Potato virus Y

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Content

1. Referat

2. Introductory part

3. Virus characteristics

3.1 Nomenclature

3.2 Physical and biochemical properties

3.3 Reproduction

3.4 Virus(es) with serologically related virions

4 Symptoms

5 Transmission

6 Diagnostic techniques for detection of Potato Virus Y

6.1 ELISA

6.2 RT-PCR

6.3 qRT-PCR

7 Control

7.1 General Recommendations

7.2 The novel gene Ny-1

8 Additions

8.1 Images

8.2 Complete genome

8.3 Gene map

8.4 Plants transformed with a cistron of a potato virus Y protease (NIa)

are resistant to virus infection

9 References



1. Referat

This course work is devoted to widely known Potato virus Y. It contains 31 pages and 9 figures.

2. Introductory part

Two of the 73 genera of plant viruses share equally more than 30% of all recognized species. Potyvirus is one of them and Begomovirus the other. Potyvirus is the largest of six genera in the family Potyviridae, and is named after its type species, potato virus Y. Many potyviruses are damaging crop pathogens. They infect species of most angiosperm taxa in all temperate and tropical climes. They are transmitted by aphids, especially species of Aphidinae, when aphids move from plant to plant and probe them in search of their preferred host species, and some are also transmitted in seeds to the progeny of infected plants.[3]

Potato virus Y (PVY) is the type member of the potyvirus group. Potyviruses, like picorna-, como-, and nepoviruses, belong to the picornavirus-like supergroup.

PVY infects many economically important plant species. These include potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill) and pepper (Capsicum frutescens L.).

Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVY(O) and PVY(N), which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVY(NTN)) and PVY(O) serotypes that cause tobacco veinal necrosis (PVY(N)-W, PVY(N:O)).[7]

3. Virus characteristics

Data collated by C. Bьchen-Osmond, 1987.

potato virus detection genome

3.1 Nomenclature

Synonyms

brinjal mosaic virus[48], datura 437 virus, potato acropetal necrosis virus, potato severe mosaic virus, tobacco vein-banding virus [24].

Acronym

PVY

Strains

potato C virus, tobacco veinal necrosis virus, potato virus Yo group (common strain), potato virus YN group (tobacco veinal necrosis strain), potato virus YC group (stipple streak strain, including potato virus C) [58].

ICTV decimal code

57.0.1.0.058

3.2 Physical and biochemical properties

Properties of particles in sap

TIP: 50-62 °C. LIV: 7-50 days. DEP: log10 minus 2-6. Infectivity of sap not changed by treatment with di-ethyl ether. Leaf sap contains few virions[18].

Particle morphology

Virions consist of a capsid. Virus capsid is not enveloped. Capsid/nucleocapsid is elongated with helical symmetry (FIG 1). Virus preparations contain one particle component[60]. The capsid is filamentous, flexuous with a clear modal length with a length of 684 nm (from purified preparations (Delgado-Sanchez and Grogan, 1966)), or 730 nm and a width of 11 nm. Axial canal is indistinct; 2-3 nm in diameter. Basic helix is obscure. Pitch of helix is 3.3 nm [57].

Electron microscopic preparation and references: Virus preparation contains few virions.

Physical properties

One sedimenting component in purified preparations; sedimentation coefficient 145 S [32]. Density 1.323 g cm-3 in CsCl (strain Yo), or 1.326 g cm-3 in CsCl (strain YN[32]). A260/A280 ratio 2.3 (corrected for light-scattering (Leiser and Richter, 1978)), or 2.9 [32],[52].

Biochemical properties

Features of the genome

The Mr of the genome constitutes 5.4-6.4% of the virion by weight[32],[52]. The genome is monopartite, only one particle size is recovered of linear, positive-sense, single-stranded RNA. Minor species of non-genomic nucleic acid are not found in virions. The complete genome is 10400 nucleotides long. Is fully and partially sequenced, complete sequence is 10400 nucleotides long [24],[38]. Sequence has the accession number ;93.6-94.6 % protein; 0 % lipid.

Genome consists of RNA; single-stranded; linear. Total genome size 10.4 kb. Genome unipartite; largest (or only) genome part 10.4 kb[39]. 5ґ terminus of RNA has a VPg. Infectivity retained when deproteinised with phenol or detergent. Poly A region present; at 3ґ end, but not essential for infectivity.

Features of proteins

The viral genome encodes structural proteins and non-structural proteins.

Virions consist of 1 structural protein(s) Mr 34000; coat protein.

Virus-coded non-structural proteins have been isolated [32] and 1 non-structural protein is found Mr 67000; cylindrical inclusion protein[44].

3.3 Replication

Upon entrance into the plant cell, the virus coat protein disassembles and releases its RNA genome. The viral RNA serves as mRNA, and although little is known about the translation thereof, it is believed that the 5' non-coding region functions as an enhancer of translation. The translated mRNA results in a polyprotein which is processed into mature proteins. Each polyprotein is then cleaved into ten different proteins which are believed to be multifunctional [18]. These proteins, along with host proteins, assemble to form a replication complex. This complex performs negative-strand RNA synthesis, using the positive strand of viral RNA as a template. Once the additional RNA copies have been produced, they code for the synthesis of various proteins, as mentioned before, as well as coat proteins [45]. These coat proteins will now enclose the newly formed genomes to give rise to new virions. It has been suggested that enclosure of the newly formed virions is initiated by the interaction of the coat proteins with the 5'terminus and that the coat protein is built up towards the 3'terminus. The entire process of viral replication occurs within the endoplasmic reticulum. These newly synthesized viral particles are subsequently transported through the plasmodesmata to adjacent plant cells via several assisting potyvirus proteins. Distribution of viruses within the plant occurs according to the source-sink relationship between maturing and growing tissues [54], [62], [28]. Virus concentration throughout the plant is high and this greatly increases the chance of uptake by aphids. Infected plants that do not show symptoms may have infected canopies and will yield lower quality products than their healthy counterparts.

Cytopathology

Virions found in epidermis; in cytoplasm and in cell vacuoles. Inclusions present in infected cells; are crystals in the nucleus [29], or pinwheels (especially in epidermal tissue in the cytoplasm [10]. Other cellular changes: mitochondria often being surrounded by filaments with a diameter of 9-10 nm but of indeterminate length [3], when infected with strain YN in Datura metel.

