Proc. Natl. Acad. Sci. USAVol. 90, pp. 3549-3553, April 1993Microbiology

VirA, the plant-signal receptor, is responsible for the Tiplasmid-specific transfer of DNA to maize by Agrobacterium

(agroinfection/maize streak virus/virA gene)

D. M. RAINERI*, M. I. BOULTONt, J. W. DAVIESt, AND E. W. NESTER**Department of Microbiology, University of Washington, Seattle, WA 98195; and tJohn Innes Institute, Institute of Plant Science Research, Norwich NR47UH, United Kingdom

Communicated by Luis Sequeira, November 30, 1992

ABSTRACT Agrobacteria exhibit marked Ti (tumor-inducing)/Ri (root-inducing) plasmid specificity in their inter-action with the Gramineae. In this study, we have used thetechnique of "agroinfection," in which Agrobacterium-medi-ated delivery of viral genomes into plants is detected by thedevelopment of viral disease symptoms, to identify the regionof the Ti plasmid which is responsible for the major differencesseen in the ability of nopaline- vs. octopine-type Ti plasmids totransfer maize streak virus (MSV) DNA to maize. Introductionof fragments of the C58 (nopaline-type) Ti plasmid into strainscontaining an octopine-type Ti plasmid showed that a fragmentcontaining the nopaline-type virA locus was able to complementthese normally non-agroinfectious strains to high levels ofMSVDNA transfer. Octopine-type virA mutant strains that expressvir genes at high levels in the absence of the plant inducingcompound acetosyringone also efficiently transferred MSVDNA. These rmdings imply a functional difference between thevirA gene products encoded by octopine- and nopaline-type Tiplasmids which has a profound effect on their ability to mediateDNA transfer to maize.

The Ti plasmids in Agrobacterium tumefaciens strains andthe Ri plasmids in Agrobacterium rhizogenes strains causecrown gall tumor and hairy root diseases, respectively. Tworegions of the Ti/Ri plasmid are required for pathogenesis:the T-DNA, which is transferred to and stably integrated inthe nuclei oftransformed plant cells, and the vir region, whichencodes the enzymes and structures required for processing,transfer, and integration of the T-DNA. The integratedT-DNA directs the production and metabolism of plantgrowth hormones as well as classes of compounds termedopines. Most Ti plasmids are classified as octopine- ornopaline-type and most Ri plasmids as agropine-, man-nopine-, or cucumopine-type based on their ability to encodethe synthesis and catabolism of these respective opines.Unlike T-DNA, the vir region is not transferred to plant cellsbut is essential forT-DNA transfer. Six operons (virA, -B, -C,-D, -E, and -G) have been characterized in detail and threeother strain-specific regions (virH, virF, and tzs) are less wellcharacterized (for reviews, see refs. 1-3).The host range of Agrobacterium, as judged by tumor

formation, was thought to be limited almost exclusively todicotyledonous (dicot) plants (4, 5). However, more recentwork reported that certain monocotyledonous plants (mono-cots) are also susceptible (6-9). Furthermore, although maizeand other graminaceous monocots have remained refractoryto tumor formation, the agroinfection technique used to studythe transfer of viral DNA to dicots (10, 11) has confirmed thatAgrobacterium is able to transfer DNA to cereal plants(12-17). Agroinfection is mediated through the same vir genepathway as is required for tumor formation in dicots (18).

This technique provides a very sensitive assay for Agrobac-terium-mediated DNA transfer, as the viral sequences escapefrom the T-DNA and are amplified as the virus replicates andspreads systemically. DNA transfer is thus detected by theformation of disease symptoms.The ability to agroinfect maize was found to be Ti plasmid-

specific (13, 16): octopine-type strains either were unable, oronly weakly able (<5%), to transfer maize streak virus(MSV) DNA, while nopaline-type strains infected 80-100%oof the inoculated plants. Agropine- and mannopine-typestrains of A. rhizogenes also efficiently agroinfected maize.With appropriate viral DNAs as markers, similar results wereobtained for other members of the Gramineae (14, 15, 17). Incontrast, Ti plasmid specificity is not typical of agroinfec-tion-dicot interactions (10, 11).A "disarmed" (T-DNA-less) nopaline strain agroinfected

