Download Integrace „hormonálních“ signálů

Integrace
„hormonálních“
signálů
příklady z novější doby
Integrace
regulací
GA s
dalšími
„fytohorm
ony“
IAA spouští zvýšenou
produkci GAs.
Narušení transportu IAA
brzdí degradaci DELLA
represorů působenou GAs.
BR a IAA
Indukují řadu
identických mRNA.
Hladiny auxinu a cytokininů inverzně korelují.
IAA inhibuje degradaci cytokininů stimulací cytokinin oxidázy,
Auxin stimuluje produkci etylenu stimulací genu pro syntézu ACC.
Etylen inhibuje polární transport auxinu.
Světlo a IAA
Světlo zasahuje do auxinem regulovaných změn v dlouživém
růstu buněk a diferenciace.
ABA interaguje antagonisticky
s GAs i IAA. Přidání ABA
může snižovat hladinu volné
IAA a zvyšovat podíl
neaktivních konjugátů;
ABA interferuje se sig.
drahami GAs.
Zvýšená hladina cytokinů a
potlačení GAs je konstitutivním
parametrem dělivého centra
apikálního meristému.
Naopak pro vývoj listových
primordií je podmínkou
zvýšená hladina GAs.
Interaguje prakticky každý s
každým a to jak na úrovni
přenosu
signálů/regulace genové exprese,
tak
také na úrovni regulace biosyntézy inhibice či
aktivace akumulace partnerského
"fytohormonu".
Plasmodesmata
rostlina jako symplastická síť
Symplastická konektivita
je dynamická, regulovaná!
Plasmodesmy vznikají při
buněčném dělení i de novo
a mohou být regulovaně
uzavřena.
Buněčně autonomní = molekula
působí jen uvnitř buňky.
Buněčně neautonomní = molekula
působí mezi buňkami.
MP = movement protein
injekce FITC-MP bílkoviny;C nepoh. mutant
G – NCAP pomáhá pohybu
dextranu.
PD=plasmodesma
NCAP = non cell
autonom. prot.
S transportem jsou spojeny
regulované změny SEL (Size
Exclusion Limit).
NCAP
=non cell
autonom. proteins
Mechanismus mezibuněčného pohybu
Dvoustupňový proces rozbalení a
vazby.
Size Exclusion Limit
je pravděpodobné, že na regulaci SEL se podílí myosin VIII.
• podobně jako při importu do organel se
jedná o interakci „adresového“ motivu s
translokačním receptorem, rozbalením (u
velkých bílkovin/RNA), změnou SEL a
posléze proběhne vlastní transport.
Floemem se pravěpodobně šíří
květní signál – bílkovina FT
HPTS= 8-hydroxypyren 1,3,6 trisulfonová kyselina
Rostlinný organismus
jako nadbuněčná
integrovaná symplastická
síť.
Systemické šíření RNA
RNA viruses can block expression of a transgene
if a copy of the transgene has been added
Tobacco plant expressing GFP protein
Infected with RNA virus with GFP gene
Virus infection travels through veins
GFP expression inhibited starting at veins.
Gene silencing and RNA viruses
share potential to produce ds RNA
Fire and Mello proved that ds RNA inhibits
expression of endogenous genes
homologous to that dsRNA
a RNA interference:
A type of gene regulation
Involving small RNA molecules
and induced by double stranded RNA
Adding a Chalcone synthase
gene from Petunia fused to
a strong viral promoter
to transgenic Petunia
interfered with expression
of the native homologous’
gene.
Gene Silencing
Plant Gene “Co-Suppression”
¾ First discovered in petunia (1990):
•
•
•
•
Increase in expression suppressed activity
Transgene mRNA degraded
Called co-suppression
Also called post-transcriptional gene silencing
(PTGS)
Gene Silencing
1. More common from strong promoters.
2. More common from inverted repeat inserts
(could make a double stranded RNA).
3. Induced by RNA viruses
(have double stranded RNA replication
intermediates).