3.4 Virus(es) with serologically related virions

Tobacco etch, henbane mosaic, potato A, pepper veinal mottle and bidens mottle viruses, but distantly [8].



4. Symptoms

Symptoms persist.

Solanum tuberosum (FIG 2, 2.1, 2.2, 2.3) - mild to severe leaf mottling, or streak or `leaf-drop streak' with vein necrosis (`stipple-streak') [13].

Capsicum spp. (FIG 3) - mild leaf mottling, but severe in complex with other viruses.

Nicotiana spp. (FIG 4) - mild mottle or veinal necrosis.

Lycopersicon esculentum - mild leaf mottling but severe in mixed infections [25].

The differences between the primary and secondary symptoms induced by potato virus Y are often uncertain because of the diversity of potato cultivars and virus strains, and effect of climatic conditions [12].

PVY has different isolates according to the symptoms they induce in various potato plant species. Extensive biological, serological and molecular variability of PVY isolates makes the classification of isolates as particular strains particularly difficult. Occurrence of a variety of symptoms and the emergence of the necrotic PVYNTN has led to a search for more reliable classification tools than simple serological identification. Traditionally three chief strains of PVY are recognized: PVYC, PVYN and PVYO. PVYC, originally known as Potato Virus C, was the first to be recognized and was identified in the 1930s. PVYC induces hypersensitive responses in a wide range of potato cultivars. These reactions include the formation of mild mosaic patterns or stipple streak. Unlike the other strains of PVY, some PVYC strains are non-aphid transmissible [34], [57] . Previous studies by Visser did not identify any of the local isolates as being PVYC but it has been reported to occur to in South Africa. A second strain of PVY is PVYN. Some notes on suspected variant of Solanum virus 2 (Potato virus Y). This strain was described in tobacco plants growing close to potato plants. PVYN results in leaf necrosis and mild or even no damage to the tubers. The ordinary strain of PVY is denoted as PVYO. Infection of a potato plant with the PVYO strain results in mild tuber damage and does not cause leaf necrosis. Both PVYN and PVYO are aphid transmissible and occur in South Africa. In Europe these two strains have been shown to have recombined to form PVYNTN. The PVYNTN has been accredited with the ability to induce potato tuber necrotic ringspot disease (PTNRD). Tubers damaged by PTNRD become unmarketable and infection by PVYNTN thus results in a larger economic impact than infection by the other strains.

PVY infection of potato plants results in a variety of symptoms depending on the viral strain. The mildest of these symptoms is production loss, but the most detrimental is 'potato tuber necrotic ringspot disease' (PTNRD). Necrotic ringspots render potatoes unmarketable and can therefore result in a significant loss of income. PVY is transmissible by aphid vectors but may also remain dormant in seed potatoes. This means that using the same line of potato for production of seed potatoes for several consecutive generations will lead to a progressive increase in viral load and subsequent loss of crop [47], [19], [57].

The intensive accumulation of H2O2 at the early stages of potato-PVY interaction is consistent with its role as an antimicrobial agent and for this reason it is a signalling molecule. Hydrogen peroxide has appeare in the necrotic area of the epidermal and mesophyll cells 24 and 48 h after the PVY infection. The most intensive accumulation of H2O2 is in the bordering cell walls of necrotic mesophyll cells and the adjacent non-necrotic mesophyll cells[34].



5. Transmission

PVY (FIG 5) may be transmitted to potato plants through grafting, plant sap inoculation and through aphid transmission. The most common manner of PVY infection of plant material in the field is through the aphid and although aphids on their own can directly damage potato plants it is their role as viral vectors which has the greatest economic impact. In cold climates aphids spend the winter either as wingless aphids giving birth to live young (viviparae) or as eggs [32]. Hosts such as weeds and other crops serve as breeding grounds for these aphids and form a temporary area of colonization before the aphids migrate to the potato fields. In moderate climates, such as in South Africa, aphids are thought to reproduce asexually on weeds, other crops, indigenous plants and garden plants. This means that there are a number of aphids present year-round. Wingless aphids have not yet been linked to the spread of PVY in potato fields [33].

The green peach aphid Myzus persicae (FIG 6) has been found to be most effective in its role as viral vector, but others such as Aphis fabae (FIG 7), Aphis gossypii, Aphis nasturtii, Macrosiphum euphorbiae (FIG 8), Myzus (Nectarosiphon) certus, Myzus (Phorodon) humuli and Rhopalosiphum insertum (FIG 9) are also strongly associated with viral transmission [25]. Twenty five species of aphids are able to function as PVY vectors. Apart from being classed according to efficiency as vectors, aphids can also be divided into two subgroups, namely colonizing and non-colonizing species. Colonizing aphids are aphids which reproduce and establish themselves on potato plants, specifically, while non-colonizing aphids do not reproduce nor establish colonies on potato plants. Colonizing aphids are better adapted to life on potato plants and are thus generally considered as better PVY vectors than non-colonizing aphids. Noncolonizing aphids do not primarily feed on potato plants but do occasionally feed on them while searching for a more suitable host [56], [34], [26]. Their lower efficiency as PVY vector is cancelled out by the sheer numbers in which they occur. Because of this, all aphids present in and around potato fields must be considered as possible vectors and their numbers carefully monitored [40].

Transmission of PVY by aphids occurs in a non-persistent, non-circulative manner which suggests a less intimate interaction between virion and vector than is the case of circulative virions. The fact that the virions are transmitted in a non-persistent fashion means that viral replication does not occur within the aphid vector and that, unless the aphid feeds on infected plants, it loses its ability to infect plants after two to three feedings. The virions attach to the aphid stylet in a matter of seconds and may remain infectious for four to seventeen hours. The distance over which the virions can be transmitted is limited due to the short period for which they remain infectious. Although the short life span outside plants inhibits long-distance viral transmission, it does not reduce the transmission efficiency bestowed by the quick rate of viral acquisition and inoculation within a field [32], [50].



6. Diagnostic techniques for detection of Potato Virus Y

6.1 ELISA

In the past, crops were inspected visually to determine whether or not they were disease free. Visual inspection was also used as a basis for seed certification. Determination of viral status through visual inspection is incredibly difficult as the symptoms may be masked or the infection latent. As a result, post season tests and inspections were introduced. These tests involved the cultivation of previously harvested material in greenhouses. The resulting plants were inspected for a more accurate estimate of viral status. Although this method of screening did offer some degree of monitoring of viral presence it was subjective and highly ineffective. Enzyme-linked immunosorbent assay (ELISA) screening of crops and seed potatoes replaced visual inspection in the early 1970s. The use of ELISA offered routine diagnostic laboratories a quick, effective and sensitive method of screening for a wide range of potato plant viruses [42].

