Phenotypes on demand via switchable target protein degradation in multicellular organisms

15 Pages·2016·4.65 MB·English

by  Frederik Faden

ARTICLE
Received12Jan 2016 |Accepted 10Jun2016 |Published 22 Jul2016 DOI:10.1038/ncomms12202 OPEN
Phenotypes on demand via switchable target
protein degradation in multicellular organisms
FrederikFaden1,2,ThomasRamezani3,4,w,StefanMielke1,2,IsabelAlmudi5,w,KnudNairz5,w,MarceliS.Froehlich6,w,
Jo¨rg Ho¨ckendorff6,w, Wolfgang Brandt7, Wolfgang Hoehenwarter8, R. Ju¨rgen Dohmen6, Arp Schnittger3,4,9,w
& Nico Dissmeyer1,2,3,4,9
Phenotypes on-demand generated by controlling activation and accumulation of proteins of
interestareinvaluabletoolstoanalyseandengineerbiologicalprocesses.Whiletemperature-
sensitive alleles are frequently used as conditional mutants in microorganisms, they
are usually difficult to identify in multicellular species. Here we present a versatile and
transferable, genetically stable system based on a low-temperature-controlled N-terminal
degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels
under physiological conditions in vivo. Thereby, developmental effects can be triggered and
phenotypesondemandgenerated.Thelt-degronwasestablishedtoproduceconditionaland
cell-type-specific phenotypes and is generallyapplicablein a wide range of organisms, from
eukaryotic microorganisms to plants and poikilothermic animals. We have successfully
applied this system to control the abundance and function of transcription factors and
different enzymes by tunable protein accumulation.
1IndependentJuniorResearchGrouponProteinRecognitionandDegradation,LeibnizInstituteofPlantBiochemistry(IPB),Weinberg3,D-06120Halle
(Saale),Germany.2ScienceCampusHalle—Plant-basedBioeconomy,Betty-Heimann-Strasse3,D-06120Halle(Saale),Germany.3UniversityGroupatthe
MaxPlanckInstituteforPlantBreedingResearch(MPIPZ),MaxDelbru¨ckLaboratory,Carl-von-Linne´-Weg10,D-50829Cologne,Germany.4Universityof
Cologne,InstituteofBotanyIII,Biocenter,Zu¨lpicherStr.47b,D-50674Cologne,Germany.5InstituteofMolecularSystemsBiology(IMSB),SwissFederal
InstituteofTechnology(ETH),Wolfgang-Pauli-Strasse16,CH-8093Zurich,Switzerland.6InstituteforGenetics,Biocenter,UniversityofCologne,Zu¨lpicher
Stra(cid:2)e47a,D-50674Cologne,Germany.7ComputationalChemistry,DepartmentofBioorganicChemistry,LeibnizInstituteofPlantBiochemistry(IPB),
Weinberg3,D-06120Halle(Saale),Germany.8ProteomicsUnit,LeibnizInstituteofPlantBiochemistry(IPB),Weinberg3,Halle(Saale)D-06120,Germany.
9De´partementMe´canismesMole´culairesdelaPlasticite´Phe´notypique,InstitutdeBiologieMole´culairedesPlantesduCNRS,IBMP-CNRS,Unite´Proprede
Recherche2357,Conventionne´avecl’Universite´deStrasbourg,12,rueduGe´ne´ralZimmer,StrasbourgF-67000,France.wPresentaddresses:TheQueen’s
MedicalResearchInstitute,MRCCentreforInflammationResearch,TheUniversityofEdinburgh,47LittleFranceCrescent,EdinburghEH164TJ,UK(T.R.);
AndalusianCentreforDevelopmentalBiology(CABD),UniversidadPablodeOlavide,Ctra.UtreraKm.1,41013Seville,Spain(I.A.);NovinoSlowdrinkGmbH,
Blu¨mlisalpstrasse31,CH-8006Zu¨rich,SwitzerlandandInselspitalBern,Freiburgstrasse10,CH-3010Bern,Switzerland(K.N.);Gru¨nenthalInnovation—Early
ClinicalDevelopment,Gru¨nenthalGmbH,Zieglerstra(cid:2)e6,D-52078Aachen,Germany(M.S.F.);UCBBIOSCIENCESGmbH,Alfred-Nobel-Stra(cid:2)e10,
D-40789Monheim,Germany(J.H.);DepartmentofDevelopmentalBiology,BiozentrumKleinFlottbek,UniversityofHamburg,Ohnhorststrasse18,D-22609
Hamburg,Germany(A.S.).CorrespondenceandrequestsformaterialsshouldbeaddressedtoN.D.(email:[email protected]).
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T
he classical way to generate temperature-sensitive alleles temperatures of 37 or 42(cid:2)C (refs 1,16,17). These high
comprises random mutagenesis followed by large-scale temperatures are beyond the physiological range of many
screensatdifferenttemperatures.Thisprocedureisusually multicellular organisms, including animals and plants18.
limited to fast-growing single-celled organisms where mutant Although there are alternative methods for conditional protein
populations can be analysed simultaneously at restrictive and shut-off via degradation, for example, the protease-mediated
permissive temperatures. Major problems in using temperature- TIPI19, the destabilizing domain (DD) systems20 and the auxin-
sensitivealleles aretheirgeneration, identification, establishment inducible degron (AID)21, their use is also limited to cells in
and leakiness. An alternative way to generate heat-sensitive culture22. The latter two systems require the addition of
mutantsistouseatemperature-inducibleN-degron(td),wherea exogenous compounds to trigger the response (Supplementary
protein of interest (POI) is fused to a portable N-terminal Note 1).
degradation cassette1. However, to date this technique has been To fill the gap of a conditional protein accumulation system
solely used in unicellular eukaryotes in a temperature range for multicellular systems under physiological conditions, we
impractical for most multicellular organisms. We show how to constructedalow-temperature(lt)N-degronandshowitsgeneral
efficientlycircumventtheselimitations byusinganovelportable applicability by studying seven POIs in five different model
td fusion protein to render the levels of active POIs conditional. systems (that is, two plant species (Arabidopsis thaliana and
Generally, degradation signals within a protein sequence that Nicotiana benthamiana (tobacco)), Drosophila melanogaster
makeitshort-livedinvivoorinvitroarecalled‘degrons’2,andan (fruit fly), Drosophila cell culture and Saccharomyces cerevisiae
‘N-degron’3 is an amino-terminal (N-terminal) degron, which (budding yeast). The design of the lt-degron not only allowed
relates the metabolic stability of a protein to the identity of its in vivo inactivation and depletion of proteins and enzymes but
amino-terminal residue depending on the N-end rule pathway4. also tuning of protein levels and enzyme activity. Depending
TheN-endrulepathwayispartoftheubiquitin(Ub)/proteasome on the POI, we demonstrated triggering of developmental
system and has been shown to be active in yeast, animals and processes on demand. We define the temporal requirement of a
plants5–9. It maintains proteostasis as a protein quality control transcriptional regulator protein mandatory for the formation of
mechanismbyremovingcleaved,damagedormisfoldedproteins trichomes, which belong to the prime model cells in plant
from the cell7,10,11. N-degrons comprise several determinants to developmental genetics. Moreover, we made use of a develop-
target a substrate to the N-end rule pathway. First, they must mental switch by stabilizing a transcription factor causing
contain a destabilizing N-terminal amino acid that can be flowering on demand, triggered protein accumulation in
recognized by N-end rule pathway-specific E3 Ub ligases N.benthamiana(tobacco),conditionallydepletedproteinsincell
(N-recognins). Second, another crucial factor is a certain culture and Drosophila flies. Therefore, the link to in-depth
flexibility and accessibility of the N-terminal amino acid enabling applications in developmental studies is addressed as well as
a proper recognition of the substrate12. Third, substrates need to the proof of concept for applications of the lt-degron in
contain at least one internal Lys in appropriate distance to the N biotechnologically relevant contexts.
