
1. Transgenic approaches - introduction of genes from other strains or species to provide resistance
2. Breeding for R genes, crossing of lines that are more resistant e.g. varieties with R3a
3. RNAinterference - aim to silence pathogen genes of P.infestans to reduce virulence
4. Chemical controls
5. Biological control - generating competition for the fungus by introducing other species e.g. trichoderma (increase resistance to other pathogens, mycotrophy grow from fungi)
122
What is the best way to engineer diversity in the field?
- Multi-resistant monoculture = each plant being resistant to multiple pathogens. And growing at the same time/rate yielding the same
-- Mosaic of resistance - can help prevent spread by having different resistances, however, for farmers flowering at different times and not having identical plants is hard
MONOCULTURE = bad easy for the pathogen to spread and infect as there is no resistance or difference
123
Give an example of how oomycetes can evade host detection
Mutation of AVR3a in P.infestans
- two amino change in the sequence renders the effector undetectable by the plant
124
How can the plant develop resistance to pathogens?
1. Mutating the immune receptor - to be able to target a variety of receptors
2. Mutating the susceptibility factor - change the target of the pathogen
e.g. MLO gene mutation providing greater resistance against powdery mildew, however can also increase susceptibility to other pathogens
e.g mutation in RAM2 which is an essential gene for lipid metabolism, preventing Phytophthora in Medicago
125
Describe the structure of bacteria
- Peptidoglycan cell wall (gram +ve or -ve)
- can have capsules
- No membrane bound organelles
- Flagellum (movement), pili (attachment and HGT)
- various secretion systems
126
What are the various secretion systems found in bacteria?
Sec+ Tat - normal for folded and unfolded proteins
GRAM POSITIVE
- SecA2 to span another membrane
- SS type VII similar to T3SS and T4SS
GRAM NEGATIVE
- Type I ABC to secrete small molecules
- T3SS needle like into the host for proteins
- T4SS needle like into the host for DNA
127
Describe the process of bacterial division
Binary Fission
1. DNA replication from origin of replication
2. Nuclear segregation
3. Cytokinesis (FtsZ for Z-ring formation) and formation of new cell wall
128
How are genes regulated in bacteria?
Operons - functionally related genes clustered together and transcribed polycistronically
Sigma factors - help target RNA pol to promoters
129
Describe the process of HGT
- Requires high cell density
Conjugation
- compaction and quorum sensing
- Pili then help transfer plasmids between bacteria
Transformation
- taken up from the environment
Transduction
- transferred via bacteriophage
130
Why is the study of bacteria difficult? What is meant by bacterial dark matter?
- Hard to culture makes them hard to analyse and assign a name, making it hard to share information about them
- HGT makes it hard to analyse their phylogeny
- Identification of species is difficult as not all are culturable so exist as 'dark matter'
131
What recent technologies have helped in the analysis of bacteria?
1. Metagenomics - analysing all the DNA and RNA in a sample and trying to assign it
2. Single cell sequencing - however, doesn't always get the full sequence
3. Culturable species - help find out more about how to culture other species
132
What are bacteriophages and what are their different lifestyles?
- virus like, obligate intracellular parasites that target bacteria
Lytic - hijack host machinery for replication then cause cell lysis
Lysogenic - integrate into the host genome as a prophage and live a slower more dormant lifestyle
133
How can bacteria defend against phages?
1. Decoy by forming a vesicle
2. Mutation of host receptors to prevent attachment and entry
3. CRIPSR-Cas to destroy foreign phage genes detected
4. Abortive infection - self sacrifice to prevent accumulation
5. Phage-exclusion by infection with another phage
134
Describe superinfection exclusion
Infection with one phage that prevents entry and provides resistance against another phage
e.g. Fruitloop encoding gp52 that interacts with the membrane protein Wag31, which is required by Rosebush and Hedgerow for infection. Odd that fruitloop is lytic and still wants to have cell all to itself
135
Describe the function of CRISPR in bacteria
Clustered Regularly Interspaced Short Palindromic Repeats
DNA library generated by protospacers that are small fragments of DNA from pathogens
- These are added to the library by the adaptation complex that recognises PAM motifs
- Cas proteins = helicases, nuclease and ligase
136
Describe how abortive infection occurs
--- Rex system - exclusion of other phages
rexa and rexb genes of λ phage
1. Detection of foreign DNA
2. Dimerisation of RexA results in activation
3. Multiple RexA required to bind to RexB an ion channel on the membrane
4. Changes in ion concentration and membrane potential
5. Dropping the level of ATP, and decreasing macromolecule synthesis
--- Lit system
- Encoded by the E.coli genome, targets T4 phage infection
- Lit protease cleave gp23 (from phage) preventing assembly
- Lit also targets player in translation, inhibiting protein synthesis resulting in cell death
137
How can bacteriophages fight back against host defences?