Biological significance of RNAi
•
Cellular immune response to viruses
In certain organism (especially plants), RNAi serves as a first line of
defense against viral infection, as viral replication typically includes
dsRNA species
•
Genetic stability
RNAi repress the mobility of transposable genetic elements in C.
elegans and S. pombe which requires the formation of dsRNA
Revealed that RNAi can also effect transcriptional gene silencing by
promoting heterochromatin formation (histone and DNA
methylation)
•
Organismal development and germline fate
Developmental processes are affected by endogenous non-coding
RNAs that function through the RNAi pathway (microRNAs)
RNA interference (RNAi)
•
RNAi is the process whereby double-stranded RNA
(dsRNA) induces homology-dependent degradation
of cognate RNA (i.e. gene silencing)
•
RNAi is central to cellular mechanisms of posttranscriptional gene silencing (PTGS) and can also
effect transcriptional gene silencing (TGS)
•
RNAi is highly conserved among eukaryotes (fungi,
protozoans, plants, nematodes, invertebrates,
mammals)
Features of RNAi
Induced by dsRNA with homology to exons
Catalytic: very small amounts of ds RNA are sufficient
Spreads: injection into gut silences genes in embryos
Small RNAs produced
What is the function of RNAi
mechanism in non-transgenic
organisms?
Protection against viruses
Keep Transposable elements inactive
Gene regulation
Transcriptional gene silencing is initiated by
RNA directed methylation of promoter regions
dsRNA homologous to promoters leads to
methylation and inactivation by recruitment of
chromatin remodeling enzymes.
RNA directed methylases also affect
methylation of genes in germline.
May be important to imprinting
RNA directed regulation of gene
expression is more common
than we thought.
How does RNAi work?
RNAi works postranscriptionally……..
in key two steps!
Overview of RNAi
•
Double-stranded RNA (dsRNA)
is processed by Dicer, an RNase
III family member, to produce
21-23nt small interfering RNAs
(siRNAs)
•
siRNAs are manipulated by a
multi-component nuclease
called the RNA-induced
silencing complex (RISC).
•
RISC specifically cleaves
mRNAs that have perfect
complementarity to an siRNA
strand
Components of the RNAi pathway
•
Dicer – u Arabidopsis 4x Dicer like = DCL
Dicer belongs to the RNase III family of dsRNA-specific ribonucleases.
Contains dsRNA-binding motifs and a PAZ domain believed to
mediate protein-protein interactions.
•
Argonaute homologs (Dicer/RISC associated) - u
Arabidopsis 10x. argonaut byl popsán jako meristémový
mutant Arabidopsis.
Argonaute family members are highly basic proteins that contain PAZ
and PIWI domains.
RISC komplex „rozmotává“ dvouřetězcové fragmenty RNA a ssRNA
RISC komplex pak působí po „hybridizaci“ s cílem degradaci RNA,
blok translace či metylaci DNA.
•
RNA-dependent RNA polymerase (RdRP)
Endogenous RdRPs influence RNAi in certain eukaryotes including
fungi, plants, C. elegans. Function to amplify the dsRNA signal.
Catalysis: RdRP copies
mRNA making more ds RNA.
Dicer cuts that generating
more siRNA
More RdRP is activated and
more dsRNA is made.
Spread: dsRNA transported
to other cells
step one:
processing the dsRNA into 21-23 nt fragments
QuickTime™ and a
GIF decompressor
are needed to see this picture.
r
34
27
21
20
16
short-interfering RNA
Dicer contains two RNAse III domains
long dsRNA
siRNAs
siRNAs have a defined structures
19 nt duplex
Rostliny
2 nt 3’ overhangs
siRNA rostlinných virů a miRNA = délka 21-22 nukleotidů
Působí DCL1,2či 4
siRNA endogenního původu (transposony, repetitivní sekv.)=
délka 24 nukleotidů. Působí DCL3
step two:
the antisense strand of the siRNA guides cleavage
Tuschl, Cell 2002
Mechanism of RNAi
Dicer binds dsRNA
And cleaves making
siRNA
siRNAs direct Risc to
copy homologous
mRNA
Dicer binds dsRNA
and cleaves it.
RNAi based methods
History:
Early 1990’s, phenomena first found by plant
scientists: co-suppression
1998, in C.elegans, formally discover dsRNA as signal
for RNA interference
1999, small RNA species derived from mRNA detected
2001, discovery of dsRNA processing enzyme Dicer
More components in RISC identified
Figure 3 | Tobacco plant phenotypes after infection with a satellite-virus-induced silencing system. Results show plant phenotypes four
weeks after infection. Phenotypes caused by the silencing of the genes that encode a | cellulose synthase, b | transketolase and c | phytoene
desaturase are shown. Images courtesy of M. Metzlaff, Ghent, Belgium. Reproduced with permission from Ref. 48 © (2002) Blackwell Publishing.
