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311
Transcriptional control of plant genes responsive to pathogens
Paul J Rushton and Imre E Somssich
∗
Transcriptional activation of genes is a vital part of the plantsdefence system against pathogens.
Cis
-acting elementswithin the promoters of many of these genes have recentlybeen defined and investigators have started to isolate theircognate
trans-
acting factors. Some of these factors havecounterparts in animals, whereas others are present only inplants, reflecting the fact that plants have developed a uniquedefence system.
Addresses
Max-Planck-Institut F¨ur Zuchtungsforschung, Department ofBiochemistry, Carl-von-Linn´e-Weg 10, D-50829 K ¨oln, Germany
∗
e-mail: somssich@mpiz-koeln.mpg.de
Current Opinion in Plant Biology
1998,
1
:311–315http://biomednet.com/elecref/1369526600100311
©
Current Biology Ltd ISSN 1369-5266
Abbreviations4CL
4-coumarate:CoA ligase
EREBP
ethylene-responsive element binding protein
JA
 jasmonic acid
MRE
MYB recognition element
PR
pathogenesis-related
PAL
phenylalanine ammonia-lyase
SA
salicyclic acid
Introduction
Perception of a pathogen by a plant triggers rapid defenceresponses via a number of signal transduction pathways.A major target of signal transduction is the cell nucleuswheretheterminalsignalsleadtothetranscriptionalactivation of numerous genes and consequently to the
de novo
synthesis of a variety of proteins and antimicrobialcompounds [1,2
•
]. Studies with different plant pathologysystems have revealed that the active response of plantsto attempted microbial ingress is associated with dramaticreprogramming of cellular metabolism [2
•
]. Expression of a large array of genes whose products are involved bothin diverse primary and secondary metabolic pathways arerapidly induced or strongly upregulated. These includegenes encoding enzymes of the shikimate and the generalphenylpropanoidpathwaysalongwithenzymesfromsubsequentbranchpathways.Anothergroupofgenes,termedpathogenesis-related(PR)genes(comprisedof classes1–10),hasoftenbeenfoundassociatedwiththedefenceresponse[3].ThefunctionofmanyPRproteinsremainsunknownbutsomeareglucanasesand chitinases.
β
-glucanases hydrolyse
β
-linked glucans,whereaschitinasesdegradechitin,themajorstructuralcomponent of the cell walls of many fungi. Additionally,some plant chitinases also have lyzozymal activity.
β
-1,3glucanase, in concert with chitinase have been shown todegrade fungal cell walls and to inhibit fungal growth both
in vivo
and
in vitro
[3].The expression of pathogen-responsive genes shows botha temporal and spatial hierarchy. Some genes undergo arapid localised activation at the site of infection, whereasothersaremoreslowlyactivatedeitherlocallyand/orsystemically. Plant hormones, including salicylic acid (SA),ethylene and jasmonic acid (JA), have been implicated inacting as secondary signals following pathogen attack andenhanced expression of many pathogen-responsive genescan be induced by these plant hormones [4
••
].
Regulatory components of pathogen-responsive genes
Differences in the expression patterns of pathogen-respon-sive genes are a result of the architecture of the promoters;pathogen-responsivepromotersbeingdissimilarinthenumber, order and type of regulatory elements present.This places the promoters at the end of one or more signaltransduction pathways as the plant responds to pathogenattack. Despite the differences in architecture, functionaldissectionofthepromoterregionsofsomeofthesegenes is starting to uncover common
cis
-acting elementsandhasprovideduswiththetoolstoidentifydistinctDNA-binding proteins required to modulate transcription.Interestingly,someofthecognatefactors,forexampleEREBPandWRKYproteins,appearuniquetoplantswhereas others such as bZIP and MYB proteins also havecounterparts in animals.
