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The NIa-proteinase of different plant potyviruses provides specific resistance to viral infection.

POTYVIRUSES are the largest group of plant viruses and infect a wide range of host plants. Potyviruses are transmitted by aphids and are non-persistent in the aphid vector. Classical breeding programs have been able to breed naturally occurring potyvirus resistance genes into commercial varieties. One of the best examples of genetic resistance to potyviruses is the Virgin A Mutant (VAM; also known as T1 1406) germplasm in tobacco. The VAM gene provides resistance to TEV, TVMV, and PVY. However, there are negative traits that are linked to the VAM locus. Cultivars carrying the VAM gene tend to be more susceptible to chewing insects and the blue mold disease, caused by Peronospora tabacina D.B. Adams, (20,24). With limited genetic variation available for virus resistance, plant biotechnology offers alternative strategies to circumvent this limitation and to avoid the problem of undesirable characteristics linked to the gene of interest.

In the last decade, pathogen derived resistance (PDR) has emerged as a new approach for providing resistance to plant pathogens. PDR offers the benefit of introducing a resistance gene into an adapted cultivar background. The greatest success with PDR has been with plant viruses. Coat protein (CP) and nucleocapsid genes have been the most widely used virus genes for PDR (30). With potyviruses in particular, CP genes provide broad spectrum resistance to several potyviruses. Stark and Beachy (26) demonstrated that transgenic tobacco plants expressing the CP of soybean mosaic virus (SMV) were resistant to TEV and PVY. Maiti et al. (16) demonstrated that plants that express the TVMV CP gene displayed a recovery-type protection to TVMV, a phenotype with reduced viral symptoms and reduced accumulation of virus in newly developing leaves. They also found that TVMV-CP carrying plants were completely resistant (no symptoms and no virus) to TEV and PVY, both closely related potyviruses. Modified potyvirus CP genes also provide virus resistance. An untranslatable CP in which a stop codon is engineered close to the start, provides resistance to the donor virus, but not to other potyviruses (6,14,15,25).

Non-structural viral genes have also been used for PDR. Replicase-mediated resistance has been demonstrated in tobacco with several taxonomic groups of viruses (4). The strategy is based on expression of full-length or modified vital replicase genes and has been used in cowpea mosaic virus (23), potato aucuba mosaic virus (11), cucumber mosaic virus (1), pepper mild mottle virus (28), and PVY (2).

The NIa, a polyprotein that contains both the VPg genome linked protein and proteinase, has been used for PDR with different results. Maiti et al. (16) found that when TVMV-NIa was expressed in tobacco, the plants were specifically resistant to TVMV, but not to TEV or PVY. Vardi et al. (29) transformed tobacco with a truncated PVY NIa that was missing the first 100 nucleotides of NIa, but included the first 251 nucleotides of NIb. They found also that plants were protected against PVY; however, protection was manifested as recovery. Swaney et al. (27) showed that plants expressing the 6-kDa protein and the VPg of TEV were protected from infection. These lines displayed both complete resistance and recovery.

These studies leave many unanswered questions. Among them is the potential for engineering resistance to several potyviruses by expression of more than one NIa gene in a single plant line. In this communication, we describe experiments intended to address this issue. Specifically, we explore the feasibility of attaining resistance to more than one potyvirus in a single plant line by introducing expressible genes encoding tandem NIa coding regions.

MATERIALS AND METHODS

Tobacco Cultivars and Viruses Used

The N. tabacum cultivar used in the transformation studies was Burley 21 (By21). Three- to 4-wk-old seedlings grown aseptically on an agar based MS medium (17) without growth regulators, provided leaf disks for transformation. [R.sub.0] transgenic seed was germinated on the same medium; however, kanamycin was added for selection at a concentration of 300 mg/L. After 3 to 4 wk of selection, surviving [R.sub.1] plantlets were transferred to soil and subsequently grown either in a greenhouse or controlled environment room. Disease screenings of transgenic plants used TVMV, [PVY.sup.0], and TEV-HAT viruses which were provided by Dr. T. Pirone (Dep. of Plant Pathology, Univ. of Kentucky). Virus stocks were maintained in the tobacco cultivar KY 14.