maize as efficiently as the wild type (13). Therefore, Tiplasmid specificity cannot be due to differences in phytohor-mone production specified by the T-DNA. One explanationfor the inability of octopine-type strains to agroinfect maizeand other graminaceous monocots is that octopine-type Tiplasmids lack an essential function present in nopaline-,agropine-, and mannopine-type Ti/Ri plasmids. Alterna-tively, octopine-type Ti plasmids might express a counter-active function.We have used the agroinfection system to characterize the

Ti plasmid requirements for transfer ofMSV DNA to maize.We observed that a DNA fragment containing the nopaline-type virA locus complemented octopine-type Ti plasmids tohigh levels of agroinfection. Furthermore, whereas preinduc-tion of octopine-type strains does not promote maize agroin-fection, octopine-type virA mutant strains, which express virgenes at high levels in the absence of the inducing compoundacetosyringone, efficiently transferred MSV DNA to maize.We discuss here the implications of these observations andpossible causes.

MATERIALS AND METHODSBacterial Strains, Plasmids, and Media. The MSV-contain-

ing binary vectors and other plasmids used in this study aredescribed in Table 1. Agrobacterium strains are listed in therelevant tables or are described in the text. Escherichia coliwas grown at 37°C in Luria broth (LB). Carbenicillin, spec-tinomycin, and kanamycin were used at 100 ,ug/ml; genta-micin and tetracycline were used at 10 ,g/ml. For selectivegrowth of Agrobacterium strains, the concentrations of gen-tamicin and tetracycline were modified to 100 ,ug/ml and 5,ug/ml, respectively. Agrobacterium strains were grown at28°C in MG/L (27).

Plasmids were introduced into Agrobacterium by triparen-tal matings (28), using pRK2073 or pRK2013 as helperplasmid, or by electroporation (29).

Abbreviations: MSV, maize streak virus; cfu, colony-forming units.

3549

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Proc. Natl. Acad. Sci. USA 90 (1993)

Table 1. Plasmids, vectors, and recombinant plasmids

Name Relevant characteristics Ref.*PlasmidspMSV-Ns-ICR MSV dimer in pIC19R 13pCGN1557 oripRiHR1 replicon, Gm 19pBIN400 IncP replicon, Sp M. BevanpDR329 IncP replicon, GmpUCD2 IncW replicon,

Cb, Tc, Km/Gm, Sp/Sm 20pDMD2 IncW replicon, Cb, TcpDR4 IncP replicon, Km D.M.R.

MSV-containing binary vectorspMSV-Ns MSV dimer in pBIN19 21pMSV-Ns-CGN MSV dimer in pCGN1557pMSV-Ns(S) MSV dimer in pBIN400pMSV-Ns(G) MSV dimer in pDR329

Recombinant plasmidspFW32 pRiA4 vir regulon, Tc, IncP 22pUCD2614 pTiC58 vir regulon,

Cb, Km, IncW 23pDR106B pDR4::pTiC58 BamHI fragment 2

(virAB'tzs, non-vir), KmpDR237B pDR4::pRiA4 Kpn I fragment 2

(virABtzs, non-vir), KmpDR106 pDMD2::pTiC58 BamHI

fragment 2, CbpDRB56 pDMD2::pTiC58 BamHI

fragment 2 virAtzs subclone, CbpDRB31 pDMD2::pTiC58 BamHI

fragment 2 virA subclone, CbpTB108 pUCD2::pTiA6 virA, Cb, Tc 24pIBlOO pUCD2::pTiA6 virA, virG, Cb 25pEB112 virA(A.s) mutant of pIB100, Cb 25pSW169 pTZ19R::pTiA6 virA, Cb 26pDR169 pTZ19R::pTiC58 virA, CbpDR172 pTZ19R::pTiC58 virA promoter-

pTiA6 virA fusion, CbpDR178 pUCD2::pTiC58 virA promoter-

pTiA6 virA fusion, Cb, Tc

Construction of vectors and recombinant plasmids is described inthe text. Cb, carbenicillin; Sp, spectinomycin; Sm, streptomycin;Gm, gentamicin; Km, kanamycin; Tc, tetracycline.*If other than this study.

