Highly Specific Antiviral State in Transgenic Gene ... · lnduction of a Highly Specific Antiviral State in Transgenic Plants: lmplications for Regulation of Gene Expression and Virus

The Plant Cell, Vol. 5, 1749-1759, December 1993 O 1993 American Society of Plant Physiologists

lnduction of a Highly Specific Antiviral State in Transgenic Plants: lmplications for Regulation of Gene Expression and Virus Resistance

John A. Lindbo,a Laura Silva-Rosales,b William M. Proebsting,'Id and William G. Dougherty a Department of Microbiology, Oregon State University, Corvallis, Oregon, 97331-3804

MBxico Departamento de lngenieria Genetica, CINVESTAV-IPN, Unidad Irapuato, Apartado Postal 629 Irapuato, Gto. 36 500

Department of Horticulture, Oregon State University, Corvallis, Qregon 97331-7304 Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-7303

Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typlcal systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenlc plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced ln the recovered tissue. The resistance was virus speclflc. Recovered plant tissue could not be lnfected wlth TEV, but was sus- ceptlble to the closely related virus, potato virus Y. The resistance phenotype was functional at the slngle-cell leve1 because protoplasts from recovered transgenic tissue did not support TEV repllcation. Surprlslngly, steady state transgene mRNA levels in recovered tissue were 12- to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studles suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular leve1 by a cytoplasmic actlvity that targets speclfic RNA sequences for inactlvation.

INTRODUCTION

The theory of pathogen-derived resistance (PDR) proposes that pathogen resistance genes can be derived from a pathogen's own genetic material (Sanford and Johnston, 1985). Numerous examples of PDR have been reported for many different plant RNA viruses in a wide range of plant species. Most examples of PDR involve transgenic plants engineered to express a viral coat protein (CP) or a segment of a replicase gene (for reviews, see Beachy et al., 1990; Wilson, 1993). As a general rule, transgenic plants accumulating one of these viral proteins are often resistant to that particular virus and closely related viruses. Although many examples of PDR have been documented, in general, the mechanism(s) underlying resistance remains to be clearly defined.

We have examined different PDR approaches to the potato virus Y (PVV; potyvirus) group. Potyviruses, assigned to the picornaviral superfamily (for review, se8 Goldbach, 1987), comprise a large and economically important group of aphid-transmissible plant viruses (for review, see Reichmann et al., 1992). Some general characteristics of potyviruses

To whom correspondence should be addressed.

include (1) flexuous rod-shaped virions (-18 x 750 nm); (2) an RNA genome with a protein (referred to as VPg) covalently attached to the 5' terminal nucleotide and a 3' polyadenylate sequence; (3) genetic information contained in a single, large open reading frame coded for by a single-stranded RNA genome (-9500 nucleotides) of plus (message) sense polarity; and (4) individual gene products expressed by proteolytic pro- cessing of the genome-derived polyprotein. Severa1 examples of PDR to various potyviruses have been reported (for review, see Lindbo et al., 1993). In most cases, no correlation between transgene product accumulation and the degree of potyvirus resistance was noted. This is in contrast to PDR studies in many other virudtransgenic plant systems in which resistance correlated with high transgene expression. Thus, the exact mechanism underlying PDR for potyviruses remains enigmatic.

We have previously described the construction of a series of transgenic Burley 49 tobacco lines producing different versions of the tobacco etch virus (TEV) CP gene sequence (Lindbo and Dougherty, 1992a). Two of these transgenic plant lines, expressing either a full-length (FL plant lines) form of the TEV CP or a form truncated at the N terminus of the TEV CP (AN29 plant lines), temporally developed typical systemic

1750 The Plant Cell

symptoms when inoculated with TEV. However, all FL andAN29 transgenic plant lines outgrew TEV infection ~3 to 5weeks after inoculation (Lindbo and Dougherty, 1992a). Werefer to this phenomenon as recovery. Other researchers havealso noted that selected transgenic plants expressing poty-virus CP sequences display a similar response after inocu-lation with other potyviruses (Ling et al., 1991; Fang andGrumet, 1993).

In this study, we examined features of recovered transgenicplant tissue. Asymptomatic, recovered tissue did not supportTEV replication, yet it supported replication of the closelyrelated potyvirus, PVY. RNA and protein analyses of recoveredtransgenic plant tissue demonstrated that both transgene RNAand protein steady state levels were markedly reduced in re-covered transgenic plant tissue. This occurred while nuclearrunoff studies suggested these transgenes were still being ac-tively transcribed. We propose that the induction of resistanceto TEV, an RNA virus that replicates in the cytoplasm, and the

decrease in RNA accumulation of the nuclear transcribed trans-gene are mediated by a common cytoplasmic activity. Cyto-plasmic regulation of gene expression may have importantimplications for a number of apparently unrelated plantphenomena.