Please close this window to return to the main article.
Note: some figures may render poorly in a web browser
miRNA genes
•
To date, 1185 miRNA genes have been identified in various
organisms, including approximately 200 in each human,
mouse, and rat genomes.
Několik set u rostlin.
•
Certain miRNAs are
highly conserved among
eukaryotic species.
MicroRNAs (miRNAs)
•
miRNAs are products of endogenous genes and function
through the RNA interference (RNAi) pathway to posttranscriptionally regulate the expression of other genes.
miRNAs in development
•
miRNAs are differentially expressed among various tissue
types and at various stages in cellular differentiation. e.g.
there exist stem cell and neuronal cell specific miRNAs.
•
Disruption of RNAi pathway results in early embryonic
lethality due to depletion of stem cells.
•
Certain miRNAs are known to control developmental
timing and fate specification in C. elegans, leaf
morphogenesis in plants, and hematopoetic lineage
differentiation in mice.
• o miRNA hovoříme tam, kde vznikají z
nedokonalé vlásenky kodované v genomu. Cílem
jejich regulace/inhibice jsou transkripční faktory a
jsou tak důležité pro regulaci vývoje rostlin.
• siRNA vzniká z celé délky dokonale homologní
dvoušroubovicové RNA (produkt RDR nebo příp.
endog. inv. opakování). Jsou produkty i
intermediáty obrany proti
invasivním/aberantním/abundantním RNA jako
jsou vir, transgeny a transposony.
Dvojí shrnující schema.
A
B
RdDM = RNA dependent DNA methylation.
RDR = RNA dependent RNA polymerázy
Stress Responses & Gene Expression
• plants must
adapt to
stresses
because of
their sedentary
lifestyle
Fig. 22.2, Buchanan et al.
Adaptation and Acclimation
• Adaptation - evolutionary changes that enable
an organism to exploit a certain
niche. These
include modification of
existing genes, as
well as gain/loss of
genes.
– e.g., thermophilic enzymes in organisms that tolerate
high temperature
• Acclimation – inducible response(s) that allows
an organism to tolerate an unfavorable or lethal (to
some plants) change in the environment.
– e.g., heat shock response
Types of Stress
Abiotic
1. heat
2. cold
3. drought
4. salt
5. wind
6. oxidative
7. anaerobic
8. heavy metals
9. wounding
10. nutrient deprivation
11. excessive light
Biotic
12. pathogens
13. herbivores
Plants respond to stresses as individual cells and as whole organisms.
Stress-induced signals can be transmitted throughout plant, making other
parts more ready to withstand stress.
Fig. 22.3, Buchanan et al.
Regulace iontové homeostasy
během reakce na zasolení.
Reakce na osmotický stress a
ABA.
Transkripční regulace v odpovědi
na chlad
Heat Stress (or Heat Shock) Response
• Discovered in Drosophila, polytene chromosome
puffing
• Specific response to temperatures ~10-15oC
above normal growth temp.
• Ubiquitous
• Conserved
• Rapid
• Transient
• Dramatic change in pattern of protein synthesis
Heat stress effects on protein synthesis in soybean seedlings (J. Key).
Heat
stress/shock
protein synthesis
in the
cyanobacterium
Synechococcus.
Generalized order of events in heat
shock response
Initial events:
1. Inhibition of protein synthesis
2. Inhibition of transcription & RNA
processing
3. Induction of new hs (hsp) mRNAs
4. Pre-existing cellular mRNAs still present
but not translated (initiation factor
eIF4A & B deactivated)
Phase II events
5. Partial restoration of protein synthesis,
mainly translation of hsp mRNAs – bez
čepičky!!
6. Accumulation of hsp (heat shock
proteins)
7. Gradual resumption of normal cellular
protein synthesis
8. Decline in hsp synthesis
Function: Thermotolerance
• Enables organisms to survive high temp.
• A sub-lethal heat shock allows organisms to
survive a lethal treatment.
• Production of hsps correlate with acquired
thermotolerance.
