Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10310-10314, October 1994 Plant Biology Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block (trnenic plant/doinat negative mutaton/ n l movement proein) DAVID L. BECK, CRAIG J. VAN DOLLEWEERD, TONY J. LOUGH, EZEQUIEL BALMORI, DAVIN M. VOOT, MARK T. ANDERSEN, IONA E. W. O'BRIEN, AND RICHARD L. S. FORSTERt Molecular Genetics Group, The Horticultural and Food Research Institute of New Zealand Ltd., Private Bag 92169, Auckland, New Zealand Communicated by George Bruening, June 23, 1994 ABSTRACT White clover mosaic virus strain 0 (WCIMV- tially difficult. New forms ofresistance active against several 0), species of the Potexvirus genus, contains a set of three different viruses or groups of viruses are being sought. One partially overlapping genes (the triple gene block) that encodes such approach involved the introduction of the gene coding nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These for rat 2'-5' oligoadenylate synthetase into the genome of proteins are necesy for cell-to-cell movement in plants but potato plants (4). Genetically engineering transgenic plants to not for replication. The WCIMV-O 13-kDa gene was mutated block virus movement, mimicking the mechanism of some (to 13*) in a region of the gene that is conserved in all viruses natural resistance genes, has been proposed (1, 5, 6) but known to possess triple-gene-block proteins. All 10 13* trans- remains mostly unexploited. genic lines of Nicodiana benthamiana designed to express the The movement function of plant viruses can be comple- mutated movement protein were shown to be resistant to mented by another, frequently unrelated, virus in double systemic infection by WCIMV-O at 1 jug ofWCIMV virions per infections (7). This apparent lack of specificity for the move- ml, whereas all plants from susceptible control lines became ment function has been demonstrated by De Jong and Ahl- systemically infected. Ofthe 13* transgenic lines, 3 selected for quist (8) who showed that the movement protein from a their abundant seed supply were shown to be resistant to Tobamovirus could support the movement requirements of a systemic infection when challenged by inoculation with three Bromovirus. This nonspecific complementation ofthe move- different WC1MV strains (0, M, and J) or with WCIMV-O ment process by unrelated viruses suggested to us that RNA at 10 pg/ml. Most plants were also resistant to systemic inhibition of movement might also be nonspecific, invoking infection at inoculum concentrations up to 250 pg of WCIMV resistance to viruses ofdifferent groups. Recently, transgenic virions per ml. In i , the three 13* tnsenic plant lines tobacco plants [a nonhost of brome mosaic virus (BMV; of were found to be resistant to systemic infection with two other the Bromovirus group)] expressing the BMV 32-kDa move- members ofthe Potexvirus group, potato virus X and narcissus ment protein were found to be resistant to infection by mosaic virus, and the Carlavirus potato virus S but not to be tobacco mosaic virus (TMV; of the Tobamovirus group), rsistnt to tobacco mosaic virus of the Tobamovius group. suggesting that expression of a heterologous movement pro- These results indicate that virus resistance can be engineered tein not adapted for a particular host can interfere with the into transgenic plants by expression of dominant negative process mediated by the homologous movement protein (5). mutant forms of triple-gene-block movement proteins. Transgenic tobacco plants expressing a mutated form of the TMV 30-kDa movement protein were found to be resistant to infection by three strains of TMV (6). However, this resis- Cell-to-cell and long-distance movement of viruses in plants tance was manifest primarily as a delay in infection. are not passive events, and most, ifnot all, plant viruses have A set of three partially overlapping genes known as the a gene or set of genes that mediate movement (1). These triple gene block encodes nonvirion proteins and is found in movement genes are an essential part of the virus-infection the genome of the Potexvirus, Carlavirus, Furovirus, and process because plant protoplasts are frequently able to Hordeivirus groups (9, 10) as well as several unclassified support the replication of viruses that are not able to infect plant viruses (11, 12) of the Sindbis virus-like superfamily the intact plant (2). Naturally occurring resistance genes may (13). The triple-gene-block proteins have been shown by function by interfering with the ability of the invading virus site-directed mutagenesis to be essential for virus movement to move from the initially infected cells to surrounding for viruses of the Potexvirus (14), Hordeivirus (15), and tissues. Although traditional plant breeding has been used Furovirus groups (16). The precise mechanism by which the successfully to control several virus diseases, natural genes triple-gene-block proteins mediate the movement process is for resistance are not always available to breeders, and