Andrew O. Jackson

Plant-Microbe Interactions: Life and Death at the Interface.

Interactions between microorganisms and plants have undoubtedly had major effects on the development of civilization since humans began to rely extensively on cultivated crops for food. Indeed, ancient chronicles of famines, plagues, and epidemics show that some of the more serious plant diseases, such as rusts, smuts, and mildews, were recognized soon af? ter the emergence of organized agriculture. Theophrastus (~371 to 287 bc) described disease symptoms on a number of plants used for food and the Romans paid tribute to appease the rust god Robigo. More recently, plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. The effects of such epidemics have been particularly devastating in situations such as the Irish potato famine of the 1840s, in which communities depended on a single crop as their primary food source. The potential for se? rious crop disease epidemics still persists today, as evidenced by recent outbreaks of Victoria blight of oats and southern corn leaf blight. These diseases result from agricultural practices that rely on monoculture crops?planting closely related crop species over wide geographical areas provides, in effect, a large Petri dish for the evolution of increasingly virulent patho? gen forms. In addition to causing food shortages, microbial interactions with plants can directly affect the health of humans and livestock. One notable example is ergot poisoning, caused by toxins in the fruiting bodies of the fungus Claviceps purpurea, which can contaminate rye flour. These toxins cause a frightening syndrome typified by hallucinations, burning sensations, miscarriages, gangrene, and, in severe cases, death. The affliction, known as St. Anthonys fire or Holy fire, was most prevalent in the Middle Ages; present day outbreaks are only prevented by the strict cultural and sanitary standards that are now applied in the regulation of grain sales. Further problems resulting from the presence of allergens, carcinogenic com? pounds, and various mycotoxins in moldy grain, peanuts, and animal feed have recently been shown to affect human health. For readers interested in more fundamental information, a broad coverage of plant pathology is provided in the text by

Mutational analysis of barley stripe mosaic virus RNA β.

Barley stripe mosaic virus (BSMV), the type member of the hordeivirus group, has a plus-stranded genome comprising RNA species designated alpha, beta, and gamma. Although RNA beta is essential for infection of whole plants, it is dispensable for infection of barley protoplasts. We have used a full-length cDNA clone of RNA beta from which infectious in vitro transcripts can be derived to construct a number of mutations in its four genes. Mutations introduced into the beta b, beta c, or beta d genes eliminated infectivity of the RNA. The coat protein and the RNA sequences encoding the coat protein were completely dispensable for infection of barley plants by BSMV, and no detrimental effect on systemic movement of the virus was observed. However, besides eliminating coat protein expression in vivo, mutations within the coat protein gene and the first intercistronic region affected a number of other phenotypes: (1) expression of a downstream gene (beta b), (2) stability of the genomic RNA during virus multiplication in planta, (3) the requirement for a trans-acting BSMV protein (gamma b), (4) symptomatology and disease development in infected barley plants, and (5) host range.

Varied Movement Strategies Employed by Triple Gene Block-Encoding Viruses

Several RNA virus genera belonging to the Virgaviridae and Flexiviridae families encode proteins organized in a triple gene block (TGB) that facilitate cell-to-cell and long-distance movement. The TGB proteins have been traditionally classified as hordei-like or potex-like based on phylogenetic comparisons and differences in movement mechanisms of the Hordeivirus and Potexvirus spp. However, accumulating data from other model viruses suggests that a revised framework is needed to accommodate the profound differences in protein interactions occurring during infection and ancillary capsid protein requirements for movement. The goal of this article is to highlight common features of the TGB proteins and salient differences in movement properties exhibited by individual viruses encoding these proteins. We discuss common and divergent aspects of the TGB transport machinery, describe putative nucleoprotein movement complexes, highlight recent data on TGB protein interactions and topological properties, and review membrane associations occurring during subcellular targeting and cell-to-cell movement. We conclude that the existing models cannot be used to explain all TGB viruses, and we propose provisional Potexvirus, Hordeivirus, and Pomovirus models. We also suggest areas that might profit from future research on viruses harboring this intriguing arrangement of movement proteins.

A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

Hordeivirus Replication, Movement, and Pathogenesis

The last Hordeivirus review appearing in this series 20 years ago focused on the comparative biology, relationships, and genome organization of members of the genus ( 68 ). Prior to the 1989 review, useful findings about the origin, disease occurrence, host ranges, and general biological properties of Barley stripe mosaic virus (BSMV) were summarized in three comprehensive reviews ( 26, 67, 107 ). Several recent reviews emphasizing contemporary molecular genetic findings also may be of interest to various readers ( 15, 37, 42, 69, 70, 88, 113 ). In the current review, we briefly reiterate the biological properties of the four members of the Hordeivirus genus and describe advances in our understanding of organization and expression of the viral genomes. We also discuss the infection processes and pathogenesis of the most extensively characterized Hordeiviruses and frame these advances in the broader context of viruses in other families that have encoded triple gene block proteins. In addition, an overview of recent advances in the use of BSMV for virus-induced gene silencing is presented.

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts

Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27 degrees but were barely detectable at 32 degrees. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.

Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA-γ

Abstract The complete nucleotide sequences of RNAγ from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5′-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3′-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNAγ ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNAγ RF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNAγ ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3′-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984 , Virology 139, 375–383). In the genomic RNAγ, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.

Biology, Structure, and Replication of Plant Rhabdoviruses

Of all the taxonomic groups of viruses recognized, only the families Rhabdoviridae and Reoviridae include members that can infect either vertebrates or plants (Matthews, 1982). Furthermore, members of both these groups are transmitted by insects, in which they also multiply. The rhabdoviruses have complex bacilliform or bullet-shaped virions composed of RNA, protein, carbohydrate, and lipid. All these viruses have striking structural similarities and, for this reason, have been classified as a single family by the International Committee on Taxonomy of Viruses (Matthews, 1982). The importance of the rhabdoviruses as disease agents and their potential danger to human, livestock, and wildlife health has been repeatedly documented (Brown and Crick, 1979). However, it is less generally recognized that many serious diseases caused by rhabdoviruses also plague plants, causing substantial crop losses. Thus, the family as a whole presents such a serious threat to the welfare of man, both directly and indirectly, that it is surprising that relatively little is known about their comparative biology.