RESULTS

Plant Studies

All TEV-infected transgenic plant lines expressing an FL orAN29 truncated form of the TEV CP recovered from TEV in-fection. During this process, initial infection and typicalTEV-induced symptoms were succeeded by progressively lesssymptomatic tissue in each newly emerging leaf. In theseemerging leaves, virus-induced symptoms were restricted to

Figure 1. TEV-lnduced Symptoms in Untransformed and Transgenic Tobacco Plants.TEV-induced symptoms are shown for a Burley 49 tobacco plant (lower left), and for a plant from a transgenic line (FL-44.4; upper left) expressingan FL version of the TEV CP. The transgenic plant has outgrown or "recovered" from TEV infection; newly emerging leaves contain less virusand virus-induced symptoms than older leaves of the same plant or than the corresponding leaf from a TEV-infected Burley 49 plant. Leavesfrom a lower, middle, or upper segment (leaves 1, 2, and 3, respectively) of these plants are presented for comparison. Photographs were taken~5 weeks after inoculation with TEV.

Virus Resistance in Transgenic Plants 1751

interveinal areas. This progressive recovery continued until leaves emerged devoid of symptoms. As a result, the recovered transgenic plants had a distinct appearance compared to TEV-infected untransformed Burley 49 tobacco plants, as shown in Figure 1.

lnoculation studies with viruses other than TEV demonstrated that the recovery phenomenon was TEV specific. These FL and AN29 transgenic plant lines were susceptible to the closely related potyviruses PVY and tobacco vein mottling virus, as well as a number of unrelated plant viruses. lnoculation of these transgenic lines with these viruses established asys- temic infection and no evident recovery (data not shown). Aside from the TEV-induced recovery phenotype, height, weight, and general morphology of transgenic plants used in this study were similar to untransformed plants.

We quantified the leve1 of TEV in transgenic and untransformed Burley 49 tobacco plants to determine if infectious TEV was present in tissue displaying the recovered phenotype. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA; Converse and Martin, 1990) was used to indirectly quantitate TEV in the various tissue types. The DAS ELISA conditions used detected only CP from replicating TEV, not TEV CP produced from the transgene. Levels of TEV CP in symptomatic leaf tissue of infected transgenic plants were usually slightly lower than those in infected untransformed Burley 49 tobacco, as shown in Table 1. However, this differ- ence was most striking when comparing TEV CP levels of TEV-infected Burley 49 tissue to levels found in the asymptomatic tissue of plants displaying the recovered phenotype. No TEV CP was detected in asymptomatic recovered leaf tissue (Table 1).

To demonstrate the presence of infectious TEV in transgenic tissue, we used bioassays involving both transmission by aphid vectors and back inoculation studies to a susceptible indica- tor host. In general, lower TEV CP levels were correlated with lower aphid transmission rates when TEV-infected transgenic tissue was used as an inoculum source by aphids (Table 1). However, aphid transmission of infectious virus from asymptomatic leaf tissue of the recovered phenotype or back inoculation of sap from this tissue (data not shown) to Burley 21 tobacco plants was unsuccessful. Therefore, using a variety of experi- mental approaches, TEV could not be detected in asymptomatic tissue of leaves showing the recovered phenotype.

These results suggested that TEV was excluded from or could not replicate in recovered transgenic tissue. Plants from the FL and AN29 lines were tested for their ability to support virus replication in a sequential inoculation series. Plants from each of the lines were “pretreated” in one of four ways prior to a challenge inoculation with TEV or PVY. The four pretreatments were (1) naive (receiving no initial treatment), (2) mock inoculation with uninfected plant sap and carborundum on a lower leaf, (3) inoculation with tobacco mosaic virus, or (4) inoculation with TEV No visible plant symptoms were evident in pretreatments 1 and 2. Pretreatment 3 resulted in the for- mation of local lesions on the inoculated lower leaf, whereas pretreatment 4 resulted in systemic TEV infection followed by

~~ ~ ~~

Table 1. TEV Concentration in TEV-lnfected Plants

Aphid

Plant Lima Typeb Tissue (mg)C Rated

Burley 49 1 1.45 616 Burley 49 2 1.5 616 Burley 49 3 1.35 516

FL-24.3 1 1 .o 516 FL-24.3 2 0.4 1 I6 FL-24.3 3 0.0 O16

FL-44.4 1 1.4 316 FL-44.4 2 0.3 2/6 FL-44.4 3 0.0 016

FL-3.3 1 8.4 316 FL-3.3 3 0.0 016

AN29-8. 1 1 .o 316 AN29-8. 2 0.5 116 A N29-8. 2 0.2 2/6

AN29-8. 8 2‘ 5.5 316 AN29-8.1e 29 0.0 O16

Leaf Virus (pg)/ Transmission

~~

a Plant nomenclature is as described in the text. Burley 49 is the untransformed tobacco germ plasm. The other lines presented are transgenic and expressing an FL form of the TEV CP ora form truncated at the N terminus (AN29).

Leaf type 1, 2, or 3 are lower, middle, or upper leaves, respectively, as shown in Figure 1. Estimation of virus concentration as a standard curve (data not

shown) was produced by adding known quantities of purified TEV to uninfected Burley 49 extracts. Tissue extracts were analyzed by DAS ELISA procedures using alkaline phosphatase-conjugated rabbit anti-TEV antibodies. lnfected plant samples (except e) were collected by randomly removing six circular (10-mm-diameter) tissue samples from a leaf, combining them, and then preparing tissue extracts. Aphid transmission rate indicates the number of test plants that be-

came infected with TEV via aphid transmission over the total number of test plants aphids were allowed to feed on. Aphid transmission tests were performed by allowing 10 aphids to feed on a particular leaf type, and then the aphids were transferred to a test plant. This was repeated six times for each leaf type. e Symptomatic and asymptomatic tissue samples from the same recovering leaf ( ia , leaf type 2, Figure 1) were analyzed. Symptomatic area.

g Asymptomatic area.

recovery. Four weeks after the original pretreatment, upper leaves of these plants were inoculated with a challenge dose of either W Y or TEV. These results are summarized in Table 2. All plants were susceptible to PVY infection. Plants receiving pretreatments 1,2, or 3 could be infected by TEV; however, those plants that had recovered from prior TEV infection were unable to support a TEV infection in the recovered tissue.