1. Mutation to avoid detection
2. Restore adherence - e.g. by breaking down capsule
3. Disguise own genome by methylating it
4. Targetting CRISPR machinery
5. Destroying the host
138
Describe the mechanism of quorum sensing
1. Intracellular production of autoinducers (IAs)
2. Extrecellular secretion of IAs until the concentration reaches a certain thresh-hold
3. Binding of IAs to receptors that regulate gene expression and can change behaviour
139
What is the role/ effect of quorum sensing?
- Promote biofilm formation for protection
- Promotes conjugation
- Important to initiate swarming
- Important for antibiotic production
140
Give an example of quorum sensing and the process (luminescence)
1. AHLs formed by LuxI
2. Diffuses out and increase in concentration leads to detection by LuxR
3. Detection leads to activation of Lux operon, encoding LuxI
= positive feedback loop
141
How can biosensing and colour be used as evidence for quorum sensing?
Chromobacteria violaceum
- Produces a purple pigment when quorum sensing
- Mutation in the external sensory cv926 meant no purple pigment was formed
- CV026 strain can be used as a biosensor, by mutating the ability to produce AHL it can be exaptated to detect environmental AHLs and produce violacein
142
What is quorum quenching? Give an example
When bacteria, fungi or plants interfere with quorum sensing
e.g. by production of enzymes that degrade signals such as AHLs
Bacillus subtilis
- stops action of gram -ve soft root bacteria by targeting AHL. Evidenced by formation of halo using C.violaceum. Also qPCR showed a reduction in cell wall degrading enzymes being transcribed
143
How can bacteria act 'multicellularly'?
1. Incomplete binary fission
- e.g. in cyanobacteria where two areas may acquire different functions such as C and N fixation
2. Filament formation by budding
3. Biofilm formation - community and differentiate
4. Myxococcus xanthus - fruiting body formation, aggregation
5. Streptomyces division of labour
144
What are some positive and negatives about bacterial multicellularity?
Positives
- Increased stress resistance
- Protection from predators seen in Flectobacillus inedible
Negatives
- Reduced motility have to move as a group
- Self sacrifice and unequal share of nutrients perhaps
145
What is kin selection in bacteria?
When bacteria exhibit co-operative strategies that benefit genetically related individuals
- promotes selection of more genetically similar, and helps suppress "cheaters"
- e.g. selection for ability for siderophore production or biofilm formation
146
Describe how P.syringae is adapted to live in the atmosphere
1. Ice nucleation proteins (INPs)
- encourage ice crystals to form at higher temperatures, to promote frost damage and entry into plants e.g. at -2°C instead of -38°C
- structure of the protein binds water in an arrangement that promotes crystal struture formation, acting as an ice nucleator
- Multimer formation of INPs, achieved by C-terminus properties is essential for organising water into crystals
- Important to form condensation nuclei and clouds that can help bacteria escape from the atmosphere
2. Resistance to dessication and UV-B (312nm)
- Evidenced by comparison to E.coli, showed greater survival and retention of the ability to synthesis INPs
- Both E.coli and P.syringae found to be susceptible to UV-C
- UV DNA repair mechanisms such as nucleotide excision repair
de Arujo et al., 2019
147
How can plants respond to ice nucleation?
- Ligning and suberin protection
- Dehydrins promote retention of water and promote the supercooling properties of pure water
- Having supercooled tissues such as the xylem
148
Describe how the conditions of the soil is harsh
- Heterogenous spread of nutrients and water
- Temporal change requires adaption e.g. seasonal changes
- Abiotic and biotic stresses are present
149
What features promote bacterial survival in the soil?
1. Mobility- achieved by chemotaxis and presence of a flagella
2. Dormancy - survive harsh conditions, carried out by 95% of bacteria
3. Self-sacrific - death of bacteria to provide nutrients for others
4. Symbiosis - relationship with plants that provide shelter and nutrients e.g. rhizobia and frankia
5. Biofilm formation - achieved by quorum sensing
6. Thick cell wall for protection
150
What are the 3 lifestyles that bacteria have evolved to deal with stressful conditions?
