Integrace „hormonálních“ signálů - Univerzita...

Integrace „hormonálních“

signálů

příklady z novější doby

Integrace regulací

GA s dalšími

„fytohormony“

IAA spouští zvýšenou produkci GAs.

Narušení transportu IAA brzdí degradaci DELLA represorů působenou GAs.

BR a IAA

Indukují řadu identických mRNA.

IAA inhibuje degradaci cytokininů stimulací cytokinin oxidázy,

Hladiny auxinu a cytokininů inverzně korelují.

Auxin stimuluje produkci etylenu stimulací genu pro syntézu ACC.Etylen inhibuje polární transport auxinu.

Světlo a IAA

Světlo zasahuje do auxinem regulovaných změn v dlouživémrůstu 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

přenosusignálů/regulace genové exprese,

tak

také na úrovni regulace biosyntézy -inhibice či

aktivace akumulace partnerského "fytohormonu".

Plasmodesmata

rostlina jako symplastická síť

Symplastická konektivitaje dynamická, regulovaná!

Plasmodesmy vznikají přibuněčném dělení i de novoa mohou být regulovaněuzavřena.

Buněčně autonomní = molekula působí jen uvnitř buňky.Buněčně neautonomní = molekula působí mezi buňkami.

MP = movement protein

PD=plasmodesmaNCAP = non cellautonom. prot.

injekce FITC-MP bílkoviny;C nepoh. mutant

G – NCAP pomáhá pohybu dextranu.

S transportem jsou spojeny regulované změny SEL (Size

Exclusion Limit).

Mechanismus mezibuněčného pohybu

NCAP=non cellautonom. proteins

Dvoustupňový proces rozbalení a vazby.

je pravděpodobné, že na regulaci SEL se podílí myosin VIII.

Size Exclusion Limit

• 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

Tobacco plant expressing GFP proteinInfected with RNA virus with GFP geneVirus infection travels through veinsGFP expression inhibited starting at veins.

RNA viruses can block expression of a transgene if a copy of the transgene has been added

Gene silencing and RNA virusesshare 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 promoterto transgenic Petunia interfered with expressionof the native homologous’gene.

Gene Silencing

Plant Gene “CoPlant Gene “Co--Suppression”Suppression”

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 stabilityRNAi 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 post-transcriptional 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 RNAimechanism 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!

• Double-stranded RNA (dsRNA) is processed by Dicer, an RNaseIII family member, to produce 21-23nt small interfering RNAs(siRNAs)

• siRNAs are manipulated by a multi-component nucleasecalled the RNA-induced silencing complex (RISC).

• RISC specifically cleaves mRNAs that have perfect complementarity to an siRNA strand

Overview of RNAi

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 meristémovýmutant 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 transportedto other cells

processing the dsRNA into 21-23 nt fragments

3427212016

short-interfering RNA

QuickTime™ and aGIF decompressor

are needed to see this picture.

step one:

r

Dicer contains two RNAse III domains

siRNAs

long dsRNA

siRNAs have a defined structures19 nt duplex

2 nt 3’ overhangs

siRNA rostlinných virů a miRNA = délka 21-22 nukleotidůPůsobí DCL1,2či 4siRNA endogenního původu (transposony, repetitivní sekv.)=délka 24 nukleotidů. Působí DCL3

Rostliny

Tuschl, Cell 2002

the antisense strand of the siRNA guides cleavagestep two:

Mechanism of RNAi

Dicer binds dsRNAAnd cleaves makingsiRNA

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 arehighly conserved amongeukaryotic species.

MicroRNAs (miRNAs)• miRNAs are products of endogenous genes and function

through the RNA interference (RNAi) pathway to post-transcriptionally 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

RdDM = RNA dependent DNA methylation.RDR = RNA dependent RNA polymerázy

B

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 StressAbiotic

1. heat2. cold3. drought4. salt5. wind6. oxidative7. anaerobic8. heavy metals9. wounding10. nutrient deprivation11. excessive light

Biotic12. pathogens13. 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 cyanobacteriumSynechococcus.

Generalized order of events in heat shock response

Initial events:1. Inhibition of protein synthesis 2. Inhibition of transcription & RNA

processing3. Induction of new hs (hsp) mRNAs4. 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 synthesis8. 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 40oC 45oC 45oC

Fig. 22.42, Buchanan et al.Soybean seedlings.