DNA Manipulations and Plant Transformation

The TVMV-NIa coding region was amplified from plasmid subclones by polymerase chain reaction (PCR). Oligonucleotides were designed to incorporate 5' XhoI and 3' SacI restriction sites for subsequent cloning. Primers also included start codons at the N termini and stop codons at the C termini in proper reading frames. Five hundred nanograms of each of two primers 5'-GCCCTCGAGGAACCATGGCAGCTGGCAAGAGTAGACGCCGACTTCAA-3' and 5'-ATGGAGCTCTTAGACGTCCCCTTGAGTGCGGACCAAAT CGTC-3'; NIa terminal sequences obtained from Genbank accession X04083), approximately 50 ng of a plasmid subclone containing TVMV NIa cDNA sequence (16), and 5 [micro]L of 10x Taq DNA polymerase buffer (1 x = 10 mM Tris-HC1 pH 8.3, 50 mM KCl, 1.5 mM Mg[Cl.sub.2], and 0.01% [v/v] gelatin) were brought to a volume of 50 [micro]l with [H.sub.2]O. Taq DNA polymerase (GIBCO-BRL, Gaithersburg, MD), 0.2 units, was added to the reaction. The NIa sequence was amplified by 35 cycles of 92oC for 1 min, 55 [degrees] C for 1 min, and 72 [degrees] C for 1 min. PCR products were ethanol precipitated, digested with XhoI and SacI, and purified by preparative agarose gel electrophoresis (21). NIa fragments were cloned into the appropriate sites of the polylinker of the plasmid pBluescript KS+ (Stratagene, La Jolla, CA) (named pTVNIa). The NIa gene was excised from pTVNIa with XhoI and SacI, ligated into the multiple cloning site of the [pKYLX71:35S.sup.2] vector (16), and digested with XhoI and SacI. Recombinant plasmids were mobilized into Agrobacterium tumefaciens strain GV3850 and used to transform By 21 as described by Schardl et al. (22).

The TEV-NIa gene was isolated in a similar manner. Primers included 5' XhoI and 3' SacI restriction sites. PCR reaction mixtures, primer concentrations, template concentrations, and cycling conditions were the same. The DNA template contained the TEV-NIa cDNA sequence. The two primers used to amplify the sequence were 5'-GCCCTCGAGGAACCATGGGGAAGAAGAATCAGAAGCACAAG-3' and 5'-ATGCACCTCTTAGGTCGACCCTTGCGAGTACACCAAT-TCATT-3'; their design was based on the sequence of TEV (Genbank accession Ml1458). TEV-NIa fragments were cloned into pBluescript (named pTENIa) and [pKYLX71:35S.sup.2] as with TVMV-NIa.

The PVY-NIa coding region was amplified from purified PVY RNA by RT-PCR. One microgram of viral RNA was mixed with 0.5 [micro]g of oligo dT and subsequently dried under vacuum. The pellet was dissolved in 5 [micro]L of 10x dNTP solution (2.5 mM of each of dATP, dCTP, dTTP, and dGTP; New England Biolabs, Beverly, MA), heated at 70 [degrees] C for 10 min, and subsequently cooled on ice. Two microliters of 5x RT-reaction buffer (GIBCO-BRL), 1 [micro]L of 0.1 M dithiothreitol (DTT), 1 [micro]L [H.sub.2]O, and 200 units of Superscript reverse transcriptase (RT; GIBCO-BR) were added to the RNA/oligo dT/ dNTP solution. The reaction was placed at 42 [degrees] C for 2 h. The PVY-NIa coding region was amplified from the cDNA with 2.5 [micro]L of the RT reaction and adding to it 50 ng of each of two primers (5'-GCCCTCGAGGAACCATGGACGTCGGGAAAAATAAATCCAAAAGAATC-3' and 5'-ATGACTAGTTTATTGCTCCACCACTACATCATGATC-3'; NIa terminal sequences were obtained from Genbank accession D00441), 5 [micro]L 10x Taq reaction buffer, and bringing the final volume to 50 [micro]L. The other PCR conditions were as described above. PVY-NIa primers included 5' XhoI and 3' SpeI restriction sites for subsequent clonings into pBluescript (named pPVNIa). Isolation of DNA fragments were as described previously. The sequence encoding PVY-NIa was excised from the pPVNIa clone using XhoI and XbaI and ligated into like sites in the polylinker of pKYLX71:35S2.