Plasmid Constructions. The MSV vector pMSV-Ns-CGN,which contains the origin of replication of the A. rhizogenesplasmid pRiHR1, was constructed by inserting the 5.4-kbtandem dimeric copy of MSV isolated from pMSV-Ns-ICRinto the EcoRI site of the binary plasmid pCGN1557. ThepMSV-Ns-CGN vector was used in combination with theIncP-derived recombinant plasmids (Table 1), which wereincompatible with the pMSV-Ns vector used in previousstudies.The spectinomycin resistance-encoding vector pMSV-

Ns(S) contained the EcoRI-Xba I dimeric fragment of MSV,isolated from pMSV-Ns, inserted into the multiple cloningsite ofpBIN400 (provided by M. Bevan, John Innes Institute,Norwich, U.K.). A gentamicin resistance-encoding vector,pMSV-Ns(G), was generated by inserting the EcoRI MSVfragment from pMSV-Ns-ICR into the single EcoRI site in theT-DNA border fragment of pDR329 (D.M.R., unpublisheddata), a gentamicin resistance-encoding binary vector de-rived from pSP329 (29). These IncP-derived, MSV-containing vectors were used in complementation studies incombination with the IncW plasmid pUCD2 or with thederivative plasmid pDMD2, carrying either Ri or Ti frag-ments of interest. Plasmid pDMD2 was generated by deletingthe Sac II-Kpn I fragment of pUCD2, creating single restric-tion sites for EcoRI and HindlIl. Comparative data were

based on studies using identical combinations ofrecombinantplasmid and MSV vector. Where this was not possible, theefficiency of agroinfection was assessed relative to the no-paline-type strain C58, which was included in all inoculationsas a positive control.Plasmid pDR178 contains a fusion of the octopine-type

virA gene from pTiA6 to the pTiC58 virA promoter. A 4.8-kbBamHI-Xho I fragment containing the pTiC58 virA gene wasligated to BamHI/Sal I-cut pTZ19R (carbenicillin resistance;United States Biochemical) to give pDR169. The pTiC58 virApromoter was excised by Xmn I digestion of pDR169 andinserted in front of a promoterless pTiA6 virA gene generatedby Xmn I digestion of pSW169. Digestion of the resultingplasmid, pDR172, with Kpn I and Hpa I released a 3.8-kbpTiC58 virA promoter-pTiA6 virA fusion fragment for inser-tion into Kpn I/Pvu II-cut pUCD2.

Plant Growth and Inoculation Conditions. Growth of Zeamays L. cv. Golden Cross Bantam and preparation and serialdilution of inocula were done essentially as described (13).Unless stated otherwise, bacterial strains were not preinducedand were used at an inoculum density of 108 colony-formingunits (cfu) per plant. Plants were examined for symptoms from5 days post-inoculation (dpi); those showing no symptoms 28dpi were scored as uninfected. Typically, MSV symptoms arelong chlorotic streaks, abundant on leaves that emerge 6-9 dpi(21). If a very dilute inoculum is used, symptom developmentmay be delayed by up to 12 days, with several asymptomaticleaves emerging in the intervening period. When delayed, thesymptoms may be confined initially to single streaks, to alimited leaf area, or to small spots widely dispersed over theleaf. Delay is likely to reflect low levels ofMSV DNA transfer(M.J.B., unpublished data).To preinduce bacterial strains, cultures were grown over-

night in MG/L, then diluted 1:20 in induction broth (24), pH5.5, containing 100 ,tM acetosyringone and further incubated18-24 hr prior to inoculation.