Further characterization of the induced resistant state involved grafting of recovered scions from various FL and AN29 transgenic plants and grafting scions from untransformed

1752 The Plant Cell

Table 2. lnduction of Recovered Phenotype in Transgenic Piants

Pre- treat- Treat- Symptoms on

Plant Lima mentb ment Upper LeavesC

Group 1 TEV (transgenic PVY plant lines) TEV

TEV TMV

TMV Mock

Mock None

None

Group 2 TEV

plant lines) TMV TMV Mock Mock None None

(control . PVY

None None TEV PVY TEV

PVY TEV

PVY TEV

PVY

None None TEV PVY TEV PVY TEV PVY

None Systemic PVY symptoms None Systemic PVY symptoms Systemic TEV symptoms

foilowed by recovered plant phenotype

Systemic P W symptoms Systemic TEV symptoms

followed by recovered plant phenotype

Systemic PVY symptoms Systemic TEV symptoms

followed by recovered plant phenotype

Systemic PVY symptoms

Systemic TEV symptoms Systemic PVY symptoms Systemic TEV symptoms Systemic PVY symptoms Systemic TEV symptoms Systemic PVY symptoms Systemic TEV symptoms Systemic PVY symptoms

aThe plant lines used in this study are presented as two groups based on their identical response to the various challenge inoculations. Group 1 plants (FL-44.4, FL-24.3, AN29-8.1, and AN29-1.9) were transgenic and displayed the recovery phenotype. The nomenclature used is as described in the text. Group 2 plants were control lines that did not dispiay the recovery phenotype and were Burly 49 tobacco and transgenic line 358-4.7 (a vector-oniy transgenic control line containing no TEV sequences).

The two sequential treatments were applied 4 weeks apart. TEV, PVY, or tobacco mosaic virus (TMV) was mechanically inoculated onto leaf tissue dusted with carborundum using a 1:lO dilution of virus- infected plant sap. For the mock inoculation studies, a 1:lO dilution of uninfected Burley 49 extract was used. C Symptoms were observed daily for 30 days afler the second inoculation treatment.

Burley 49 plants onto TEV-infected Burley 49 tobacco rootstock. Scions from transgenic plants displaying the recovered phenotype remained symptom and virus free, whereas untransformed Burley 49 scions became infected and showed typical systemic TEV symptoms within 10 to 15 days after the graft was established. Resistance was absolute in the recovered tissue and symptom development was never observed in the recovered scions of more than 50 grafted plants. The results of one grafting experiment using FL-44.4 transgenic tobacco plants are presented in Table 3. Recovered FL or AN29 scions grafted onto PVY-infected Burley 49 rootstock did become infected and

suppofted P W replication (data not shown). Attempts were made to induce TEV resistance in unchallenged FL and AN29 lines and in Burley 49 tissue. Various graft combinations were established, typically by grafting untransformed Burley 49 tissue or an unchallenged FL or AN29 scion onto a recovered FL or AN29 rootstock. In nearly all cases, the scion became infected within 10 to 20 days after grafting. This suggested that virus moved readily through recovered tissue and that a translocatable signal was not involved in the induction of resistance.

The TEV resistance phenotype of progeny plants from TEV- recovered AN29 and FL lines was also examined. Plants

Table 3. TEV lnfection of FL-44.4 Grafted Plants

TEV Symptomsb No. of Sciona Grafted Scion Rootstock Plants Rootstock

r 1 - FL-12.6(r) r FL-44.4 TEV

FL-44.4 FL-12.6(r) TEV

FL-12.6(r) TEV FL-44.4

2 r r -

- 1 - FL-12.6(r)

FL-44.4 TEV + r

FL-44.4(r) r - 5 FL-44.4 TEV

5 FL-44.4(r) FL-44.4 TEV

- 2 - FL-44.4(r) TEV

FL-44.4 -

2 - FL-44.4(r) TEV Burley 49 -

-

FL-44.4(r) Burley 49 TEV

Burley 49 TEV

FL-44.4(r)

FL-44.4 35S-4.3 TEV

2

+ - 1 r

3 r - +

a The scionlrootstock combination is presented. The nomenclature used is as described in Table 1, The designation (r) indicates whether the rootstock or scion was from a plant showing the recovery phenotype. The TEV designation indicates whether rootstock or scion was mechanically inoculated with TEV. The symptoms evident 30 days after infection with TEV are pre-

sented. The presence (+) or absence (-) of TEV-induced systomic symptoms or tissue displaying the recovery phenotype (r) is noted.


