David W. Thornbury

Site-directed mutations in the potyvirus HC-PRO gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants

Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions (283/284) and (368/369), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and (368/369) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the (283/284) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.

Purification and characterization of potyvirus helper component

Helper component (HC) was purified from tobacco vein mottling (TVMV)- and potato virus Y (PVY)- infected tobacco plants by sucrose gradient fractionation followed by affinity chromatography on oligo(dT)-cellulose and by gel electrophoresis. The subunit apparent molecular weights (M(r)) of the purified HCs were 53,000 (53K) and 58K for TVMV and PVY, respectively. Antisera to these purified polypeptides specifically blocked the activity of the homologous HC, as determined by aphid transmission assays, and specifically precipitated 75K products of the cell-free translation of the homologous RNA. The molecular weight of undissociated, biologically active TVMV or PVY HC, as determined by high-pressure liquid chromatography (HPLC)-gel permeation chromatography was found to be between 100K and 150K, suggesting that the active molecule is a dimer.

Cell-free translation of tobacco vein mottling virus RNA. II. Immunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion, and helper component proteins.

The genomic RNA of tobacco vein mottling virus (TVMV) was translated in a cell-free system derived from rabbit reticulocytes. Antisera against TVMV coat protein, TVMV cylindrical inclusions, the helper component required for aphid transmission of TVMV, and the 49- and 54-kd nuclear inclusion proteins of tobacco etch virus (TEV) were used to characterize the translational products. Each of the five antisera precipitated a distinctive pattern of polypeptides. Specificity of immunoprecipitation was shown by competing with the various proteins to which antisera were raised and by sequentially precipitating with two different antisera. These experiments showed that the five antisera define five different nonoverlapping regions of the TVMV genome.

Helper components of two potyviruses are serologically distinct.

The specificity of antisera to helper component (HC) from tobacco vein mottling virus (TVMV)- or potato virus Y (PVY)-infected tobacco plants was tested in immunoprecipitation and immunoabsorption chromatography experiments. Treatment with the homologous antiserum abolished or drastically reduced the activity of either TVMV-HC or PVY-HC, as measured by their ability to effect aphid transmission of purified tobacco etch virus, while the heterologous antiserum had little or no effect on HC activity. Loss of TVMV-HC and PVY-HC activity in the immunoabsorption chromatography experiments was associated with the removal of a 53- and a 58-kDa polypeptide, respectively. The results indicate that serologically distinct HC proteins are produced in response to specific potyvirus infection and suggest that HC is virus coded.

Immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus Y

The serological relationships of the products of in vitro translation of the RNA of various potyviruses were analyzed by using antisera to helper component (HC) from tobacco plants infected with either tobacco vein mottling virus (TVMV) or potato virus Y (PVY). The PVY-HC antiserum immunoprecipitated a specific PVY-RNA translation product; this product was not reactive with antisera to PVY-induced cylindrical inclusion protein or capsid protein or to the two tobacco etch virus nuclear inclusion proteins. The antiserum to PVY-HC did not immunoprecipitate significant amounts of any translation products of 16 other potyviruses including TVMV. In contrast the antiserum to TVMV-HC efficiently immunoprecipitated a specific product(s) of four different potyviruses, some isolates of which are poorly transmitted or nontransmissible by aphids, and less efficiently a product(s) from 12 other potyviruses, including PVY. Distinct serotypes were resolved among the major in vitro translation products of 17 different potyviral RNAs by the antisera to TVMV-HC and PVY-HC. There appears not to be a correlation between the serological reactivities of HC-related polypeptides and the ability of different HC-virus combinations to effect aphid transmission of the virus.

Telomere-targeted retrotransposons in the rice blast fungus Magnaporthe oryzae: agents of telomere instability.

The fungus Magnaporthe oryzae is a serious pathogen of rice and other grasses. Telomeric restriction fragments in Magnaporthe isolates that infect perennial ryegrass (prg) are hotspots for genomic rearrangement and undergo frequent, spontaneous alterations during fungal culture. The telomeres of rice-infecting isolates are very stable by comparison. Sequencing of chromosome ends from a number of prg-infecting isolates revealed two related non-LTR retrotransposons (M. oryzae Telomeric Retrotransposons or MoTeRs) inserted in the telomere repeats. This contrasts with rice pathogen telomeres that are uninterrupted by other sequences. Genetic evidence indicates that the MoTeR elements are responsible for the observed instability. MoTeRs represent a new family of telomere-targeted transposons whose members are found exclusively in fungi.

Nucleotide sequence of a strain of tobacco etch virus that does not cause Tabasco pepper wilt

Infection of Tabasco pepper (Capsicum annuum L., cv. Tabasco) by tobacco etch virus (TEV) typically causes wilting due to root necrosis (1). A recently isolated strain of TEV, designated TEV non-wilting (NW), causes mosaic symptoms but not wilting in Tabasco pepper. In order to find the mutation(s) responsible for the inability of TEV NW to cause wilting, the complete nucleotide sequence was determined. TEV NW was single-lesion passaged through Chenopodium amaranticolor for three generations. Virus within a lesion from the final passage was tested on Tabasco pepper for infectivity and lack of the wilting response, and was inoculated onto KY 14 tobacco plants for propagation. Viral RNA was prepared by a modified SDS-phenol extraction of purified virus (2). TEV NW cDNAs were synthesized by reverse transcription-polymerase chain reaction (RT-PRC) of the viral RNA. Vent ® DNA polymerase (New England Biolabs) was used for PCR amplification of the first strand cDNA. Oligonucleotide primers for RT-PCR were designed according to the TEV HAT sequence (3). The

Subtelomere Organization, Evolution, and Dynamics in the Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae is a filamentous, ascomycete fungus best known as the causal agent of a devastating disease of rice known as blast. In addition to being a pathogen of rice, it also causes diseases on other important crops, including wheat, millets, and forage grasses, and on turf grasses such as perennial ryegrass and St. Augustinegrass. Despite the species’ broad host range, fungal isolates from one host genus usually are unable to infect other host genera. Such specificity can also be manifested at the subspecies level, such that an isolate from one rice cultivar is often unable to infect other cultivars.