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. Class I cytoplasmic2. Class II cytoplasmic3. Chloroplast localized4. 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.

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 areassigned to 3 classes and 14 groups. Particularly striking is the finding of anew 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 well-characterized tomato Hsfs. These are putative motifs for nuclear importand export and transcriptional activation (AHA motifs). There is intriguingflexibility of size and sequence in certain parts of the otherwise stronglyconserved 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.

HSFs u Arabidopsis

PCD rostlinné buňky

a vakuola

Geneticky a vývojově řízená sebevraždaGeneticky a vývojově řízená sebevraždabuňky.buňky.

Programmed Cell DeathProgrammed Cell Death = = PCDPCDprogramovaná buněčná smrtprogramovaná buněčná smrt

•• Concept developed in animal systemsConcept developed in animal systems•• Each cell has intrinsic genetic program Each cell has intrinsic genetic program

for ordered cell deathfor ordered cell death•• Cell suicide pathway encoded by genome Cell suicide pathway encoded by genome

of dying cellof dying cell•• SelfSelf--execution and dismantling execution and dismantling in the in the

context of the organismcontext of the organism..

Funkce PCDFunkce PCD

•• Control development and Control development and morphogenesismorphogenesisof of multicellularmulticellular organisms.organisms.

•• Remove unwanted or displaced cells, Remove unwanted or displaced cells, without exposing cell contents to trigger without exposing cell contents to trigger inflammatory responseinflammatory response

•• Defense mechanism against pathogens, Defense mechanism against pathogens, cancer, and environmental insultscancer, and environmental insults

ApoptosisApoptosis-- specifický typ PCDspecifický typ PCD

•• Characterized in animal systemsCharacterized in animal systems•• Term coined in 1972 by KerrTerm coined in 1972 by Kerr•• Has a distinct set of morphological featuresHas a distinct set of morphological features

Specifické symptomy aSpecifické symptomy apoptospoptosee

•• Shrinkage and Shrinkage and vesiculationvesiculation of cytoplasmof cytoplasm•• Condensation and shrinkage of nucleusCondensation and shrinkage of nucleus•• Condensation of chromatinCondensation of chromatin•• EndonucleaseEndonuclease cleavage of chromatin in to cleavage of chromatin in to

300 and/or 50 300 and/or 50 bpbp fragmentsfragments

•• Cleavage at DNA linker sites between Cleavage at DNA linker sites between nucleosomesnucleosomes, resulting in DNA fragments , resulting in DNA fragments multimersmultimers of 180 of 180 bpbp

•• Results in DNA ladderingResults in DNA laddering

•• TUNELTUNEL assayassay-- ((Terminal Terminal deoxynucleotidyldeoxynucleotidylTransferaseTransferase BiotinBiotin--dUTPdUTP Nick End LabelingNick End Labeling))

•• uses terminal uses terminal deoxynucleotidyldeoxynucleotidyl transferasetransferase ((TdTTdT) ) to transfer biotinto transfer biotin--dUTPdUTP to these strand breaks of to these strand breaks of cleaved DNA. The biotincleaved DNA. The biotin--labeled cleavage sites are labeled cleavage sites are then detected by reaction with HRP conjugated then detected by reaction with HRP conjugated streptavidinstreptavidin and visualized by DAB showing and visualized by DAB showing brown color. brown color.

Důležitou roli hrají Důležitou roli hrají mitochondrie mitochondrie

•• Leakage of Leakage of cytochromecytochrome C C from from mitochondria mitochondria into cytoplasm precedes deathinto cytoplasm precedes death

a výtok cytochromu C z nich.

•• Formation of apoptotic bodiesFormation of apoptotic bodies-- blebbingblebbing of of DNA into membraneDNA into membrane--bound bodies and bound bodies and engulfment by engulfment by phagocytosisphagocytosis to prevent an to prevent an inflammatory responseinflammatory response

Živočišná vs. rostlinná buňka = autofágie.