• Some mutants (yeast) and transgenic plants with
altered expression of certain hsps don’t show
thermotolerance.
28oC
Soybean seedlings.
40oC Æ 45oC
45oC
Fig. 22.42, Buchanan et al.
Heat Shock Proteins (hsp)
• ~100, ~90, ~70, and ~60 kDa
• Low molecular weight (LMW) hsp: ~27, ~20-22,
~15-18 kDa
• all induced within 30 min.
• more LMW hsp in plants
• 2-Dimensional gel electrophoresis and
molecular
cloning indicates most hsps are
families of
related proteins, particularly hsp70 and the LMW
hsps
HSP functions
• 1 LMW hsp is ubiquitin, which tags
proteins to be degraded by the proteasome
• hsp90, hsp70, and hsp60 involved in protein
folding: "molecular chaperones"
• hsp100 may promote translation of hsp
mRNAs, via CAP-independent
mechanism Dominantní regulátor
termotolerance u Arabidopsis.
HSP70, a chaperonin
• Essential gene
• Homologues found in cytoplasm, ER lumen,
mitochondria, and chloroplasts
• function in protein targeting and assembly in
normal
(non-stressed) cells, hydrolyze ATP
• Constitutive & heat-induced (cytoplasmic) forms
– the heat-induced form first appears in the nucleolus, then
goes to cytoplasm (may protect pre-ribosomes from heat
stress?)
• Also, some hsp70s are light-induced; chloroplast hsp70 helps
protect PSII from light/heat damage in Chlamy
HSP60 (cpn60)
• first one termed "molecular chaperone“
• Discovered as the RuBPCase LS binding protein
that participates in RuBPCase assembly
• in eucaryotes, only in mitochondria and plastids
• similar to E. coli GroEL gene
• exists as abundant 720 kD complex with two
subunits of 61 and 60 kDa (ATPase)
• associates with cpn10 (GroES homologue)
• facilitates folding/ assembly of other proteins
LMW HSPs
• highly heat-induced
• 4 nuclear gene families:
1.
2.
3.
4.
Class I cytoplasmic
Class II cytoplasmic
Chloroplast localized
Endomembrane localized (ER)
• found in organelles only in plants
• function mostly unknown
• aggregate in vivo into "heat shock granules"
HSP regulation
• most work on LMW hsp in plants
• induction is mainly transcriptional but
also translational control (hsp mRNAs
preferentially translated)
• genes induced coordinately, but not
equally in all tissues
• light can also induce some LMW hsps
Cis-acting transcriptional regulatory
elements
• HSE (heat shock elements) in the 5' regions:
– ~10-15 bp partial palindromes
– multiple copies required
– also found in other HS genes (e.g., hsp 70)
– similar to HSEs in animals
HSEs in
plants
and
animals.
Heat-shock transcription factor (HSF)
•
•
•
•
•
studied mostly in animals and yeast
Binds to HSEs
Contains leucine zipper motifs
Binds DNA as a trimer
Activity is induced by heat, and
phosphorylation
• Activity Repressed by HSP70
Fig. 22.43, Buchanan et al.
Fig. 22.44, Buchanan et al.
HSFs u Arabidopsis
Sequencing of the Arabidopsis genome revealed a unique complexity of
the plant heat stress transcription factor (Hsf) family. By structural
characteristics and phylogenetic comparison, the 21 representatives are
assigned to 3 classes and 14 groups. Particularly striking is the finding of a
new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs
identified from frequently found expressed sequence tags of tomato,
potato, barley, and soybean. Evidently, this new type of Hsf is well
expressed in different plant tissues. Besides the DNA binding and
oligomerization domains (HR-A/B region), we identified other functional
modules of Arabidopsis Hsfs by sequence comparison with the wellcharacterized tomato Hsfs. These are putative motifs for nuclear import
and export and transcriptional activation (AHA motifs). There is intriguing
flexibility of size and sequence in certain parts of the otherwise strongly
conserved N-terminal half of these Hsfs. We have speculated about
possible exon-intron borders in this region in the ancient precursor gene of
plant Hsfs, similar to the exon-intron structure of the present mammalian
Hsf-encoding genes.
PCD rostlinné
buňky
a vakuola
Geneticky a vývojově řízená sebevražda
buňky.