RESULTSAU Ti/Ri Plasmid Requirements for Maize Agroinfection

Reside Within the pTiC58/pRiA4 vir Regulons. To determinewhether portions of the nopaline-type Ti plasmid pTiC58 andthe agropine-type Ri plasmid pRiA4 could complement anoctopine-type Ti plasmid for maize agroinfection, large re-gions of these plasmids were mobilized into strain A348 (C58chromosome; pTiA6). In addition, clones ofthe entire pRiA4and pTiC58 vir regulons were mobilized into strain LBA4011(30) or the LBA4011-derived rec-deficient mutant LBA4301(31), Ti plasmid-less derivatives of the octopine-type strainAch5 (Ach5, octopine-type, chromosome). Finally, the MSVbinary vector was introduced into each of the recombinantplasmid-containing strains.Clone pFW32, containing the entire pRiA4 vir regulon,

promoted MSV DNA transfer from LBA4011 into 38% of theinoculated plants. Strain LBA4301 containing a clone of thepTiC58 vir regulon, pUCD2614 (Fig. 1), mediated diseasesymptoms in 83% of the inoculated plants, similar to the78-89o range of infectivity obtained when pUCD2614 wasintroduced into the cured nopaline-type strain A136 (Table 2).Clones of non-vir regions of the Ti and Ri plasmids did notpromote MSV DNA transfer.

Subcloning ofthe vir regions showed that BamHI fragment2 from pTiC58 promoted maize agroinfection when intro-duced into A348 and the octopine-type virA mutant strainMX226 (34). Infectivities were consistently higher for thecomplemented virA mutant [MX226(pDR106B)] than for theparental strain [A348(pDR106B)] (Table 2). The equivalentregion of pRiA4 (subclone pDR237B) promoted MSV DNAtransferfrom A348 into 62% ofthe inoculated plants, whereasother regions of the Ti and Ri vir regulons failed to promotemaize agroinfection (data not shown). The complementing

3550 Microbiology: Raineri et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 3551

vir regulon

KpnIL4 5 | 11i j13bI | 9

i.-fi=3kb

|13a pUCD2614

ItzsI | virA | virBBssnil 2 1 ~~~~~~~~~=lkb~Nde I

pDR106pDRB56pDRB31

FIG. 1. Map of the pTiC58 vir regulon (32) showing regionscontained within pUCD2614 and the complementing subclones,whose designations are given at right. All fragments were tested inboth orientations.

subclones encompassed a common region that included thevirA and tzs loci as well as a flanking, non-vir region of -5 kb.

Efficiencies of MSV Infection Mediated by pTiC58 and theComplemented Octopine-Type Ti Plasmids Are Similarly Af-fected by Inoculum Dilution, but Those Mediated by a virFMutant Are Not. Symptom formation mediated by the com-plemented octopine-type Ti plasmids was not markedly de-layed, suggesting that MSV DNA transfer from these strainswas efficient. Titration of C58(pMSV-Ns) showed that 100%agroinfection could be obtained with as few as 107 cfu/ml, or2 x 105 cfu per plant (13). The gradual decline in MSVinfectivity seen with lower cell concentrations of the comple-mented A348 strain closely paralleled that obtained with C58,both strains being capable of efficient transfer at a 1:10,000dilution of the normal inoculum concentration (Fig. 2).Jarchow et al. (16) proposed that the limitation to maize

agroinfection by octopine-type strains lies in the octopine-type Ti plasmid-specific virF locus (35). We also have ob-served that A348-derived virF mutants promote maizeagroinfection (36). However, compared with C58, MSVinfectivities ofthe virF mutants were low (only 0-15% greaterthan those mediated by strain A348), streaking was lesssevere, and symptom appearance was significantly delayed.We concluded that virF plays some minor role but is not themajor contributor to the limitation of the octopine-type Ti

plasmids (36). To address this discrepancy, inoculum titra-tion studies were used to quantitate the agroinfection effii-ciency of the octopine-type virF mutant LBA1517 used byJarchow et al. (16).A 100-fold dilution of the LBA1517 inoculum completely

abolished infectivity (Fig. 2), whereas 22 of the 23 C58-inoculated plants showed symptoms at this dilution.