terminus,whichmayserveaspolyubiquitinationsite3.TheN-end
rulepathwaytargetsbothcytosolicandnuclearsubstratesbutalso Results
proteins localized inthe membrane13. N-degron functionality in multicellular organisms. To assess
Conditional protein expression via heat-sensitive N-degrons N-degron functionality in multicellular organisms, we chose
was designed as a genetic tool to generate conditional, easily scorable, quantitative and irreversible biological read-outs
temperature-inducible alleles in budding yeast1. The system is after temperature shifts in vivo, like the development of
basedonrapid,reversibledepletionoraccumulationofPOIsand Arabidopsis trichomes (leaf hairs) and flower induction. We first
targetstheentireproteinforproteasome-dependentproteolysisat asked whether the established yeast td degron containing the
a restrictive temperature. Therefore, it can be used as a rapid single-mutated DHFRP67L (termed ‘K1’), exhibiting a restrictive
ON/OFF system for reversible accumulation of active proteins temperature of 37–42(cid:2)C (refs 1,16,17,23) is functional in plants.
(Fig.1a,bandSupplementaryFig.1).HereanN-degronservesas Our DHFR was initiated by phenylalanine (Phe), a strongly
a destabilizing N-terminal tag (26kDa) fused to the POI as a destabilizingresidueinplants,whichispresumablyrecognizedby
portabletdcassette. It consistsof threevital parts:(1)Ubthatis the bona fide N-end rule E3 Ub ligase PROTEOLYSIS1
co-translationally removed from the fusion by deubiquitinating (PRT1)24–26. As the first test system, we used the formation of
enzymes (Ub-fusion technique14); (2) a thermo-labile mouse Arabidopsistrichomes,whichareawell-establishedmodelincell
dihydrofolate reductase (DHFRts) that triggers protein anddevelopmentalbiology27–29,andanattractivetargetforplant
degradation at the restrictive (high) temperature; and (3) a metabolicengineering.30WD40proteinTRANSPARENTTESTA
destabilizing N-terminal residue at the Ub–DHFRts junction, GLABRA1 (TTG1) is an essential regulator for trichome
which is exposed after deubiquitination of the full fusion development31, ttg1 mutants are devoid of trichomes (Figs 1c
construct, which serves as dormant N-degron and can be and 2a) but the mutant phenotype can be rescued by expressing
recognized by N-end rule E3 Ub ligases (Supplementary Fig. 1). TTG1 under control of the constitutive cauliflower mosaic virus
Even if a potential degron (that is, a motif theoretically (CaMV) 35S promoter (Pro35S)32. In this evaluation system, the
recognized by a N-end rule pathway E3 Ub ligase) is present on read-out for a functional N-degron is the number of trichomes
the surface of a target, its conformational rigidity may prevent formed per leaf after the shift from permissive to restrictive
its recognition due to steric hindrance. A conformationally temperature. Under permissive conditions, the degron is non-
destabilizedproteinmaycontainformerlyburied(cryptic)surface functional, that is, the POI will accumulate and form trichomes,
degrons (that is, dormant degrons), which were masked in a whereas under restrictive conditions, the degron is active and
stabilizedversionoftheprotein15.Intheclassicalyeasttddegron, leads to the degradation of the fusion protein, and leaves will be
both happens according to the presented model1: a previously devoid of trichomes.
rigid surface degron (N-terminal) undergoes conformational To this end, transgenic ttg1 mutant Arabidopsis plants were
relaxation and previously buried degrons, such as internal Lys generated that expressed TTG1 fused to degron cassette K1
side chains, are exposed. (Supplementary Fig. 2). We set the presumptive maximal
However,todate,thissystemhasonlybeenusedatthesingle- restrictive temperature treatment to 29(cid:2)C, which is near but
cell level in yeast and cell culture where it requires restrictive notabovetheupperendofthephysiologicaltemperaturerangeof
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a c
Degron cassette ttg1 Pro35S::K2:TTG1
13 °C 29 °C
DNA
construct 5′-35S-Ω Ub F-eL DHFRts HAT GOI -3′
“K“
Degron cassette
Fusion N- Ub F-eL DHFRts HAT POI -C
protein “K“
b
Cassette K2
(Phe-1)
d e
ttg1 Pro35S::K2:TTG1 ttg1 Pro35S::K2:TTG1
13 °C 29 °C 13 °C 25 °C
Min h Min h
WT
t 0 10 30 60 90 2.53.5 4.5 24 t 0 10 30 1 2 4 8 1216 24 3240
IB: α-HA
IB: α-HA
K2:TTG1 70 kDa 70 kDa
K2:TTG1
Loading 55 kDa
(Ponceau) Loading
55 kDa
(Ponceau)
29 °C 13 °C
IB: α-HA t 0 5 10 30M4in5 6090 120 24 h Min 13 °Ch WT
K2:TTG1 70 kDa t 0 10 30 1 2 4 8 12 16 24 32 40
IB: α-HA
Loading 55 kDa 70 kDa
(Ponceau) K2:TTG1
Loading 55 kDa
f ttg1 Pro35S::K2:TTG1 (Ponceau)
prt1 WT
C C C C
13 ° 29 ° 13 ° 29 °
IB: α-HA
K2:TTG1
70 kDa
Loading 55 kDa
(CBB)
Figure1|Conditionalproteindepletion/accumulationcausedevelopmentalphenotypesondemandinvivoandcanbeusedtodeveloptrichomecells.
(a,b)Lt-degronarchitecture.ThemechanismofconditionalN-degroncontainingproteindepletionisshowninSupplementaryFig.1,degronconstruct
designisshowninSupplementaryFig.2.(c)ttg1K2:TTG1plantsgrownatpermissiveorrestrictivetemperatures.Arrowheadsindicatetrichomesininlets.