1. Persistors - replicate slowly and can withstand low nutrient conditions by having sideorphores and high affinity transporters
2. Opportunists - replicate rapidly and make the most of any nutrients that become available quickly, short-term thinking. Sporulate and show seasonality
3. Competitors - large genomes and great catabolic diversity
151
What factors regulate which lifestyle is carried out by the bacteria?
Genome size - small size means low metabolic diversity and adaptability and vice versa
Abiotic/Biotic factors - providing stress for evolution
152
Give examples of how bacteria can be so metabolically diverse
1. Methylocapsa gorgona - encodes a methane monoxygenase (MMO) that can oxidise and fix methane
- Also able to use N, H, CO, CO2 and O for nutrition (can grow in air alone). Only encodes one MMO that is responsible for fixation in low and high conc (Tveit et al., 2019)
2. Phototrophy and Chemotrophy - cyanobacteria fixing CO2 in oxygenic conditions, while GSB in anoxic conditions
3. Fixation of nitrogen and nitrate e.g. rhizobia and frankia
153
Describe the formation of endospores
Bacillus (aerobic ) and Clostridia (anaerobic), produce endospores that are the most resistant form of life on planet Earth
1. Stress results in increase in sigma factor H encoded by RPoH
2. Phosphorelay system activated by KINA
3. Pi of master regulator SP0A
4. Asymmetric division and spore formation, cortex (peptidoglycan) and coat (keratin-like molecules) that provide resistance to stress
154
In what ways are Streptomyces unique? How is this reflected in their genome?
1. Bacteria that has linear instead of circular chromosome
- Divided into core and arms (dynamic region, undergoes more recombination) (Bury-Mone et al., 2023)
- Evidenced by new 3C technology (chromosome conformation capture)
2. Genome content
- Known for antibiotic production e.g. streptomycin
- Geosmin for attracting insects
3. Ecological plasticity (diversity)
- mutualism - produces auxin, promotes nodulation
- parasitism - Carbohydrate active enzymes (CAZymes), pathogenicity islands, necrogenic proteins
4. Dispersal
- No flagella or pili, Rodlin to attach to nematodes
- Geosmin attract insects for spore dispersal
155
How can bacteria establish themselves in a niche, provide an example?
- Directing water and sugar transport to a specific area e.g. the apoplast
1. Pseudomonas toxin promotes stomatal opening for bacterial entry
2. Release of another toxin controlling ABA signalling to promote stomatal coslure to increase water retention
- AvrE1 and HOPM1 act was water channels promoting movement, also regulator of ABA
- In Xanthomonas TAL effectors also promote the expression of SWEET
156
Describe the lifestyle of phytoplasma. How is this reflected in their genome?
- No cell wall, preventing PTI
- Obligate biotrophic pathogen, requiring a host
- Spread via insects and plant
- Co-evolved with plants, to prevent rejection
Genome
- REDUCTIVE EVOLUTION = Undergone a lot of gene loss (smallest bacterial genome)
- unable to synthesise ATP synthase and cell wall proteins
- Increase in sugar and amino acid transporters
- Retain Sec for effector secretion
157
Name some effectors from phytoplasma (3)
Aster yellow witches broom (AY-WB) phytoplasma
- has increased branching and yellowing leaves
1. SAP11
- Destabilises TCP, e.g. TCP4 that is responsible fo JA biosynthesis (via LOX2)
- causes curly leaves Sugio et al., 2011
2. SAP54
- intereferes with APT1 and SEP3 regulating normal flower development
- RAD23 targets these MADS-box TF for degradation
- Generates leaf-like flowers that promote insect reproduction, favoured by leafhoppers MacLean et al., 2014
3. SAP05
- promotes extended juvenile phase in the plant by targeting GATA and SLPs (binding with RPN10 resulting in degradation), with younger plants being more likely to invest in development of leaves, which bacteria can exploit due to more nutrients
158
Describe the origins and conditions for N-fixation symbiosis
100Mya
- Shares pathway with AMS
- Requires low conc of O2 for nitrogenase enzyme function
- with rhizobia a root nodule is formed
159
What are the two ways in which establishment can occur in N-symbiosis?