Insertion of a Fragment Containing the Nopaline-Type virALocus into A348 Promotes Maize Agroinfection. To localizethe complementing activity, subclones ofBamHI fragment 2from pTiC58 were generated in the IncW plasmid pUCD2 orpDMD2 (Table 1).

Digestion of BamHI fragment 2 with HindIII generated a5.6-kb BamHI-HindIII fragment encompassing the virA andtzs loci (subclone pDRB56, Fig. 1) and a 5.0-kb HindIIIfragment encompassing the non-vir region. pDRB56 pro-moted DNA transfer from A348 with MSV infectivities of38-68%, compared with 40-60% for clone pDR106 (Table 2),which contains BamHI fragment 2 in the same IncW plasmid(Fig. 1). Further subcloning localized the complementingactivity to a 3.1-kb Nde I fragment containing the pTiC58 virAlocus (subclone pDRB31; Fig. 1 and Table 2). Other regionsof the 5.6-kb vir fragment failed to promote DNA transfer(data not shown).

In contrast, pUCD2 containing the octopine-type virAlocus alone (pTB108) or in combination with the octopine-type virG locus (pIB100) resulted in only slight enhancementsin MSV DNA transfer when introduced into A348 and theoctopine-type virA mutant strain A1030 (Table 3). In bothcases, symptom formation was delayed by up to 12 days andstreaking was initially much less severe than that observedwith C58 or the complemented A348 strains.To investigate the possibility that differences in the virA

promoter region might be responsible for specificity in maizeagroinfection, the pTiC58 virA promoter was cloned in frontof a promoterless pTiA6 virA gene (pDR178) and introducedinto A348 and octopine-type virA mutant backgrounds. Theresulting strains were not complemented for MSV DNAtransfer to maize (Table 3) but were competent for tumorformation on dicots (data not shown), indicating that func-tional VirA was being expressed from the pTiC58-pTiA6virA fusion.

Table 2. Complementation studies

Complementing Chromosome/Ti Opine No. of plants Infectivity,plasmid Strain plasmid family inoculated %

None A348 C58/pTiA6 oct 201 0-5None C58 C58/pTiC58 nop 340 95-100None A4 A4/pRiA4 agr 40 80

Nopaline pTiC58 vir regulonpUCD2614 LBA4301 Ach5/- (oct) 18 83pUCD2614 A136 C58/- (nop) 75 78-89

Nopaline pTiC58 BamHI fragment 2pDR4 A348 C58/pTiA6 oct 117 0-7pDR106B A348 C58/pTiA6 oct 166 53-82pDR106B MX226 C58/pTiA6::virATn5 oct 56 83-100

Nopaline pTiC58 BamHI fragment 2 and subclonespDMD2 A348 C58/pTiA6 oct 67 0-3pDR106 A348 C58/pTiA6 oct 111 40-60pDRB56 A348 C58/pTiA6 oct 48 38-68pDRB31 A1030 C58/pTiB6806::virATn5 oct 66 53-73In addition to the plasmids listed, each strain carried one of the MSV binary vectors. Plasmids and

vectors are described in Table 1. Specific infectivities represent data from a single experiment, rangesof infectivities represent data from a minimum of four independent experiments. Whenever possible,pTiA6-derived vir mutants were used for complementation studies. When this was not possible, virmutants of pTiB6806, an octopine-type plasmid with >87% homology by hybridization analysis topTiA6 (33), were used. agr, Agropine; oct, octopine; nop, nopaline. Where Ti plasmid-less strains wereused the opine designation is given in parentheses.

Microbiology: Raineri et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

60-

'>50-/ /

40

30i

20 -

I0-

0

lxio' Ix,I 1X106 lx107 lx1l0

# of bacteria in inoculumi (20pI)

FIG. 2. Effect of inoculum dilution on MSV DNA transferefficiency from LBA1517 (e), C58 (e), and A348(pDR106B) (A).Percent infectivity equals the number of infected plants divided bythe number of inoculated plants and multiplied by 100.