The GCC Box and EREBPs
The GCC Box (AGCCGCC; Figure 1) is found in thepromoterregionsofmanypathogen-responsivegenes[5,6
•
]. This
cis
-acting element has been shown to functionasanethyleneresponseelement[5].Thebiosynthesisofethyleneincreasesrapidlyduringplant–pathogeninteractionsandethylenethenactsthroughtheGCCBoxtoinducetheexpressionofpathogen-responsivegenes [5,7
••
]. Interestingly, although similar elements alsoactaslowtemperatureresponseelementsandwaterdeficitresponseelements,theGCCBoxhasyettobe found in the promoters of ethylene-regulated genesinvolved in some of the other ethylene responses, suchas fruit ripening [8
•
,9]. It seems, therefore, that ethyleneperception involves distinct regulatory mechanisms.AnumberofproteinsthatbindtoGCCBoxeshavebeen isolated and found to be members of the EREBP(ethylene-responsiveelementbindingprotein)familyof DNA-binding proteins [5,6
•
,7
••
,8
•
]. EREBPs containaDNA-bindingdomainthatisalsopresentintheAPETALA2 family of proteins [6
•
]. The homeotic gene
 APETALA2
playsacentralroleintheregulationof 
 
Plant–microbe interactions
312
Figure 1
P 
ATGTATAHG HH
      G      G     /     /     H     H     B     B     F     F     1     1      G      G     /     /     H     H     B     B     F     F     1     1
P Pv vCCH HS S1155
ATGTATA
W1WWRRKKYY W2WWRRKKYY
P Pcc R R11--11
ATGTATA
LPBBPPFF--11
P PccP PAALL11
ATGTATAGCC
EERREEBBPP
GCC
N Nt tGGl lnn22
ATGTATA
MRE? MRE?MMYYBB11
N Nt tP PR R--11aa
MMYYBB11 EERREEBBPP Current Opinion in Plant BiologyMYBMYB
     K     A     P   -     1     K     A     P   -     2
Schematic representation of pathogen responsive
cis
-acting elements(W-, P-, L-, G-, H-boxes, GCC-MRE-elements) and their cognateinteracting factors (WRKY, BPF-1, MYB, G/HBF1, KAP1/2, EREBP).The promoter regions of the
Petroselinum crispum
PR1-1 (
PcPR1-1
)and phenylalanine ammonia-lyase 1 (
PcPAL1
), the
Phaseolus vulgaris
chalcone synthase (
PvCHS15
), and the
Nicotiana tabacum
glucanase2 (
NtGln2
), and PR1 (
NtPR-1a
) genes are shown. The relativepositions of the interacting partners in relation to their respectiveTATA-boxes and the translation start sites (ATG) are given but arenot drawn to scale. bZIP factors, such cs G/HBF1, are known to bindas dimers. Thus, a homodimer of G/HBF1 is indicated binding to theG-box although it cannot be excluded that G/HBF1 binds insteadwith an unknown second factor as a heterodimer to this element.Arrows point to additional elements which may be contacted by therespective factors as described in the text.
flower development in
Arabidopsis thaliana
[10]. The firstEREBPstobeisolatedweretobaccoEREBP-1,-2,-3and -4 [5]. All four bind specifically to GCC Boxes andareethylene-inducibleinleaves.Morerecently,CBF1,anEREBPfrom
A. thaliana
,wasisolatedbyayeastone-hybrid screen using a dehydration response element(GCCGAC)asthe‘bait’DNA[8
•
].ThissuggeststhatmembersoftheEREBPfamilymayplayawiderroleintheresponsetovariousstresses.Inaddition,CBF1is capable of transactivation in yeast and this requires afunctional
cis
-acting element [8
•
]. Further evidence for arole for EREBPs in plant defence has come from yeasttwo-hybrid experiments using the tomato resistance gene
 Pto
asa‘bait’protein[7
••
].
Pto
confersresistanceto
 Pseudomonas syringae 
strainscarryingthecorresponding
avrPto
avirulence genes. Pto is a protein kinase and wasshown to interact with three proteins: Pti4, Pti5 and Pti6.Pti4/5/6areEREBPsandbindspecificallytoGCCboxes. In tobacco carrying a
Pto
transgene, expression of an EREBP gene was specifically enhanced upon infectionwithabacterialstrainharboringthe
avrPto
avirulencegene.Thissuggestsadirectlinkbetweenaresistancegene and the specific activation of plant defence genesandimpliesthatPtoconfersresistancebyactivatingasignallingpathwaythatleadsthroughEREBPstoactivation of PR genes containing GCC boxes.
