CONSTRUCTION AND BIOLISTIC INOCULATION OF AN INFECTIOUS CDNA CLONE OF APPLE CHLOROTIC LEAF SPOT TRICHOVIRUS
H SATOH1< /sup>, N YOSHI KAWA1 AND T TAKAHASH l1
1Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020, Japan
Background and objectives
Apple chlorotic leaf spot trichovirus (ACLSV) is the type member of the genus Trichovirus and the causal virus of apple topworking disease in Japan. The virus has very flexuous filamentous particles, 740-760 nm in length and contains apolyadenylated plus-sense, ssRNA with an Mrof 2.48 x 106 and asingle coat protein of 22K. The genome of ACLSV (isolate P-205) from apple consists of 7552 nucleotides (nt) excluding poly (A) tail and encodes three open reading frames . In this study, we report the cloning of full-length cDNA of ACLSV genome downstream of acauliflower mosaic virus 35S RNA promoter and its application in successfully infecting Chenopodium quinoa1> and apple plants by mechanical and biolistic inoculation.
Materials and methods
The 5'-terminal region of the genome of ACLSV (P-209) was amplified by Vent DNA polymerase and the product was ligated to aStul site of cloning vector containing 35S RNA promoter as described previously . The resulting clones were used to produce the full-length cDNA clone (PCLSF) by adding the fragments from ACLSVcDNA clones. pCLSF was purified and then used for mechanical inoculation and inoculation by particle bombardment using the Biolistic PDS-1 000 Particle Delivery System (Bio-Rad).
Results and conclusions
C.quinoa plants mechanically inoculated with pCLSF (0.1m g/m 1) developed chlorotic and necrotic spots on inoculated leaves 5-7 days after inoculation and mosaic symptom on upper leaves 10-13 days after inoculation. Immunoblot analysis using antisera against virus paricles and the 50 kDa protein encoded by ORF2, bands of the coat ptotein and the 50 kDa protein were detected in PC LS F-inoculated plants. Northern blot analysis of RNA from PCLS F-inoculated plant leaves also revealed the RNA band corresponding to the genomic RNA of AC LSV In immunoelectron microscopy of extracts from PCLS F-inoculated plant leaves, filamentous virus particles decorated with an antiserum against AC LSV were observed. These results demonstrated that pCLSF was infectious in plants. pCLSF was diluted serially from 2.5m gm1 to 4ng/m and inoculated onto C.quinoa plants by mechanical inoculation. All plants were infected at the concentration of more than 0.1 ~ glaf Inoculation by particle bombardment resulted in 100% infection in .quinoa at 100 ng of pCLSF I macrocarrier and also 85 % infection in Apple seedlings at 1 m g of pCLSF Imacrocarrier.
1. Sato K, Yoshikawa N, Takahashi T, 1993. Journal of General Virology 74, 1927-31.
2. Terauchi, H, Magome H, Yoshikawa N, Takahashi T, 1997. Ann Phytopath. Soc. Japan 63 (in