Table 4. Analysis of TEV and PVY Replication in Protoplasts Derived from Unchallenged or Recovered Transgenic Plant Tissue

~

Recovery of Percent of lnfectious TEV Protoplasts from Protoplasts Suppotting Virus lnfected with

lnoculum

Protoplast Sourcea Replicationb TEV RNAC

also analyzed for the presence of infectious particles in back- inoculation studies. The results are presented in Table 4. Pro- toplast studies correlated with whole-plant studies; protoplasts from “recovered tissue did not support TEV replication, whereas PVY replicated in protoplasts from either recovered or unchallenged leaf tissue. Therefore, both the protoplast and whole-plant studies suggested that the recovered phenotype was dueto the inability of TEV to replicate in recovered leaf tissue.

TEV PVY No RNA RNA RNA Analysis of Transgene Expression

FL-24.3 88 59 o + FL-29.3 96 65 O + AN29-8.1 82 30 O +

FL-24.3 (r) o 57 o - FL-29.3 (r) o 57 o - AN29-8.1 (r\ O 69 O - a Results from three different lines are presented. Transgenic plant nomenclature is as described in text; (r), leaf tissue with the recovered plant phenotype.

Protoplasts were transfected with purified TEV RNA, PW RNA, or with no RNA, as described in the text. Results are presented as a percentage of protoplasts infected as determined in protoplast blot- ting assays. Monoclonal anti-TEV CP antibodies or anti-PVY polyclonal antisera were used to detect vira1 antigen. Approximately 200 viable protoplasts (per sample) were counted.

Protoplasts that had been transfected with TEV RNA were pelleted and lysed; extracts were inoculated onto Burley 49 tobacco leaves. Plants were examined 1 week later for the appearance of TEV-induced systemic symptoms.

showing the recovered phenotype were allowed to self-pollinate and set seed. Progeny plants were grown and then inoculated with TEV-infected plant sap. These plants reacted to virus infection in a fashion identical to parenta1 plants; plants became infected systemically and then recovered from TEV infection. Therefore, progeny had not acquired the induced TEV-resistant state from their parent but had inherited the ability to recover after TEV inoculation.

Protoplast Studies

Studies were conducted to determine if the TEV-induced resistance was functional at the single-cell levei. Protoplasts from both asymptomatic tissue of TEV-infected plants displaying the recovered phenotype and unchallenged FL and AN29 transgenic leaf tissue were transfected with either PVY or TEV RNA. After incubation ( ~ 2 4 to 48 hr), the percentage of protoplasts supporting virus replication was determined by analyzing transfected protoplasts for the presence of TEV CP in a protoplast printing assay (Jung et al., 1992). Transfected protoplasts were

Recovered and unchallenged transgenic leaf tissues were analyzed for transgene transcript and translation product accumulation. Steady state levels of transgeneencoded RNA and protein were examined by RNA and protein gel blot analyses, respectively. Surprisingly, transgene CP could not be detected in recovered tissue, as shown in Figure 2A, and transgene mRNA levels from recovered tissue were greatly reduced, as shown in Figure 2B. Quantitation of transcript levels revealed a 12- to 22-fold reduction in transgene mRNA levels in recovered tissue, as compared to transgene mRNA levels in unchallenged transgenic plant tissue at an equivalent Stage of development. As an internal control, actin mRNA levels were also analyzed in these same RNA samples. This analysis revealed that actin mRNA levels were approximately constant in the different RNA samples (Figure 2C).

Nuclear run-off assays were used to estimate the relative transcriptional activities of TEV CP transgenes in nuclei isolated from TEV-recovered or unchallenged transgenic plant tissue, as shown in Figure 3. In this assay, actin transcription rates were also monitored as an internal control. CP transgene transcription rates were normalized to actin gene transcription rates. This analysis revealed that the relative transcription rates of the TEV CP transgene were roughly equivalent in TEV- recovered and unchallenged transgenic plant tissue or that CP transgene transcription rates may even be slightly higher in recovered transgenic plant tissue.

DlSCUSSlON

We observed a unique, TEV-resistant state induced in transgenic plants expressing an FL form ora form truncated at the N terminus of the TEV CP gene. When initially challenged with TEV, these transgenic plants displayed typical TEV-induced symptoms, but gradually outgrew infection by -3 weeks postinoculation. We refer to this process as recovery. As a result of this process, recovered transgenic plants consist of lower symptomatic leaves (which contain virus) and upper asymptomatic leaves (which are virus free). Only TEV induced this phenotype; mock inoculation or inoculations with other viruses did not induce the recovered plant phenotype. Using avariety of approaches, we examined the ability of recovered tissue

1754 The Plant Cell

1 2 3 4 5 6 7 8

B

f t

Figure 2. Analysis of Steady State Levels of Transgene RNA and Pro-tein from the Asymptomatic Areas of Recovered Leaves and fromUnchallenged Transgenic Plant Tissue.