Transgenic plants were confirmed by PCR of genomic DNA. For DNA extraction, 100 mg of tissue was placed in a 1.5-mL microcentrifuge tube, frozen with liquid N2 and ground with a plastic disposable grinder. Four hundred microliters of CTAB extraction buffer (100 mM Tris-HCl pH 7.5, 0.7 M NaC1, 50 mM EDTA pH 8.0, 27 mM hexadecyltrimethylamonium bromide [CTAB], 143 mM 2-mercaptoethanol) was added to the powder, vortexed briefly, and incubated at 65 [degrees] C for 90 min. After incubation, 400 [micro] L of chloroform/isoamyl alcohol (24:1 v/v) was added and mixed by inversion. After centrifugation, the upper phase was removed and another 400 [micro] L of chloroform/isoamyl alcohol was added. The upper phase was removed, mixed with 400 [micro] L of cold 2-propanol, allowed to precipitate at room temperature for 15 min, and centrifuged at 15 000 x g for 10 min. The pellet was washed with 76% (v/v) ethanol/0.2 M sodium acetate for 20 min on ice and then centrifuged. The pellet was then washed briefly in 76% (v/v) ethanol/10 mM ammonium acetate, then dried by vacuum centrifugation. The pellet resuspended in 50 [micro] L of milli-Q (Millipore Corp., Bedford, MA) water. Fifty nanograms of genomic DNA was used in the PCR reactions. PCR conditions were the same as those described above.

RNA for RT-PCR reactions was isolated with the RNeasy Plant mini prep kit (Qiagen Inc., Chatsworth, CA). Five micrograms of total RNA was used for RT reactions, using reaction conditions recommended for SuperScript RT (GIBCO-BRL). Three microliters of the RT reaction was used in the PCR reactions. PCR reaction conditions and primer concentrations were as described above.

Construction of Dual NIa Genes

The TEV NIa-PVY NIa gene fusion was derived by taking advantage of a Sa/I site included in the C-terminal primer used to amplify the TEV-NIa sequence. The pTENIa clone was restriction digested with XhoI and Sa/I, and the TEVNIa fragment was purified by preparative gel electrophoresis. The PVY-NIa was PCR amplified from pPVNIa with primers that incorporated a Sa/I restriction site at the 5' end and a stop codon at the 3' end (5'-GCGTCGACCGGGAAAAATAAATCCAAA-3' and 5'-ATGACTAGTTTATTGCTCCACCACTACATCATGATC-3'). PCR products were ethanol precipitated, digested with XhoI and Sa/I, and purified by preparative agarose gel electrophoresis. The digested PVYNIa fragment was then ligated into the digested pTENIa plasmid (pTE-PVNIa) and transformed into the Escherichia coli strain TB1. The TEV-PVY fusion was then cut with XhoI and XbaI, gel purified, and ligated into the XhoI and XbaI digested site of [pKYLX71:35S.sup.2]. Further purification, ligations, and transformations were as described above.

The TEV-TVMV NIa fusion was constructed by first amplifying the TVMV-NIa sequence with primers that included 5' SalI and 3' SacI restriction sites. The PCR conditions were the same as described above except for the two primers, 5'GCGTCGACCGGCAAGAGTAGACGCCGA-3' and 5'ATGGAGCTCTTAGACGTCCCCTTGAGTGCGGACC-AAATCGTC-3', and pTVNIa was used as a template. The TVMV-NIa PCR fragment was digested with SalI and SacI to produce the fusion TEV-TVMV NIa (pTE-TVNIa). Further purification, ligations, and transformations were as described above. The fusion was cut with restriction enzymes XhoI and SacI and ligated into like sites of [pKYLX71:358.sup.2].

The TVMV-PVY NIa fusion was created by engineering a PstI site in the 3' primer for TVMV-NIa. The PCR included the two primers, 5'-GCCCTCGAGGAACCATGGCAGCTGGCAAGAGTAGACGCCGACTTCAA-3' and 5'-ATGCTGCAGCCCTTGAGTGCGGACCAAATCGTC-3', and pTVNIa as a template. The PCR fragments were digested with XhoI and PstI, gel purified, and ligated into pBluescript [pTVNIa(PstI)]. This clone was digested with PstI and SpeI and subsequently gel purified as above. PVY NIa was amplified with primers designed with 5' PstI and 3' SpeI restriction sites. PCR reactions included two primers (5'-ATGCTGCAGGGGAAAAATAAATCCAAAAGAATC-3' and 5'ATGACTAGTTTATTGCTCCACCACTACATCATGAT- C-3'), and pPVNIa as a template. The PCR fragment was then digested with PstI and SpeI then ligated into the cut pTVNIa(PstI) plasmid. The TVMV-PVY NIa fusion was isolated by restriction digestion with XhoI and SpeI. This fragment was ligated into XhoI and XbaI sites of the [pKYLX71:35S.sup.2] polylinker.