Programmed Cell Death = PCD
programovaná buněčná smrt
• Concept developed in animal systems
• Each cell has intrinsic genetic program
for ordered cell death
• Cell suicide pathway encoded by genome
of dying cell
• Self-execution and dismantling in the
context of the organism.
Funkce PCD
• Control development and morphogenesis
of multicellular organisms.
• Remove unwanted or displaced cells,
without exposing cell contents to trigger
inflammatory response
• Defense mechanism against pathogens,
cancer, and environmental insults
Apoptosis- specifický typ PCD
•
•
•
Characterized in animal systems
Term coined in 1972 by Kerr
Has a distinct set of morphological features
Specifické symptomy apoptose
•
•
•
•
Shrinkage and vesiculation of cytoplasm
Condensation and shrinkage of nucleus
Condensation of chromatin
Endonuclease cleavage of chromatin in to
300 and/or 50 bp fragments
• Cleavage at DNA linker sites between
nucleosomes, resulting in DNA fragments
multimers of 180 bp
• Results in DNA laddering
• TUNEL assay- (Terminal deoxynucleotidyl
Transferase Biotin-dUTP Nick End Labeling)
• uses terminal deoxynucleotidyl transferase (TdT)
to transfer biotin-dUTP to these strand breaks of
cleaved DNA. The biotin-labeled cleavage sites are
then detected by reaction with HRP conjugated
streptavidin and visualized by DAB showing
brown color.
Důležitou roli hrají
mitochondrie
• Leakage of cytochrome C from
mitochondria into cytoplasm precedes death
a výtok cytochromu C z nich.
• Formation of apoptotic bodies- blebbing of
DNA into membrane-bound bodies and
engulfment by phagocytosis to prevent an
inflammatory response
Živočišná vs. rostlinná buňka = autofágie.
Iniciace PCD je regulována
preteázami - kaspázami
• Regulated by caspases- cysteine proteases
• Very specific proteases known- usually no
more than 1 or 2 breaks substrate
• Orchestrates cell death- eg. Caspase-3 an
executioner caspase that starts a cascade
• Regulated by pro and anti-apoptotic
proteins• BLC-2 like proteins- cytoplasmic proteinsregulate caspase activation
• Trigger or suppress PCD, can also act on
mitochondrial permeability
• Highly conserved among animal kingdom
• Ceramide signalling also involved
INHIBITORY PCD
•
•
•
IAP proteins- inhibitors of apoptosis
Suppress PCD by deactivating caspases
p35 from a baculovirus
Účast PCD na vývoji rostlin
příklady
• Aleurone cells- aleurone in monocots form
a secretory tissue that releases hydrolases to
digest the endosperm and nourish the
embryo. Die after germination is complete.
• Root cap cells -protect the root apical
meristem during seed germination and
growth. Root cap are continually displaced
to the root periphery by new cells.
• Happens when growing in water, not a
caused by abrasion.
• TUNEL positive
•
•
•
Tracheid elements- part of xylem
Functional cells are dead
After they elongate, deposit cell wall
components, including lignin, then undergo
autolysis.
• Actinomycin D or cycloheximide block cell
death
• Leaf senescence- induced by ethylene- a
way of recapturing cellular material for use
in other organs- i.e. to the roots.
• Membrane integrity and cellular
compartmentalization are maintained until
late into the process, so there is little leakage
• Hypoxia- results in aerenchyma formationempty cells with internal air spaces to allow
transfer of O2 from aerial organs to
waterlogged stem bases and roots.
• Can be blocked by mutants in ETR
(ethylene responsive gene)
Hypersensitivní Reakce
hypersens. resp. (HR)
• A hallmark of resistance- non-host, host
specific
• the rapid death of plant cells in
association with the restriction of
pathogen growth
• Effective against biotrophic fungi,
bacteria, and viruses
…je nespecifická a širokospektrá
JE HR FORMOU PCD ?
• Do plant cells show PCD or apoptotic
characteristics during infection?
• Ryerson and Heath 1998showed TUNEL and DNA
laddering in resistant reaction
with soybean rust.
ANO
• PCD seen in cells treated with mycotoxin
• Wang et al. 1996. Tomato treated with AAL
toxin (Alternaria alternata lypcopersici).