Detection of pathogens using ELISA relies on the interaction between the antigen and specific antibodies and has become a popular and cost-effective means of routine detection. In an ELISA the solid phase can be coated with the sample of interest containing the antigen. The efficiency to which the antigen binds to the solid phase is dependent on temperature, length of exposure as well as concentration. Solid phases used include nitrocellulose membranes, paper, glass, agarose and polystyrene or polyvinylchloride microtiter plates. Microtiter plates are the most widely used solid phase due to the fact that they are easy to handle, allow for automation and for analysis using microtiter plate readers. A drawback of these plates is that they are highly absorptive and this increases the incidence of non-specific binding of components used in the ELISA. Non-specific binding to the plates is reduced through the use of buffers containing proteins such as casein and non-ionic detergents such as Tween 20. After coating, excess sample is removed and the plate typically treated with a 1% casein containing solution. Subsequent to this the solid phase is treated with antibodies raised against the antigen of interest. After each incubation step the plate is washed with Tween 20 containing PBS. These washing steps are aimed to wash away any non-specifically bound components. Nonspecifically bound components are less strongly bound than the specific bound ones. Detection is achieved either through the addition of an enzyme-coupled antibody or the addition and detection of a biotinylated antibody. In a system using an enzyme-coupled antibody the subsequent addition of an appropriate substrate results in the formation of a colour proportional to the amount of antigen.[46] Alternatively the plate can be coated with antibody followed by incubation with the sample that is to be detected. This, in turn, can be detected as described above and is then referred to as the double antibody sandwich (DAS) ELISA [15]. Both of these systems, however, have a disadvantage in that coupling of the enzyme to the antibody may result in stearic hindrance which in turn may result in a loss in function of the antibody and/or the enzyme. This may be overcome through the use of a biotin-avidin or biotin-streptavidin bridge. In this type of system biotin is coupled to the antibody. The biotin molecule has no influence on the working of the antibodies and is easily detectedusing avidin or streptavidin conjugated to a suitable enzyme. Streptavidin has an extremely high affinity for biotin which results in even a higher degree of specificity than a system in which the enzyme is coupled directly the antigen. To establish whether or not the antigen is present, a substrate specific for the enzyme used is added. The enzyme then converts the substrate to a coloured product and the colour intensity can be correlated to the amount of antibodies bound and thus the amount of antigen present. A DAS-ELISA has the advantage that it can increase the specificity of the ELISA and reduce the occurrence of non-specific binding. As a result the DAS-ELISA principle is commonly employed in ELISA's for the detection of plant pathogens in plant sap without prior purification of the pathogen [24].

The ELISA is considered to be a safe, inexpensive and rapid method for detection of plant viruses. The inexpensive nature and relative simplicity thereof allows for it to be used as a workhorse within the agricultural sector and is used to screen thousands of samples per year. Unfortunately ELISAs are not completely failsafe. Virus levels within potato tubers, which are screened by ELISA for use as seed potatoes, are normally low while the tubers are dormant [45]. ELISA detection of viruses in these potatoes is difficult and absorbance values may fall below the set cut-off value. For this reason, seed tuber screening is performed on sprouting rather than dormant tubers. Although this results in more reliable readings than direct tuber testing, it does delay the certification of seed potatoes. Another disadvantage of an immuno-based detection method is that changes at the gene level may have an influence on the immunogenicity of the antigen to be detected. In terms of potato plant viruses, mutations within the CP gene may cause the CP to undergo conformational changes rendering antibodies produced against the previously present virus less effective.

6.2 RT-PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) has become a powerful and effective method for detection of potato plant viruses within potato plant material and even dormant potatoes [21]. Only a minute piece of plant material is required for analysis using RT-PCR. Considering the protocol described within this thesis, 0.1 g of plant material is enough for 14 500 separate reactions. During a RT-PCR specific target RNA sequences are amplified exponentially into DNA copies. For this to occur, however, the RNA of the virus must first be transcribed to DNA by means of a reverse transcriptase polymerase. This polymerase synthesizes a DNA strand using the RNA as template. This results in a DNA/RNA complex. For synthesis of a DNA strand from the RNA template only the reverse primer is required since the RNA is a single strand arranged from 5' to 3'. Subsequently the newly synthesized DNA strand is used as a template for traditional PCR.

Different types of reverse transcriptase polymerases are available to suite different needs and reaction conditions. Reverse transcriptase enzymes commonly used include AMV RT, SuperScriptTM III, ImProm-IITM, Omniscript, Sensiscript and Tth RT [54]. At the end of the RT step the polymerase enzyme is heatactivated. It could also be that the reverse transcriptase polymerase and DNA polymerase is one and the same enzyme and that the enzyme only requires a DNA polymerase activation step after the RT step. An example of such an enzyme is Tth polymerase. This enzyme has both RNA-dependent reverse transcriptase and DNA-dependent polymerase activity. However, the active center of the DNA polymerase is covered by dedicated oligonucleotides, called Aptamers. At temperatures below the optimal reaction temperature of the DNA-dependent polymerase component of Tth remains covered by the Aptamers. At these temperatures the Tth enzyme only synthesizes a DNA copy of the RNA template. Once the reaction temperature is raised to 95°C, the Aptamers are removed and the DNA-dependent polymerase component will start to amplify the target sequence [43].

PCR amplification of the DNA target occurs in three steps: denaturation, annealing and extension [48]. Each of theses steps occur at a specific temperature for a fixed period of time. Denaturation is normally allowed to occur between 90 and 95°C and leads to the dissociation of DNA strands. After this the reaction is cooled to between 40 and 70°C to allow the primers to associate with their respective target sequences. This step is known as the annealing step and is primer specific. The temperature at which the primers anneal is critical. Too high temperatures would not allow the primers to associate with the DNA, resulting in no or poor amplification. Too low annealing temperature would ultimately lead to non-specific binding of the primers and non-specific amplification.[46] Primers bound to the regions flanking the target DNA provide 3'-hydroxyl groups for DNA polymerase catalyzed extension. The most commonly used DNA polymerase is Taq, a thermo-stable enzyme isolated from the thermophilic bacterium, Thermus aquaticus. The DNA polymerase synthesizes new DNA strands along the template strands, using the primers as starting points. During the extension step the strands are amplified beyond the target DNA. This means that each newly synthesized strand of DNA will have a region complimentary to a primer [34]. There is an exponential increase in the amount of DNA produced as the three above mentioned steps are repeated in a cyclic fashion. In a traditional PCR these steps might be repeated 20 to 55 times. A problem, however, with PCR amplification is that the temperature required for DNA strand dissociation also results in DNA polymerase denaturation. This is partially overcome by the bioengineering of polymerases which are more thermal stable and have longer half-lives [2].

