Construction of infectious clones from two strains of Papaya ringspot virus (PRSV) and comparative studies among different isolates of PRSV
Date Issued
2012
Date
2012
Author(s)
Yang, Jui-Chun
Abstract
Papaya ring spot caused by Papaya ringspot virus (PRSV) is considered to be the most important disease impacting papaya industry. This disease has occurred in Taiwan for decades since 1975. After the long-term genomic evolution in the field, there are 3 major pathological strains of PRSV in Taiwan at present time such as SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. DF is a predominant strain of PRSV in the field of Taiwan now, and SMN should be the most destructive strain causing quick decline in papaya plants especially when the season changes. SM, DF and SMN strains have highly similar nucleotide sequences, but their incited symptoms in papaya hosts are different. To investigate the relationship between molecular and pathological characters, this thesis was dedicated to construct the infectious clones for SM, DF and SMN strains of PRSV. Each intact infectious clone was derived from the splicing of 3~4 amplified cDNA fragments with two-step RT-PCR. The artificial full-length PRSV RNA transcripts could be harvested from the infectious clones through in vitro transcription, and they were used in the inoculation tests in either papaya or Chenopodium quinoa hosts. The results showed that the PRSV RNA transcripts derived from infectious clones could successfully infect papaya plants. However, the symptoms caused by the transcripts appeared 1 week later than those caused by natural PRSV inocula. The developing time of symptoms produced by the transcript-infected sap was similar to those caused by natural PRSV. The present SMN strain might incite “fern-leaf” in addition to original symptoms, which indicated mutation probably occurred in the genome of PRSV-SMN. Based on the alignment of full-length genomic nucleotide sequences among 3 different PRSV strains, SM and DF have 97.3% homology; SM and SMN have 96.5% homology; and SM and DF have 96.5% homology. Individual gene analyses revealed that they were 95.8-96.7%, 94~95% and 93.0-94.2% similarity of nucleotide sequences in P3, P1 and 5’-UTR respectively. The rest of genes showed 96-99% similarity among 3 strains. The further alignment of full-length genomic nucleotide sequences among several PRSV isolates from different countries demonstrated that all isolates from Taiwan are close to those from Korea and they can be categorized into a phylogenic group with those from China and Thailand. The PRSV isolates from America and South Asia form another phylogenic group. It indicates that strain-diversity of PRSV corresponds with geographical difference. Constructions of infectious clones of 3 PRSV strains will be helpful to study key genes or regions associated with symptom determinants (such as 5’-UTR, P1, P3, HC-Pro and CI) through molecular recombination and substitution. Besides, a new primer pair specific to the DF strain was developed in this thesis to improve the sensitivity and specificity and avoid non-specific amplification in the RT-PCR assay of PRSV-SM. It will provide a more reliable method for the identification among different PRSV strains.
Subjects
papaya ringspot potyvirus
infectious clone
virus strain
Type
thesis
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