Molecular Breeding 6: 87–93, 2000. 87 © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat protein genes Sheng-Zhi Pang1;†, Fuh-Jyh Jan1;†, David M. Tricoli2, Paul F. Russell3,KimJ.Carney3, John S. Hu1, Marc Fuchs1, Hector D. Quemada3 & Dennis Gonsalves1;∗ 1Department of Plant Pathology, Cornell University, NYSAES, Geneva, NY 14456, USA (∗author for corres- pondence; Fax: 315-787-2389; e-mail: [email protected]); 2Seminis Vegetable Seeds, 37437 State Highway 16, Woodland, CA 95695, USA; 3Formerly of Asgrow Seed Company; †These authors contributed equally to this research. Received 2 February 1999; accepted in revised form 19 July 1999 Key words: cosuppression, field test, gene silencing, pathogen-derived resistance, SqMV, transgenic squash Abstract The approach of pathogen-derived resistance was investigated as a means to develop squash mosaic comovirus (SqMV)-resistant cucurbits. Transgenic squash lines with both coat protein (CP) genes of the melon strain of SqMV were produced and crossed with nontransgenic squash. Further greenhouse, screenhouse and field tests were done with R1 plants from three independent lines that showed susceptible, recovery, or resistant phenotypes after inoculations with SqMV. Nearly all inoculated plants of the resistant line (SqMV-127) were resistant under greenhouse and field conditions and less so under screenhouse conditions. Plants of the recovery phenotype line (SqMV-3) were susceptible when inoculated at the cotyledon stage but leaves that developed later did not show symptoms. The susceptible line (SqMV-22) developed symptoms that persisted and spread throughout the plant. Plants were also analyzed for transcription rates of the CP transgenes and steady state transgene RNA levels. Results showed that the resistant line SqMV-127 displayed post-transcriptional silencing of the CP transgene as evidenced by high transcription rates but concomitant low accumulation of transgene transcripts. This is the first report on the development of transgenic squash that are resistant to SqMV. Abbreviations: CP, coat protein; SqMV, squash mosaic comovirus; CPMV, cowpea mosaic comovirus Introduction proteins are derived by proteolytic cleavages [9]. The virus capsid is composed of two distinct polypeptides Squash mosaic comovirus (SqMV) is a member of the with molecular weight of 42 kDa and 22 kDa, which genus Comovirus, with isometric virus particles about are encoded as a 64 kDa precursor by M-RNA [14]. 30 nm in diameter [4]. SqMV infects almost all spe- Nucleotide sequences of the coat protein (CP) genes cies in the Cucurbitaceae family and is transmitted by have been reported for a melon strain of SqMV [15]. beetles and through seeds [5]. The viral genome of Recently, cDNA clones of RNA-2 from two SqMV comoviruses consists of two single-stranded, positive- isolates were constructed and the full-length sequence sense RNA molecules identified as middle-component were determined [13]. RNA (M-RNA or RNA-2) and bottom-component Control of SqMV currently relies on the use of RNA (B-RNA or RNA-1) of about 4200 and 6000 virus-free seeds and insecticides for the control of nucleotides, respectively. Both M-RNA and B-RNA beetles [18]. Due to lack of SqMV resistant host genes are polyadenylated at the 30 end and have a genome- that can be incorporated into cucurbits by conventional linked protein (VPg) at the 50 end [9]. The RNAs are breeding [22], pathogen-derived resistance [24] could translated into polyproteins from which the functional provide an alternative to control SqMV and other co- 88 moviruses. Nida et al. [19] reported the production of transgenic tobacco expressing the 60 kDa precursor of the two capsid proteins of cowpea mosaic comovirus (CPMV) but resistance was not observed. Recently, reports from Sijen et al. [25, 26] have shown that Nico- tiana benthamiana plants expressing the full-length replicase, movement protein or CP genes were res- istant to CPMV. However, transgenic cucurbits with resistance to comoviruses have not been reported. We report here that transgenic squash plants ex- pressing the chimeric genes encoding the two SqMV CP genes are resistant to SqMV under greenhouse, screenhouse and field conditions. Furthermore, the protection we observed was RNA-mediated via post- transcriptional transgene silencing [3, 21, 29]. Materials and methods Cloning and squash transformation Figure 1. Agrobacterium binary vector pGA482G/SqMV. The loc- ations of important genetic elements within the binary vector are indicated: BR, right border; BL, left border; nos-npt, plant express- Two full-length CP genes from the melon strain of ible neomycin phosphotransferase gene; cos, cos site; tet, bacterial squash mosaic comovirus (SqMV m-88) were PCR tetracycline resistance gene; and gent, bacterial gentamicin resist- ance gene. Both 22 kDa and 42 kDa CP transgenes are driven by amplified and cloned in the sense orientation into a 0 plant expression vector pUC18cpexp [27], as pre- CaMV 35S promoter (35S-P), CMV-C 5 -untranslated leader and 35S terminator (35S-T). viously described by Hu et al. [15]. The result- ing pUC18cpexp-22 kDa and -42 kDa CP plasmids were partially digested by HindIII (the CP open ELISA, Northern blot, nuclei isolation and nuclear reading frames contain internal HindIII sites), and run-on transcription assay both SqMV 22 kDa and 42 kDa CP expression cas- settes were cloned into the transformation vector Double-antibody sandwich enzyme-linked immun- pGA482G, a derivative of pGA482 described by An osorbent assay (DAS-ELISA) [10] was used to detect [2]. The original binary vector pGA482 contains the the accumulation of the two CP polypeptides and the right and left T-DNA borders of pTi37 which flank nptII enzyme in transgenic plants using antibodies the plant expressible neomycin phosphotransferase II against SqMV virions [15] and an nptII ELISA kit (5 gene (nptII), restriction enzyme polylinker, and bac- Prime to 3 Prime, Boulder, CO), respectively. Total teriophage λcos site. To improve the use of this vector, plant RNAs were isolated according to the procedure the bacterial gentamicin-(3)-N-acetyl-transferase gene described by Napoli et al. [17]. Total RNA (10 µg [1] was cloned into the SalI site located outside the per lane) was separated on a formaldehyde-containing T-DNA region, generating pGA482G. The plant ex- agarose gel [23] and the agarose gels were stained with pressible SqMV CP genes were inserted as tandem ethidium bromide to monitor the uniformity of total repeats and were oriented in the same direction as plant RNAs in each lane. Hybridization conditions the nptII gene (Figure 1). The resulting binary vec- were described previously [20]. Isolation of nuclei and tor was transferred into the disarmed T-DNA deletion nuclear run-on transcription assays were essentially derivative of the Agrobacterium tumefaciens strain performed as described by Dehio and Schell [7]. The C58. same amount of labeled RNA was used for hybridiz- Transgenic squash plants were obtained by in- ation to replicated dot blot membranes (BioRad, Her- oculating leaf tissues from an Asgrow inbred yellow cules, CA) that contained 0.2 µgof22kDaCP,42kDa crookneck squash line using the procedure essentially CP, actin and nptII genes. Images of some autoradio- described by Tricoli et al. [28]. grams were photographed with a COHU CCD camera, Model 4915-2000 (COHU, San Diego, CA). Signals 89 were quantitated using the US National Institutes of Eleven R0 transgenic squash plants were gener- Health Image program version 1.59. ated and crossed with an untransformed inbred line while only five of them were able to produce seeds. Inoculation of transgenic plants R1 plants from these five lines were initially ana- lyzed at Kalamazoo, MI by inoculating seedlings and The strain SqMV m-88 was used in inoculation tests. monitoring the symptom development over a 35 day It was obtained from infected melon seeds [15] and period. Transgenic R1 seeds were germinated and as- typed as the melon strain by biological and serological sayed by nptII ELISA to identify the nontransgenic comparisons with the type strain [18]. Inocula were segregants from the R1 populations. All inoculated prepared by propagating the virus in zucchini squash transgenic plants of line SqMV-22 showed typical (Cucurbita pepo L.) and grinding infected leaves in symptoms about 10 days after inoculation and symp- 0.01 M potassium phosphate, pH 7.0. In most cases, toms were severe throughout the 35-day observation the cotyledons of small squash plants (ca. 10 days after period. These symptoms were similar to those of non- germination) were dusted with 400 mesh carborundum transformed controls. The R1 transgenic plants of line and rubbed with 1:15 dilution of SqMV m-88-infected SqMV-127, however, initially had moderate symp- squash leaf extract. Systemic infection were recorded toms but the symptoms either were not persistent or every day for at least 35 days. new leaves were symptomless by 21 days after in- oculation. At 35 days after inoculation, none of the Field evaluation inoculated plants were symptomatic. The R1 plants of line SqMV-3 developed symptoms which persisted The R seeds of three transgenic squash lines as well 1 throughout the observation period but were, on the as those of nontransformed controls were germinated average, less severe than the nontransgenic plants. A on 9 June, seedlings were screened by nptII ELISA, percentage of the R plants of lines SqMV-136 (17%) and nptII positive seedlings were inoculated twice 1 and SqMV-69 (39%) were symptomless throughout with SqMV m-88 on 20 June and 22 June 1994. The the 35-days observations period. Three transgenic 35 inoculated R plants of the three transgenic squash 1 squash lines, SqMV-3, SqMV-22, and SqMV-127, lines and the untransformed control were planted in were selected for further investigations because their the field 3 feet within the row, and 6 feet between rows R plants exhibited differential symptom reactions.