Characterization of grapevine vein clearing virus expression strategy and development of caulimovirus infectious clones

Abstract

Grapevine Vein Clearing Virus (GVCV) is a newly discovered DNA virus in grapevine that is closely associated with grapevine vein clearing syndrome observed in vineyards in Missouri and surrounding states. However, Koch's postulates have never been completed. Four chapters discussing four projects related to GVCV are included in this dissertation. This is a step further toward efficient management of the grapevine vein clearing syndrome in the future. Chapter 2 focuses on GVCV promoter characterization and GVCV mRNA transcript mapping. Portions of the GVCV large intergenic region were cloned and assessed for promoter activity, and the segment between nucleotides 7,332 and 7,672 was sufficient to drive expression of downstream ORF. 5' RACE and 3' RACE revealed that transcription was initiated predominantly at nucleotide 7,571 and terminated at nucleotide 7,676. Additional transient expression analysis studies were supportive of a ribosomal shunt model for expression of ORF1 of GVCV Chapter 3 is about Cauliflower mosaic virus (CaMV) and its P6 protein. P6 and P6-GFP were examined for the ability to complement a defect CaMV isolate that contains a lethal mutation in its P6 coding region. P6-GFP was able to perform all the functions of P6 and support coat protein expression and virion assembly. The co-agroinfiltration assay of Nicotiana benthamiana developed in this chapter was used to evaluate the infectivity of an infectious clone of GVCV. Chapter 4 describes the construction of a terminally redundant clone of GVCV. The GVCV genome was assembled via three overlapping DNA fragments amplified from GVCV-infected tissues and the terminally redundant clone, designated pGVCV-1, was inserted into an Agrobacterium binary vector for delivery into plant cells. The co-agroinfiltration assay described in chapter 3 was applied, and pGVCV-1 was shown to be capable of replication and encapsidation. Furthermore, a systemic veinal chlorosis symptom was observed in several of the N. benthamiana plants agroinoculated with pGVCV-1, indicating that the virus clone was infectious. Two types of virus-like particles, long flexuous rods and bacilliform particles, were purified from either pGVCV-1 infiltrated N. benthamiana leaves or GVCV-infected grape leaves showing typical vein clearing symptoms, and further research is planned to characterize the nature of the flexuous rods in these plants. Two species of mealybugs were tested for their ability to acquire and transmit Grapevine vein clearing virus in chapter 5. The longtailed mealybugs were collected from a cycad plant in Tucker greenhouse (University of Missouri), whereas a mixed population of longtailed and citrus mealybugs were collected from infested grapevines in the Ashland-Gravel greenhouses (University of Missouri). Both populations were able to acquire GVCV after short feeding periods of as little as three days. However, the tucker population was not able to persist on grapevines and was not tested for transmission of GVCV. The Ashland-Gravel population was tested for transmission but none of the plants developed symptoms indicative of GVCV, and PCR assays showed GVCV was not transmitted to any of the 31 grapevine plants. I conclude that the citrus and longtailed mealybugs are unlikely to be the vectors for GVCV.