W Boxes and WRKY proteins
There are two types of well studied W Box [11] elements,the first contains the hexamer sequence TTGACC whilstthesecondconsistsofthetwotetramerhalfsitesTGAC-N
x
-GTCA. A W Box in the parsley PR-10 classgene,
PR1-1
,isthesiteofafungalelicitor-inducibleDNA-binding activity [12]. A similar activity also bindstoaWBoxinthepromoterofthetobaccochitinasegene
CHN50
[13]. Experiments have shown not only thatW Boxes are functional elicitor response elements in theparsley
PR1
genes [11], the maize PR-1 class gene
PRms
[14], and the tobacco class I chitinase gene
CHN50
[13,15]but also that they are sufficient to direct elicitor responsiveexpression in transient expression systems [11,14]. Otherpathogen-responsive promoters contain W Box sequenceswithin functional areas of the promoters. These includethe potato glutathione
S 
-transferase
gst1
(
 prp1
) [16], PR10genes from asparagus (
 AoPR1
) [17] and potato (
 PR-10a
)[18], and the stilbene synthase gene,
Vst1
, from grapevineencodinganenzymeofphytoalexinbiosynthesis[19].Thus, W Boxes may be a general feature of a large subsetof pathogen-responsive gene promoters.WBoxesarebindingsitesfortheWRKYfamilyof DNA-binding proteins [11,20]. In parsley, three WRKYproteins, WRKY1, -2 and -3, bind specifically to functionalW Boxes in the
PR1-1
and
PR1-2
promoters [11]. Theoptimalbindingsiteforthe
A thaliana
WRKYproteinZAP1alsohastwoWBoxes[20].WRKYproteinsaredefinedbythepresenceoftheWRKYdomain.Thiscontainstheaminoacidsequence
WRKY 
GQKthatisconservedinallWRKYdomainssofaranalysed.Onthe carboxy terminal side of the protein to this is whatappears to be a novel zinc finger-like structure [11,20,21].Zinc-fingers are protein regions containing cysteine and/orhistidine residues which are predicted to form a fingerstructure by binding a single zinc atom. In several cases,it has been demonstrated that the zinc-finger region andzinc are required for DNA binding of the protein. In othercases, however, the zinc-finger constitutes an interface forinteraction with other specific proteins [22]. Outside of the WRKY domain, most WRKY proteins are dissimilar,thoughtheydopossessfeaturesfoundintranscriptionfactors.Indeed,ZAP1cantransactivate,bothinplantcells and in yeast and this transactivation is dependentonthepresenceoftheWBox[20].Furthermore,theparsleyWRKY1proteinistargetedtothenucleus(TEulgem and IE Somssich, unpublished data) and fungal
 
Transcriptional control of plant genes responsive to pathogens
Rushton and Somssich313
elicitor induces rapid and transient changes in the mRNAlevels of WRKY1, -2 and -3 [11]. Thus, WRKY proteinsappear to be transcription factors that play a role duringplant defence by binding to W Boxes in the promoters of pathogen-responsive genes.
MRE-like sequences and MYB factors
Anotherclassof 
cis
-actingelementsisrepresentedbyBoxes P and L from the parsley phenylalanine ammonia-lyase(PAL)and4-coumarate:CoAligase(4CL)genesandtheH-boxfromthebeanchalconesynthasegene
Chs15 
[23,24].TheseelementsfitthetypeIIMYBconsensussequenceA(A/C)C(A/T)A(A/C)C,suggestingthat they are MYB recognition elements (MREs). BoxesPandLwereinitiallyidentifiedwithintheparsley
 PAL1
promoterasthesitesoffungalelicitor-inducibleDNA–proteininteractions[25]andweresubsequentlyshown to bind a MYB-like protein, BPF-1 [26,27
•
]. BPF-1mRNAaccumulatesrapidlyinelicitor-treatedparsleycells suggesting that it participates in the plant defenceresponse [26]. Boxes P and L from the parsley
PAL1
,
4CL1and 4CL2
promoters can be specifically bound by MYBproteins [27
•
]. Furthermore, expression of the tobacco
4CL
gene, along with other MRE-containing genes encodingenzymes of phenylpropanoid metabolism, was significantlyrepressedintransgenictobaccoplantsoverexpressingan
Antirrhinum majus
MYB protein [28
•
]. Additionally, aflower-specificMYBcanactivatetranscriptionfromthe
 A. thaliana gPAL2
promoterthroughsimilarelements[29].FunctionalstudieswiththeH-boxindicatedthatitcannotfunctiontoahighlevelalone.Gainof functionexperiments,however,showthatitisactiveincombinationwithaG-Boxelement(seebelow)intransgenic tobacco plants in establishing the characteristictissue-specificpatternofexpressionandmutationsineithertheH-boxorG-Boxreducedtheresponsetotobacco mosaic virus (TMV) infection [24,30].Finally,thetobacco
MYB1
geneisinducedbyTMVduring the hypersensitive response and the developmentofsystemicacquiredresistance[31].ApplicationofSAinduced expression of 
MYB1
within 15 minutes. Althoughthe MYB1 protein can bind
in vitro
to a MYB consensusbinding site found in the tobacco
PR1-a
promoter [31],itremainsunclearwhetherthisDNAelementisof functional importance for
PR1-a
expression and whetherit represents the
in vivo
target site for MYB1.