Octopine-Type Acetosyringone-Independent Signaling (Ais)virA Mutants Promote Maize Agroinfection at High Frequency.The octopine-type virA(Aip) locus carried by pEB112 (25)mediates vir gene expression in the absence of inducingstimuli. Introduction of pEB112 into A348 and the octopine-type virA mutant A1030 resulted in MSV infectivities of75-89% and 75%, respectively, in the absence of acetosy-ringone (Table 4). Two other virA(Ais) mutant plasmids,pEB129 and pEB137, were also tested. These mutants exhibit2.5-fold (pEB137) and 7-fold (pEB129) lower levels of ace-tosyringone-independent vir gene expression than pEB112(25) but still resulted in significant numbers of MSV-infectedplants when introduced into strain A348 (Table 4).

DISCUSSIONAgrobacterium exhibits marked Ti/Ri plasmid specificity inits interaction with the Gramineae (13-17). In this study wehave used the agroinfection technique to identify the regionofthe Ti plasmid which is responsible for the specificity in theagroinfection-maize interaction.We identified a region of the nopaline- and agropine-type

Ti/Ri plasmids which complemented octopine-type Ti plas-mids for maize agroinfection. Since there is no inhibition ofMSV DNA transfer from the nopaline-type strain C58 byoctopine-type Ti plasmid sequences (36), we concluded thatnopaline- and agropine-type strains contain an essential func-tion for maize agroinfection which is either lacking, nonfunc-tional, or specifically inhibited in octopine-type strains.

Subcloning of the complementing region of the nopaline-type Ti plasmid pTiC58 localized the complementing activityto the virA locus. By contrast, Jarchow et al. (16) found thatthe nontransferring octopine-type strain LBA4301(pTiA6)could be complemented with a plasmid containing all of thevir genes of pTiC58 except tzs, virA, and part of the virB

operon. The IncW-derived plasmid used by those investiga-tors, pTH60, is estimated to give four to five copies perAgrobacterium cell (37) whereas the Ti plasmid has a copynumber of 1-2. Therefore, the differences in the data mightbe explained by a "multicopy effect" in which the need forvirA-mediated induction is bypassed by increased expressionof virG (37, 38).An octopine-type virA gene under control of the nopaline-

type virA promoter was not complemented for MSV DNAtransfer to maize. This indicated that the VirA coding regionaccounts for the specificity in maize agroinfection, perhapsreflected in the greater similarity between nopaline- andagropine- than between nopaline- and octopine-type VirAproteins (90% and 73% amino acid identities, respectively;refs. 39 and 40).Higher levels of maize agroinfection were mediated by the

complemented octopine-type virA mutant than by the com-plemented wild-type strain. Based on recent findings whichindicated that VirA is multimeric (Shen Pan, personal com-munication), the most likely explanation for the decreasedMSV DNA transfer when octopine-type VirA was present isthat heteromultimers of nopaline- and octopine-type VirAhad intermediate phenotypes.

Octopine-type virA(AiS) mutants efficiently agroinfectedmaize. These findings support our contention that a functionor property of the virA locus, which can be altered by amutation conferring an acetosyringone-independent pheno-type, is responsible for the octopine-type Ti plasmid-specificdefect in agroinfection. The final level of vir gene expressionmediated by the virA(AiS) mutants in the absence of acetosy-ringone is lower than that mediated by the wild-type virA atsaturating levels of acetosyringone (25). The only apparentdifference between the wild-type virA and the virA(Ais) mu-tants is that virA(Ais)-mediated vir gene expression occurs inthe absence of acetosyringone, acidic conditions, and induc-ing monosaccharides. Therefore, the reason for the inabilityof octopine-type strains to agroinfect maize may be thatoctopine-type VirA activity is uninduced or inhibited bymaize extracts.

Induction of octopine-type vir genes by extracts fromwounded maize seedlings has been demonstrated (16, 18).However, those studies were done in vitro after pH adjust-ment of the extracts and so may not reflect the situation inplanta. This is especially relevant since Turk et al. (41) haveshown that octopine- and nopaline-type VirA proteins differin their pH requirements for optimal vir induction. Thus, itmay be that cereal sap is more compatible with the function-ing of nopaline-type VirA than octopine-type VirA. Anincrease in copy number of virA and virG can partially relievethe pH dependence of vir gene induction (42), perhapsexplaining the increased MSV infectivity mediated by oc-topine-type Ti plasmids when the octopine-type virA and virGwere inserted onto a higher-copy-number plasmid.