Scalebar,1cm,5mm(inlet).(d)InvivoproteindepletiontimecourseofK2:TTG1fromtransgenicttg1Pro35S::K2:TTG1mutantplants.(e)Invivodimmingof
K2:TTG1levelsbyapplicationofsemi-permissivetemperatures.Plantsweresubjectedtotemperature-shiftexperimentsfrom13to25(cid:2)C.Westernblotsof
plantsshiftedtosemi-restrictive(25(cid:2)C)andkeptatconstitutivelypermissivetemperature(13(cid:2)C)areshown.Plantswereshiftedatt¼0from13to25(cid:2)C
(upperpanel)orkeptat13(cid:2)C(lowerpanel).(f)StabilizationofK2:TTGinprt1.TheK2:TTGfusionproteinstabilizesintheprt1mutantbackgroundunder
restrictivetemperatureindicatingthattheE3ligasePRT1istheprincipalcomponentresponsibleforK2degradation.K2:TTGalsopartiallyaccumulates
underpermissivetemperaturesintheprt1mutantbackgroundcomparedwiththeWTindicatingthatalsohere,thefusionproteinisnotfullystabilized.All
samplesderivedfromthesameexperiment,processedinparallelandleavesofthesamedevelopmentalstagewerecompared.Datawereconfirmedby
analysisofatleastthreebiologicalreplicates.EqualloadingwasfurtherconfirmedbyPonceauSstainingofblottedandimmunostainedmembranes.
Arabidopsis18andfoundthatK1:TTG1stablytransformedplants screen for destabilizing point mutations within DHFR (Supple-
developed trichomes also at this temperature indicating that the mentary Note 2)33. However, ttg1 mutants containing K3:TTG1
fusion protein is not sufficiently destabilized (Supplementary neverformedtrichomesunderanytestedconditions.Theprotein
Table 1). appearedtobeunstableaftergrowingtheplantsatconstitutively
Tolowertherestrictivetemperature,wegeneratedanN-degron permissive or restrictive temperatures or shifting them between
variant containing a triple mutated DHFRT39A/P67L/E173D (K3) the two conditions (Supplementary Fig. 3 and Supplementary
with two additional substitutions (T39A/E173D). We had Table 1). Lack of trichomes under low temperatures correlated
previously identified the latter mutations in a yeast mutagenesis with levels of K3:TTG1 protein undetectable by western blot
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a Wild type ttg1
Leaf 1/2 Leaf 3/4 Leaf 1/2 Leaf 3/4
Constitutive
29 °C
16 °C
for 72 h
Constitutive
16 °C
b ttg1 Pro35S::K2:TTG1
(temperature pulses: 29 °C > 16 °C > 29 °C)
Leaf 1/2 Leaf 3/4 Epidermis Trichomes
Constitutive
29 °C
16 °C for
2 h
8 h
16 h
48 h
72 h
Constitutive
16 °C
c ttg1 Pro35S::K2:TTG1 (temperature shifts)
16 °C 29 °C 16 °C > 29 °C 29 °C > 16 °C
Figure2|Spatiotemporaldevelopmentoftrichomesastestsystemforconditionalproteinaccumulation.(a)Phenotypeofttg1mutantscomparedwith
theWT.Cryoscanningelectronmicrographsweretakenfrom4-week-oldplantsgrownconstitutivelyat29or16(cid:2)C.Scalebar,1mm.(b,c)Conditional
complementationofttg1withK2:TTG1dependingontemperatureandtime.SEMsof4-week-oldttg1K2:TTG1plants.(b)Temperature-pulsingofK2:TTG1
proteininttg1byshortshiftsfromtherestrictivetothepermissivetemperature(16(cid:2)C).Controlplantsweregrownconstitutivelyatrestrictive(29(cid:2)C,
upperrow)orpermissive(16(cid:2)C,bottomrow)temperatures.Centre:plantsgrownatrestrictiveandshiftedtopermissivetemperatureforthetimeindicated
(2–48h).Redlines:bordersoftrichomeinitiationzonemovingfromlateraltomedialwithdurationoftheK2:TTG1pulse.Greencircles:fullymature
trichomesandepidermalpatterningappearingafter48h.Arrowheads:lopedcircumferenceattrichomebases.Scalebars,1mm(left),500mm(second
andthirdcolumns)and100mm(right).(c)WT-andttg1-likeglabrousleavesofttg1K2:TTG1grownatpermissiveandrestrictivetemperatures(lefttwo
panels)oraftertemperatureup-anddownshiftsduringleafdevelopment(righttwopanels).Arrows:directionofproximo-distalleafdevelopment
highlightingthesectoraffectedbythetemperatureshift.AphenotypiccomparisonofdifferentdegronvariantsisshowninSupplementaryFig.3,
expressionlevelsofalldegronfusiongenesunderrestrictiveandpermissiveconditionsarepresentedinSupplementaryFig.4.Moleculardynamics
simulationsandmodelsoftheDHFRvariantsusedarepresentedinSupplementaryFig.5.
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analysisalthoughitstranscriptwasfound(SupplementaryFig.3). and allowed fading out the POI to achieve intermediate levels at
ThesefindingsindicatedthatproteinscarryingtheK3degronare semi-restrictive temperatures (Fig. 1e). For this experiment,
too unstable at lower temperatures to provide a permissive plants were shifted at t¼0 from 13 to 25(cid:2)C or kept at 13(cid:2)C.
phenotype. Protein degradation appeared slower when shifted to 25(cid:2)C
comparedwithrestrictivetemperature(Fig.1d),andlowlevelsof
POIwerestilldetectableafter40h.Thedecreaseoflt-proteinover
Novel lt N-degron for in vivo application. Finally, we fused
TTG1 to DHFRT39A/E173D (K2), which only contains the two timecouldbeobservedreachingalevellowerthanatpermissive
but higher than at restrictive temperatures. To test for the
destabilizing point mutations T39A/E173D identified in the
requirement of a functional N-end rule pathway in the host
mutagenesis screen. The expression of K2:TTG1 caused a con-
organism to efficiently degrade the Phe-initiated lt-degron, we
ditional(thatis,temperature-dependent)restorationoftrichomes
introgressed ttg1 K2:TTG into the prt1 mutant. Proteins with
inthettg1mutantbackground(Figs1cand2b,c,Supplementary
initiating Phe at their N terminus are expected to be recognized
Fig. 3 and Supplementary Table 1). In time-course experiments,
by one of the two known plant N-recognins, namely PRT1. We
decreasing and increasing levels of K2:TTG1 protein could be
observed that the fusion protein is stabile in the prt1 mutant
tracked as a consequence of temperature up- or downshifts
background also at restrictive temperature (Fig. 1f).