Extracts from the same tissues are in identical lanes in (A), (B), and(C), except where noted. Protein ([A]) or RNA ([B] and [C]) sampleswere used from the following tissues: lanes 1, samples from Burley49 tobacco tissue to which 10 ng of purified TEV ([A]) or 100 pg ofan RNA transcript containing the TEV coat protein sequence was added([B]); lanes 2, samples from Burley 49 tobacco tissue; lanes 3, sam-ples from a transgenic FL-24.3 plant that was not infected with TEV;lanes 4, samples from a FL-24.3 transgenic plant that had recoveredfrom TEV infection; lanes 5, samples from a transgenic FL-44.4 plantthat was not infected with TEV; lanes 6, samples from a transgenicFL-44.4 plant that had recovered from TEV infection; lanes 7, samplesfrom a transgenic AN29-8.1 plant that was not infected with TEV; lanes8, samples from a AN29-8.1 transgenic plant that had recovered fromTEV infection.(A) Protein gel blot analysis of transgenic and Burley 49 plant tissueisolated from plants described above. Total protein was extracted andelectrophoresed on a 10% polyacrylamide gel containing SDS andthen transferred to a nitrocellulose membrane. The membrane wasreacted with rabbit anti-TEV polyclonal sera, goat anti-rabbit IgG (alka-line phosphatase-conjugated), and the chromogenic substrates p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indoyl phosphate todetect bound TEV CP molecules. The membrane was incubated withthe substrate for 1 hr. The position to which full-length TEV CP mi-grates is indicated by the arrow on the left. Proteins that cross-reactwith our anti-TEV serum are present at the top and serve as an inter-nal control for the amount of protein loaded in each lane.(B) and (C) RNA gel blot hybridization analyses of Burley 49 and trans-genic plant tissue. Total RNA was extracted from plant tissue describedabove and analyzed. RNA samples were separated by electrophore-sis on denaturing (formaldehyde) agarose gels and transferred tonitrocellulose. TEV CP transgene transcripts ([B]) or actin sequences([C]) were detected by hybridizing filters with the appropriate 32P-labeled RNA probe. The position to which the 1100-nucleotide TEVCP transgene transcript migrates is indicated by the arrow on the leftof (B). Filters were exposed to x-ray film and photographs of the au-toradiograms are presented. Note that no sample was loaded in lane1 of the autoradiogram in (C).

to support TEV replication. In whole-plant studies, recoveredtissue could not be infected with TEV; but FVY, a closely related(~47°/o total nucleotide sequence homology; ~63°/o CP genesequence homology) potyvirus, established a normal systemicinfection. Comparable results were obtained in protoplaststudies; protoplasts from recovered tissue did not support TEVreplication but did support PVY replication.

For the induction of the TEV-resistant state, we observedthat the infecting virus (TEV) must first establish a systemicinfection. This suggested that stimulation (or infection) of cellsin the apical meristem was necessary. It appears that a spe-cific interaction involving TEV, the transgene, and the host plantis necessary for the induction of the resistance phenotype. Thedata suggest that the transgene transcript and the replicatingTEV genome act additively to trigger a natural cellular responsewhich down-regulates or inactivates the transgene RNAs inthe cytoplasm. Stimulation of this system apparently resultsin a highly resistant phenotype to TEV, because TEV RNA se-quences are inactivated.

The induced resistance observed in this study is distinct fromthe phenomenon of systemically acquired resistance (for re-view, see Kuc, 1982). Systemically acquired resistance can beinduced by a variety of pathogens after a necrotic local lesionresponse to a pathogen; it also shows broad spectrum effects

I 4TEVCP

pTL-37 NPTIIActin

Figure 3. Estimation of Transgene Transcription Levels in TEV-Recovered and Unchallenged Transgenic Plant Tissue.

Nuclei were isolated from TEV-recovered and unchallenged transgenicplant tissue. Nascent transcripts were labeled by in vitro nuclear run-off transcription reactions in the presence of a-32P-CTP. Labeledtranscripts were hybridized to nitrocellulose filters to which DNA se-quences of the plasmid pTL-37 (as a negative control, lanes 1), theTEV CP gene (lanes 2), the neomycin phosphotransferase II (NPTII)gene (lanes 3), and an actin cDNA sequence (lanes 4) had been bound.The relative transcriptional activities of the TEV CP and NPTII genewere estimated by normalizing all signals to the levels of actin genetranscription.(A) Slot blot probed with run-off transcripts from nuclei isolated fromunchallenged transgenic plant tissue (AN29 plant line 8.1).(B) Slot blot probed with run-off transcripts from nuclei isolated froma TEV-recovered transgenic plant tissue (AN29 plant line 8.1).


against different pathogens (viruses, fungi, and bacteria), is active in untransformed germ plasm (does not require the pres- ente of a particular transgene), and has been linked to the induction of a number of genes with the involvement of sali- cylic acid as a translocatable signal (Ward et al., 1991). In contrast, the induced TEV-resistant state in FL and AN29 plants that resulted from “outgrowing” a systemic infection was TEV specific. The induced TEV-resistant state also required the pres- ente of a TEV-derived transgene. An endogenous signaling molecule did not appear to be involved because the phenotype could not be transmitted in grafted plant studies. The involvement of endogenous host genes activated by viral or viral-induced signals has not been determined in our system.

Coincident with the TEV-resistant state in recovered plant tissues, no transgene CP was detected and transgene mRNA accumulation levels were reduced 12- to 22-fold compared to transgene mRNA levels in unchallenged transgenic plant tissues of the same developmental stage. Nuclear run-off assays suggested that the relative transcription rates of the transgene were essentially identical in these two tissue types. Collec- tively, these observations suggest that the reduction in steady state transgene RNA levels is due to a post-transcriptional mechanism of control and not to inactivation of the transgene promoter in TEV-recovered transgenic plant tissue.

