Studies on genome sequence, rapid detection and RNAi-mediated control for calla lily potyviruses
Date Issued
2009
Date
2009
Author(s)
Hu, Wen-Chi
Abstract
Abstractalla lily (Zantedeschia spp.), belonging to the family Araceae, are perennial bulbous flowers. Because calla lily is a favorite ornamental flower, it becomes an important economic flower crop worldwide. Calla lily original from Africa, and have been introduced into Taiwan more than 10 years. Tissue culture technique has been developed for calla lily propagation and many calla hybrids have been bred in Taiwan. However, the viral disease control is important in calla lily cultivation. There are already 18 viruses have been reported which belonging to 9 genus. Potyirus is first reported and mainly calla lily-infecting virus in the field. Potyvirus is a positive sense stranded RNA virus with ~10 kb genome. Four viruseses are important in Taiwan, including Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Zantedeschia mosaic virus (ZaMV), and Zantedeschia mild mosaic virus (ZaMMV). For detection and molecular characterization of potyviruses, the largest plant virus, full-length cloning and sequencing strategy was developed. The RT-PCR-based methods for detection and identification of virus are based on the use of degenerate primers for RT-PCR amplification, combined with modified 5’RACE by using terminal transferase to modify the 5’ end sequence of potyviruses genome. The complete sequence would be identified by cloning and sequencing. The degenerate primers were designed from conserved sequences in the viral genome. According to the sequence alignment, theNIbF1, CIF2/CIR2, and HCF4/HCR4 were designed from potyviral NIb, CI and HC-Pro-coding regions. Expected PCR products were amplified by these primers from seven potyviruses. Complete genome sequences of ZaMMV and ZaMV, were successfully characterized. The sequence analysis reveals that ZaMV is the same with the Konjac mosaic virus (KoMV). On the other hand, in order to save time and simultaneous detection of DsMV, TuMV, ZaMV and ZaMMV in field, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3’ terminal region of respective viruses. To prevent the false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used as an internal control of RT-PCR. After specific and sensitivity test, the multiplex RT-PCR can rapidly detect multiple targets in one single assay, and the detection sensitivity of multiplex RT-PCR was 25-625 times higher than that of I-ELISA depending on the virus. Furthermore, a control method that prevents the virus infection using the mechanism of RNA silencing was investigated. A dsRNA expression and screening system was used to obtain highly efficient interference fragments from the consensus regions of four calla lily-infecting potyviruses, DsMV, TuMV, ZaMMV and ZaMV. The viruses were chosen for multiple sequence alignment. Several TuMV specific primers were designed within the conserved regions of HC-Pro, NIa and CP genes. Different fragments were PCR amplified and cloned into LITMUS 38i vector to produce viral dsRNA. Mechanical inoculation of different dsRNA transcripts with target virus on tobacco plants induced different levels of interference with virus infection. Two of the most effective fragments (located in HC-Pro and NIa genes) were further analyzed by using Agrobacterium tumefaciens infiltration (agoinfiltration) as transient expression system for hairpinRNA (hpRNA) expression. The successful interferences of TuMV infection were observed in the infiltrated leaves. Besides, Bean yellow mosaic virus (BYMV), another calla lily-infecting virus, was interfered by TuMV hpRNAs transiently expressed in plants. Several lines of transgenic Nicotiana benthamiana plants transformed with hpHC and hpNIa fragments were obtained. Over 80% of T0 and T1 transgenic plants revealed resistance response to TuMV infection.
Subjects
calla lily
potyvirus
detection
RNAi
File(s)![Thumbnail Image]()
Loading...
Name
ntu-98-D91633002-1.pdf
Size
23.53 KB
Format
Adobe PDF
Checksum
(MD5):857f8b4f60e7fd4bbb49370ff8534d80