causing depletion versus accumulation (Fig. 1d). Neither the
endogenous TTG1 protein without the lt-degron nor the
K2:TTG1 transcript levels responded to the temperature shifts Triggering trichome induction on demand. The tunability of
whentestedinatransgenicttg1TTG1-HAcomplementationline K2:TTG1 and the conditional induction of trichomes in the
(Supplementary Figs 3a and 4a). mutantbackgroundallowedustoperformpreviouslyimpossible
For the classical temperature-induced yeast N-degron, it was experiments,thatis,todissectthetemporalrequirementofTTG1
hypothesized that after heat-induction, a dormant N-terminal function during trichome establishment and maturation in great
degron becomes active after conformational changes and that detail(Fig.2b,c).Takingthepatternofwild-type(WT)trichome
internal Lys side chains get exposed1. We tested this hypothesis distribution into account35, we found that a pulse of K2:TTG1
for the point mutations present in the sequences of the DHFR protein is needed for at least 2h to establish a trichome fate
variants of the classical td (K1) and our novel lt-degron (K2) by duringtheinitialinductionphase(Fig.2b,c).Afterapulseof2h
stability predictions and molecular dynamics simulations at permissive temperature of 16(cid:2)C, the front of the trichome
(Supplementary Table 2 and Supplementary Note 3). Modelling initiation sites started to move proximal to the midvein and
of the point mutations within the DHFR crystal structure trichomes began to differentiate (branching). Notably, initiation
suggested that E173D contributes to a higher conformational sites develop proximal to the mid-vein as spots with enlarged
flexibility and increases the exposure of Lys side chains on the precursor cells, suggesting that different leaf areas have a
surface under elevated temperatures. This may lead to a better differential predisposition to form trichomes. After 8h, further
accessibility to the ubiquitination machinery (Supplementary branchpointsareformedandfewthree-branched,albeitnotfully
Fig. 5a–d). developed, trichomes appeared. Many trichomes formed a loped
circumference, which is typical for de-differentiation if initial
N-terminal accessibility and N-end rule requirements. We maintenanceofcellfateisnotcompleted36.After16h,aspacing
hypothesized that a possible hyper-destabilization of K3:TTG1 pattern was formed indicating robust trichome maintenance.
could be due to the flexibility introduced by the short linker eL Forty-eight hours of TTG1 function were needed for developing
(2-His-Gly-Ser-Gly-Ile-6;SupplementaryFigs2,3)betweenDHFR WT-like three-branched trichomes. Expansion and partial
and the destabilizing Phe residue compared with the classical restoration of a WT-like distribution pattern, as well as
yeastdegron34.BydeletingeLfromthedegroncassettesandthus morphology of trichomes, is fully accomplished after 72h of
making the destabilizing N-terminal Phe less accessible for the TTG1 action (Fig. 2b). Here the lt-degron allowed us to
N-end rule Ub ligases, we tested if a conditional phenotype can determine the temporal requirement of TTG1 in the context of
now be observed as a way to further optimize, that is, decrease, the development of a highly specific cell type.
the restrictive temperature of the system. However, transgenic
plants expressing K3:TTG1 lacking eL showed very similar
Switchingfloralinductionondemand.Asa secondtestsystem
phenotypes as the previously tested eL-containing versions.
with an easily scorable biological read-out, we chose WT
Similarly, only expression of K2:CO containing the linker led to
Arabidopsis plants that conditionally overexpress the transcrip-
a temperature-dependent response, the constructs lacking the tionfactorCONSTANS(CO),akeyregulatoroffloweringtime37.
linkerdidnot(SupplementaryTable1andSupplementaryFig.3).
Overexpressing CO causes a rapid shift from the vegetative to
As control, cassette K4 was engineered. It contains the same reproductive growth mode and thereby an early flowering38,39.
DHFR variant as K2 but starts with a methionine and can
Thus, at permissive temperatures, plants expressing a functional
therefore not be recognized by N-end rule E3 ligases due to the
degron–CO fusion protein (CO-td) were expected to flower
lacking destabilizing N-terminal residue (Supplementary Figs 2
earlier than plants grown under the restrictive temperature. The
and 3). The T39A and E173D mutations were previously shown
read-outishencethedaystillfloweringatpermissivetemperature
to cause degradation by a protein quality control pathway at
incomparisonwiththeWT.SimilarlytotheTTG1-tdconstructs,
higher temperatures (30(cid:2)C)33. The K4:TTG1 phenotypes were
neither K1:CO nor K3:CO showed a temperature-dependent
highly variable but not temperature-dependent as expected from
phenotype (Supplementary Table 1). In contrast, the lt-degron
a conditionally stable TTG1-td (Supplementary Fig. 3c,d and
system very efficiently worked as expression of K2:CO at the
Supplementary Table 1). K4:TTG1 plants developed trichomes
permissive temperature caused early floral induction. Here the
regardless of the growth temperature. These data demonstrated
inducible formation of flower meristems occurred about 14 days
that the destabilizing N-terminal Phe residue is a critical feature
earlier than in the WT when grown at the same permissive
of the conditional lt K2 degron.
temperature (Fig. 3a). No differences were found when plants
were grown at the restrictive temperature as in a time-course
Tuning of protein levels. Indeed, protein accumulation by this experiment, the onset of flowering was observed from day 15
novel lt-degron containing the K2 cassette was even tuneable onwardsforbothWTandK2:COplants(SupplementaryFig.6).
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a b
17 °C 27 °C Pro35S::K2:CO
C C WT
I II 13 ° 29 °
IB: α-HA 100 kDa
pe K2:CO 70 kDa
y
d t
Wil
55 kDa
Loading
(CBB)
17 °C 27 °C
c
III IV prt1
Pro35S::K2:CO
O C C
2:C 13 ° 29 °
S::K IB: α-HA 100 kDa
5 K2:CO
o3 70 kDa
Pr
Loading 55 kDa
(CBB)
Figure3|SwitchableflowermeristemsbyconditionalaccumulationofCO-td.(a)ConditionalexpressionofCO-tdfusionleadstoprematureflowering
invivo.Four-week-old(IandII)WTand(IIIandIV)K2:COtransgenicplantsgrownunderpermissiveandrestrictivetemperaturesandshort-dayconditions.
(III)IntransgenicK2:COplants,plantsboltedearlierthanintheWT(I).Scalebars,5cm.(b)K2:COproteinlevelsfromtransgenicplantsgrownat
permissiveorrestrictivetemperature.(c)StabilizationofK2:COinprt1.TheK2:COfusionproteinstabilizesintheprt1mutantbackgroundunderrestrictive
temperatureindicatingthattheE3ligasePRT1isrequiredforK2degradation.FordetailsonN-degrontechnology,seeSupplementaryFig.1;forconstruct
layout,seeSupplementaryFig.2.Fortranscriptlevelsunderrestrictiveandpermissiveconditions,seeSupplementaryFig.4.Adevelopmentaltime-course
experimentforCO-tdisshowninSupplementaryFig.6.
This phenotype correlates with decreased levels of K2:CO under K2:GUSproteinlevelsincreasedandcorrelatedwithhighenzyme
restrictiveconditionswhiletranscriptlevelsremainedunchanged activity after shift to permissive temperature and vice versa if
(Fig.3bandSupplementaryFig.4b).K2:COintrogressedintoprt1 shifted to restrictive conditions (Fig. 4f,g). K2:GUS western blots
lead to stabilization of the fusion protein also at restrictive showed two bands of different sizes (Fig. 4d,f). To identify the
temperature(Fig.3c).HereconditionallystableCO-td,enabledus K2:GUS population with a temperature-dependent hydrolase
tocontroltheonsetof floweringandto generatefloralmeristem activity, which accumulates at permissive temperature, we
cells, that is, specialized cell types, on demand. analysed the bands by mass spectrometry (Supplementary
Fig. 7). This revealed that only the K2:GUS population, which
accumulatedatthepermissivetemperaturecontainsthelt-degron
Invivoinactivationanddepletionofproteinsandenzymes.To
partandishighlyactive(Fig.4e,g).Atthesametime,K2:GUSis
furtherassesstheapplicationspectrumofthelt-degroninplants,
stabilized also at restrictive temperature after the addition of
wechosegreenfluorescentprotein(GFP),b-glucuronidase(GUS)
proteasome inhibitor indicating the degradation of the fusion
and tobacco etch virus protease (TEV) as additional target
protein via the Ub/proteasome system (Supplementary Fig. 4f).
proteins to be tested when stably transformed in Arabidopsis.