One explanation for our observations was that decreased transgene steady state levels and the induced TEV-resistant state were unlinked, induced by two separate mechanisms. In this scenario, a reduced rate of transport of transgene RNA from the nucleus to the cytoplasm could be responsible for the low steady state levels of transgene RNA, and the induced TEV-resistant state could be mediated by another cellular process. While cognizant of this possibility, we propose that the two characteristics common to all recovered tissue tested (i.e., a decreased accumulation of a nuclear-encoded TEV mRNA transcript and the inability of TEV, an RNA virus, to replicate in the cytoplasm) are linked. With this caveat, we have developed a working model for our observations.

The model predicts that the molecular basis of the recovered phenotype is a cytoplasmic event in which specific RNA sequences (in this study, TEV CP sequences) are targeted, functionally suppressed, and eventually eliminated from the cell. Mechanistically, the model suggests that a protein or nucleic acid factor binds to a specific RNA sequence, rendering this RNA functionally inactive and targeting it for elimination. A schematic drawing of the model is presented in Figure 4.

What are the components that could comprise such a system and how might a system that recognizes foreign, over- expressed, or aberrant RNAs operate? lnactivation and elimination of specific RNAs could be mediated by either protein or nucleic acid factors. In the scenario in which a protein factor governs specificity, an RNA binding protein that recognizes a specific primary sequence or secondary or tertiary struc- ture is induced, synthesized, and binds to the RNA species. The RNA would be inactivated and targeted for elimination by an RNase that recognizes this RNA-protein complex. Such a system would require a significant amount of genetic

Elevated transgene expression

TEV RNA Ly LY

-1 /- 4. Degradation I ‘ - --

.I-

, ’I - I

Figure 4. Schematic Presentation of the Working Model Proposed to Account for RNA Degradation and the Antivirai State.

The induced resistance is proposed to be acell-mediated event. Plant cells are able to (1) sense elevated or aberrant RNA levels in a manner not understood. These sequences are then (2) targeted and (3) inactivated by a cellular factor that may be a protein or nucleic acid. The complex formed between the target RNA sequence (host and/or viral) and the cellular factor will direct cellular enzymes to (4) degrade the RNA, resulting in its elimination from the cytoplasm. The cellular factor is represented by the open ovals and RNAs are presented as rectangular boxes. RNA sequences shared between transgene transcript and viral RNA and targeted by the proposed cellular system are filled in.

information to code for the large repertoire of RNA binding factors needed to identify specific sequences. An alternative model implicates RNA as the factor mediating the process. Plant cells are known to contain RNA-dependem, RNA-polymerase (RdRp), an enzyme with a molecular weight of 130,000, that lacks specificity in cell-free reactions, and that will copy most input template RNA into small complementary 2s to 5s RNAs (<100 nucleotides) (Schiebel et al., 1993). Cellular RdRp levels increase approximately two- to threefold (although up to 20-fold stimulationhas been reported; van der Meer et al., 1984) during viroid and virus infection. RdRp activity has not been analyzed during elevated transgene expression. In the model, RdRp would copy a small segment or segments of an RNA that had accumulated to unacceptably elevated levels. These small RNAs would then hybridize with the target RNA, rendering the RNA nonfunctional, and RNases would target the partially double-stranded messenger or viral RNA complex for degradation. The small complementary RNA species

1756 The Plant Cell

generated by RdRp would circulate freely in the cytoplasm and rebind to new target RNA (viral or transgene) sequences. Al- ternatively, RdRp, once primed, would constitutively express these RNAs. Subcellular compartmentalization of messenger or viral RNA may also be important in eliciting the response. A system mediated by RNA has appeal in its relative simplic- ity, specificity, and the limited amount of genetic information required to code for implicated cellular factors.

The induction of a cytoplasmic activity that inactivates and eliminates specific RNA sequences from a cell may explain a number of apparently disparate and unrelated plant cell phenomena. For example, we suggest that certain examples of cross-protection between infectious agents, the effect of some defective interfering RNAs (DI RNAs), selected examples of sense or cosuppression of transgenes, and the recently described examples of RNA-mediated virus resistance may be explained by such a mechanism.

In the typical demonstration of cross-protection (for review, see Fulton, 1986), a plant is infected systemically with a mild isolate of a particular virus. The infected plant, when challenged with a more virulent isolate of that same virus, does not support replication of the second virus. As a result, severe symptoms induced by the second virus do not develop. A host response, similar to that proposed for recovered trangenic plants (i.e., enhanced viral RNA turnover), may be activated and prevent the second challenging virus from replicating. Such a mechanism would not only be functional for RNA viruses, but also could explain the cross-protection phenomenon observed with viroids (Niblett et al., 1978; Branch et al., 1988).

DI RNAs reduce virus replication levels in a number of different virus-host systems via mechanisms proposed to be competitive with virus replication machinery. Recent work may be interpreted to suggest that some DI RNAs also mediate their effect via a host cell RNA regulatory system. Elegant studies with brome mosaic virus (BMV) have shown that certain minus strand DI RNAs quite effectively inhibit BMV replication in protoplasts (Huntley and Hall, 1993). However, inhibition is clearly not via the complementary pairing of DI RNA and virus RNA. An elevated cellular RdRp activity that transcribed these truncated viral RNAs was also noted in this study. The BMV DI RNA analysis may provide insight into the length and composition of sequences needed to elicit the putative cellular response.