The multiple NIa constructs were tested by in vitro translation for efficiency of proteolytic processing. The pBluescript plasmids containing each NIa construct were used as templates for making mRNA; the orientation of the NIa constructs in the vectors allowed for the use of a T3 polymerase promoter. RNA was made with T3 RNA polymerase according to the protocol of the manufacturer of the kit (Epicentre Technologies Corp., Madison, WI). The RNA was purified by phenol/ chloroform extraction and ethanol precipitation. RNA was translated with wheat germ translation kit (Ambion, Austin, TX), again according to the manufacturer's specifications. Proteins were radiolabeled with [sup.35]S-L-methionine (NEN Life Science Products, Boston) and separated by 11% (w/v) polyacrylamide gel containing SDS. The gels were dried onto Whatmann 3MM filter paper and exposed to X-Omat film (Kodak).

Transgenic plants containing these constructs were produced and analyzed for the presence and expression of the transgene as described in the preceding section.

Inoculation and Evaluation of T1 Transgenic Plants

Three to 4 wk after germination on selection medium (or, when untransformed Burley 21 was used as a control for virus infectivity, media without kanamycin), 10 R1 seedlings were transferred to soil and grown in either a controlled environment room or greenhouse. These tobacco plants were grown for 2 wk, and the youngest, most fully expanded leaf was used for mechanical inoculation. Virus inoculum was prepared by grinding samples of infected stock plants (tobacco) in a 1:2 wt of tissue/volume of water. Leaves were dusted with carborundum and mechanically inoculated with a 1:50 dilution of the sap. Two plants from each line were used for mock-inoculated controls (treated as were virus-inoculated plants, but with virus inoculum replaced with distilled water). Plants were evaluated daily for appearance of disease symptoms, with each experiment extending for 28 to 35 days post inoculation (dpi). Some plants displayed disease phenotypes different from infected control plants and thus were categorized by the criteria in Table 1.
Table 1. Definitions used to classidy responses to
virus infections.

                 Definition of symptom phenotypes

Resistant   Virus accumulation and symptoms could not be detected
            by protein blot analysis or visually, respectively.

Recovered   Normal disease symptoms became apparent 5-7 dpi
            similar to the control plants. However, symptoms
            were either diminished or non-existent on new
            leaves that appeared and developed therafter.
            Immunoblots also showed a reduction in viral
            antigen.

Susceptible  Disease severity and accumulation of viral
             antigen were indistinguishable from wild-type
             and vector-only control plants.


In the case of the lines with the dual NIa genes, 20 [R.sub.t] seedlings from each line were transferred to soil. Ten plants were grown in a controlled environment room and ten plants were grown in a greenhouse. One challenge virus was inoculated in the controlled environment room and the other inoculated on the plants in the greenhouse. This was done to avoid inadvertent infection by both viruses. Two plants from each line, in each environment, were used for mock inoculated controls. In these studies, the courses of infection with each of the viruses were similar in the greenhouse and controlled environment room.

Inoculated plants (as well as inoculated controls) were also evaluated for the accumulation of viral coat protein. At the end of each screening period, approximately 28 dpi, five leaf disks (6-mm diam., about 50 mg per disk) were collected from the sixth leaf above the inoculated leaf. Samples were homogenized in 200 [micro]L of SDS-PAGE extraction buffer (0.0625 M Tris-HC1 pH 6.8, 10% [v/v] glycerol, 2% [w/v] SDS, 10% [v/v] 2-mercaptoethanol), boiled at 98 [degrees] C for 5 min, centrifuged at 14000 x g for 5 min, and 12 [micro]L loaded onto a 12.5% (w/v) polyacrylamide gel containing SDS. After electrophoresis, proteins were transferred to nitrocellulose and probed with antiserum raised against the respective virus (21). The antisera to each of the viruses that was used in the immunoblot assays was provided by Dr. David Thornbury (Dep. of Plant Pathology, University of Kentucky). These sera were raised in rabbits against purified viruses and recognize the coat protein of the respective virus. These antibodies display specificities of at least 10-fold with respect to cross-reactivities with other potyviruses and very weak cross reactivity with extracts prepared from uninfected plants (3); in addition, they do not react with host proteins similar in size to the virus coat proteins (as analysis of the mock-inoculated controls indicates; data not shown). Antisera dilutions used in immunoblotting in this study were anti-TVMV, 1/1000 dilution; anti-TEV, 1/1000 dilution; and anti-PVY, 1/1000 dilution.