•• Regulated by Regulated by caspasescaspases-- cysteinecysteine proteasesproteases•• Very specific proteases knownVery specific proteases known-- usually no usually no

more than 1 or 2 breaks substratemore than 1 or 2 breaks substrate•• Orchestrates cell deathOrchestrates cell death-- egeg. . CaspaseCaspase--3 an 3 an

executioner executioner caspasecaspase that starts a cascadethat starts a cascade

Iniciace PCD je regulována preteázami - kaspázami

•• Regulated by pro and antiRegulated by pro and anti--apoptotic apoptotic proteinsproteins--

•• BLCBLC--2 like proteins2 like proteins-- cytoplasmiccytoplasmic proteinsproteins--regulate regulate caspasecaspase activationactivation

•• Trigger or suppress PCD, can also act on Trigger or suppress PCD, can also act on mitochondrialmitochondrial permeabilitypermeability

•• Highly conserved among animal kingdomHighly conserved among animal kingdom•• CeramideCeramide signallingsignalling also involvedalso involved

•• IAP proteinsIAP proteins-- inhibitors of apoptosis inhibitors of apoptosis •• Suppress PCD by deactivating Suppress PCD by deactivating caspasescaspases•• p35 from a p35 from a baculovirusbaculovirus

INHIBITORY PCD

Účast PCD na vývoji rostlinÚčast PCD na vývoji rostlinpříkladypříklady

•• AleuroneAleurone cellscells-- aleuronealeurone in monocots form in monocots form a a secretorysecretory tissue that releases tissue that releases hydrolaseshydrolases to to digest the endosperm and nourish the digest the endosperm and nourish the embryo. Die after germination is complete.embryo. Die after germination is complete.

•• Root cap cells Root cap cells --protect the root apical protect the root apical meristemmeristem during seed germination and during seed germination and growth. Root cap are continually displaced growth. Root cap are continually displaced to the root periphery by new cells. to the root periphery by new cells.

•• Happens when growing in water, not a Happens when growing in water, not a caused by abrasion.caused by abrasion.

•• TUNEL positiveTUNEL positive

•• TracheidTracheid elementselements-- part of xylempart of xylem•• Functional cells are deadFunctional cells are dead•• After they elongate, deposit cell wall After they elongate, deposit cell wall

components, including lignin, then undergo components, including lignin, then undergo autolysisautolysis. .

•• ActinomycinActinomycin D or D or cycloheximidecycloheximide block cell block cell deathdeath

•• Leaf senescenceLeaf senescence-- induced by ethyleneinduced by ethylene-- a a way of recapturing cellular material for use way of recapturing cellular material for use in other organsin other organs-- i.e. to the roots. i.e. to the roots.

•• Membrane integrity and cellular Membrane integrity and cellular compartmentalization are maintained until compartmentalization are maintained until late into the process, so there is little leakagelate into the process, so there is little leakage

•• HypoxiaHypoxia-- results in results in aerenchymaaerenchyma formationformation--empty cells with internal air spaces to allow empty cells with internal air spaces to allow transfer of Otransfer of O2 2 from aerial organs to from aerial organs to waterlogged stem bases and roots.waterlogged stem bases and roots.

•• Can be blocked by mutants in ETRCan be blocked by mutants in ETR(ethylene responsive gene)(ethylene responsive gene)

HypersensitivHypersensitivníní ReReakce akce hypersenshypersens. resp. (HR). resp. (HR)

•• A hallmark of resistanceA hallmark of resistance-- nonnon--host, host host, host specificspecific

•• the rapid death of plant cells in the rapid death of plant cells in association with the restriction of association with the restriction of pathogen growthpathogen growth

•• Effective against Effective against biotrophicbiotrophic fungi, fungi, bacteria, and virusesbacteria, and viruses

…je nespecifická a širokospektrá

JE JE HR HR FORMOUFORMOU PCD ?PCD ?

•• Do plant cells show PCD or apoptotic Do plant cells show PCD or apoptotic characteristics during infection?characteristics during infection?

•• RyersonRyerson and Heath 1998and Heath 1998--showed TUNEL and DNA showed TUNEL and DNA laddering in resistant reaction laddering in resistant reaction with soybean rust.with soybean rust.

A N O

•• PCD seen in cells treated with PCD seen in cells treated with mycotoxinmycotoxin•• Wang et al. 1996. Tomato treated with AAL Wang et al. 1996. Tomato treated with AAL

toxin (toxin (AlternariaAlternaria alternataalternata lypcopersicilypcopersici).).