Even though RT-PCR is technically more difficult to perform and more expensive than ELISA, it has the ability to allow for the detection of low viral loads. RT-PCR is considered to be 102 to 105 fold more sensitive than traditional ELISA. RT-PCR also allows for the detection of several viral targets in the same reaction through the use of several primer combinations. This is called multiplexing [61]. Although multiplexing is technically more demanding than a traditional simplex reaction it allows for a higher throughput in that a single sample can be tested for several viral strains in a single reaction. Primers used for multiplexing are chosen in such a manner that they result in amplicons of various sizes. This allows for post RT-PCR analysis using gel electrophoresis. Although RT-PCR saves time, allows for multiplexing and is more sensitive than ELISA, the reagents and instrumentation needed are expensive and require a higher level of technical expertise. Also, end product analysis using gel electrophoresis is laborious, relatively more expensive, time consuming and does not lend itself to automation. For these reasons the use of RT-PCR for routine screening is not feasible and has not replaced ELISA [23]. It does, however, provide the industry with the opportunity to screen borderline cases, especially in the case of seed potato certification.

6.3 qRT-PCR

In most traditional PCRs the resulting products are analyzed after the PCR has been completed. This is called end-point analysis and is normally qualitative of nature rather than being quantitative. For this sort of analysis, products are mostly analyzed on an agarose gel and visualized using ethidium bromide as a fluorescent dye [5]. Direct correlation between signal strength and initial sample concentration is not possible using end-point analysis since PCR efficiency decreases as the reaction nears the plateau phase [18]. Real-time PCR, however, offers an accurate and rapid alternative to traditional PCR [15]. Real-time PCR offers the researcher the opportunity to amplify and analyze the product in a single tube using fluorescent dyes. This is known as homogenous PCR. During a real-time PCR the increase in fluorescence is correlated with the increase in product. Through the use of different specific, dyes real-time PCR can be used to distinguish between different strains of a virus and even to detect point mutations. The major advantage of real-time PCR is that analysis of resulting products using gel electrophoresis is not required. This means that realtime PCR can be implemented as a high-throughput technique for sample screening [45].



7. Control

7.1 General Recomendations

In temperate areas, perennials rarely act as virus reservoirs in nature. Potato plants as "ground-keepers" are a reservoir host. In tropical and subtropical areas, weeds such as Solanum atropurpureum and other Solanum spp., may act as important virus sources [9].

Use of insecticides to control virus spread through vectors have been ineffective. The main control methods are:

(1) avoidance of infection, i.e. growing crops when vectors are absent or numbers are low;

(2) not growing crops near established crops of the same species;

(3) destroying haulms of seed-potato crops before maturity to restrict virus spread at the end of the growing season;

(4) spraying with mineral oils to reduce frequency of transmission [45];

(5) breeding for resistance, if sources of durable resistance can be obtained;

(6) use of reflective surface and sticky yellow sheets which can reduce virus spread [12];

(7) destroying volunteer potatoes which may harbour the virus;

(8) growing crops where aphid vector populations are few or absent (higher altitude or windswept coastal areas) [52].

7.2 The novel gene Ny-1

Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. Ny-1 - a novel hypersensitivity gene which can cause a resistance to Potato virus Y (PVY). This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. The gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes.

Nucleotides 5812-7260 of the potato virus Y (PVY) genome - is the fragment contains all but the first 100 5' terminal bases of the cistron encoding one of the PVY proteases (NIa) as well as the first 251 bases of the next cistron (NIb). Nicotiana tabacum cv. SR1 plants were transformed with this fragment. The presence of the NIa sequences in transformed plants сauses resistant to PVY [11].



8.Additions

8.1 Images

FIG 1 (Virion PVY)

FIG 2 (Solanum tuberosum)

FIG 2.1 (Solanum tuberosum)

FIG 2.2 (Solanum tuberosum)

FIG 2.3 (Solanum tuberosum)

FIG 3 (Capsicum spp.)

FIG 4 (Nicotiana spp.)

FIG 5 (Electron micrograph (PVY))

FIG 6 (Myzus persicae)

FIG 7 (Aphis fabae)

FIG 8 (Macrosiphum euphorbiae)



FIG 9 (Rhopalosiphum insertum)

8.2 Complete genome

DEFINITION Potato virus Y, complete genome.

KEYWORDS aphid transmission factor; capsid protein; genome; polyprotein;

protease; RNA polymerase; RNA-dependent RNA polymerase.

SOURCE Potato virus Y

ORGANISM Potato virus Y

Viruses; ssRNA positive-strand viruses, no DNA stage; Potyviridae;

Potyvirus.

ORIGIN [47]