K2:GFP robustly followed the accumulation/depletion regime
(Fig. 4a,b and Supplementary Fig. 4c). Moreover, for K2:GFP, Tuningofactivitylevels.Tofurtherstrengthenourclaimonthe
we have designed two different lt-degron cassettes to test for temperature-dependent tunability of the lt-degron system, we
degradationviatwoknownbranchesoftheN-endrulepathway. performed temperature-shift assays with ProUBQ10::K2:GUS
We used either the previously mentioned Phe or, as another addressing temperature-dependent changes in both protein and
potentially destabilizing residue, Arg, which results as neo-N- activitylevelsatvarioustemperatures(Fig.4h).Incontrasttothe
terminal after deubiquitination of the translated K2:GFP fusion in vivo dimming of K2:TTG1 (Fig. 1e), this represents a more
protein.Theseresiduesareexpectedtoberecognizedbythetwo appropriate tuning experiment because here, the activity of the
knownN-recognins,thatis,ArgviaPRT6andPheviaPRT1.The conditional lt-POI could be quantified in relation to the lt-POI
response of these two constructs was compared under standard protein levels and totalprotein content of thesample. Thelevels
growth conditions (Supplementary Fig. 4c) and in time-course ofactiveK2:GUSfusionproteinwerestabilizedanditsactivity,as
experiments (Supplementary Fig. 4d) to demonstrate robustness. monitoredbythequantitativeGUSassay,followedtheincreased
Both destabilizing N termini gave comparable, strongly ordecreasedproteinlevelsaccordingtotheappliedtemperatures
temperature-dependent results and revealed that the lt-degron is in the permissive to restrictive range. Moreover, K2:GUS
functional by both branches of the N-end rule pathway. activity levels can be efficiently regulated by means of different
HistologicalstainingforK2:GUSrevealedactivityofthefusion temperatures. All of this indicated a high flexibility of the
proteininvivoatpermissiveandasignificantlyreducedactivityat lt-degron regardless of temperature adjustment from permissive
restrictive temperature (Fig. 4c). K2:GUS protein levels and to restrictive temperature or vice versa.
activity were strongly decreased for individuals grown constitu-
tively at restrictive temperature (Fig. 4d,e) with transcript levels Application of lt-degrons inN. benthamiana. Next, we applied
unchanged(SupplementaryFig.4e).Intime-courseexperiments, ourlow-temperatureapproachtoTEVasatarget,whichiswidely
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a Pro35S::K2:GFP b GFP Brightfield Merge c
ProUBQ10::K2:GUS Pro35S::GUS Wild type
C C
13 ° 29 ° I II III
IB: α-HA C
K2:GFP 55 kDa GFP13 ° 13 °C
2:
K
Loading 55 kDa o35S:: IV V VI
(CBB) Pr29 °C 29 °C
d ProUBQ10:: Pro35S:: e 0.5 *** 50 f ProUBQ10::K2:GUS
K123 °:GCU29S °C 1G3 °UCS29 °C 13 W°CT29 °C ein per s) 0.4 1239* *°°CC 40 IB: α-HAt(h)R0T 4 8 12162103 2°C4R0T 4 812 16 2209 2°4C
KIB2:: Gα-UHSA > 100 kDa US activityg total prot 00..23 2300 K2:GUS > 100 kDa
Gμ
er 0.1 10 Loading 55 kDa
IB: α-GUS ol p (CBB)
m
K2:GUS > 100 kDa (p Pro0UBKQ21:0:G:US Wild type 35S::GUS 0
g
ProUBQ10::K2:GUS 13 °C
Loading 0.35 29 °C
(CBB) 55 kDa GUS activityμmol per g totalprotein per s) 00..00021...55321
p
( 0.05
0
0 4 8 12 16 20 24
Time of shift (h)
h i Pro35S::K2:TEV
0.6
n per s) 0.5 ProUBQ10::K2:GUS 14 IB: α-HA 13 °C 29 °C
GUS activityμer g total protei 000...234 1248 25.2 1169.8.6 2129.4.6 22.4 16.8 1169..86 22.4 192.26.4 2156..28 1248 KPIB2S:: TTαAE-IVRE
ol p 0.1 25.2 28 25.2 loading
m
p
( 0
0 24 48 96 120 144 168 192 216 240 264
Time of shift (h)
Figure4|Conditionalmolecularphenotypesarelinkedwithproteindegradationanddeactivationoflt-degronfusionproteins.(a)Conditional
accumulationofK2:GFPfusionproteininwholeseedlingsand(b)functionalityofK2:GFPinroottips.AlsoArg-initiatedK2:GFPisconditionallydepleted
(SupplementaryFig.4c).Scalebar,50mm.(c)InvivohydrolaseactivityoftransgenicProUBQ10::K2:GUScomparedwithPro35S::GUS,overexpressionlines
andtheWTconstitutivelygrownatpermissiveorrestrictivetemperature.Scalebar,1cm.(d)K2:GUSproteinlevelsofseedlingsconstitutivelygrownatthe
indicatedtemperatures.Membraneswereimmunostainedwitha-HAantibodyfollowedbyasecondstainingwitha-GUSantibodyafterstripping.The
asteriskindicatesK2:GUSprotein.(e)QuantitativeGUSassaysofplantsconstitutivelygrownat13versus29(cid:2)C.Sixbiologicalreplicateswereperformed
forK2:GUSandthreebiologicalreplicatesforWTand35S::GUS.Allsamplesweremeasuredasthreetechnicalreplicates.PvaluesforStudent’st-test
(two-sidedt-test,type3)areK2:GUS:6.44766(cid:2)10(cid:3)7(***);35S::GUS:0.004365663(**).Allerrorbarsinthispanelrepresents.d.,asterisk:significant.
(f)WesternblotsofProUBQ10::K2:GUSseedlingsconstitutivelygrownatambienttemperature(roomtemperature(RT))shiftedtopermissiveorrestrictive
temperature.(g)Quantitationofenzymeactivityoff.Seedlingsweregrownunderstandardlong-dayconditions(16-hlight,8-hdark,21(cid:2)C)asepticallyon
MSplates.One-week-oldseedlingswereshiftedtopermissiveorrestrictiveconditions,respectively.Inall,5–10individualseedlingswerecollectedand
pooledevery4h.Threebiologicalreplicateswereperformedwithtwotechnicalreplicateseach.Allerrorbarsinthispanelrepresents.d.(h)Degrontuning
assaymonitoringK2:GUSactivitythroughtemperatureshift.PlantsexpressingProUBQ10::K2:GUSweregrownatcold(permissive)orwarm(restrictive)
temperatureandthenshiftedtotherespectiveoppositetemperatureandbackoverthecourseof10days.Samplesweretakenevery24h,followedbya
temperatureshiftof2.8(cid:2)C.ActivityoftheK2:GUSfusionproteinclearlyfollowsthetemperatureshift.Thenumbersnexttothedatapointsrepresent
thesamplingtemperature,n¼3.Allerrorbarsinthispanelrepresents.d.MSanalysisofK2:GUSpopulationsisdocumentedinSupplementaryFig.7.