1. Fixation thread nodules
- Bacteriod surrounded by the cell wall and retained in the apoplast
2. Symbiosome
- Internalised into the cytoplasm and retained within vesicle like structures
160
Describe the genome of N fixing symbiotic bacteria
- Sym plastid that encodes symbiotic related genes and nitrogenase
- large genome as it switches between a free-living aerobic bacteria to a symbiotic anaerobic bacteria
- one or more megaplasmids (separate core from symbiotic genes)
161
Describe the dialogue between plants and bacteria in N -symbiosis
1. Flavonoid secretion by the plant to promote symbiosis
2. Bacteria secrete Nod factors (LCOs) that are detected by LysM receptors
3. Plants attenuate their defence mechanisms
4. Nodulation inception (NIN) is triggered to form a new organ
5. Infection thread and root hair curling forms, forming cytoplasmic bridge which promotes bacterial entry
6. Formation of a root nodule, by cell division, with bacteria infection
162
Describe the difference between symbiotic nodules and lateral roots
1. Purpose
- Symbiotic to house bacteria
- Later roots for nutrient uptake
2. Inducing factors
- Symbiotic via Nod and LCOs (driven by high cytokinin and low auxin)
- Lateral by hormones such as high auxin and ABA
3. Structural differences
- Symbiotic compact and divide from cortical cells
- Lateral divide from pericycle cells, more branched
163
What genes have been found to be essential for nodulation?
LSH1/2
- important for nodule colonisation
MtNoot1/2
- important to maintain nodule identity
LHK1
- for detection of cytokinin to promote nodule growth
164
How does the plant help the bacteria in N-symbiosis?
- production of leghemoglobin that binds to oxygen for bacterial respiration, also leads to pink colour of nodule
- plant provides sugars for the bacteria to carry out the energetically costly N-fixation reaction
++ bacteria undergoes changes that renders it unable to synthesis proteins and carbon, and only N2 fixation ++
- plants can also provide other nutrients to help bacterial growth
- Creates low oxygen environment in the vesicle/ symbiosome
165
Describe the importance of viruses targeting coccolithophores
Emiliana huxleyi (algae, coccoliths/haptophyte) that helps with climate regulation
- Releases Dimethyl sulfide (DMS) that helps with cloud formation
- Captures carbon and forms coccoliths that it can shed
VIRUS - EhV-201
- infection leads to shedding of protective layers
166
Why are viruses used as a biocontrol against bacteria?
1. To prevent bacteria from developing multi-drug resistance from use of antibiotics
2. Prevent pollution of the environment with chemical drugs
3. Bacteriophages are highly specific
167
How are viruses used as a biocontrol against bacteria?
- Use a phage cocktail to target bacteria
- Multiple targets puts a lot of stress on the bacteria and evolve resistance with weakened essential traits = EVOLUTIONARY SUICIDE
- Can also be used to target biofilm formation and increase susceptibility to antibiotics
168
What are the disadvantages of using phages as biocontrols?
- Low efficacy, due to host specificity
- Regulations in the EU are very strict so takes a lot of time to be approved in the market
169
How can viruses be used against fungi?
- Rendering fungus less infectious, for example for controlling blight diseases
- Then these viruses can be spread via hyphal anastomosis
- However, fungi can be important, so targeting all may be detrimental for the ecosystem
170
How can viruses be used against viruses?
Cross-protection (vaccine-like)
- Use a milder virus to prime the RNA silencing machinery in preparation for true infection
Disadvantages
- Strains could mutate and immunity may fade
- Labour intensive to infect all plants
- mild strain may not be enough to trigger a good response
171
What is genomic bottlenecking?
Reduction in genetic diversity within a population or organelle genome
- Caused by selective pressures and evolutionary events such as endosymbiosis
- Selection for genes with essential functions, others either lost or moved to nucleus
= Functional efficiency, and for other genes, nucleus co-ordinated regulation
172
Why might plastid genomes be similar or different?
Similar
- Loss/ movement of other genes and retain core genes such as ones encoding photosynthetic machinery, ones that encode proteins that can't be imported, and certain ribosomal RNA
Differences
- Different algae arisen from different endosymbiotic events, secondary events engulfing different archaeplastids (green/red)
- Horizontal gene transfer may occur, bacteria transferring to algae
- Genetic bottlenecking adapt to selection pressure and reduce gene size. e.g. Helicosporidium (green algae) parasitic unable to carry out photosynthesis. another example are apicomplexans
173
Describe the function of CRN8s from P.infestans
- Oomycete cytoplasmic effector
- CRINKLED, N-termini important role in cell translocation
- CRN8 is a kinase, with 5 serine residues
- Localises in the nucleus, however, the phosphorylation targets are unknown
- Carries out autophosphorylation
174
What are 3 ways to prevent bacteriophage adsorption?