RESULTS

Introduction and Expression of Different Potyvirus NIa Genes in Transgenic Tobacco

The NIa genes of TEV, TVMV, and PVY were cloned with primers as shown in Fig. 1A. The design included start and stop codons that were in the correct reading frame. Two extra amino acids were included at the C-terminal end of both TEV and TVMV-NIa genes; these were added to assure appropriate translation and to facilitate further cloning steps. The genes were cloned into the [pKYLX71:35S.sup.2] vector so that their expression in plants would be under control of a CaMV 35S promoter with a duplication of the enhancer (16). Approximately 30 independent transgenic plants were generated for each construct in each genotype. After roots developed on the initial transformants, the [R.sub.0] plants were transferred to soil. The presence of the transgene in [R.sub.0] plants was verified by PCR with the same primers that were used for the initial cloning of the NIa genes. Transcription of the genes was verified by RT-PCR of leaf total RNA of [R.sub.0] plants; however, transcript levels were not quantified. Twelve of 24 lines tested proved positive for the presence of the TVMV-NIa transcript, 15 of 25 lines for the PVY-NIa transcript, and 15 of 30 lines for the TEV-NIa transcript.

[Figure 1 ILLUSTRATION OMITTED]

Different combinations of these three NIa genes were also expressed as single polyproteins, each capable of processing into the respective NIa proteins (Fig. 1B). For this, three additional amino acids were included as restriction sites at the N-terminus of the downstream NIa coding region (Fig. 1B). The effect of these amino acids on NIa activity was not determined. However, cleavage activity of the construct was verified by in vitro translations (data not shown). As described above, the presence of the appropriate transgene in the transgenic [R.sub.0] plants was confirmed by PCR with genomic DNA, and gene expression was evaluated by RT-PCR.

[R.sub.0] plants were inoculated with the respective virus from which the NIa gene was derived. In this screen, 28 lines, representative of all of the constructs made here, were observed to be resistant to virus infection and 51 lines displayed a recovery phenomenon (see Table 1 for a summary of terminology used in this paper). Primary transformants were allowed to self-pollinate and produce seed. On the basis of the RT/PCR and [R.sub.0] inoculation results, a number of the resulting [R.sub.1] lines were selected for further study; these lines were positive for the presence of the respective NIa gene and transcript, and exhibited either recovery or resistance in the initial screenings.

Properties of Plants That Contain Different Potyvirus NIa Genes

To determine the effectiveness of the TVMV, TEV, and PVY NIa genes for PDR, several (8-12) independent transgenic lines were examined for their susceptibility to infection by one of the three viruses used in this study. For this, 5- to 6-wk-old plants were inoculated with a quantity of virus that was at least 50-fold greater than what was needed to infect 100% of the control plants (untransformed By21 or By21 transformed with a 35S-chloramphenicol acetyltransferase [CAT] gene). The appearance and progression of the virus disease was evaluated visually at various times after inoculation. The number of plants within each line that exhibited symptoms similar to the inoculated controls was noted and plotted. In this study, an individual plant that showed any significant reduction or modification of disease symptoms, in comparison to inoculated controls, was considered to be protected (and thus not scored as infected). Immunoblot analysis was used to correlate accumulation of viral antigen with visual symptoms; in these analyses, antiserum specific for the respective viral coat protein was used. Untransformed By21 and By21 containing a CAT gene were used as control lines in all of the experiments.

