Detection, diagnostics, and characterization of virus-like organisms and conformational disease-like proteins in plants

Abstract

The projects in this thesis all investigated virus-like organisms in agronomically important plants. Chapter one consists of reports describing viruses identified in new hosts or locations. Tobacco rattle virus (TRV) was identified in symptomatic Phryma leptostachya L, a native perennial, from plants in an uncultivated habitat and suggests that TRV may be endemic to North America. Since TRV is the causal organism of corky ringspot of potato this study raises the possibility that native perennial plants could serve as a potential reservoir to cause disease in potato. Canna yellow mottle virus was isolated for the first time from symptomatic Canna indica in Kenya. Cut flowers are a major agronomic crop of Kenya and growers should plant only virus indexed plants to limit losses from virus infection. Orchid fleck virus was confirmed by microscopy and sequence analysis for the first time in the United States in Phalaenopsis hybrida. Asymptomatic P. hybrida tested by one step reverse transcription polymerase chain reaction (RT-PCR) did not yield the expected product while a two step RT-PCR, creating cDNA first, yielded the expected product. This indicates the one step RT-PCR diagnostic test can yield false negatives for asymptomatic plants. Chapter two describes the production of polyclonal antibodies for the detection of Orchid fleck virus (OFV). OFV is a mite transmitted virus and has been reported word wide. The previous project identified a need for a reliable, inexpensive method to detect OFV in plants use for propagation, breeding, conservatories, or virus indexing projects. Polyclonal antibodies were produced in rabbits against Escherichia coli expressed OFV phosphoprotein and matrix protein. The resulting antiserums were assayed in PTA-ELISA and DAS-ELISA. OFV phosphoprotein antisera in PTA-ELISA readily differentiated between healthy and OFV infected orchid (Phalaenopsis hybrida) tissue. OFV matrix antisera in PTA-ELISA detected bacterially-expressed protein but did not differentiate between healthy or OFV infected tissue. The OFV phosphoprotein antiserum can be used by in PTA-ELISA to reliably detect OFV. Chapter three describes the molecular and biological characterization of a new Nepovirus causing a leaf mottling disease in Petunia hybrida. The sequence of the majority of the genome was determined by next generation sequencing and the sequence of remainder of the genome was obtained using a 5’ RACE amplification and RT-PCR using poly-A tail and virus specific primers. Due to phylogenetic relationship and sufficient genome dissimilarity to characterized viruses I propose the name of Petunia Chlorotic Mottle Virus for a new Nepovirus. The fourth chapter describes characterization of filamentous virus-like particles in members of the Asteraceae plant family including sunflower, chrysanthemum, coneflower, gerbera daisy, and zinnia. The filaments were 7-10 nanometers in diameter and could exceed 3,000 nm in length. The N-terminal sequences of the major proteins associated with purified filaments from several species were nearly identical and shared homology with the kunitz soybean trypsin inhibitor (KTI) family of proteins. CID MS/MS sequencing of the major proteins of purified sunflower filaments also shared homology with a KTI sequence. A Western blot using antiserum prepared against recombinant sunflower KTI protein labeled the observable protein bands from sunflower filaments. Filaments composed of a major protein of KTI have been found across the Asteraceae family but have not been observed in dandelion, thistle, or lettuce.