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Construction of infectious full-length cDNA clones of apple viruses and plant viral vector development

Zhang, Lei

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Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV) are important viral pathogens in apple. The aim of the present work was to construct full-length cDNA clones of ACLSV and ASPV and to agroinoculate apple seedlings with the constructed infectious clones using a newly developed vacuum infiltration method. A further goal was to explore and create viral vectors based on the obtained infectious full-length cDNA clones of ACLSV.

In the thesis, the full-length cDNA clones of ACLSV and ASPV were constructed using different methods. The methods contained circular polymerase extension cloning (CPEC), Gibson assembly and In-Fusion cloning. In total 17 full-length cDNA clones of ACLSV and ASPV were obtained. Four of the 17 clones were infectious on Nicotiana occidentalis 37B, i.e. pIF3-15, pIF3-19, pIF14-23 and pIF4-4. The viral genomic cDNAs in these infectious clones were completely sequenced, and the sequence data were analyzed by alignment with published sequences in NCBI. The results indicated that three isolates of ACLSV were rescued: ACLSV isolate 38/85A (pIF3-15), 38/85B (pIF3-19) and (36)/88 (pIF14-23). One ASPV isolate was rescued: ASPV 40/87.

A protocol of agroinoculation of apple seedlings by vacuum infiltration was developed to inoculate the infectious clones (pIF3-15, pIF3-19, pIF14-23 and pIF4-4) to apple seedlings. In the protocol, the treatment of seedlings, preparation of inocula and parameters of vacuum infiltration were evaluated. The highest PCR-positive rate (infection) of 78% (11/14), 100% (11/11), 25% (2/8) and 50% (9/18) were observed for the infectious clones of pIF3-15, pIF3-19, pIF14-23 and pIF4-4, respectively. The infection of virus was determined by RT-PCR. The existence of viral particles in PCR-positive plants was determined by immunosorbent electron microscopy.

To explore and develop plant viral vectors based on ACLSV, marker genes of Emerald GFP, mCherry or iLov were inserted into the genomic cDNA of ACLSV. Nine plasmids with marker genes were constructed using three different strategies, including pIF13-9, pIF18-2, pIF25-7, pG11-15, pIF24-6, pIF23-1, pIF16-1, pIF20-16 and pIF27-10. After agroinoculation of N. occidentalis 37B with the constructed plasmids, it was found that deletion of marker genes in pIF13-9 and pG11-15 occurred due to homologous recombination between duplicated fragments of ACLSV genomic cDNA. Typical ACLSV symptoms were observed on the test-plants inoculated with pIF13-9 and pG11-15. By western blot, viral proteins of CP of identical size with wild type virus (pIF3-19) were detected for the two constructs in symptomatic plants. In one trial in winter, the pIF25-7 caused systemic infection in one plant. The other plasmids of pIF18-2, pIF24-6, pIF23-1, pIF16-1, pIF20-16 and pIF27-10 did not cause local or systemic infection in any test-plant.

Item Type: Dissertation
Supervisor: Jelkmann, Prof. Dr. Wilhelm
Date of thesis defense: 18 September 2017
Date Deposited: 21 Sep 2017 10:13
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
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