As previously noted, tobacco plants that express the TVMV NIa gene (noted in this report as TVMV-NIa By21 lines) are resistant to TVMV infection (16). To extend this report, and to provide a control suited for studies with plants that contain combinations of NIa genes (see the following section), several additional TVMV-NIa-containing plant lines were generated and studied. The responses of two of the eight lines tested (Lines 264 and 287) to inoculation with TVMV were indistinguishable from the inoculated control plants (Fig. 2). Conversely, nearly all (7/8 and 8/8, respectively) of the TVMV-inoculated plants from two other lines, 184 and 265, failed to develop symptoms by 27 dpi and failed to accumulate virus, judging from immunoblots using anti-TVMV antiserum (Fig. 2). All but one of the TVMV-inoculated plants from an additional two lines (114 and 144), developed disease symptoms, as did inoculated control plants (Fig. 2); however, in a majority of the symptomatic plants, symptoms abated during the course of the experiment, such that more than 50% of inoculated plants displayed very mild or no symptoms at the end of the experiment. In these two lines, virus accumulation in upper leaves correlated with the symptom rating (e.g., normal vs. attenuated) at the end of the experiment (Fig. 2).

[Figure 2 ILLUSTRATION OMITTED]

Individuals from Lines 149 and 203 fell into one of two categories (Fig. 2): those that failed to develop symptoms in the course of the experiment (resistant) and those that developed symptoms and then recovered. The recovery phenotypes varied among individual plants. At the end of the experiment, some individual plants had no disease symptoms and no detectable virus in their uppermost leaves. Other individuals displayed localized symptoms as well as symptom-free zones in the same leaves. Immunoblot analysis using anti-TVMV antiserum showed that some virus was present in the symptomatic zones but not in the symptomatic-free regions (data not shown).

The efficacy of the TEV-NIa as a source for resistance to TEV was evaluated by screening nine TEV-NIa By21 lines for their response to TEV. As was seen with the TVMV-NIa lines, a range of phenotypes, ranging from resistance to recovery to susceptibility was seen in the lines analyzed. Also, similar to observations with TVMV-NIa Lines 149 and 203, both recovery and resistant responses could be discerned among individuals of some lines (e.g., Lines 143 and 233 in Fig. 3). The majority of inoculated plants in Lines 231,240, and 257 developed disease symptoms indistinguishable from those observed in control plants, but these plants invariably recovered by 20 dpi, such that significant reductions in symptom severity were seen (Fig. 3). All plants in Line 223 also developed disease symptoms, as did controls, but these plants displayed a noticeable reduction in disease symptoms in the upper leaves (rather than complete disappearance of symptoms) after 20 dpi. In two of the plant lines examined (Lines 193 and 230), 100% of the inoculated plants developed disease symptoms, as did controls, but only a fraction of these recovered with time (Fig. 3). One of the TEV-NIa BY21 lines (Line 226) was indistinguishable from the controls in its response to TEV (Fig. 3A). Virus accumulation in all of these plants reflected the presence or absence of symptoms, judging from immunoblot analysis using antiTEV antiserum (Fig. 3).

[Figure 3 ILLUSTRATION OMITTED]

In a manner analogous to the studies with the TVMV and TEV NIa genes, we evaluated whether a full length PVY-NIa gene could provide resistance to PVY in transgenic tobacco. In the nine lines selected (PVYNIa lines), only two individual plants did not develop symptoms after inoculation with PVY. PVY-inoculated plants of Line 180 remained indistinguishable from the inoculated controls through the duration of the experiment (Fig. 4). Different percentages of PVY-inoculated plants in the other lines displayed a recovery. However, recovery was rarely complete; instead, PVY symptoms became localized to the outer edges of newly formed leaves. As was the case with TVMV-NIa and TEV-NIa lines, the accumulation of PVY was also reduced in the majority of the individuals (Fig. 4), as determined by immunoblot analysis using anti-PVY antiserum.

[Figure 4 ILLUSTRATION OMITTED]

Properties of Lines That Carry Multiple NIa Genes

Nine By 21 [R.sub.1] lines (TEP21) that contained the TEV NIa-PVY NIa gene were selected and examined for their responses to TEV and PVY. (We would point out that these experiments did not involve dual or mixed inoculations.) As above, TEV symptoms on control plants (pIDK8 plants) were apparent 5 dpi, and PVY-inoculated control plants became symptomatic by 7 dpi. Moreover, 100% of inoculated controls invariably became infected, and symptoms persisted for the duration of the experiment. With one exception (Line 357), a majority (but never 100%) of the TEP21 plants that were inoculated with TEV developed symptoms within 1 to 2 d after the appearance of symptoms on the inoculated controls (Fig. 5). However, beginning at about 18 dpi, recovery could be observed in most of the symptomatic plants (with individuals in Lines 350 and 354 being an exception to this). With the exception of one line (352), there was an excellent correlation between the presence of symptoms and virus accumulation (Fig. 5). In Line 352, TEV could be detected in a few recovered plants; this reflects inadvertent sampling of symptomatic parts of recovered plants.

