Characterization of the promoter of Grapevine vein clearing virus.

Grapevine vein clearing virus (GVCV) is a recently discovered DNA virus in grapevine that is closely associated with the grapevine vein clearing syndrome observed in vineyards in Missouri and surrounding states. The genome sequence of GVCV indicates that it belongs to the genus Badnavirus in the family Caulimoviridae. To identify the GVCV promoter, we cloned portions of the GVCV large intergenic region in front of a GFP gene present in an Agrobacterium tumefaciens binary vector. GFP expression was assessed by ELISA 3 days after agroinfiltration of Nicotiana benthamiana leaves. We found that the GVCV DNA segment between nts 7332 and 7672 directed expression of GFP and this expression was stronger than expression using the Cauliflower mosaic virus 35S promoter. It was revealed by 5' and 3' RACE that transcription was initiated predominantly at nt 7571 and terminated at nt 7676.

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms.

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

Uncoupling resistance from cell death in the hypersensitive response of Nicotiana species to cauliflower mosaic virus infection.

Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the resistance of N. edwardsonii to W260 infection was conditioned by a gene derived from N. glutinosa, whereas cell death was conditioned by a gene derived from N. clevelandii. An analysis of pathogenesis-related (PR) protein expression in response to W260 infection revealed that elicitation of PR proteins was associated with resistance rather than with the onset of cell death.

Agroinfiltration of Cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species.

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.

Light-dependent systemic infection of solanaceous species by cauliflower mosaic virus can Be conditioned by a viral gene encoding an aphid transmission factor.

Gene II of cauliflower mosaic virus (CaMV), which encodes an 18-kDa protein originally identified as an aphid transmission factor (ATF), influences host specificity in a light-dependent manner. A point mutation within the ATF gene that occurs in several CaMV strains was responsible for conditioning light-dependent systemic infections. A point mutant of CaMV strain W260 that carried the single mutation within the ATF gene was able to systemically infect Nicotiana bigelovii at low light intensity (100-180 micromol m-2 sec-1), but not at a higher light intensity level (350-450 micromol m-2 sec-1), while the wild-type W260 virus could systemically infect N. bigelovii under both light conditions. The same point mutation also affected the stability of the amorphous CaMV inclusions and previous studies have shown that it abolishes transmission of CaMV by aphids. The point mutation within the ATF gene that mediated light-dependent infections was complemented by transgenic N. bigelovii plants that express the CaMV gene VI product, a viral protein that has been identified as a translational transactivator. The complementation studies indicated that the ATF gene may influence systemic infections through an interaction with the CaMV gene VI product. The ATF gene of CaMV may contribute to viral infections by regulating expression of downstream genes or by influencing cell-to-cell or long distance movement within the plant.

Unique Immunogenic Proteins in Heterodera glycines Eggshells.

Polyclonal antibodies were raised against Heterodera glycines eggshells to determine the feasibility of developing an immunoassay for H. glycines eggs. An indirect enzyme-linked immunosorbent assay (ELISA) was developed from anfisera collected 10 weeks after the initial injection. From serial dilutions of sonicated eggshells or whole eggs, a sensitivity of detection to 5 ng/ml sonicated eggshells or 1 egg of H. glycines was determined. The method of eggshell preparation had no effect on the antibodies produced; however, the antibodies cross-reacted with sonicated J2 of H. glycines and eggs of Meloidogyne incognita and H. schachtii. Most of the proteins in both life stages of H. glycines and eggs of M. incognita and H. schachtii had similar migration properties when separated on SDS-PAGE gels and stained with Coomassie blue. Western blot analysis, with antisera adsorbed with homogenized J2 of H. glycines, showed proteins that were specifically localized to eggshells of H. glycines. Monoclonal antibodies might provide a useful immunoassay where polyclonal antibodies lack sufficient specificity.

Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure.