(i)K2:TEVinstablytransformedArabidopsisplants.Westernblotofmaterialcollectedfromseedlingsgrownatpermissiveandrestrictivetemperatures.
EqualloadingwasfurtherconfirmedbystainingofblottedmembraneswithCoomassieBrilliantBlueG225afterimmunostainingora-PSTAIREantibody
detectingCYCLIN-DEPENDENTKINASEA;1(CDKA;1).Fortranscriptlevelsunderrestrictiveandpermissiveconditions,seeSupplementaryFig4.
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used for cleaving proteins in vitro and in vivo. The protease can for 4h (Fig. 7a,b). K2:TEV was depleted from macrophage-like
be expressed for site-specific protein cleavage in many different Schneider 2 (S2) cells after a shift from 16 to 29(cid:2)C (Fig. 7c).
hostorganismswithoutcausingadverseeffects40.InArabidopsis, AstabletransgenicDrosophilalineexpressingK2:TEVunderthe
K2:TEV transcript levels were unaffected by temperature shifts control of the Actin5c promoter proved the principle in vivo.
(Supplementary Fig. 4g), but the fusion protein accumulated After shifting the living flies from the permissive (18(cid:2)C) to the
exclusively at permissive temperature (Fig. 4i). restrictive (29(cid:2)C) temperature, we observed that K2:TEV
N. benthamiana (tobacco) plants are widely used expression becomesinstable(Fig.7d).Thisrevealedthatthelt-degronhasa
hosts for plant-made pharmaceuticals and high-value proteins. broad spectrum of applications and can be used to modulate
Therefore, we tested the lt-degron in tobacco, which was proteinabundancenotonlyinplantsandyeastbutalsoinanimal
transiently transformed with K2:TEV. The fusion protein was cell culture and living insects.
depleted from plants shifted to 29(cid:2)C and accumulated in
individuals grown at 13(cid:2)C (Fig. 5a). A conditional TEV offers
Discussion
opportunities for downstream processing of recombinant pro-
Tight modulation of protein activities is still a major challenge,
teins by intracellular cleavage. In addition to K2:TEV, K2:GUS
especially in multicellular organisms. We introduce here a
and a fusion of K2 with the agriculturally important herbicide
transferable lt-degron for obtainingconditional mutants coupled
resistance proteins phosphinothricin-N-acetyltransferase (PAT)
to tunable protein accumulation in entire plants, insects and cell
were also expressed in a conditional manner in tobacco
culture.Thelt-systemallowsarapidandreversiblemodulationof
(Fig. 5b,c).Togetherthesefindingsestablished thatthelt-degron
proteinfunctionandenzymaticactivities.Thiswasdemonstrated
approach was also suitable for the manipulation of tobacco and
in the case of seven different targets in five model systems,
therefore is likely applicable for many more plant species.
suggesting that it is generic and versatile.
Conditional protein depletion in S. cerevisiae. To test whether Pronounced and unique technical advantages of the method
thelt-degronsystemisalsofunctionaloutsideofplantspecies,we over comparable systems include non-invasive functionality in a
used orotidine 50-phosphate decarboxylase (URA3) as a reporter wide range of multicellular organisms, the lack of technically
in S. cerevisiae. URA3 functionality and half-life were tested at difficult or costly applications of exogenous chemical triggers,
17.5and27(cid:2)CintheWT,inubr1-DlackingthefunctionalN-end thepossibilityoftheformationofcellandtissuetypesondemand
rule pathway E3 Ub ligase UBR1 and in ump1-D, with impaired by direct action on protein activity, and the perspective of
proteasome function (Fig. 6a). Experiments in WT (JD47-13C), applicability in large scale, for example, in entire growth
ubr1-D (JD55) and ump1-D (JD59) strains revealed that K1 and containments, as the trigger is temperature.
K2conferuracilprototrophicgrowthintheubr1-Dandump1-D In today’s age of recent technological advances where site-
mutants at all temperatures that were generally growth- specificgeneinactivationisbecomingroutine,clearlymethodsfor
permissive for these strains, and at lower temperatures in WT there-introductionoftightlyandrapidlycontrollableconditional
cells (Supplementary Fig. 2c). Increasing the temperature, how- genes will allow additional functional analyses. In combination
ever, resulted in growth inhibition of the WT strain expressing with CRISPR/Cas9, ZFNs or TALENs, our method allows
these constructs on medialacking uracil. Moreover, inWT cells, directedgenerationofnovelconditionalallelesofgenes,including
astrongdecreaseoffusionproteinwasobserved30minfollowing essential ones, at the organismal and tissue-specific level. The
ashiftto27(cid:2)C(Fig.6b).Thus,thelt-degronallowsasignificantly lt-degron is technically well suited for the application in
lower restrictive temperature by about 10(cid:2)C compared with the poikilothermal animals such as fishes, amphibians, reptiles,
classical temperature-controlled DHFRP67L variant. insects and other invertebrates, facilitating the design of
conditional knockoutanimals inmany modelsystems todissect,
Conditional protein depletion in Drosophila. We then tested for example, developmental programmes.
the lt-degron in D. melanogaster using K2:GFP and K2:TEV as We recently discussed the classical heat-inducible N-degron
modelPOIs.K2:GFPwasdestabilizedafterashiftfromstabilizing with other techniques for conditionally altering protein abun-
15to29(cid:2)CinembryonicKccellssubjectedtoatemperatureshift dance and generating phenotypes on demand (Supplementary
a b c
Pro35S:: ProUB10:: Pro35S::
K2:TEV K2:GUS K2:PAT
13 °C 29 °C 13 °C 29 °C 13 °C 29 °C
IB: α-HA IB: α-HA IB: α-HA
55 kDa
100 kDa
K2:POI K2:POI K2:POI
40 kDa
Loading 55 kDa Loading 55 kDa Loading 55 kDa
(CBB) (CBB) (CBB)
RT–PCR RT–PCR RT–PCR
500 bp 500 bp 500 bp
DHFR DHFR DHFR
250 bp 250 bp 250 bp
ACTIN ACTIN ACTIN
Figure5|Thelt-degroninN.benthamianaasaproteinaccumulationsystem.(a)StabilityandtranscriptlevelsofK2:TEVintransientlytransformed
N.benthamiana(tobacco)plants.(b)ConditionalexpressionofK2:GUSand(c)ofK2:PAT.Equalloadingwasconfirmedbystainingofblottedmembranes
withCoomassieBrilliantBlueG225afterimmunostaining.