[Figure 5 ILLUSTRATION OMITTED]

These responses were similar to those of the TEVNIa lines to TEV inoculation in most respects (Fig. 3). In contrast, the responses of the TEP21 lines to PVY infection (Fig. 5) were rather different from those of PVY-NIa lines (Fig. 4). The majority of PVY-inoculated TEP21 plants developed symptoms with at most a two day delay, compared with the control lines (Fig. 5). With only two of these lines (Lines 358 and 360) was recovery seen in a majority of inoculated plants (Fig. 5). The rest of the inoculated plants remained symptomatic for the duration of the experiment. In agreement with these observations, PVY could be detected in all symptomatic plants by immunoblot analysis using anti-PVY antiserum (Fig. 5). However, in several such plants, virus accumulation was significantly lower than that seen in inoculated controls. Interestingly, those three lines that showed significant protection against PVY (357, 358, and 360) were among the most resistant to TEV.

Eight TEV-TVMV NIa By21 (TET21) lines were evaluated for their responses to TEV or TVMV infection. In TEV or TVMV inoculated control plants (pIDK8), disease symptoms were apparent 5 dpi and persisted for the duration of the study. Six of the TET21 lines (Lines 372, 390, 401,407,411, and 414) were significantly protected against both TEV and TVMV (Fig. 6); in these lines, a majority of inoculated plants either failed to develop symptoms or recovered from an early incidence of disease. The degree of protection of Line 375 to TEV and TVMV was low (Fig. 6), with 62.5% of the TEV-inoculated plants and 87.5% of the TVMV-inoculated plants displaying a disease indistinguishable from inoculated controls. A majority of TVMV-inoculated individuals from Line 394 recovered from an initial infection, while a minority of TEV-inoculated plants of this line recovered. For the most part, there was an excellent correlation between the presence of symptoms and virus accumulation (determined by immunoblot analysis using the appropriate anti-virus antibody; Fig. 6). However, in some lines (390, 401, and 407), virus could be detected in some recovered plants. As stated above, this probably refiects inadvertent sampling of symptomatic parts of recovered plants. Overall, six of eight TET21 lines were protected against both TEV and TVMV.

[Figure 6 ILLUSTRATION OMITTED]

Nine TVMV-PVY NIa By21 (TVP21) lines were chosen and examined for their responses to TVMV and PVY. With the exception of one line (463), all TVMV-inoculated TVP21 plants developed an initial infection (Fig. 7). Subsequently, a majority of plants in all nine lines recovered from this infection. In these plants, there was a general trend towards lower TVMV accumulation after 28 dpi (Fig. 7). However, most of the inoculated plants still harbored virus, which was localized to symptomatic regions of recovered leaves (not shown).

[Figure 7 ILLUSTRATION OMITTED]

The responses of the TVP21 lines to PVY inoculation were more varied. Plants of one line (468) failed to develop symptoms throughout the course of the experiment and failed to accumulate any detectable virus (Fig. 7). Plants of Lines 447, 457, and 463 recovered from initial infection by 35 dpi (Fig. 7) while Line 443 contained inoculated plants that were both resistant and recovered. Interestingly, while the initial disease symptoms in the TVP21 lines were similar to those seen in controls, and while the recovery phenotype was visually indistinguishable from that noted in the PVY-NIa and TEP21 lines (e.g., symptoms were only present along the outside edges of leaves on PVY recovery plants), PVY-inoculated TVP21 lines usually accumulated large quantities of virus, irrespective of the symptomology of the plant (Fig. 7).

DISCUSSION

This study was initiated to address several questions regarding the utility of potyvirus NIa genes as agents of pathogen-derived resistance. One question dealt with the general utility of NIa coding sequences as sources of protection against potyviruses. Our results indicate that NIa-mediated protection against three different potyvirus pathogens of tobacco could be obtained. However, the nature of the observed protection varied with the source of the NIa gene and the challenge virus. Lines in which a majority of inoculated plants failed to develop TVMV symptoms were readily obtained with the TVMV NIa gene (Fig. 2), whereas lines that developed and then recovered from a virus infection were the rule with the TEV and PVY NIa genes (Fig. 3 and 4). In this regard, our results are similar to those reported by Maiti et al. (16), Vardi et al. (27), and Swaney et al. (27). The reasons for the differences seen with different NIa genes and challenge viruses are not clear. Nonetheless, our observations indicate that NIa genes of different potyviruses may be considered as reliable agents for PDR.