1 aattaaaaca actcaataca acataagaaa aacaacgcaa aaacactcat aaacgctcat

61 tctcactcaa gcaacttgct aagtttcagt ttaaatcatt tccttgcaat tctctagaac

121 aatattggaa accatttcaa ctcaacaagc aatttcatca cttccaacca atttcagatc

181 ctcaatggca acttacatgt caacaatctg ttttggttcg tttgaatgca agctaccata

241 ctcaccagcc tcttgcgagc atattgtgaa ggaacgagaa gtgccggctt ccgttgatcc

301 tttcgcagat ctggaaacac aacttagtgc acgattgctc aagcaaaaat atgctactgt

361 tcgtgtgctc aaaaacggta cttttacgta ccgatacaag actgatgccc agataatgcg

421 cattcagaag aaactggaga ggaaggatag ggaagaatat cacttccaaa tggccgctcc

481 tagtattgtg tcaaaaatta ctatagctgg cggagatcct ccatcaaagt ctgagccaca

541 agcaccaaga gggatcattc atacaactcc aaggatgcgt aaagtcaaga cacgccccat

601 aataaagttg acagaaggcc agatgaatca cctcattaag cagataaaac agattatgtc

661 ggagaaaaga gggtctgtcc acttaattag taagaaaacc actcatgttc aatataagaa

721 gatacttggt gcatactccg cagcggttcg aactgcacat atgatgggtt tgcgacggag

781 agtggacttc cgatgtgata tgtggacagt tggacttttg caacgtctcg ctcggacgga

841 caaatggtcc aatcaagtcc gcactatcaa catacgaagg ggtgatagtg gagtcatctt

901 gaacacaaaa agcctcaaag gccactttgg tagaagttca ggaggcttgt tcatagtgcg

961 tggatcacac gaagggaaat tgtatgatgc acgttctaga gttactcaga gtattttaaa

1021 ctcaatgatc cagttttcga atgccgacaa tttttggaag ggtctggacg gtaattgggc

1081 acgaatgaga tatccttcgg atcacacatg tgtagctggt ttacctgtcg aagattgtgg

1141 tagggtagct gcattgatgg cacacagtat ccttccgtgc tataagataa cttgccccac

1201 ctgtgctcaa cagtatgcca gcttgccagt tagcgatctg tttaagctat tgcataaaca

1261 tgcaagagat ggtttgaatc gattgggagc ggataaagac cggtttatac atgttaataa

1321 gttcttgata gcgttagagc atctaactga accggtggac ctgaatctcg agcttttcaa

1381 tgagatattt aaatccatag gggagaaaca gcaagcaccg ttcaagaatt taaatgtctt

1441 aaataatttc ttcctgaaag gaaaagaaaa tacagctcat gaatggcagg tagctcaatt

1501 gagtttgctc gaattagcaa ggttccagaa gaacagaact gataacatca agaaaggtga

1561 tatatctttc ttcagaaata aattatctgc caaggcaaac tggaatctgt atttgtcgtg

1621 cgacaaccag ctggataaaa atgcaaactt cctctgggga caaagggagt atcatgctaa

1681 gcggtttttc tcaaacttct ttgaggaaat tgatccagca aagggatact cagcatatga

1741 aatccgcaag catccaagtg gaacaaggaa gctctcaatt ggtaacttag ttgtcccact

1801 tgatttagct gagtttaggc agaagatgaa aggtgactat aggaaacaac caggggtcag

1861 caaaaagtgc acgagttcga aagatggtaa ttatgtgtat ccctgttgtt gcacaacact

1921 tgatgatggt tcagccattg aatcaacatt ctatccacca actaaaaagc accttgtaat

1981 tggcaatagt ggtgaccaaa aatttgttga tttaccaaaa ggggattcgg agatgttata

2041 cattgccaag cagggttatt gttatattaa cgtgtttctt gcaatgctga ttaacattag

2101 cgaggaggat gcaaaggatt tcacaaagaa agttcgcgac atgtgtgtgc caaagcttgg

2161 aacctggcca actatgatgg atttggcgac cacttgtgct caaatgagaa tattctatcc

2221 tgacgtacat gatgcagaat tgcccagaat attggttgac catgacactc aaacgtgtca

2281 tgtggttgac tcatttggct cgcagacaac tggatatcat attctaaaag catccagcgt

2341 gtctcaactt atcttgtttg caaatgatga attagaatct gatataaaac attatagagt

2401 tggtggtgtt cctaatgcta gccctgaact tgggtccaca atatcacctt tcagagaagg

2461 aggagttata atgtctgagt cggcagcgct gaaactgctt ttgaagggaa tttttagacc

2521 taaggtgatg agacagttgc tgttagatga gccttacctg ttgattctat caatactatc

2581 ccctggcata ctgatggcta tgtataataa tgggattttt gaacttgcgg tgaggttgtg

2641 gattaatgag aaacaatcca tagctatgat agcatcgcta ctatcagctt tagccctacg

2701 agtgtcagcg gcagaaacac tcgtcgcaca gaggattata attgatgctg cagctacaga

2761 cctccttgat gctacgtgtg atgggttcaa cctacatcta acgtacccca ctgcattgat

2821 ggtgttgcaa gttgttaaga atagaaatga atgtgatgat accctattca aggcgggttt

2881 tccaagttac aacacgagcg tcgtacagat tatggaaaaa aattatctaa atctcttgaa

2941 cgatgcttgg aaagatttaa cttggcgaga aaattatccg caacatggta ctcatacaga

3001 gcaaaacgct ctatccactc ggtacataaa acccacagaa aaggcagatt tgaaagggtt

3061 atacaacata tcaccacaag cgttcttggg ccgaagcgcc caggtggtca aaggcactgc

3121 ctcaggattg agcgagcgat ttaataatta tttcaatact aagtgtgtaa atatttcatc

3181 ctttttcatt cgtagaatct ttaggcgttt gccaaccttt gtcacttttg ttaactcatt

3241 attagttatt agtatgttaa ccagcgtagt ggcagtgtgt caggcaataa ttttagatca

3301 gaggaagtat aggagagaaa tcgagttgat gcagatagag aagaatgaga ttgtctgcat

3361 ggagctatat gcaagtttac agcgcaaact tgaacgcgat ttcacatggg atgagtacat

3421 tgagtatttg aagtcagtaa accctcagat agttcagttt gctcaagcgc agatggaaga

3481 atatgatgtg cgacaccagc gttccacacc agttgttaaa aatttggaac aagtggtagc

3541 atttatggct ttagtcatca tggtgtttga tgctgaaagg agtgattgcg tgttcaaaac

3601 tctcaataaa tttaagggtg tcctttcctc actggattat gaagttagac atcagtcctt

3661 agacgatgtg atcaagaatt ttgatgagag gaatgagatt attgattttg aattgagtga

3721 ggacacaatt cgaacttcat cagtgctaga tacaaagttt agtgattggt gggatcgaca

3781 aatccagatg ggacatacac ttccacatta cagaactgag gggcacttca tggaatttac

3841 aagagcaact gctgttcaag tggctaatga cattgcccat agcgaacacc tagacttttt

3901 agtacgggga gctgttgggt ctggaaagtc aactgggttg cctgttcatc ttagtgtggc

3961 cggatctgtg cttttaattg aaccaacgcg accactagcg gagaacgttt tcaaacagct

4021 atctagtgaa ccattcttca agaagccaac actgcgtatg cgtggaaata gtatatttgg