•• A A sphinganinesphinganine analog analog mycotoxinmycotoxin•• Saw DNA laddering and TUNEL in AALSaw DNA laddering and TUNEL in AAL-- treated treated

protoplasts and leaf cellsprotoplasts and leaf cells•• Also showed TUNEL in root cap cells and leaf Also showed TUNEL in root cap cells and leaf

tracheidstracheids•• VictorinVictorin also causes apoptotic response, including also causes apoptotic response, including

mitochondrialmitochondrial alterations (Curtis and alterations (Curtis and WolpertWolpert, , 2002)2002) VictorinVictorin,, thethe hosthost--selectiveselective toxintoxin producedproducedbyby Cochliobolus victoriaeCochliobolus victoriae,, the causalthe causal agentagent of of victoria blight of oatsvictoria blight of oats, has, has been demonstratedbeen demonstrated totobindbind toto the mitochondrialthe mitochondrial PP--proteinprotein and also and also inducesinduces aa form ofform of PCD .PCD .

(FCCP je mit.uncoupl.)

Evidence Evidence propro PCD PCD vv HRHR

•• Lesion Lesion mimicksmimicks of of ArabidopsisArabidopsis--•• SingleSingle--gene mutations in regulatory gene mutations in regulatory

pathway results in phenotype similar to HRpathway results in phenotype similar to HR•• mimickmimick the effect of infection in the absence the effect of infection in the absence

of the pathogen. Thus, plants contain an of the pathogen. Thus, plants contain an intrinsic genetic program that initiates and intrinsic genetic program that initiates and carries out cell death sentences on infected carries out cell death sentences on infected cells.cells.

•• LsdLsd mutantsmutants-- LsdLsd 11--55 ((llesion esion ssimulating imulating ddisease) isease)

•• AcdAcd mutants (mutants (aaccelerated ccelerated ccell ell ddeath)eath)•• LlsLls mutants (mutants (llethal ethal lleaf eaf sspot) in maizepot) in maize

Lsd1 Lsd1 mutantmutant

Lsd3 Lsd3 mutantmutant

LlsLls mutantmutant

Funkční analýza LSD1Funkční analýza LSD1

•• LSD1LSD1-- gene has been cloned. A zincgene has been cloned. A zinc--finger finger protein needed to protein needed to restrict lesion sizerestrict lesion size during during HR. HR.

•• May be a transcription factor that down May be a transcription factor that down regulates or dampens cell deathregulates or dampens cell death--a negative a negative regulator of PCDregulator of PCD

•• This proThis pro--death pathway requires ROSdeath pathway requires ROSLSD1 = Negativní regulátor HR lezí.

•• EppleEpple et al. 2003et al. 2003-- found a found a paralogueparalogue of of LSD1LSD1-- called called LOL1, acts as a positiveLOL1, acts as a positiveregulatorregulator of cell deathof cell death

•• Both affect Both affect superoxidesuperoxide dismutasedismutase (ROI)(ROI)•• May regulate SODMay regulate SOD-- changes changes superoxidesuperoxide to to

HH22OO22, and the balance of H, and the balance of H22OO22 and NO and NO control HR.control HR.

Rop GTPázy všude – i v PCD

funkce dalších funkce dalších lokusů lokusů PCDPCD

•• LlsLls-- codes a putative codes a putative dioxygenasedioxygenase required required to limit the spread of cell death in leavesto limit the spread of cell death in leaves--may degrade SA or other may degrade SA or other phenolicphenoliccompoundcompound

•• MloMlo resistance gene from barleyresistance gene from barley-- encodes a encodes a transmembranetransmembrane proteinprotein

•• Absence of this protein causes a leaf lesion Absence of this protein causes a leaf lesion phenotype and increased resistancephenotype and increased resistance

Účastní se Účastní se kaspázy kaspázy PCDPCDrostlin rostlin ??

•• No homologueNo homologue to animal to animal caspasescaspases have been have been foundfound-- looked through genome of looked through genome of ArabidopsisArabidopsis..

aleale

•• ChichkovaChichkova et al. 2004et al. 2004-- identified a identified a metacaspasemetacaspase in tobaccoin tobacco

•• Used a substrate of human Used a substrate of human caspasecaspase--3 (VirD2 3 (VirD2 from from AgrobacteriumAgrobacterium) as a target, and found ) as a target, and found an enzyme that specifically cleaved this in an enzyme that specifically cleaved this in HRHR--induced with TMVinduced with TMV

•• CLPsCLPs

•• CaspaseCaspase inhibitors, effective in animal inhibitors, effective in animal systems, also block PCD in plantssystems, also block PCD in plants

•• Lam and del Lam and del PozoPozo, 2000, Del , 2000, Del PozoPozo and Lam, and Lam, 20032003-- used p35 from used p35 from baculovirusbaculovirus, to inhibit , to inhibit caspasecaspase and HR when expressed in tobacco.and HR when expressed in tobacco.