We demonstrate that recombinant viruses formed between a wild-type virus and a viral transgene can be isolated from transgenic plants under conditions of moderate to weak selection pressure. We inoculated cauliflower mosaic virus (CaMV) strain W260 to transgenic Nicotiana bigelovii plants that expressed a copy of CaMV gene VI derived from CaMV strain D4, a gene that determines systemic infection of solanaceous species, including N. bigelovii. Because W260 infects nontransformed N. bigelovii systemically, a recombinant virus formed between W260 and the D4 transgene would be expected to have little selective advantage over the wild-type W260 virus W260 was inoculated to approximately 100 plants each of nontransformed and transgenic N. bigelovii and it systemically infected nearly all of the plants. An analysis of viral DNA recovered from 23 transgenic plants infected with W260 revealed that 20 infections resulted from the systemic movement of the wild-type W260 virus, while a recombinant between W260 and the D4 transgene was detected in three of the infections. To determine the percentage of recovery of recombinant viruses under strong selection pressure, we inoculated approximately 100 nontransformed and 100 D4 gene VI transgenic plants with CaMV strain CM1841, a virus that is unable to infect nontransformed N. bigelovii. CM1841 infected 36% of the transgenic plants systemically, but none of the nontransformed controls. An analysis of 24 infected plants showed that a recombination event occurred in every plant, demonstrating that under strong selection conditions, the recovery of CaMV recombinants from transgenic plants can be very high.

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene VI coding sequence was placed under the control of the galactose-inducible promoter GAL1, which is presented in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL1 promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.

Expansion of Viral Host Range through Complementation and Recombination in Transgenic Plants.

We have shown previously that gene VI of cauliflower mosaic virus (CaMV) strain D4 governs systemic infection of Nicotiana bigelovii and that transgenic N. bigelovii expressing the D4 gene VI product can complement at least one CaMV isolate for long-distance transport. We have now found that DNA of two other isolates of CaMV recombine with the gene VI coding sequence present in the transgenic plants. The formation of recombinant viruses occurs as a consequence of CaMV replication, involving two template switches during reverse transcription of the CaMV RNA to DNA. The first template switch occurs at the 5[prime] end of the 35S RNA to the gene VI mRNA produced by the transgenic plants. A second switch occurs at the 5[prime] end of the gene VI mRNA back to the 35S RNA. We also demonstrate that CaMV can acquire sequences from transgenic plants that alter the symptomatology and host range of the virus, an observation that may have important risk assessment implications for strategies using pathogen-derived resistance to protect plants against virus diseases.

Identification of domains within gene VI of cauliflower mosaic virus that influence systemic infection of Nicotiana bigelovii in a light-dependent manner.

Gene VI of cauliflower mosaic virus strains D4 and W260 is an important determinant of systemic infection in solanaceous species (Qiu and Schoelz, 1992). To investigate whether D4 and W260 share any sequences within gene VI that determine their solanaceous host range, we characterized more completely the regions of gene VI involved in systemic infection of Nicotiana bigelovii. We found that two domains within gene VI, which corresponded approximately to the 5' third and middle third of gene VI, influenced systemic infection of N. bigelovii. Exchange of these domains between D4, W260, and CaMV strain CM1841, a strain which is unable to systemically infect any solanaceous plant, revealed different virus combinations that could specify systemic infection of N. bigelovii when plants were grown under two different lighting conditions. Systemic infection of N. bigelovii by D4/CM1841 chimeric viruses required only the 5' third (domain 1) of gene VI of D4. In contrast, systemic infection of N. bigelovii by W260/CM1841 chimeric viruses required both the domain 1 and middle third of gene VI (domain 2) of W260, as well as two other regions primarily containing W260 genes I and II, and gene IV. The genetic requirements for systemic infection by chimeric viruses were not as stringent when plants were grown under low light conditions. Specifically, domain 2 of gene VI of D4 contained sequences sufficient for D4/CM1841 chimeric viruses to systemically infect N. bigelovii at low light intensity. We sequenced gene VI of strain W260 and compared differences in the deduced amino acid sequences between W260 and the previously published sequences of D4 and CM1841. There was only one amino acid position in domain 1 of gene VI, and no sites in domain 2, in which W260 and D4 agreed with each other and differed from CM1841. Consequently, the host range studies and sequence information indicate that different sequences within gene VI of CaMV strains W260 and D4 are responsible for systemic infection of N. bigelovii.

Three regions of cauliflower mosaic virus strain W260 are involved in systemic infection of solanaceous hosts.

We have identified regions of CaMV strain W260 involved in systemic infection of Nicotiana bigelovii and Datura stramonium by constructing chimeric viruses between W260 and CM1841, a strain that is unable to systemically infect any solanaceous host. All of the chimeric viruses systemically infected turnips, demonstrating the viability of the chimeric viruses in a host that is susceptible to both CM1841 and W260. Three regions of W260, containing primarily genes I, IV, and VI, influenced the ability of that virus to induce systemic symptoms in the solanaceous hosts. The involvement of the regions containing gene I, and to a lesser extent gene IV, were affected by environmental conditions. When infected plants were grown under conditions of low light, low temperatures (18 degrees), and short days (9.5-hr day), the source of genes I and IV no longer influenced whether a chimeric virus moved systemically. As light intensity and day length were increased, the genetic requirements became more stringent and genes I and IV, as well as gene VI, had to be derived from W260.

