An apple chlorotic leaf spot virus (ACLSV) isolate was detected by TAS-ELISA and RT-PCR in an ornamental dwarf flowering almond (Prunus glandulosa Thunb.). This plant, maintained at the Centre for Plant Health, Sidney, B.C., Canada, has been showing transient leaf symptoms during the spring seasons. A 390-bp fragment and a 1,350-bp product, in the RNA polymerase and the coat protein viral coding regions, respectively, were amplified by RT-PCR from the infected plant. A sequence comparison of the 390-bp fragment of this ACLSV isolate (designated as AL1292) with other published isolates, revealed a similarity of 81% to 84% at the nucleotide level and 88% to 100% at the amino acid level. In contrast to other ACLSV isolates, AL1292 has an exceptionally narrow range of experimental herbaceous and woody hosts, as determined by mechanical and graft inoculation assays. These standard bioassays may not be effective for the detection of the AL1292 isolate because of its limited host range. The results we report in this paper confirm P. glandulosa as a natural host of this virus. Currently it is not known how ACLSV is spread, other than by bud-grafting and possibly by root grafts. The use of virus-tested source plants for the preparation of planting material will minimize its spread.
Sara Spiegel, Dan Thompson, Aniko Varga, and Delano James
Zongrang Liu, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James
Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.