1. Secretion of extracellular matrix
2. Alteration to bacteriophage receptors
- inclusing phase variation
3. Secrete competitors or inhibitors to prevent binding
175
Name overarching mechanisms that bacteria use to prevent bacteriophage infection
1. Preventing adsorption
2. Preventing DNA phage entry
- Superinfection exclusion
3. Cutting phage nucleic acids
4. Abortive infection system
176
Give examples of extracellular matrix secretions from bacteria to prevent phage infection
1. Alginate
- increased phage resistance in Azobacter, however phage can evolve to encode alginate lyase
2. Hyaluronic acid
- important to protect bacteria from immunity sensing and phage infection. Barrier can be degraded by hyaluronidases, which can be found in prophages in other bacteria
177
How do phages overcome the bacterial methods to prevent adsorption?
1. Extracellular matrix secretion, overcome by encoding lyases and hydrolases that are either found on the surface of the phage or released during lysis
2. Blocking phage receptors can be overcome by being able to bind to more than one receptor, although with lower efficiency
3. Restriction-Modification system can be overcome by mutations in the endonuclease recognition site. Or by encoding methylases to prevent detection as foreign
4. CRISPR-Cas can be overcome by point mutations in the targeted proto-spacer
5. Abortive infection systems can be overcome by encoding proteins that still ensure lysis and spread of phage
178
Give an example of the production of competitive inhibitors preventing phage entry
Microcin J25 an antimicrobial that binds to the FhuA iron transporter on E.coli, preventing coliphage binding
179
Describe the mechanism of phase variation that allows bacteria to prevent phase adsorption
Bordetella uses phase variation, where it exists as two different phases and presents different receptors on its surface.
In the Bvg+ it presents the Pertactin autotransporter (Prn) that is required for infection by Bordetella phase
- However in the Bvg- phase, infection efficiency is lower, however, still present suggesting it can use other transporters
180
How have ectomycorrhizal fungi and other evolved to be biotrophic?
1. Repurposing of previous genes- that are now essential for symbiosis. Achieved by reduplication and changes in the promoter or coding sequence e.g. in other place no longer pathogenic effect
2. Evolution/ HGT of symbiosis related genes
3. Gene loss e.g. of cell wall that acts as a DAMP
181
What are the benefits and challenges associated with endosymbiosis? e.g. of bacteria
Benefits
- development of new chemical defences
- access to different nutrients, changes to metabolism e.g. nitroplast in B.bigelowii that fixes N
Challenges
- overcoming immune response
- growth synchronisation in growth
- integration into metabolism
182
Give an example of an oomycete effector that causes cell death
CRN8 from P.infestans responsible for cell death
- Has a kinase activity and 5 serine residues that can be phosphorylated
- Phosphorylation of these residues is essential for cell death, however, kinase activity of CRN8 isn't
- Effector requires nuclear translocation
- Kinase-inactive CRN8 was found to have a dominant negative function on CRN8, reducing virulence, while CRN8 increased virulence
183
Discuss non-canonical gene expression strategies utilised by plant viruses
Non-standard techniques
1. Cap-snatching e.g. Tomato spotted wilt virus (TSWV)
2. Frameshift/ Transcriptional Slippage
3. Internal Ribosome Entry Sites
4. Ambisense RNA e.g. Tospovirus tomato spotted wilt virus (TSWV)
5. Translational read-through - leaky stop codon e.g. TMV
6. Subgenomic RNA production - express proteins later e.g. TMV
+ Adv, more proteins encoded in small genome, temporal control of expression, evasion of host defences
184
How has oomycete pathogen genomics have accelerated plant disease resistance breeding?
1. Genome sequencing has generated libraries to locate effectors
2. Comparative genomics
3. Functional genomics (mutant analyses, GUS and CRISPR)
4. Indirect analyses looking at host-induced response
185
How is resistance to P.infestans commonly characterised? Where have resistant strains been found?
- Hypersensitive response (HR) causing cell death
- Detected by R genes known as Resistance against P.infestans (Rpi)
- Resistant strains found in wild cultivars in Mexico and Guatemala
186
Compare and contrast the life cycle of oomycetes and fungi
Similarities
- Both able to carry out asexual and sexual reproduction
- Both release pheromones which are small peptides to attract the opposite mating type
- Both produce spores
Differences
- Spore morphology differs, oomycete is biflagellated and motile, while fungal is non motile
- Fungi can temporally control plasmogamy and karyogamy
187
What are the three main aims for pathogen effectors?