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Note 1)22. In contrast, the system presented here is expected to to be taken into consideration when adopting this technique:
work for many proteins under physiological conditions in (1) POIs must tolerate N-terminal fusions of about 26kDa,
multicellular, intact organisms, however, different aspects have a C-terminal fusion is not feasible; (2) POIs must at least
a
T (°C) 17.5 27 30 37
Empty vector
pe F-DHFRwt:URA3
y
d t K1:URA3
Wil
K2:URA3 SD
(cid:2) K1:URA3 -Trp,
1- -Ura
br K2:URA3
u
(cid:2) K1:URA3
1-
mp K2:URA3
u
Empty vector
e
yp F-DHFRwt:URA3
Wild t K1:URA3
K2:URA3 SD
(cid:2)1- K1:URA3 -Trp
ubr K2:URA3
(cid:2)1- K1:URA3
p
m K2:URA3
u
b Vector K2:URA3
Wild type ubr1-(cid:2)
T (°C) 27 17.5 27 27
MW (kDa) 0 30 60 90 0 30 60 90 0 30 60 90 t (min after CHX
130 - chase)
100 - IB: α-HA/α-Cdc11
< Cdc11 (α-Cdc11)
55 -
< K2:URA3 (α-HA)
35 -
Figure6|Thelt-degronsignificantlylowerstherestrictivetemperatureforconditionalallelesinS.cerevisiae.(a,b)Modulationofenzymeactivityand
proteindegradationinS.cerevisiae.YeastcellsweretransformedwithF-DHFRwt:URA3,K1:URA3,K2:URA3undercontrolofthecopper-inducibleP
CUP1
promoter.(a)SpottestsofWT,ubr1-Dorump1-Dcellsinserial(1:5)dilutionsgrownfor3daysattheindicatedtemperatures.Platesweregrownat17.5(cid:2)C
for5days.ump1-Dshowsageneralgrowthdefectatelevatedtemperature.(b)Cycloheximide(CHX)chaseofK2:URA3.Equalloadingwasconfirmedby
simultaneouslyprobingagainstCdc11(redsignal).
a b c d
ProActin5c::K2:GFP ProActin5c:: ProActin5c::K2:TEV ProActin5c::K2:TEV
K2:GFP
F M
15 °C 29 °C d 15 °C 29 °C
el C C C C
CHX – + – – + – ht fi F M F M 18 ° 29 ° 18 ° 29 °
Mock – – + – – + Brig IB:α-HA IB:α-HA
IB:α-HA K2:TEV 55 kDa
K2:GFP 55 kDa GFP 15 °C K2:TEV> 55 kDa
IB:α-TUB IB:TUB
Loading 55 kDa Loading 55 kDa Loading 55 kDa
(CBB) ge
er
M
Figure7|ConditionalproteindepletionincellcultureandlivingDrosophilaflies.(a–d)ModulationofproteinabundanceinD.melanogaster.Twoof
themostcommonlyusedDrosophilacelllines,aswellasstablytransformedlivingflieswereusedasfollows.(a)K2:GFPstabilityinembryonicDrosophilaKc
cellswith24-hrecoveryaftertransfectionandafteratemperatureshiftfor4hfrompermissive(15(cid:2)C)torestrictivetemperature(29(cid:2)C).CHXchase
wasperformedwith100mgml(cid:3)1inDMSO.(b)K2:GFPfunctionalitydetectedasgreenfluorescenceatpermissivetemperature.Scalebar,2mm.
(c,d)DepletionofK2:TEVdependingonadestabilizingN-terminalresidue(F:phenylalanine)accordingtotheN-endrule.Methionine(M)ascontrol.
(c)TransfectionintoDrosophilaSchneider2(S2)cellswith24-hrecoveryaftertransfectionand60-hpost-transfectiontemperatureshiftfrom16to29(cid:2)C.
(d)DrosophilaflieswerestablytransformedwithK2:TEVinitiatedbyaPhe(F)residueoraM-K2:TEVcontrol.Fliesweresubjectedtoshiftstopermissive
(18(cid:2)C)andrestrictivetemperatures(29(cid:2)C).Equalloadingwasfurtherconfirmedbystainingofblottedmembraneswithprobingagainsttubulin.CBB,
CoomassieBrilliantBlue.
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temporally localize to the nucleus or cytosol, the compartments trichome-lacking mutant or induced development of flowering
with high activity of the N-end rule pathway or—in case for tissuecanberegardedasthedesignofareactioncompartmenton
membrane localized proteins—have accessible N termini13; demand, a tool to possibly form in vivo bioreactors.
(3) a pre-selection via phenotype and/or western blotting after
transformation helps to identify lines responding to the
Methods
temperature alterations. The functionality of the system in
Plantwork.A.thaliana(L.)Heynh.plantsweresownonsoilmixture1(eightbags
organelles other than the cytosol or the nucleus where the
ofMiniTray(70lperbag,BalsterEinheitserdewerk);add50lofwatercontaining
actionoftheN-endrulepathwayisunclearsuchasendoplasmic 800mlOsmocoteStart(ScottsInternational)and250gBioMu¨kk(Sautterand
reticulum and other membrane systems, mitochondria and Stepper)),oronsteamed(pasteurizedforatleast3hat90(cid:2)C)soilmixture2
chloroplasts is still to be determined. (EinheitserdeClassicKokos(45%whitepeat,20%clay,15%blockpeat,20%coco
fibres;10-00800-40,EinheitserdewerkePatzer);25%Vermiculite(grainsize
Plantgrowth anddevelopment areknownto beinfluenced by 2–3mm;29.060220,Ga¨rtnereibedarfKamlott)and300–400gExemptorperm3soil
temperature18,41–44 and these topics are in the focus of current substrate(100gkg(cid:3)1thiacloprid,802288,HermannMeyer).Afterstratificationof
research. Detrimental side effects of temperature treatment may 4to5daysat4(cid:2)Cinthedark,seedsweregerminatedandplantsgrownunder
represent a downside of a temperature-induced approach, the standardlong-day(16/8hlight/dark)orshort-day(8/16hlight/dark)greenhouse
conditionsbetween18and25(cid:2)C.N.benthamiana(tobacco)plantsweregrownon
impactof29(cid:2)Casarestrictivetemperatureonplantgrowthwas
soilmixture2.
documented (Figs 1 and 2, and Supplementary Figs 3 and 6). Forstrictlycontrolleddevelopmentsuchasduringtemperature-shift
Effects of the plant high-temperature adaptation responses such experiments,plantsweregrowneitheringrowthcabinets(AR-66L2andAR-66L3,
ashypocotylandpetioleelongation,increasedleafsizeandearlier PercivalScientific,CLFPlantClimatics)orinwalk-inphytochambers(Johnson
Controls,equippedwithESC300softwareinterface)atahumidityof60%
floweringhavetobetakenintoconsideration.Effects ofelevated
dependingontherequirements,andwateredwithpre-warmedorpre-cooled
temperature on plant development and genetic control of plant tapwater.
