Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 96-100).

Graduation date: 2000

Beet yellows virus (BYV) is a filamentous, positive-strand RNA virus that belongs to the family Closteroviridae. BYV particles encapsidate a 15.5 kb RNA and posses complex polar architecture. A long virion body is formed by the major capsid protein(CP), whereas the minor capsid protein (CPm) assembles a short tail that encapsidates the 5'-terminal region of BYV RNA. In addition to proteins required for viral RNA replication and encapsidation, BYV encodes four proteins whose role in the virus life cycle was unknown. These proteins include a small, 6-kDa, hydrophobic protein (p6), a homolog of the cellular 70-kDa heat shock proteins (Hsp7Oh), a 64-kDa protein (p64), and a 20-kDa protein (p20). It was found recently that Hsp7Oh, p64, and p20 are incorporated into BYV virions, and that Hsp7Oh is required for the virus movement from cell to cell. In this study, we characterized genetic requirements for BYV assembly and cell-to-cell movement, and determined relationships between these two processes. It was demonstrated that in addition to Hsp7Oh, p6, p64, CP, and CPm are each essential, but not sufficient for virus movement. These results indicated that five-component movement machinery of BYV is the most complex among plant viruses. Extensive mutational analysis of CP and CPm revealed strong correlation between abilities of BYV to assemble tailed virions and to move from cell to cell, suggesting that formation of functional virions is a prerequisite for virus translocation. We have found that CPm, Hsp7Oh, and p64 are necessary for the efficient virion tail formation. Assembly of the virion tails and bodies was shown to occur independent of each other and likely to involve two separate packaging signals within the genomic RNA. Our work demonstrated that BYV encodes one conventional movement protein, p6, whose only known function is to mediate virus movement. The other four movement associated proteins of BYV, CP, CPm, Hsp7Oh, and p64 are the virion components, each of which is required for assembly of the tailed, movement-competent virions. Based on these and other data, we propose that BYV and other closteroviruses evolved virion tails as a specialized device for the directional cell-to-cell movement of large RNA genomes. / Graduation date: 2005 / Best scan available.

More than half of the recognized genera of positive strand RNA viruses employ polyprotein processing as one of the strategies for their genome expression. Normally, this processing is mediated by virus-encoded proteinases that belong to the trypsin-like or papain-like family. In particular, papain-like, leader proteinases were found in diverse families of human, animal, plant, and fungal positive strand RNA viruses. In addition to autocatalytic processing, these proteinases play a variety of roles in the virus life cycle. In plant potyviruses, a papain-like helper component-proteinase (HC-Pro) was implicated in genome amplification, cell-to-cell movement, long distance transport, and suppression of host defense. The p29 proteinase encoded by a fungal hypovirus CHV1 was found to be dispensable for virus replication, but it was identified as a major determinant of viral pathogenicity. In an animal equine aterivirus (EAV), a papain-like proteinase nspl was demonstrated to possess a putative zinc finger domain, which functions in subgenomic RNA synthesis, although it is not essential for virus replication. The Lab proteinase of the foot and mouse disease virus (FMDV) is involved in inhibition of cellular mRNA translation and in virus spread in infected animals. In general, it appears that functional plasticity of the papain-like leader proteinases played an important role in the evolution of viral diversity. Here, we examined the functions of a papain-like leader proteinase (L-Pro) in the life cycle of the beet yellows closterovirus (BYV). It was found that L-Pro is required for autoproteolytic processing, genome amplification, virus invasiveness and cell-to-cell movement for BYV. The gene swapping experiments involving several closterviruses, a potyvirus, as well as CHV1, FMDV, and EAV revealed complex functional profiles of the papain-like leader proteinases. The possible mechanisms that underlie L-Pro functions are discussed. / Graduation date: 2002

In total, 801 samples of potato leafroll disease were collected and tested for potato leafroll virus (PLRV) and beet western yellows virus (BWYV) in 1986, 1987, and 1988 using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and virus-specific monoclonal antibodies. The samples represented 32 cultivars and originated in eight Canadian provinces and 12 American states. None of the samples tested positive for BWYV, whereas 772 (96.4%) tested positive for PLRV. Neither PLRV nor BWYV could be recovered, with aphid transfers to indicator hosts, from 28 of the 29 samples that tested negative for both viruses. PLRV was recovered from one sample that originally tested negative by TAS-ELISA; the indicator plant tested positive for PLRV by TAS-ELISA. Nucleic acid spot hybridization (NASH) using random primed and cloned cDNA probes was compared with double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and TAS-ELISA, and aphid transmission tests for detection and identification of PLRV and BWYV in 165 potato leafroll disease samples. All of the samples tested negative for BWYV with each of the assay procedures. PLRV was detected in all of the samples with TAS-ELISA, NASH with a cloned cDNA probe for PLRV, and with aphid transmission to ground cherry (Physalis pubescens). Both DAS-ELISA and NASH using random primed cDNA produced one false-negative result. Shepherd's purse (Capsella bursa-pastoris) was a host for 72% (119/165) of the PLRV isolates. The susceptibility of potato to BWYV was tested by inoculating Russet Burbank with three isolates of BWYV from Canada and four from the United States. Two of the isolates were in a mixed infection with PLRV. None of the isolates were transmitted by Myzus persicae to virus-free potato plants, either by themselves or in association with PLRV. Common weeds were surveyed in the potato-producing areas of British Columbia for PLRV and BWYV. In total, 10,098 weed samples, representing 98 species in 22 plant families, were collected and tested by TAS-ELISA from 1986 to 1989. BWYV was detected in 1% of the samples; the hosts were: chickweed, common groundsel, heart-podded hoary cress, hedge mustard, little western bittercress, prickly lettuce, shepherd's purse, and wild radish. PLRV was detected in three volunteer potato plants, two samples of shepherd's purse, and one black nightshade plant. The low incidence of PLRV in plants other than potato indicates that weeds are of minor importance in the epidemiology of potato leafroll disease in British Columbia. / Land and Food Systems, Faculty of / Graduate

A new disease of sugar beet, named Fusarium yellowing decline, was recently found in in the Red River Valley of MN and ND. This disease is caused by a novel pathogen named Fusarium secorum. Pathogens such as F. secorum secrete proteins during infection called ‘effectors’ that help establish disease. Since pathogenicity and disease development may depend on effector proteins produced by F. secorum during infection, effector protein identification furthers our understanding of the biology of this important pathogen. A list of 11 candidate effectors was generated previously. In this study, to characterize putative effectors, we developed a transformation system using polyethylene glycol–mediated transformation. Several mutant lines were created with an effector deleted from the genome using a split-marker knock-out strategy. To explore their role in pathogenicity, mutant strains have been inoculated to sugarbeet and compared to WT F. secorum.