Life Sciences Commons^™

A Unique Role For The Host Escrt Proteins In Replication Of Tomato Bushy Stunt Virus, Daniel Barajas, Yi Jiang, Peter D. Nagy

Plant Pathology Faculty Publications

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I …

Viral Vectors Useful In Soybean And Methods Of Use, Said A. Ghabrial, Chunquan Zhang, Honcgang Gu

Plant Pathology Faculty Patents

The invention provides Bean pod mottle virus (BPMV) vectors useful for expression of heterologous proteins in plants such as soybean. The BPMV vectors are also useful for virus-induced gene silencing. The invention also provides methods for expressing a heterologous polypeptide in a plant such as soybean. The invention additionally provides methods for virus-induced gene silencing, particularly in a soybean plant, which can be used to determine the function of a gene of interest.

Defective Interfering Rnas: Foes Of Viruses And Friends Of Virologists, Kunj Bihari Pathak, Peter D. Nagy

Plant Pathology Faculty Publications

Defective interfering (DI) RNAs are subviral RNAs produced during multiplication of RNA viruses by the error-prone viral replicase. DI-RNAs are parasitic RNAs that are derived from and associated with the parent virus, taking advantage of viral-coded protein factors for their multiplication. Recent advances in the field of DI RNA biology has led to a greater understanding about generation and evolution of DI-RNAs as well as the mechanism of symptom attenuation. Moreover, DI-RNAs are versatile tools in the hands of virologists and are used as less complex surrogate templates to understand the biology of their helper viruses. The ease of their …

Inhibition Of Rna Recruitment And Replication Of An Rna Virus By Acridine Derivatives With Known Anti-Prion Activities, Zsuzsanna Sasvari, Stéphane Bach, Marc Blondel, Peter D. Nagy

Plant Pathology Faculty Publications

BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.

METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect …

The Genome Of Nectria Haematococca: Contribution Of Supernumerary Chromosomes To Gene Expansion, Jeffrey J. Coleman, Steve D. Rounsley, Marianela Rodriguez-Carres, Alan Kuo, Catherine C. Wasmann, Jane Grimwood, Jeremy Schmutz, Masatoki Taga, Gerard J. White, Shiguo Zhou, David C. Schwartz, Michael Freitag, Li-Jun Ma, Etienne G. J. Danchin, Bernard Henrissat, Pedro M. Coutinho, David R. Nelson, Dave Straney, Carolyn A. Napoli, Bridget M. Barker, Michael Gribskov, Martijn Rep, Scott Kroken, István Molnár, Christopher Rensing, John C. Kennell, Jorge Zamora, Mark L. Farman, Eric U. Selker, Asaf Salamov, Harris Shapiro, Jasmyn Pangilinan, Erika Lindquist, Casey Lamers, Igor V. Grigoriev, David M. Geiser, Sarah F. Covert, Esteban Temporini, Hans D. Vanetten

Plant Pathology Faculty Publications

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 …

Evolution Of A Subtilisin-Like Protease Gene Family In The Grass Endophytic Fungus Epichloë Festucae, Michelle K. Bryant, Christopher L. Schardl, Uljana Hesse, Barry Scott

Plant Pathology Faculty Publications

BACKGROUND: Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors.

RESULTS: Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus Epichloë festucae. Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis …

Enhanced Disease Susceptibility 1 And Salicylic Acid Act Redundantly To Regulate Resistance Gene-Mediated Signaling, Srivathsa C. Venugopal, Rae-Dong Jeong, Mihir Kumar Mandal, Shifeng Zhu, A. C. Chandra-Shekara, Ye Xia, Matthew Hersh, Arnold J. Stromberg, Duroy Navarre, Aardra Kachroo, Pradeep Kachroo

Plant Pathology Faculty Publications

Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or …

Phylogenetic Analyses Reveal Monophyletic Origin Of The Ergot Alkaloid Gene Dmaw In Fungi, Miao Liu, Daniel G. Panaccione, Christopher L. Schardl

Plant Pathology Faculty Publications

Ergot alkaloids are indole-derived mycotoxins that are important in agriculture and medicine. Ergot alkaloids are produced by a few representatives of two distantly related fungal lineages, the Clavicipitaceae and the Trichocomaceae. Comparison of the ergot alkaloid gene clusters from these two lineages revealed differences in the relative positions and orientations of several genes. The question arose: is ergot alkaloid biosynthetic capability from a common origin? We used a molecular phylogenetic approach to gain insights into the evolution of ergot alkaloid biosynthesis. The 4-γ,γ-dimethylallyltryptophan synthase gene, dmaW, encodes the first step in the pathway. Amino acid sequences deduced …

A Discontinuous Rna Platform Mediates Rna Virus Replication: Building An Integrated Model For Rna-Based Regulation Of Viral Processes, Baodong Wu, Judit Pogany, Hong Na, Beth L. Nicholson, Peter D. Nagy, K. Andrew White

Plant Pathology Faculty Publications

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo …

Role Of P33 In Tombusvirus Replication, Jozsef Stork

University of Kentucky Doctoral Dissertations

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and …

Identification And Characterization Of Host Factors Involved In Tombusvirus Replication, Yi Jiang

University of Kentucky Doctoral Dissertations

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI …

Towards Elimination And Genetic Manipulation Of Ergot Alkaloid Production In Fungal Endophytes, Simona Florea

University of Kentucky Doctoral Dissertations

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three …