single unclear; however, in all instances the protein encoded by the genes for resistance can be overcome by rapidly changing 5'-most gene ofthe triple gene block has domains conserved virus populations. As a consequence, genetically engineered in RNA helicases. All of the proteins encoded by the central protection against viruses in transgenic plants has been gene ofthe triple gene block have two hydrophobic domains adopted as an alternative control strategy. This strategy has suggestive of transmembrane proteins (9, 10). been shown to have great potential, but most of the trans- The triple gene block of white clover mosaic virus genic plants produced to date show resistance against only a (WClMV), a member of the Potexvirus group, encodes pro- limited numberofviruses orvirus strains (3). Many important teins of26 kDa, 13 kDa, and 7 kDa (10, 17). A WCIMV mutant crop species, for example potato and rice, are infected by expressing a mutated 13-kDa gene (13*) was unable to numerous different viruses or virus strains, making the multiply to detectable levels in intact plants but produced development of superior plant lines resistant to each virus wild-type levels of viral RNA species and coat protein when based on these narrow-spectrum resistance factors poten- Abbreviations: WCIMV, white clover mosaic virus; TMV, tobacco The publication costs ofthis article were defrayed in part by page charge mosaic virus; NMV, narcissus mosaic virus; PVX, potato virus X; payment. This article must therefore be hereby marked "advertisement" PVS, potato virus S. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 10310 Downloaded by guest on September 29, 2021 Plant Biology: Beck et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10311 inoculated to protoplasts (14), suggesting that the mutations ml for rooting to occur. Only one shoot per leaf disc was introduced into the 13* gene inactivated the function of the transferred to rooting medium to ensure that independent Ro encoded protein. The mutated 13* gene was transferred into generation plant lines were established. the genome of Nicotiana benthamiana (a systemic host of Inheritance of kanamycin resistance was determined by WCIMV) to determine whether this gene could confer non- germinating self-pollinated seed (R1 generation), harvested specific virus resistance. This paper describes the analysis of from regenerated transgenic plants (Ro generation), on me- transgenic plants expressing the 13* gene. Resistance was dium containing 300 ,g ofkanamycin per ml. Approximately observed against systemic infection by three different strains 2-week-old kanamycin-resistant R1 seedlings were trans- of WCIMV, two other Potexvirus species, and one Carlavi- ferred to soil and grown under containment glasshouse con- rus, but not against a Tobamovirus. ditions. Ten plant lines (13*1_lo) transformed with the mutated WC1MV 13* gene plus 7-kDa sequences were established. MATERIALS AND METHODS Control lines, C1_3, were similarly established after transfor- Virus Isolates. WC1MV strains 0 and M (WCLMV-O and mation with the unmodified binary vector pART27 (20). WClMV-M) were obtained from individual white clover Analysis ofTransgenic Plants. Northern (RNA blot), West- plants from the South Island and North Island of New ern (immunoblot), and ELISA analyses of uninoculated and Zealand, respectively (18). WCIMV strain J from Japan was inoculated leaves of R1 plants were performed by standard supplied by C. Hiruki (Edmonton, AB). The WCIMV strains techniques (24, 25). For Northern blot analysis, 32P-labeled were maintained in pea, and virions were purified as de- RNA complementary to WClMV-O sequences 3997-5802 scribed (18). Potato virus X (PVX, type species of the (the triple gene block plus coat-protein gene) was used. Potexvirus group) strain NZ1 was obtained from field- Western blot analysis involved rabbit anti-coat protein serum infected potato cv. Ilam Hardy, maintained in Nicotiana produced by injecting New Zealand White rabbits with tabacum cv. Xanthi, and virions were purified as described purified WClMV-O virus particles. For analysis ofresistance (19). Narcissus mosaic virus (NMV; member of the Potex- to viral infection, approximately 6-week-old kanamycin- virus group) was obtained from field-infected narcissus, resistant R1 plants were challenged by mechanical inocula- maintained in Nicotiana clevelandii and purified as described tion either with preparations of virus particles or with virus for PVX. Concentrations ofpurified virion preparations were RNA. Three carborundum-dusted leaves of each plant re- estimated by absorbance at 260 nm (A260). An A260 of 3.0 was ceived 30-50 u4 of inoculum per leaf (depending upon leaf considered equal to 1 mg ofvirus per ml. Virions were diluted size). Plants inoculated with PVX, NMV, or PVS were in 0.25% sodium pyrophosphate buffer (pH 8.0) containing examined daily for symptom development. Because WCIMV 0.25% Celite and 0.25% bentonite and mechanically inocu- does not produce definitive symptoms on N.