1. Entry into the host
2. Establishment in the host
3. Sustenance
4. Reproduction sometimes also
+ Co-expression can lead to greater plant susceptibility e.g. by targeting different immunity pathways.
188
How do algae regulate gene expression?
Commonly post-translational modifications
e.g. RNA editing in dinoflagellates, and polycistronic RNA that is chopped up
- PPR proteins (post endosymbiotic innovation) bind to specific sequence repeats on the RNA to target or suppress enzymatic action and modifications
(Not fully known if has same function in plants due to diversity)
189
Describe in more detail the findings from Ustick et al., 2021 paper on data derived from Tara oceans
- Looked at changes in the presence of genes important for N, Fe and P stress
e.g. increase in nitrate, nitrite, ammonium and urea uptake genes in conditions of N stress
e.g. increase in genes encoding siderophores, however, in extreme Fe stress also lose genes that require Fe
- Classed nutrient stress into three categories high, medium and low
- Data followed some known patterns but also helped generate detail on the extent of nutrient stress, as well as highlight new areas
- Fe stress was found in the equatorial Pacific and temperate regions in the N and S Atlantic Ocean, and in the Arabian Sea.
= Using genetics could roughly map the spread of nutrients
190
How has lifestyle affected the genome of fungi and oomycetes?
1. Genome size
- Can increase or decrease
- Biotrophy increase as don't need to conserve energy e.g. AM
- Necrotrophy - increase to promote evolutionary advantage more duplications of genes
- Parasitic e.g. microsporidia(fungus), decrease of metabolic pathways reatin nutrient transporters Stajich 2017
2. Genome content
Biotrophic - gene loss of lytic, pathogen associated genes. Increase in repetitive elements. Co-opt to promote colonisation
Necrotrophic - duplications of effectors (lytic and transporter genes), increase in variety
Saprotrophy - retain lignin degradation
3. Genome architecture
Two-speed genome - gene sparse repeat rich to promote evolution of effectors
Conditionally Dispensable chromosomes - can be lost without losing fungal viability
Gene movement to sub-telomeric (Avr-Pita) increase recombination
191
How can climate change effect viral epidemics?
1. Geographical Range
- Direct - temperature restriction (optimal working temps)
- Indirect - changes to location of cultivation of viral hosts
2. Spread
- Changes in temp => susceptibility of plants (stomata more open)
- Changes in rainfall => stress response in plants, spread and survival of vectors
- Changes in wind => promote wounding of plant greater entry
3. Viral replication
- Rate of replication due to changes in temperature
192
How have viruses been shown to be mutualistic and beneficial to plants?
- Infection with plant virus has led to increased resistance to drought
- e.g. TMV, CMV infection of N.benthi and rice
- Some studies found an increase in SA production (in drought and non-drought) Gonzalez et al., 2020
- Other studies have found that there were greater conc of antioxidants and osmoprotectants (from GC-MS) Xu et al., 2008
+ Viral infection of fungus also important to improve the role of fungus in providing drought tolerance. Plant produced more melanin and trehalose when interacting with viral-infected fungus.
193
How has the genome of the biotrophic oomycete Hyalopernospora arabidopsis adapted to obligate biotrophy?
Baxter et al. 2010
Reduction in
- RXLR effectors
- Genes encoding cell wall degrading enzymes
- Metabolic processes such as inorganic nitrogen and sulfur assimilation
- NLPs that trigger cell death (maybe also expansion and change in function)
- Genes encoding PAMPs to facilitate 'stealth entry'
194
Describe the ingenuity of bacterial genomes
- Avg 88% of the DNA is coding, so mutations changes protein function
- Growth in large population means even smallest advantages can be selected for and deleterious removed
- Propagation of few cells has been shown to lead to a build up of deleterious mutations
- Gene birth by mutations, and recombination, and integration of phage genetic material e.g. type VI secretion system adapted from phage
- Adaptability to different environments leads to either gene expansion or streamlining
195
What changes to gene expression occur in rhizobia bacteria once in symbiosis with a legume?
- Shut down of most aspects of cell growth and division, synthesis of ribosomal proteins, nucleic acid synthesis and repair, outer membrane proteins, and peptidoglycan
- Surrendered control over biosynthesis of essential compounds
- even mutants of ammonia assimilation require amino acids, but can fix N2 so symbiosis is sustained
196
What are protists?
Eukaryote that isn't a plant, animal or fungus
e.g. oomycete, apicomplexans
197
What are the effects of host jumping on immunity development? What can make host jumping easier (e.g. for oomycetes?