• A sphinganine analog mycotoxin
• Saw DNA laddering and TUNEL in AAL- treated
protoplasts and leaf cells
• Also showed TUNEL in root cap cells and leaf
tracheids
• Victorin also causes apoptotic response, including
mitochondrial alterations (Curtis and Wolpert,
2002) Victorin, the host-selective toxin produced
by Cochliobolus victoriae, the causal agent of
victoria blight of oats, has been demonstrated to
bind to the mitochondrial P-protein and also
induces a form of PCD .
(FCCP je mit.
uncoupl.)
Evidence pro PCD v HR
• Lesion mimicks of Arabidopsis• Single-gene mutations in regulatory
pathway results in phenotype similar to HR
• mimick the effect of infection in the absence
of the pathogen. Thus, plants contain an
intrinsic genetic program that initiates and
carries out cell death sentences on infected
cells.
• Lsd mutants- Lsd 1-5 (lesion simulating
disease)
• Acd mutants (accelerated cell death)
• Lls mutants (lethal leaf spot) in maize
Lsd1 mutant
Lsd3 mutant
Lls mutant
Funkční analýza LSD1
• LSD1- gene has been cloned. A zinc-finger
protein needed to restrict lesion size during
HR.
• May be a transcription factor that down
regulates or dampens cell death-a negative
regulator of PCD
• This pro-death pathway requires ROS
LSD1 = Negativní regulátor HR lezí.
• Epple et al. 2003- found a paralogue of
LSD1- called LOL1, acts as a positive
regulator of cell death
• Both affect superoxide dismutase (ROI)
• May regulate SOD- changes superoxide to
H2O2, and the balance of H2O2 and NO
control HR.
Rop GTPázy všude – i v PCD
funkce dalších lokusů PCD
• Lls- codes a putative dioxygenase required
to limit the spread of cell death in leavesmay degrade SA or other phenolic
compound
• Mlo resistance gene from barley- encodes a
transmembrane protein
• Absence of this protein causes a leaf lesion
phenotype and increased resistance
Účastní se kaspázy PCDrostlin ?
• No homologue to animal caspases have been
found- looked through genome of
Arabidopsis.
ale
• Chichkova et al. 2004- identified a
metacaspase in tobacco
• Used a substrate of human caspase-3 (VirD2
from Agrobacterium) as a target, and found
an enzyme that specifically cleaved this in
HR-induced with TMV
• CLPs
• Caspase inhibitors, effective in animal
systems, also block PCD in plants
• Lam and del Pozo, 2000, Del Pozo and Lam,
2003- used p35 from baculovirus, to inhibit
caspase and HR when expressed in tobacco.
• Disrupted N-mediated resistance- TMV
became systemic
• Mutated versions of p35, impaired in
caspase inhibition, were not effective
VPE - vakuolární kaspázy
(Casp.LikeProt.) u rostlin
Vacuolar Processing Enzyme (VPE) je vakuolární cysteinová
proteáza, která štěpí substráty specifické pro kaspázy.
Rostliny s potlačenou expresí
VPE mají částečně potlačen také
kolaps vakuoly při HR.
Ale PCD je celkem normální
u kombinovaných mutantů bez
VPE proteáz…
A co známe živočišné
regulátory PCD – fungují u
rostlin?
• Regulators in animal systems- the Blc-2
family (Bcl-2 and Bax)- have not been found
in plants.
• However, when expressed in plants, affect
PCD
Existují homologní regulátory…
• DAD1- defender against apoptotic cell
death)-highly conserved among both animal
and plant kingdom
• A universal negative regulator of PDC,
homolgues have been found in Arabidopsis,
citrus, apple, tomato
Plant programmed cell death and the point of no return
Wouter G. van Doorn
The point of no return during programmed cell death (PCD) is
defined as the step beyond which the cell is irreversibly
committed to die. Some plant cells can be saved before this
point by inducing the formation of functional chloroplasts. A
visibly senescent tissue will then become green again and live
for months or years. The mechanism of this reversal is only
partially known. The point of no return in fungi and animals is
often associated with lack of mitochondrial function. In plant
cells that do not regreen, there is no evidence for PCD reversal
that results in a long life. It is unclear why chloroplastcontaining cells, in contrast to those with only mitochondria,
have long lives after PCD reversal.