A second question concerned the potential of engineering protection against more than one potyvirus in a single line, by assembling genes capable of expressing (as RNA and protein) the Nlas from two different potyviruses. Our results demonstrate the feasibility of such an approach; we were able to identify at least three lines with each of the dual NIa constructs that were protected against both potyviruses (Table 2). Some interesting differences between the three different sets of plants were also apparent. For example, there was a relatively good correlation between the "levels" of protection against TEV and TVMV in the TEV-TVMV NIa plants, such that lines that yielded the fewest symptomatic TVMV-infected individuals also yielded the fewest TEV-infected plants (Fig. 6). This was also seen with the TEV-PVY NIa plants (Fig. 5) but not with the TVMV-PVY NIa lines (Fig. 7). In addition, a greater number of TVMV-PVY NIa lines could be classified as protected against PVY (Fig. 7) than could the TEVPVY NIa (Fig. 5) lines. Although these trends cannot be evaluated statistically for significance, they do raise the possibility of subtle differences between plant lines that express different combinations of virus genes. The TVMV-PVY lines we studied were unusual in one additional respect. Specifically, while many individual plants in several lines apparently recovered from initial infections, this recovery was not accompanied by a concomitant decrease in virus levels (Fig. 7).

There have been few studies that evaluated the use of multiple genes for resistance to multiple viruses. Lawson et al. (10) demonstrated that plants that contain the CP genes of both PVY and PVX were protected against both viruses. Several instances of protection against several viruses in lines transformed with a single viral gene have been reported. Expression of a TMV replicase that has been modified by nucleotide insertion in the methylase and helicase domains yields plants resistant to several tobamoviruses (5). The CP genes of several potyviruses have been reported to offer protection against many potyviruses when expressed in plants (1,16,18,19,26). The present study provides an example of resistance against multiple viruses in plants that express non-structural virus genes. Given the possibility of trans-encapsidation in transgenic plants that express CP genes (7,12), the use of non-structural genes is of great possible significance.

Our studies do not directly address the issue of mechanism of NIa gene-mediated protection. Swaney et al. (27) proposed an RNA-based mechanism for the protection observed in plants that carried a 6K-VPg gene, on the basis of similarities in the protected phenotypes with potyvirus CP-containing plants (6,8,13,25). Such a hypothesis is consistent with the remarkable similarity in the protected phenotypes observed with plants that carry such different forms of the NIa gene as have been described (16,27,29; this study). In contrast, a mechanism based on the involvement of NIa or VPg proteins is more difficult to reconcile with the disparate natures of the various genes used in these different studies to effect protection. In any case, the means by which potyvirus NIa genes act to protect plants against virus infection are compatible with the use of multiple NIa genes for protection against more than one virus, an observation that offers new strategies for the engineering of resistance against multiple plant viruses.

ACKNOWLEDGMENTS

We thank Carol Von Lanken and Ray Stevens for excellent technical support during the course of this project, and Mark Nielsen, Joe Chappell, and Said Ghabrial for helpful suggestions in the course of this work.

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Abbreviations: NIa, nuclear inclusion a; NIb, nuclear inclusion b; dpi, days post inoculation; CP, coat protein; TEV, tobacco etch virus; TVMV, tobacco vein mottling virus; PVY, potato virus Y; PDR, pathogen derived resistance.

John P. Fellers, Dep. of Botany, North Carolina State Univ., Box 7612, Raleigh, NC 27695-7612. Glenn B. Collins and Arthur G. Hunt, Univ. of Kentucky, Dep. of Agronomy, Lexington, KY 40546-0091. Contribution 97-06-147 of the Univ. of Kentucky Agric. Exp. Stn. Supported in part by USDA NRI grants #94-37303-0470 and #9037262-5519. Received 15 Sept. 1997. (*)Corresponding author (aghunt00@pop.uky.edu).
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Author:Fellers, John P.; Collins, Glenn B.; Hunt, Arthur G.
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Date:Sep 1, 1998
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