4081 ctcttctcca atctccgtca tgactagcgg atttgcgcta cactacttcg ccaataatcg

4141 ctctcaatta gctcagttca actttgtaat atttgatgag tgtcatgttc tggatccttc

4201 cgcgatggcg ttccgcagtc tgctgagtgt ttatcatcaa gcatgcaaag tattaaaagt

4261 gtcagctact ccagtgggaa gagaggttga attcacaaca cagcagccag tcaagttaat

4321 agtggaggac acactgtctt tccaatcatt tgttgatgca caaggttcta aaactaatgc

4381 tgatgttgtt cagtttggtt caaacgtact tgtgtacgtg tcgagctaca atgaagttga

4441 caccttggcc aagctcctaa cagacaagaa tatgatggtc acaaaggttg atggcagaac

4501 aatgaagcac ggttgcctag aaattgtcac aaaaggaacc agtgcgagac cacattttgt

4561 tgtagcaacc aacataattg agaatggagt gactttggac atagacgtgg ttgtggactt

4621 tgggttgaaa gtctcaccgt tcttggacat tgacaatagg agcattgctt acaataaggt

4681 gagtgttagc tatggtgaga gaattcaaag gctgggtcgt gttggacgct tcaagaaagg

4741 agtagcattg cgcattggac acactgaaaa gggaattatt gaaattccaa gcatggtcgc

4801 tactgaggcg gctcttgctt gctttgcata taacttgcca gtgatgacag gaggcgtttc

4861 aactagtctg attggcaatt gtactgtgcg ccaggttaaa acaatgcagc aatttgaatt

4921 gagtcccttc tttatccaga atttcgttgc tcatgatgga tcaatgcatc ctgtcataca

4981 tgacattctt aaaaagtata aacttcgaga ttgtatgacg cctttgtgcg atcagtctat

5041 accatacagg gcatcgagta cttggttatc ggttagtgaa tatgagcgac ttggagtggc

5101 cttagaaatt ccaaagcaag tcaaaattgc attccatatc aaagagatcc ctcctaagct

5161 ccacgaaatg ctttgggaaa cggttgtcaa gtacaaagac gtttgcttat ttccaagcat

5221 tcgagcatcg tccatcagca aaatcgcata cacattgcgt acagatctct tcgccatccc

5281 aagaactcta atattggtgg agagattgct tgaagaggag cgagtgaagc agagccaatt

5341 cagaagtctc atcgatgaag ggtgctcaag catgttttca attgttaact taaccaacac

5401 tctcagagct agatatgcaa aagattacac cgcagagaac atacaaaaac ttgagaaggt

5461 gagaagtcaa ctaaaagaat tctcaaattt ggatggttct gcatgtgagg agaatttaat

5521 aaagaggtat gagtcgttgc agttcgttca tcaccaagct gcgacgtcac ttgcaaagga

5581 tctcaagttg aaggggattt ggaacaagtc attagtggct aaagacttga tcatagcagg

5641 cgctgttgca attggtggaa taggactcat atatagttgg ttcacacaat cagttgagac

5701 tgtgtctcat caagggaaaa ataaatccaa aagaatccaa gccttgaagt ttcgccatgc

5761 tcgtgacaaa agggctggct ttgaaattga caacaatgat gacacaatag aggaattctt

5821 cggatctgca tacaggaaaa agggaaaagg taaaggtacc acagttggta tgggtaagtc

5881 aagcaggagg ttcatcaaca tgtatgggtt tgatccaaca gagtactcat tcatccaatt

5941 cgttgatcca ctcactgggc ggcaaataga agaaaatgtc tatgctgaca ttagagatat

6001 tcaagagaga tttagtgaag tgcgaaagaa aatggttgag aatgatgaca ttgaaatgca

6061 agccttgggt agtaacacga ccatacatgc atacttcagg aaagattggt gtgataaagc

6121 tttgaagatt gatttaatgc cacataaccc actcaaagtt tgtgacaaaa caaatggcat

6181 tgccaaattt cctgagagag agctcgaact aaggcagact gggccagctg tagaagtcga

6241 tgtgaaggac ataccagcac aggaggtgga gcatgaagct aaatcgctca tgagaggctt

6301 gagagacttc aacccaattg cccaaacagt ttgtaggctg aaagtatctg ttgaatatgg

6361 ggcatcagag atgtacggtt ttggatttgg agcatacata gtagcgaacc accatttatt

6421 taggagttac aatggttcca tggaggtgca atccatgcac ggtacattca gggtgaagaa

6481 tctacacagt ttgagcgttc tgccaattaa aggtagggac atcatcctca tcaaaatgcc

6541 gaaagatttc cctgtctttc cacagaaatt gcatttccga gctcctacac agaatgaaag

6601 aatttgttta gttggaacca acttccaaga gaagtatgct tcgtcgatca tcacagaaac

6661 aagcactact tacaatatac caggcagcac attctggaag cattggattg aaacagataa

6721 tggacattgt ggactaccag tggtgagcac cgccgatgga tgtatagtcg gaattcacag

6781 tctggcaaac aatgcacaca ccacgaacta ctactcagcc ttcgatgaag attttgaaag

6841 caagtacctc cgaaccaatg agcacaatga atgggtcaag tcttgggttt ataatccaga

6901 cacagtgttg tggggcccgt tgaaacttaa agacagcact cccaaagggt tattcaaaac

6961 aacaaagctt gtgcaagatc taatcgatca tgatgtagtg gtggagcaag ctaagcattc

7021 tgcatggatg tttgaagcct tgacaggaaa tttgcaagct gtcgcaacaa tgaagagcca

7081 attagtaacc aagcatgtag ttaaaggaga gtgtcgacac ttcacagaat ttctgactgt

7141 ggatgcagag gcagaggcag aggcattctt caggcctttg atggatgcgt atgggaaaag

7201 cttgctaaat agagatgcgt acatcaagga cataatgaag tattcaaaac ctatagatgt

7261 tggtgtcgtg gatcggatgc atttgaggaa gccatcaata gggttatcat ctacctgcaa

7321 tgtgcacggc ttcaagaagt gtgcatatgt cactgatgag caagaaattt tcaaagcgct

7381 caacatgaaa gctgcagtcg gagccagtta tgggtgcaaa aagaaagact attttgagca

7441 tttcactgat gcagataagg aagaaatagt catgcaaagc tgtctgcgat tgtataaagg

7501 tttgcttggc atttggaacg gatcattgaa ggcagagctc cggtgtaagg agaagatact

7561 tgcaaataag acgaggacgt tcactgctgc acctctagac actttgctgg gtggtaaagt

7621 gtgtgttgat gacttcaata atcaatttta ttcaaagaat attgaatgct gttggacagt

7681 tgggatgact aagttttatg gtggttggga taaactgctt cggcgtttac ctgagaattg

7741 ggtatactgt gatgctgatg gctcacagtt tgatagttca ctaactccat acctaatcaa

7801 tgctgttctc accatcagaa gcacatacat ggaagactgg gatgtggggt tgcagatgct

7861 gcgcaattta tacactgaga ttgtttacac accaatttca actccagatg gaacaattgt

7921 caagaagttt agaggtaata atagtggtca accttctacc gttgtggata attctctcat

7981 ggttgtcctt gctatgcatt acgctctcat taaggagtgc gttgagtttg aagaaatcga

8041 cagcacgtgt gtattctttg ttaatggtga tgacttattg attgctgtga atccggagaa