(i) no examples of plant PCD conform to the
apoptotic type
(ii) many examples of PCD during plant
development agree with the autophagic type,
and
(iii) that other examples are apparently neither
apoptotic nor autophagic.
Hlavní nástroj genetické
transformace rostlin je derivátem
biotické interakce mezi
rostlinami a Agrobakteriemi.
Genetic engineering of plants with
Agrobacterium tumefaciens
• A. tumefaciens: used
extensively for genetic
engineering of plants.
• Contains T-DNA
(bacterial plasmid)
• Genes colud be integrated
into the plant
chromosomes when the
T-DNA is transferred.
Tumor induced by
A. tumefaciens
http://courses.washington.edu/z490/gmo/natural.html
Biology of A. tumefaciens
Well known to induce crown gall tumor
A.tumefaciens lives around
root surfaces (in rhizosphere)
where it using nutrients
that leak from the root tissues
infects only through wound sites
and actively chemotactic to them
www-genvagar.slu.se/teknik/ djup/plasm.htm
Bacterial T-plasmid produces
receptors for acetosyringone
Plant wound produces
acetosyringone
The basis of Agrobacteriummediated genetic engineering
• T-DNA of A. tumefaciens is excised and integrates into
the plant genome as part of the natural infection process.
• Any foreign DNA inserted into the T-DNA will also be
integrated.
Important genes encoded by
Ti plasmid
1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
2. Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and
preventing aging)
3. Enzymes for synthesis and release of novel plant
metabolites:
the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives) .
Nopaline
Opines and agrocinopines are NUTRIENTS for A.tumefacies.
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
Opines are nutrients that are also
for quorum sensing
The plant cells start to secrete
the opines
from transferred bacterial T DNA
opine diffuses
into the surrounding cells
and serves as a signal molecules
for the conjugation
of the agrobacterium
(Quorum sensing)
Ti Plasmid
T-DNA
region
Tumorproducing
genes
Opine catabolism
Virulence region
ORI
DNA between
L and R borders is
transferred to plant
as ssDNA;
T-DNA encoded
genes can be
substituted by
target genes
Ti-plasmid based vectors
Binary systems
Co-integrated vectors
Needs 3 vectors
Needs 2 vectors:
Disarmed Ti plasmid
with gene of interest
(no vir genes)
Helper vector
for infection
(with vir genes)
Form
co-integrated plasmid
after homologous
recombination on T-DNA
Disarmed Ti plasmid
capable for infection
Intermediate vector
with T-region
and gene of interest
(transferred by conjugation)
Helper vector
for transfer of
intermediate plasmid into A.tum
Co-integrated vectors
(hybrid ti-plasmids)
Right now
rarely used
DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E. coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use
2) Relatively inefficient gene transfer compared to the binary vecto
Ti plasmid vector systems
are often working as binary vectors
T DNA region removed
Gene of interest
Plant selectable marker
Bacterial selectable
marker
Disarmed
Ti
plasmid
ori for E.coli
Virulence
region
ori for A. tumefaciens
HELPER
plasmid
ori for A. tum
DISADVANTAGE: Depending on the orientation,
plasmids with two different origins of replication may be unstable in E. coli
ADVANTAGE: small vectors are used, which increases transfer efficiency
from E. coli to Agrobacterium.
No intermolecular recombination is needed
Promoters used for expression in
transgenic plants
35S, cauliflower mosaic virus 35S promoter
CaMV is a circular dsDNA genome virus
CaMV 35S is a strong promoter
that is active
in essentially all dicot plant tissues.
s vyjímkou pylu!
Procedure for creation a transgenic plant
1. Both plasmids are transfected into A.tumefaciens
2. Plant cell culture is infected with A.tumefaciens
3. Products of Vir genes excised gene of interest within T-DNA
and transfer it to plant chromosome
T-DNA Repeat
Polylinker
Kan-resistance gene
T-DNA Repeat
Gene of interest
4. Plant cells are selected on kanamycine
5. Presence of transgene confirmed by PCR
6. Whole plant could be grown from transformed cells !!!
U Arabidopsis je možná vysoce
účinná transformace ponořením
mladých květenství do suspenze
Agrobacteria. Dochází posléze k
transformaci zárodečných vaků a
vzniku heterozygotních
transformantů po opylení.
Účinnost je kol. 2%.