We suggest that a cytoplasmic activity, similar to that proposed for our virus-induced state, may be operational for some examples of sense or cosuppression. The phenomenon of sense suppression has been described for a number of different genes in transgenic plants (Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990; de Carvalho et al., 1992; Hart et al., 1992). Attempts to overexpress a particular gene product via the introduction of a redundant transgene often result in suppression of both the endogenous gene and exog- enous transgene. In a number of cases, methylation of DNA sequences correlates with reduced gene expression (Matzke et al., 1989; Hobbs et al., 1990,1993; Matzke and Matzke, 1991;

Ottavianai et al., 1993). However, in some cases, sense suppression appears to be mediated post-transcriptionally (de Carvalho et al., 1992), and a cytoplasmic system may account for the suppression of transcript levels.

Finally, we suggest that the antiviral state in RNA-mediated resistant transgenic plants (de Haan et al., 1992; Lindbo and Dougherty, 1992b; van der Vlugt et al., 1992; Pang et al., 1993) and the recovered CP-producing transgenic plants function by a similar mechanism. We have previously described transgenic tobacco plants containing a TEV CP gene modified by introduction of three stop codons immediately downstream of the AUG start codon (Lindbo and Dougherty, 1992b). Such transgenic plant lines express an untranslatable TEV CP mRNA. Approximately 40% of transgenic plant lines expressing this RNA are highly resistant to TEV infection but remain susceptible to PVY, tobacco vein mottling virus, and other viruses. The other n~60% of the transgenic lines express the Same untranslatable TEV CP mRNA but are susceptible to TEV as well as other RNA plant viruses. Results obtained with whole plants are reproduced at the cellular leve1 in protoplasts. We also observed that resistance to TEV did not correlate with transgene mRNA accumulation levels in these transgenic plants. We propose that the highly resistant plants expressing the untranslatable RNA have a cytoplasmic activity that is per- manently stimulated and functions in a manner analogous to the TEV-induced system in recovered FL and AN29 transgenic plants. Activation of this RNA sequence-specific, cellular response renders the plants completely resistant to subsequent TEV infection but susceptible to potyviruses with similar yet distinct sequences. Activation of such a system may also explain why resistance and RNA accumulation levels in these plants are not correlated. Steady state levels of transgene transcripts would be a function of RNA synthesis and turnover rates. In highly resistant lines, the system would be activated. RNA accumulation in these lines may be lower than in susceptible transgenic lines in which the system has not been activated and only synthesis is being monitored. Hence, one might ex- pect to find a negative or no correlation between RNA accumulation and resistance.

Our analysis and interpretation of the PDR literature suggest that no one unifying mechanism exists; rather, a number of different mechanisms will be functional with different PDR strategies. The model proposed only accounts for our observations with potyvirus-derived resistance. There are a number of obvious predictions as well as many unanswered questions implicit in the model. The intent of the model and its extension to other plant-related phenomena is to stimulate discussion and perhaps provide a new conceptual framework to examine a number of important yet perplexing plant phenomena.

METHODS

Construction of Transgenic Plants

Construction, selection, and analyses of transgenic plants used in this study have been described previously (Lindbo and Dougherty, 1992a).


The transgenic plant lines used in this study accumulate either a full- length (FL) or N terminally truncated (AN29) form of the tobacco etch virus (TEV) coat protein (CP). The AN29 transgenic plant lines express a form of the TEV CP missing the N-terminal 29 amino acids.

Vlrus lsolates

TEV-H (highly aphid transmissible) strain was originally obtained from Dr. Tom Pirone (University of Kentucky, Lexington). The potato virus Y (PW) isolate used in this study was obtained from Dr. Guy Gooding (North Carolina State University, Raleigh). Viruses were maintained in Nicotiana tabacum cvs Burley 49 or Burley 21. Virus was purified as described by Dougherty and Hiebert (1980). Potyviral RNA was obtained from purified virus preparations by adding an equal volume of proteinase K solution (50 mM Tris-HCI, pH 8.0, 1 mM CaCI2, 1% SDS, 100 pg/mL proteinase K) to virus preparations. The solution was vor- texed, incubated at 45OC for 10 min, and then extracted with an equal volume of phenol/chloroform (1:l). RNA was precipitated as described by Sambrook et al. (1989) and resuspended in double-distilled water.

Whole-Plant lnoculation Experiments

Plant leaves were lightly dusted with carborundum and virus inoculum (50 pL) was applied with a cotton swab. Virus inoculum was a 1:lO dilution (wh) of virus-infected plant tissue in 100 mM sodium phosphate buffer, pH 7.8.

Analysis of Transfected Protoplasts

Protoplasts were analyzed by protoplast printing as described by Jung et al. (1992). Approximately 200 viable protoplasts were pipetted onto nitrocellulose and air dried. Bound viral antigen was detected using standard immunoblotting techniques with mouse antiTEV monoclo- na1 antibodies, alkaline phosphatase-conjugated goat anti-mouse antibodies, and the chromogenic substrates p-nitro blue tetrazolium chloride and 5-bromo-44chlore3indoyI phosphate ptoluidine salt (Bie Rad). Back inoculation of transfected protoplasts to test plants was performed as described previously (Lindbo and Dougherty, 1992b).