8101 agagagcatt ctcgatagaa tgtcacaaca tttctcagat cttggtttga actatgattt

8161 ttcgtcgaga acaagaagga aggaggaatt gtggttcatg tcccatagag gcctgctaat

8221 cgagggtatg tacgtgccaa agcttgaaga agagagaatt gtatccattc tgcaatggga

8281 tagagctgat ctgccagagc acagattaga agcgatttgc gcagctatga tagagtcctg

8341 gggttattct gaactaacac accaaatcag gagattctac tcatggttat tgcaacagca

8401 accttttgca acaatagcgc aggaagggaa ggctccttat atagcaagca tggcactaag

8461 gaaactgtat atggataggg ctgtggatga ggaagagcta agagccttca ctgaaatgat

8521 ggtcgcatta gatgatgagt ttgagcttga ctcttatgaa gtacaccatc aagcaaatga

8581 cacaattgat gcaggaggaa gcaacaagaa agatgcaaaa ccagagcagg gcagcatcca

8641 gccaaacccg aacaaaggaa aggataagga tgttaatgca ggcacatctg ggacacatac

8701 tgtgccgaga atcaaggcta tcacgtccaa aatgagaatg cccacaagca agggagcaac

8761 cgtgctaaac ttagaacatt tgcttgagta tgctccacaa caaattgata tttcaaatac

8821 tcgggcaact caatcacagt ttgatacgtg gtatgaggca gtgcggatgg catacgacat

8881 aggagaaact gagatgccaa ctgtgatgaa tgggcttatg gtttggtgca ttgaaaatgg

8941 aacctcgcca aatgtcaacg gagtttgggt tatgatggat gggaatgaac aagttgagta

9001 cccgttgaaa ccaatcgttg agaatgcaaa accaaccctt aggcaaatca tggcacattt

9061 ctcagatgtt gcagaagcgt atatagaaat gcgcaacaaa aaggaaccat atatgccacg

9121 atatggttta attcgaaatc tgcgggatat gggtttagcg cgttatgcct ttgactttta

9181 tgaggtcaca tcacgaacac cagtgagggc tagggaagcg cacattcaaa tgaaggccgc

9241 agcattgaaa tcagcccaac ctcgactttt cgggttggac ggtggcatca gtacacaaga

9301 ggagaacaca gagaggcaca ccaccgagga tgtctctcca agtatgcata ctctacttgg

9361 agtcaagaac atgtgatgta gtgtctctcc ggacgatata taagtattta catatgcagt

9421 aagtattttg gcttttcctg tactactttt atcataatta ataatcgttt gaatattact

9481 ggcagatagg ggtggtatag cgattccgtc gttgttagtg accttagctg tcggttctgt

9541 attattaagt cttagataaa aagtgccggg ttgttgttgt gtgactgatc tatcgattag

9601 gtgatgctgt gattctgtca tagcagtgac tatgtctgga tttagttact tgggtgatgc

9661 tgtgattctg tcatagcagt gactgtaaac ttcaatcagg agac

8.3 Gene map

Monopartite, linear, ssRNA(+) genome of 10 kb in size. 3' terminus has a poly (A) tract. 5' terminus has a genome-linked protein (VPg).

8.3 Plants transformed with a cistron of a potato virus Y protease (NIa) are resistant to virus infection



ABSTRACT

An oligonucleotide caryin signals for translation initiation in plants was engineered upstream to a cDNA clone containing nucleotides 5812-7260 of the potato virus Y (PVY) genome. This faent contains all but the first 100 5' terminal bases of the cistron encoding one of the PVY proteases (NIa) as well as the first 251 bases of the next cistron (NIb). Nicotana tabacum cv. SR1 plants were ransformed with this fragment. The presence of the NIa sequences in transformed plants was determined by hybridization or PCR, and its expression was ascertained by reverse tanscription coupled to PCR. Plants expressing NIa were self-pollinated, and the R, kanamycin-resistant progeny were rechecked for NIa expression. Several of these plants were found to be resisant to PVY infection, inasmuch as they did not develop symptoms for at kast 50 days (the duration of the experiments), and no viral accumulation could be detected in their leaves by ELISA. All of the descendents of resistant homozygous R2 plants were also resistant. Several of the plants transformed with the last three cistrons of PVY (bases 5812-9704; Nla-NIb-coat protein) were also resistant to PVY. None ofthe transformed plants exhibited rsistance to tobacco mosaic virus. Exposure of the plants to 35°C for 48 hr prior to inoculation lowered, but did not abolish, resistance.

Potyviruses constitute the largest known group of plant viruses, inflicting heavy economic damage (1, 2). The organization of their large (9-10 kb) RNA genome is well understood (2). One of their major haracteristics is that their genome comprises a single, large open reading frame, encoding a large primary polyprotein. The mature virusencoded proteins are produced by self-proteolysis, carried out by the products of three of the viral cistrons (3-8). Several nonstructural viral proteins tend to accumulate in infected tissues, some in the form of inclusion bodies (2, 9). The nuclear inclusion body comprises aggregates of two virus-encoded proteins: NIa and NIb. NIa is the major potyvirus protease, cleaving at all proteolytic sites except the first two in the N-terminal region (2, 4, 7). NIb is thought to be associated with replication, due to some sequence homology with the polio virus replicase (10-12). Since potyviruses express a fair amount of nonstructural viral proteins that aggregate into stable forms, they offer a unique opportunity to study the expression and function of these proteins. As part of an effort to study the factors controlling NIa expression and function, we transformed plants with the NIa cistron. Unexpectedly, as reported below, these plants exhibited a high degree of resistance to potato virus Y (PVY) infection.