thermomorphogenesisarecurrentlymatterofintenseresearch45. SeedswereplatedeitheronsoilorasepticallyinvitroonplasticPetridishes
However,alltestedorganismswereabletodegradetheK2fusion underlong-dayregime(16/8hlight/dark)on0.5%Murashige&Skoog(MS;
DuchefaBiochemicals,M0221)containing1%sucroseand8gl(cid:3)1phytoagar
proteinsatrestrictivetemperature,andtranscriptlevelsremained
(DuchefaBiochemicals,P10031)ingrowthcabinets.Forasepticculture,dryseeds
stable over permissive and restrictive conditions. This suggests a
weresterilizedwithchlorinedioxidegasproducedfrom75%EaudeJavel(Floreal
lowvulnerabilityofourlt-degronsystemtosideeffectsrelatedto Haagen)and25%HCl.SelectiveMSmediacontained10mgl(cid:3)1DL-
growth differences caused by temperature. phosphinotricin(PPT,Basta,glufosinate-ammonium,DuchefaBiochemicals),
In this study, the lt-degron enabled us to determine the 50mgl(cid:3)1kanamycinsulfate,5.25mgl(cid:3)1sulfadiazinesodiumsalt(Sigma-Aldrich,
S6387)or20mgl(cid:3)1hygromycineB(DuchefaBiochemicals,H0192).Basta-
temporal requirement of the transcriptional regulator TTG1 in
resistantplantswereselectedinsoliumincotyledonstagebyspraying150mlper
the context of trichome development. With a switchable CO trayofa1:1,000dilutionofBasta(contains200gl(cid:3)1glufosinate-ammonium;
transcription factor, we controlled timing of flowering and BayerCropScience)intapwater,whichwasrepeatedthreetimesina2-day
generated specialized cell types and meristematic tissue of the interval.
PlantsusedinthisstudywereallinthebackgroundoftheColumbia-0
flower on demand. An engineered conditional TEV protease
accession(Col-0)andeitherWTplants,T-DNAinsertionmutantsforTTG1(ttg1,
offersopportunitiesfor,forexample,afacilitateddownstreaming GABI_580A05,NASCstockID:N455589,kindlyprovidedbytheGABI-Kat50
of recombinant proteins by intracellular self-cleavage directly in teamviaNASC—TheEuropeanArabidopsisStockCentre,arabidopsis.info),ethyl
the expression host. K2:GUS temperature-shift experiments methanesulfonatemutantsforPRT1(prt1HygSorprt1-1,kindgiftofAndreas
Bachmair,MaxF.PerutzLaboratories,Vienna,Austria)24–26and‘TTG1-HA’is
coupled to GUS activity measurements revealed that the
ttg1-13Pro35S::TTG1-3xHA(kindgiftofMartinaPesch,UniversityofCologne,
lt-degronopens completelynewavenuesby,for example,testing Germany.ttg1-13isadeletionlinefromfast-neutronbombardment,originally
the influence of various levels or pulses of conditionally isolatedfromDavidOppenheimer,UniversityofAlabama,Tuscaloosa31.),which
accumulatedproteinsofinterestinonestablegeneticbackground carriestheTTG1CDSinthebackboneofpAM-PAT-GW-3xHA(seebelowfor
plasmiddetails)undercontroloftheCaMV35Spromotor(Pro35S)andcarrying
without the need to generate a plethora of transgenic events.
thebarresistancemarker(LaurentD.Noe¨landJaneParker,MaxPlanckInstitute
Trichomes are very well-studied specialized model cells29
forPlantBreedingResearch,Cologne,Germany)),whichwasusedasanegative
present on the surfaces of many plants and capable of controlforatemperature-dependentdestabilizationofTTG1.Leaves3/4ofthettg1
synthesizing and either storing or secreting large amounts of mutantshowfewundifferentiatedstichel-liketrichomesorinitiationsitesatthe
specialized metabolites30. The use of the intrinsic metabolic rimoftheleaf,butneitherinitiationsitesproximaltothemidveinnorfurther
differentiatedtrichomes(Figs1cand2a).Thecentralpartsremainglabrous31.
capacity of trichomes to produce secondary metabolites or to
AllplantstrainsusedarelistedinSupplementaryTable3.
enhancetranscriptlevelsofgenesencodingenzymesrequiredfor Forcryoscanningelectronmicrographs(SEMs),5-to7-day-oldplants(pulses;
biosynthesis of small molecules within trichomes has been Fig.2bor2-week-oldplants(shifts;Fig.2c)weretreatedfortheindicatedtime.
discussed46–49. First,leaveswerenotyetvisibleatthetimeofthepulseexperiments.Beforeprotein
isolation,transgenicplantsweresubjectedtotemperatureshiftsbyremovingthe
A special feature of using the heat-inducible N-degron system
pottedplantsconstantlygrownattheindicatedstartingtemperature(16(cid:2)Cin
in living organisms is that it allows conditional build-up of this Fig.2bor29(cid:2)CinFig.2c)andshiftingthemtothecorrespondingdestination
cellular trichome compartment in the K2:TTG1 system. This temperature(29(cid:2)CinFig.2bor16(cid:2)CinFig.2c).Toachievearapid
couldbeusedasageneticbackgroundtogloballyswitchON/OFF acclimatization,plantswereimmediatelywateredwithwaterpre-warmed
orpre-cooledtothefinaltemperature.
a regulatory protein and establish a specific cellular reaction
compartment or micro reactor. Such a cell population or tissue
couldharbouranenzymaticreactioncascadeneededtobuild-up DNAworkanddegronconstructdesign.DNAcloningwasperformedfollowing
standardproceduresusingEscherichiacolistrainDH5a(Invitrogen).Thecon-
a molecule of interest, which is not necessarily a protein, which
structsweregeneratedbyfusionPCR(Pfupolymerase,fermentas)usingtheprimer
could potentially directly be influenced by the fusion to the combinationsaccordingtoSupplementaryTable4.Allindividualconstructs
lt-degron. Compartment-specific expression can be achieved by designedforthisstudyareschematicallyrepresentedinSupplementaryFig.2.
usingcompartment-specificpromoters.Thus,themanufactureof DNAampliconswerepurifiedwithExoSap-IT(USB)precedingfusionPCRs.All
fusionswereflankedbyGatewayattB1/attB2sitesandrecombinedbyBPreactions
biomolecules represents a possible scalable technical application
intopDONR201(Invitrogen).
asaproduction platforminthecontext ofsyntheticbiology.We ConstructsK1–K3startwiththestrongCaMV35Spromoter,a30-tobacco
are convinced that the lt-degron is a powerful tool both for Omega(O)leadersequencefortranslationenhancingfollowedbyasequence
fundamental research in cell biology and developmental genetics encodingforasyntheticcodon-optimizedUb,atripletforthedestabilizingbulky
andhydrophobicaminoacidPhe(F),ashortlinkerpeptide(eL;translatesto
but also has strong potential in biotechnological contexts like
HGSGI)34tofurtherexposetheN-terminalresidue,oneofthethreedifferent
molecular farming of proteins or for metabolic engineering of temperature-sensitivemousedihydrofolatereductasesequences(DHFRts)
small molecules. The conditional formation of trichomes in a triggeringtheproteinunfoldingresponse,atriplehemagglutinin(HA)-tag(HAT)
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