G-Boxes,
as-1
-like elements and bZIP factors
G-Boxes(CACGTG)functionduringtheregulationof diversegenesbyenvironmentalcues,suchasabscisicacid (ABA), light, UV radiation and wounding, as well aspathogen signals [32]. G-Boxes are members of the familyof ACGT-containing
cis
-acting elements and have beenimplicated in the expression of a number of genes duringpathogen attack [33]. Often they function in concert withother
cis
-acting elements as in the case of the G-Box andH-box from the bean
Chs15 
promoter [24].Another class of elements implicated in the plant defenseresponse,consistsof 
as-1
-likeelements.The
as-1
(or
ocs
)element(CTGACGTAAGGGATGACGCAC)wasinitiallyidentifiedinthe
35S 
promoterofcauliflowermosaic virus and the
nos
and
ocs
promoters of 
Agrobacteriumtumifaciens
[34,35]. The
as-1
element confers responsive-ness to several signals, including SA, auxin, jasmonatesand hydrogen peroxide [4
••
]. Although the
as-1
elementis responsible for the activation of a number of pathogengenes, it is not clear whether functional
as-1
-like elementsare found widely in plant genes [34].BoththeG-Boxandthe
as-1
or
ocs
elementareboundbybZIPproteins,awellcharacterisedclassof transcriptionfactorsalsofoundinanimalswheretheyregulate, both positively and negatively, various cellularprocesses. bZIP proteins appear to play important rolesduring the activation of a number of genes during plantdefence.AbZIPproteinfromsoybeanbindstotheG-Box in the bean
Chs15 
promoter [36
•
]. This protein,G/HBF-1,canalsobindtotheadjacentH-box.Thesignificance of this relaxed binding specificity is unclear.Although the mRNA and protein levels of G/HBF-1 donot increase during the induction of its putative targetgenes, the protein itself is rapidly phosphorylated and
invitro
phosphorylation enhances binding to one (H-box III)out of the three H-boxes present in the
Chs15 
promoter.A serine kinase capable of phosphorylating G/HBF-1 wasalso identified and this kinase is rapidly and transientlystimulated in elicitor-treated cells [36
•
]. B¨ uttner and Singh[6
•
] showed that an
ocs
element-binding protein (OBF4)interacts with an EREBP suggesting that cross-couplingbetween EREBP and bZIP factors may be involved inthe regulation of gene expression during the plant defenceresponse.Apart from being a binding site for bZIP proteins, theG-Boxisalsoapotentialbasichelix-loop-helixbHLHproteinbindingsite(CACNTG)[37].bHLHfactorsarepresentinplants[38]butalthoughMYB/bHLHinteracting partners control flower pigmentation [39
•
], norole has yet been assigned to them during plant defence.
Additional regulatory elements and DNA-binding proteins
Salicylic acid (SA) is an important signal in plant defence[4
••
]andaSA-responseelement(SARE)hasbeenidentified in the tobacco
PR2-d 
gene. A 76bp fragmentconferred a 20-fold induction by SA in transgenic tobaccoplants and protein binding studies indicated that the coresequenceofthisSAREisTTCGACCTCC[40].Thenature of the binding factor is unknown.Matton
et al.
[18] identified an elicitor-responsive regionwithin the potato
PR-10a
gene that is recognised by twonuclear factors, PBF-1 and PBF-2. Both wounding andelicitor treatment induce the phosphorylation of PBF-1[41].NeitherPBF-1norPBF-2hasbeencloned.Theregion contains the sequence TGAC-N
6
-GTCA, however,
of 00

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