•• Disrupted NDisrupted N--mediated resistancemediated resistance-- TMV TMV became systemicbecame systemic

•• Mutated versions of p35, impaired in Mutated versions of p35, impaired in caspasecaspase inhibition, were not effectiveinhibition, were not effective

VPE - vakuolární kaspázy (Casp.LikeProt.) u rostlin

Vacuolar Processing Enzyme (VPE) je vakuolární cysteinováproteáza, 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 A co známe živočišné regulátoryregulátory PCDPCD –– fungují u fungují u

rostlinrostlin??•• Regulators in animal systemsRegulators in animal systems-- the the BlcBlc--2 2

family (family (BclBcl--2 and 2 and BaxBax))-- have not been found have not been found in plants.in plants.

•• HoweverHowever, when expressed in plants, affect , when expressed in plants, affect PCDPCD

Existují Existují homologní homologní regulátory…regulátory…

•• DAD1DAD1-- ddefender efender aagainst gainst aapoptotic cell poptotic cell death)death)--highly conserved among both animal highly conserved among both animal and plant kingdomand plant kingdom

•• A universal negative regulator of PDC, A universal negative regulator of PDC, homolgueshomolgues have been found in have been found in ArabidopsisArabidopsis, , citrus, apple, tomatocitrus, apple, tomato

Plant programmed cell death and the point of no returnWouter 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 thispoint by inducing the formation of functional chloroplasts. Avisibly senescent tissue will then become green again and livefor 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 chloroplast-containing 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.

http://courses.washington.edu/z490/gmo/natural.html

Tumor induced byA. tumefaciens

Biology of A. tumefaciens

www-genvagar.slu.se/teknik/ djup/plasm.htm

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 sitesand actively chemotactic to them

Plant wound produces acetosyringone

Bacterial T-plasmid produces receptors for acetosyringone

The basis of Agrobacterium-mediated 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.

1. Cytokinins(plant hormone for cell plant division and tumorous growth)

2. Enzymes for indoleacetic acid (auxin) synthesisAnother 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) .

Opines and agrocinopines are NUTRIENTS for A.tumefacies. They can not be used by other bacterial species

It provides unique niche for A.tumefaciens

Important genes encoded by Ti plasmid

Nopaline

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

Tumor-producing genes

Virulence region

Opine catabolism

ORI

T-DNAregion

DNA between L and R borders istransferred to plantas ssDNA;

T-DNA encoded genes can be substituted by target genes

Ti-plasmid based vectors

Binary systems Co-integrated vectors

Needs 2 vectors: Needs 3 vectors

Disarmed Ti plasmidwith gene of interest(no vir genes)

Helper vectorfor infection(with vir genes)

Disarmed Ti plasmidcapable for infection

Intermediate vectorwith T-region and gene of interest (transferred by conjugation)

Form co-integrated plasmidafter homologous recombination on T-DNA

Helper vectorfor transfer of intermediate plasmid into A.tum

Co-integrated vectors (hybrid ti-plasmids)

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

Right now rarely used

Ti plasmid vector systems are often working as binary vectors

Virulence region

T DNA region removed

ori for A. tum

Gene of interest

Plant selectable marker

Bacterial selectable marker

ori for A. tumefaciensori for E.coli

HELPER plasmidDisarmed

Tiplasmid

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 35S is a strong promoter that is active

in essentially all dicot plant tissues.

CaMV is a circular dsDNA genome virus

s vyjímkou pylu!

Procedure for creation a transgenic plant1. 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-DNAand transfer it to plant chromosome

Polylinker Kan-resistance geneT-DNA Repeat T-DNA Repeat

Gene of interest

4. Plant cells are selected on kanamycine5. Presence of transgene confirmed by PCR

6. Whole plant could be grown from transformed cells !!!

U Arabidopsis je možná vysoce účinná transformace ponořenímmladých květenství do suspenzeAgrobacteria. Dochází posléze k transformaci zárodečných vaků a

vzniku heterozygotních transformantů po opylení.

Účinnost je kol. 2%.