MATERIALS AND METHODS

Cloning of the PVY Cistrons and Their Transfer to Plants. 3'PVY3.9 (bases 5812-9704 of PVY), carrying the NIa-NIbcoat protein (CP) cistrons, was isolated from the EcoRI fragments of a full-length PVY-cDNA clone (Fig. 1; ref. 13). PVY1.4NIa, carrying most of NIa cistron (Fig. 1), was an EcoRI/Bgl II fragment of 3'PVY3.9 (bases 5812-7260 of PVY). An oligonucleotide carrying Lutcke's consensus signal for ranslation initiation in plants (GACAAIG; ref. 16) between EcoRI and Xba I restriction sites was synthesized and ligated to the EcoRI site of 3'PVY3.9. The engineered 3'PVY3.9 construct was inserted at the Xba I site of the binary plasmid pGA643, and the engineered construct of PVY1.4NIa was inserted between the Xba I and Bgl II sites of pGA643. Thus, both were inserted between the 35S promoter and a terminator, linked to a kanamycin (Km)-resistance gene and placed between the border sequences of transferred DNA (Fig. 1; ref. 17). The engineered binary plasmids (pG3PVY3.9 and pGNIal.4, respectively) were transferred to Agrobacterium tumefaciens (EHA 101), which was then used to transform plants. Tobacco leaf disks were inoculated with the transformed Agrobacterium (18) and maintained on a regeneration medium containing Murashige and Skoog (MS) salt mixture (19), 2% sucrose, 1% mannitol, 1% Noble agar, 2 mg of zeatin per liter, and 0.1 mg of indoleacetic acid per liter (pH 5.8), supplemented with 300 ,ug of Km per ml. Shoots that developed under these conditions were transferred individually to tissue culture flasks containing MS salts, 3% sucrose, 1% Noble agar, and 400 ,ug of Km per ml. Rooted plantlets were transplanted to utoclaved soil and grown in a greenhouse. All work was carried out at 24-26°C. The primary transformed plants were designated the Ro generation. Selected Ro plants (see below for selection criteria) were self-pollinated, and their seeds were collected and germinated on Km-containing agar. The resultant R1 generation segregated for Km sensitivity or Km resistance. The Km-resistant plantlets were kept for further studies as was a Km-sensitive specimen, which served as a negative control for each transformation event. R1 plants were also selfpollinated and R2 plants were treated and selected as above. The copy number of the inserted neomycin phosphotransferase II (NPTII; Km+) gene was determined from the segregation of R1, and the NPTII homozygous state was determined from the segregation of R2. Analysis of Transformed Plants. pGNIal.4 was subcloned into the plasmid pBluescript KS (Stratagene) and transcripts directed by a T7 promoter were used as probes in hybridization assays ofplant material. DNA (20) and RNA (21) were extracted from leaf tissue. Successful transformation of Kmresistant plants was verified by Southern blot analysis or by DNA-PCR (34). pG3PVY3.9-transformed plants were assayed for expression by Western lot analysis, using CP antibodies. The unavailability of NIa antibodies, however, precluded Western blot analysis in this case. Hence, expression of NIa in transgenic plants was tested by RNA-PCR (reverse transcription coupled with PCR; ref. 35). The synthetic PCR primers were designed to distinguish between RNA and DNA as described below. The primer sequences are indicated:

Primer 1 (sense): 5'-CTATCCTTCGCAAGACCCTTC-3'

Primer 10 (sense): 5'-TTTGGATCTGCATACAGGAAGAAAGGG-3'

Primer 11 (antisense): 5'-CCGACTATACATCCATCGGCGGTG-3'.

The DNA, either extracted or obtained from RNA by reverse transcription, was submitted to 30 cycles of PCR: denatuation (45 sec; 94°C), annealing (45 sec; 60°C), and extension (60 sec; 72°C).

Virus Assays. PVY was purified from infected tobacco leaves according to Tanne et al. (14). Two leaves per plant were mechanically inoculated with 30 pg of PVY per ml (in 10 mM Tris HCI, pH 7.0). The plants were kept in a growth chamber at 26°C and inspected daily for symptom appearance. Actual viral accumulation was determined by ELISA. Every 4-5 days, leaf disks (5 mm in diameter) were removed from the third leaf above the inoculated ones. The leaf disks were homogenized in 1.5-ml Microfuge tubes with a Teflon pestle (five disks in 500 ,ul of phosphate-buffered saline/Tween/polyvinylpyrrolidone). Sap as applied to a microtiter plate (20 Ml per well). Each plate included viral standards, ranging from 1 to 1000 ng of PVY per ml. The ELISA was performed according to Sela (15) using antiserum developed against the purified PVY.

FIG. 1. (A) Schematic diagram of the organization of the PVY genome.

The size of the proteins of each of the eight cistrons is marked at the bottom. Cistrons with specific names are marked as follows: HC, helper omponent; CI, cytoplasmic inclusion; NIa and NIb, nuclear inclusions a and b; CP. The relevant EcoRI (E) and Bgl II (B) sites are marked. UTR, untranslated region. (B and C) Maps of the binary vectors pG3PVY3.9 and pGNIal.4. The PVY sequences were inserted between the cauliflower mosaic virus 35S promoter and the transcription-termination signal of gene 7 ofpTiA6 (13). The sequence at the top of each illustration is that of a translation-initiation signal ligated to the PVY clone. Numbers indicate the base number in the PVY sequence (12). NPTII, gene for NPTII. BR and BL, right and left border sequences of ransferred DNA.

RESULTS

Analysis of Transormed Plants. Two hundred Km-resistant Ro plants were analyzed by Southern blot; 164 of them were unambiguously found to carry sequences ofPVY1.4NIa (data not shown). Following self-pollination, R1 progeny were selected for Km resistance. Ten R1 progeny of each of 50 randomly selected Ro plants (total of 500 plants) were further tested. One Km-sensitive R1 plant (NIal) was also maintained for each of the 50 independent transformation events, to serve as a negative control descending from the same parent plant. All 500 selected R1 plants (forming 50 lines: V1-V50) were tested for PVY resistance as described below. All member plants of48 lines showed PVY symptoms by day 7 from inoculation, whereas 4 plants of line V2 and 5 of line V3 remained symptomless to the end of the experiment (day 50).

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