Enzyme-Linked lmmunosorbent Assay

The presence of viral antigen (CP) in wild-type and transgenic tissue was examined by standard double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) procedures (Converse and Martin, 1990) using rabbit antiTEV polyclonal sera. The assay approached saturation with TEV-infected tissue 1 hr after the addition of substrate. TEV-free transgenic plants, expressing different versions of the TEV CP, required a 4- to 8-hr incubation period with substrate before a reading significantly above background was detected. Hence, transgene CP did not interfere with the DAS ELISA analyses of TEV-infected plants.

RNA and Protein Gel Blot Analysis

RNA and protein gel blot analysis of plant tissues was performed as previously described (Lindbo and Dougherty, 1992a, 1992b).

Grafting Studies

Rootstocks were prepared by removing the shoot of the rootstock above at least two healthy basal leaves and making a vertical cut, 3 to 4 cm long, in the center of the internode. Leaves larger than 4 cm in length were removed from the scion, and the base was trimmed to a wedge. The cambia of stock and scion were aligned along the length of the cuts, secured with paraffin film, and covered with a polyethylene bag for 7 days.

Protoplast Preparation

Protoplasts were isolated from transgenic or wild-type Burley 49 tobacco leaves by lightly abrading the abaxial surface of leaves with carborundum and a cotton swab. Abraded leaf pieces were incubated overnight (15 to 18 hr) in 1.5% cellulase Onozuka R-10 (Yakult Honsha Co., Tokyo, Japan) and 0.2% macerase in mannitol-Mes (0.6 M mannitol, 0.1% [w/v], 2-[N-morpholino]ethanesulfonic acid [Mes], pH 5.7). After enzyme treatment, protoplasts were floated on a 0.6-M sucrose cushion, collected, and washed two to three times in mannitol-Mes.

Protoplast Transfection

The protocol of Jones et al. (1990) was used for protoplasts and transfection, except that the PEG CaCI2 solution was replaced with PEG-Mg/CMSsolution (1 mL of 50% [w/v] PEG 1500 in 75 mM Hepes, pH 8.01 [Boehringer Mannheim], to which 15 pL of 1 M MgC12 and 100 pL of 1 M Ca[NO&, pH 7.9, had been added). After transfection with 2 pg of viral RNA, protoplasts were transferred to incubation me- dia (Luciano et al., 1987).

Aphid Transmission Experiments

Green peach aphids (Myzuspersicae) were raised on mustard spinach plants. Prior to use in transmission experiments, aphids were collected and fasted for severa1 hours. The aphids were then exposed to infected leaf tissue for 3 to 5 min and then transferred to test plant seedlings and allowed tofeed overnight (8 to 12 hr). Aphidswere then killed with an insecticide. Symptoms on test plants were allowed to develop for 10 days.

Nuclear Run-off Assays

lsolation of nuclei from transgenic plant tissue and in vitro labeling of run-off transcripts was as described by Cox and Goldberg (1988). Transcripts from nuclei from recovered or unchallenged transgenic plant tissue were labeled with U-~~P-CTP (3000 pCilmmo1). Labeled transcripts were isolated by the following modification of the protocol of Cox and Goldberg (1988). After DNase and proteinase K treatment of the in vitro-labeling reaction, the reaction mix was extracted two times with phenol/CHCI, (1:l). Transcripts were precipitated with 0.4 volumes 5 M ammonium acetate and 2.5 volumes EtOH. The final pellet was resuspended in 200 pL double-distilled water. The number of in- corporated counts per labeling reaction was determined by DE81 filter analysis as described by Sambrook et al. (1989).

Labeled run-off transcripts were hybridized to linearized plasmid DNA bound to nitrocellulose. The plasmid pTL-37 (a derivative of Promega pGEM3), plasmids containing the DNA sequence of the TEV CP gene or an actin cDNA sequence (both in a pGEM-3 vector back- bone), and the plant transformation vector pPEV (Lindbo and

1758 The Plant Cell

Dougherty, 1992a), containing the neomycin phosphotransferase II gene were bound to nitrocellulose(0.1 or 1 vg per slot). Nitrocellulose filters were then prehybridized and hybridized according to Sambrook et al. (1989). During hybridization, an equal number of counts per milliliter of hybridization solution was used in each blot (4 to 10 x 106 cpmlmL of hybridization solution). Blots were hybridized (>4 hr) at 55OC, washed in 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate) at room temperature for 10 min, and then in 0.2 x SSC at 55OC for 60 min (two times). Washed nitrocellulose filters were air dried and exposed to Kodak X-Omat film with an intensifying screen at -7OOC. The amount of radioactivity bound per slot was estimated by densito- metric analysis of exposed x-ray films with aZeineh soft-laser scanning densitometer (model SL-DNA; Biomed lnstruments Inc., Fullerton, CA.).

ACKNOWLEDGMENTS

We thankT. Dawn Parks for critical reading of the manuscript and Jim Hay and his greenhouse staff for assistance with transgenic plants. This work was supported in part by a Tartar Research Fellowship to J.A.L. and a grant from the U.S. Department of Agriculture National Research lnitiative Competitive Grants Program (No. 92-37303-7893) to W.G.D. This is Oregon Agricultura1 Experiment Station technical pa- per No. 10,345.

Received August 20, 1993; accepted October 8, 1993.

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DOI 10.1105/tpc.5.12.1749 1993;5;1749-1759Plant Cell

J. A. Lindbo, L. Silva-Rosales, W. M. Proebsting and W. G. Doughertyof Gene Expression and Virus Resistance.

Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation

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