- Require broader set of R genes to confer resistance
- For biotrophic oomycets- having hosts with similar morphology can make it easier, also changing to naive host that hasn't got ETI adapted to the oomycete e.g. Hyalopernospora
198
How are RXLR effectors secreted by oomycetes?
Released at the haustoria
1. Produced in the cytoplasm
2. Travel to the ER and golgi where packaged into vesciles
3. Exocytosed from the cell
4. RXLR motif is responsible for entry into the host
4. Clathrin mediated endocytosis (CME) by RXLR binding to receptors
199
What is the evidence that suggests that RXLR effectors undergo proteolytic modifications?
1. Similar to PEXEL that is known to undergo cleavage by two proteases
- Variations in RXLR motif occur around the Rs and not the L, suggesting importance of conserving the component for cleavage
2. Mass-spec analysis finding no RXLR motif in the final functional effector
3. Protease inhibitors - showed delayed RXLR effector processing
(Mutation in RXLR prevents translocation but not function)
200
What is cisgenics? and an example
Modification of a recipient plant with a natural gene from a crossable plant
e.g. used to introduce generate resistance against potato late blight, by introducing Rpi-sto1 and Rpi-vnt1
Jo et al., 2014
201
What is the hypothesis as to why primary plastid acquisition has not been repeated multiple times?
1. Endosymbiosis likely primed by the release of effector proteins in the host cytosol, from intracellular Chlamydiales pathogens.
- genes imprinted from Chlamydiales found in Archaeplastids
2. Very specific niche and complex interaction
202
What are some examples of susceptibility factors in plant-microbe interactions?
1. SYMRK and POLLUX - used in AM symbiosis, but also important for haustoria formation and colonisation by Hpa. Mutant had multilobed haustoria and reduced reproductive success. Interestingly seemed to be specific to Hpa and not other powdery mildew fungi
2. SWEET genes - sugar transporters, commonly induced by Xanthomonas, via TALENs
203
How might climate change alter ocean nutrient distribution? How might this effect future distribution of algae?
1. More upwellings due to wind and ocean stratification. Nutrient rich cold water moves up, but then nutrients are lost. Diatoms are well adapted to rapid changes and low Fe conditions
2. Warming moving populations towards to poles due to heat stress and greater stratification e.g. cyanobacteria
3. Change in nutrients and stress - dinoflagellates are heterotrophs so might do well
4. Ocean acidification - negative effect on the abundance of haptophytes as it lowers the amount of carbonate ions for coccolith shell formation.
204
Give examples of virus that produces a protein that interferes with plant immunity (3)
1. Helper Component Proteinase (HCPro) from potyviruses
Targets RNA silencing by:
- Suppressing ARGONAUTE
- Suppressing the production of siRNAs
- potentially sequestering siRNAs
2. Cucumber MV (CMV 2b)
- also suppresses RNA silencing
3. Potat virus x
- tgb1 triple gene block suppresses RNA silencing
205
Describe the interplay between viruses and algae (link to ecology)
- Algal blooms, formed by phytoplankton, important in regulating the amount of dissolved organic matter (DOM)
- Large E.huxleyi blooms are terminated by EhV201
- Infection leads to changes in metabolism that promotes fatty acid synthesis and upon lysis these are released into the ocean
Ecology - effect DOM, fate of C in ocean and climate
206
How can plant-microbial interactions influence plant physiology?
1. AMF + Rhizobia promotes nodule formation, changing root morphology
2. Rhizobia + CMV infection can alter plant volatile emissions that reduce herbivory and promote pollination
3. TMV infection in rice and benthi can increse drought resistance Rossnick et al., 2011
4. AgBac - alter plant growth and causes formation of a gall, overproduction of cytokinins
5. phytoplasma - effectors SAP05 causes leaf curling, SAP11 changes to inflorescence and SAP54 - an extended juvenile phase
207
How can VLPs be loaded with cargo? (4)
1. Self assembly
2. Infusion
3. Genetic engineering
4. Bioconjugation
- Load chemical, drug, proteins, nucleotides
208
What are the challenges with using plant viruses for mammalian therapeutics?
1. Activation of hypersensitive response
2. Getting regulatory approval
3. Reluctance of pharmaceuticals in changing manufacturing protocols
209
What are photodynamic therapies, how can these be delivered using viruses?