Boulton et al. (13) and Jarchow et al. (16) demonstratedthat preinduction of octopine-type strains does not promoteagroinfection, leading them to conclude that the limitationwas not due to lack of vir gene induction. However, Alt-Moerbe et al. (43) demonstrated that removal ofinducer leadsto an immediate stop in the formation of vir products and

Table 3. virA complementationsComplementing Chromosome/Ti Opine No. of plants Infectivity,

plasmid Strain plasmid family inoculated %pTB108 A348 C58/pTiA6 oct 33 11pTB108 A1030 C58/pTiB6806::virATn5 oct 49 9pIB100 A348 C58/pTiA6 oct 45 20pDR178 A348 C58/pTiA6 oct 53 4pDR178 A1030 C58/pTiB6806::virATnS oct 49 9

For a description of recombinant plasmids, see Table 1. For other details, see legend to Table 2.

3552 Microbiology: Raineri et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 3553

Table 4. Transfer efficiencies of octopine-type virA mutants exhibiting acetosyringone-independent expression of thevir regulon

Complementing Chromosome/Ti Opine No. of plants Infectivity,plasmid Strain plasmid family inoculated %pEB112 A348 C58/pTiA6 oct 89 75-89pEB112 A1030 C58/pTiB6806::virATn5 oct 39 75pEB129 A348 C58/pTiA6 oct 18 40pEB137 A348 C58/pTiA6 oct 16 51pEB137 A1030 C58/pTiB6806::virATn5 oct 22 43

For description of recombinant plasmids, see Table 1. For other details, see legend to Table 2.

T-DNA intermediates. In summary, these observations aloneare not sufficient evidence that induction of octopine-type virgenes by maize occurs in planta.

It is also possible that inhibitory substances, presumablyspecific to graminaceous plants, specifically block octopine-type VirA activity. The observation that octopine-typevirA(Ais) mutants can promote agroinfection might be ex-plained by the inability of maize inhibitors to block thesignal-independent induction of vir gene expression by themutant sensor proteins.

It has been suggested that the limitation to maize agroin-fection by octopine-type Ti plasmids lies in virF (16). Titra-tion ofinocula of strains containing octopine-type Ti plasmidsand the complementing region from pTiC58 gave an infec-tivity gradient analogous to that obtained with wild-type C58.In contrast, we rapidly reached an end-point dilution of thevirF mutant LBA1517 (35). Furthermore, VirF did not sup-press agroinfection mediated either by nopaline-type Ti plas-mids or by the complemented octopine-type Ti plasmids (16,36). Thus, we concluded that virFdoes not play the major rolein determining the specificity of maize agroinfection as sug-gested by Jarchow et al. (16).The simplest interpretation of the data presented above is

that the block to maize agroinfection by octopine-type Tiplasmids involves a function or property of the virA locus.VirA acts as an environmental sensor of plant-derived in-ducer molecules and transmits this information to the level ofvir gene expression. We propose that slight variations at thevirA locus could have a profound effect on T-DNA transferefficiency. An understanding of the differences between theoctopine- and nopaline-type virA gene products will provideimportant information on the requirements for efficient DNAtransfer it.o maize. We assume that the observations madeon agroinfection of maize will hold for agroinfection ofgraminaceous monocots in general.

We thank C. Kado for kindly providing pUCD2614, P. Bottino forhelp with ELISAs, and Lin Luong, S. Bowra, and D. King forexcellent technical support. This work was supported by RockefellerFoundation Grant RF90031108 and National Institutes of HealthGrant 5ROlGM32618-20. D.M.R. is the recipient of a North AtlanticTreaty Organization/Science and Engineering Research Councilpostdoctoral fellowship. M.I.B. is supported by a grant from theRockefeller Foundation.

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