Genetic analysis of determinants of disease severity and virus concentration in cauliflower mosaic virus.

Cauliflower mosaic virus (CaMV) strains CM1841 and W260 produced markedly different symptoms when inoculated onto turnips (Brassica campestris L. 'Just Right'). The CM1841 strain induced a mild degree of stunting of infected plants while strain W260 caused moderate to severe stunting. Although CM1841 was significantly milder than W260, it accumulated to a significantly higher concentration than W260 in systemically infected leaves. We constructed a series of hybrid viruses in order to map regions of W260 responsible for enhanced disease severity relative to CM1841 and to map regions of CM1841 responsible for higher virus accumulation. We found that the characteristic degree of stunting caused by a CaMV isolate is determined in a complex manner by viral genes that influence viral gene expression and viral genes that disrupt host metabolism. Genes I and VI influenced both virus concentration and stunting severity, suggesting that these regions affected disease severity primarily through their effect on gene expression. In addition, an interaction between genes IV and VI was observed which further indicated that stunting severity was influenced by differential accumulation of virus. In contrast, three regions of W260 influenced the stunting phenotype but had no effect, or a negative effect, on virus concentration. The three regions contained (1) portions of genes II and III, (2) gene IV, independent of gene VI, and (3) the 3' half of gene V and the 19 S promoter. These regions may influence stunting severity primarily by disrupting host metabolism. Additionally, some of the chimeric viruses induced systemic necrosis on leaves, a symptom that is not characteristic of either CM1841 or W260. The necrotic flecking symptom was caused by an interaction between a W260 DNA segment containing gene I and the 5' half of gene II and a CM1841 DNA segment containing the 3' half of gene II, gene III, and gene IV.

Tobacco mosaic virus RNA enters chloroplasts in vivo.

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35 degrees C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.

Tobacco mosaic virus particles contain ubiquitinated coat protein subunits.

Virions of tobacco mosaic virus (TMV) are composed of a single strand of RNA, encapsidated in about 2130 copies of a coat protein of MW 17,500. Asselin and Zaitlin [Virology 91, 173-181 (1978)] demonstrated that virion preparations also contained small amounts of a second protein of MW 26,500, which they termed "H protein." H protein, detectable to an average frequency of one per virion, was thought to be a protein of host origin. Subsequent studies [Collmer, Vogt, and Zaitlin, Virology 126, 429-448 (1983)] showed the H protein was comprised of a backbone of TMV coat protein, linked by a postulated isopeptide bond to a small protein that probably was of host origin. The host-derived moiety of H protein is shown here to be ubiquitin, most probably coupled to the coat protein at lysine 53. This finding is based on microsequencing of the H protein, and is substantiated by immunoblotting analysis with antibodies to human ubiquitin. Conjugated ubiquitin was detected in virions of all five strains of the virus tested. To our knowledge, this is the first report of a ubiquitinated viral structural protein.

Evidence for shorter average O-polysaccharide chainlength in the lipopolysaccharide of a bacteriophage Felix 01-sensitive variant of Salmonella anatum A1.

Prolonged culturing in the laboratory has resulted in the formation of a stable derivative of the smooth Group E bacterial strain, Salmonella anatum A1, that is sensitive to both the R-core-specific bacteriophage Felix 01 and O-polysaccharide-specific bacteriophage epsilon 15. The variant strain, designated S. anatum A1-1, exhibits a normal number of irreversible binding sites for epsilon 15 but the relative quality and/or accessibility of those sites appears to be diminished. Infectious epsilon 15 phage particles are released more rapidly from S. anatum A1-1 than from its parent under acidic pH conditions known to interfere with the phage DNA ejection step. The purified lipopolysaccharide (LPS) of S. anatum A1-1 exhibits a reduced rhamnose/heptose ratio in chemical assays. Fractionation of this LPS on SDS-urea-polyacrylamide gels followed by silver staining reveals a narrower range of O-polysaccharide chain lengths relative to that of the parent (0 to 20 vs. 0 to 40 repeating units, respectively).