PDTs can be targeted to certain areas by viruses, for example to tumours
- PDT uses photosynthesisers to produce ROS when activated by specific wavelengths
- Recent experiments have used TMC and CMV to try and treat melanoma
- PDT has also been used to treat biofilm formation by Staph.areus using Cowpea Chlorotic Mosaic Virus (CCMV) as a delivery system
(Czapar + Steinmetz 2017)
210
Describe the process of nodule organogenesis in response to rhizobia and low nitrate conditions
Oldroyd et al., 2011
- Requires high cytokinin and low auxin levels (opposite to lateral roots)
- CRE1/LHK1 cytokinin receptors are essential, with mutants unable to form proper nodules
- Requires hormonal balance between cytokinin (+VE) and ABA (-VE)
211
What is terminal genomic differentiation?
Sociomicrobiology idea that suggests that fitness changes in populations outweighs fitness changes in an individual
- for example in Streptomyces, mutability that leads to reduced genetic fitness increases population diversity, for example ability to produce antibiotics = this is more favourable (Bury-Mone et al., 2023)
212
Describe the life style of Magnaporthe orzyae
Fernandez + Orth 2019 - hemibiotrophic fungus targeting rice
- Destruction of 10-30% of rice harvested globally
1. Appressorium forms with great turgor pressure, requires ROS build up.
- mutation in MoNADPH oxidases led to defect appressorium
2. Forms invasive hyphae
3. Avirulent effectors (Avr-pita etc. ) and Biotrophy-associated secrete protein (BAS1) accumulate and are cytoplasmic
- secretes monoxygenase (Abm) that disables JA signalling
4. Switch to necrotrophy by CWDEs
213
What are the roles of CMV2b?
1. Perturbs miR159 for immunity
2. Suppress RNA silencing (binding to AGO)
3. Altering aphid interaction
214
How can viruses be used for therapeutics?
1. Drug delivery - cisplatin in CPMV and TMV
2. Photodynamic therapy delivery
- photosensitisers that require light to produce ROS, CCMV targeted biofilm from Staph.aureus
3. Biosensor - fluorescent tag help with tumour localisation
4. Vaccines - expression of antigen on capsid surface, or biopharming, increasing antigen content by transient expression in N.benthi (Influenza Hg1 Goulet et al., 2019)
e.g. CPMV (Cowpea) vaccine targeting S1009 gene. This gene binds to Ca2+ and is upregulated in various cancer types (Komane et al., 2025)
5. Target bacteria - instead of antibiotics, use phages to target bacteria avoid resistance
215
How is lignin broke down?
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
216
What evidence suggests that UCYN-A has become a nitroplast in B.bigelowii?
- genome reduction
- 258 of the 368 B. bigelowii–encoded proteins enriched in UCYN-A, which accounted for 62.6% of all B. bigelowii protein quantified in UCYN-A
- synchronised division with the host haptophyte
- functional integration, changes to matrix and association with mitochondria
Coale et al., 2024
217
Why does host jumping occur? What effect does it have on the pathogen?
Occurrence
- Effector innovations, physiological similarities, stress, redundancy of effectors
Effect on genome
- Increase diversity, to adapt and resist to new R genes
- Gene loss, due to greater stress, resulting in reduced resistance to previous plant
- Fitness-trade off, reduce pathogenicity to promote colonisation of host, lack of balance promotes continued host jumping
218
Describe the ancestral form of fungi
From a common ancestor 700Mya hyphal fungi have diversified giving rise to a variety of clades such as Dikarya and Mucuromycota
- Driven by heterogenous nutrient environments in the soil that requires exploration strategies
'Hyphal fungi could not have diversified before the origin of food sources that are big enough to sustain the exploration strategies' Berbee et al., 2017
- Its retention in most fungal phyla is a testament to its versatility and efficiency
219
How can fungal genomes under changes and plasticity?
Starship (large mobile element) that carry 'genome ships'
ROLES: niche adaptation, host interactions and virulence
- voyager contains OCH1 = N-linked glycosylation, cell wall remodelling and hyphal growth
Gluck-Thaler et al., 2022
Sato et al., 2025 - (necrosis-and ethylene-inducing peptide) NLP6 formed through fusion of NLP3 and non-coding starship element
220
Describe the termitomyces symbiotic relationship
- termitomyces symbiotic fungus present in termites
- helps them degrade plant material via lignocellulases (type of CAZymes)
- termites get nutrients that are more easily broken down and fungi get a habitat and substrate
221
Describe how viruses can be vertically spread?
Embryonically and non-embyronically
- in embryo or on seed coat/ endosperm
- e.g. partitiviruses that are are cryptic and transmitted strictly vertically.
Part II
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