Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant 'potex-like' viruses.

This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3' terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant 'potex-like' viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant 'potex-like' coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant 'potex-like' virus group, distinct from BVF.

Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus.

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.

An improved polyclonal antiserum for detecting Ryegrass mosaic rymovirus.

Ryegrass mosaic virus (RGMV) is considered the most serious and widespread virus infecting temperate pasture grasses. The use of visible symptoms to diagnose infection is unreliable and ELISA analysis requires antibodies with broad cross-reactivity. Here we describe the production of a polyclonal antiserum (PAb-cp3'Delta) using a bacterially expressed RGMV coat protein fragment. The PAb-cp3'Delta antiserum is specific for RGMV and recognises RGMV strains from each major phylogenetic cluster. PAb-cp3'Delta may be used in ELISAs for fast, accurate and inexpensive detection of RGMV.

Genetic Characterization of Differential Reactions Among Host Group 3 Common Bean Cultivars to NL-3 K Strain of Bean common mosaic necrosis virus.

ABSTRACT A previously unrecognized recessive resistance gene (or allele) was identified in three host group (HG) 3 common bean (Phaseolus vulgaris) cvs. Olathe, Victor, and UI 37, based on genetic analysis of plants from five populations screened with the NL-3 K strain of Bean common mosaic necrosis virus (BCMNV). The gene (or allele) was associated with resistance to leaf stunting and deformity and reduction in plant height. The gene (or allele) provides similar, but slightly better resistance than the bc-1(2) gene that is characteristic of HG 3 cultivars. Traditional HG 3 cultivars like Redlands Greenleaf B with bc-1(2) are susceptible to NL-3 K, whereas this newly identified gene (or allele) conditions resistance to NL-3 K. Other slight variations in disease reaction pattern to a wide array of bean common mosaic (BCM)-inducing strains were noted among HG 3 differentials, indicating that additional resistance to BCM exists in common bean that remains to be exploited. To gauge the full breeding value of this newly identified gene (or allele), allelism tests with existing genes, namely bc-1(2), and further characterization of responses to all Bean common mosaic virus (BCMV) and BCMNV strains need to be conducted. Meanwhile, breeders should consider introgressing this more effective gene (or allele) into susceptible cultivars while plant pathologists continue to decipher the genetic variability present among HG 3 differential cultivars.

A Quantitative Method to Screen Common Bean Plants for Resistance to Bean common mosaic necrosis virus.

ABSTRACT A quantitative method to screen common bean (Phaseolus vulgaris) plants for resistance to Bean common mosaic necrosis virus (BCMNV) is described. Four parameters were assessed in developing the quantitative method: symptoms associated with systemic virus movement, plant vigor, virus titer, and plant dry weight. Based on these parameters, two rating systems (V and VV rating) were established. Plants from 21 recombinant inbred lines (RILs) from a Sierra (susceptible) x Olathe (partially resistant) cross inoculated with the BCMNV-NL-3 K strain were used to evaluate this quantitative approach. In all, 11 RILs exhibited very susceptible reactions and 10 RILs expressed partially resistant reactions, thus fitting a 1:1 susceptible/partially resistant ratio (chi(2) = 0.048, P = 0.827) and suggesting that the response is mediated by a single gene. Using the classical qualitative approach based only on symptom expression, the RILs were difficult to separate into phenotypic groups because of a continuum of responses. By plotting mean percent reduction in either V (based on visual symptoms) or VV (based on visual symptoms and vigor) rating versus enzyme-linked immunosorbent assay (ELISA) absorbance values, RILs could be separated clearly into different phenotypic groups. The utility of this quantitative approach also was evaluated on plants from 12 cultivars or pure lines inoculated with one of three strains of BCMNV. Using the mean VV rating and ELISA absorbance values, significant differences were established not only in cultivar and pure line comparisons but also in virus strain comparisons. This quantitative system should be particularly useful for the evaluation of the independent action of bc genes, the discovery of new genes associated with partial resistance, and assessing virulence of virus strains.

Trans-complementation of long-distance movement of White clover mosaic virus triple gene block (TGB) mutants: phloem-associated movement of TGBp1.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Both cell-to-cell and long-distance movement of White clover mosaic virus in which individual, combinations, or all movement functions were mutated could be rescued by transgenic Nicotiana benthamiana expressing complementary viral products. To address the importance of TGB functions in vascular transport, we used an experimental system based on grafted plants and trans-complementation, to define co-translocated viral products and the minimal requirements for viral exit from the plant vasculature. Evidence is presented that TGBp1 is co-translocated with viral RNA and CP and that, once viral RNA is loaded into the phloem translocation stream, it can exit in sink tissues and replicate in the absence of TGBp2-3. These results are discussed in the context of the recent finding that TGBp1 can mediate the suppression of signaling involved in systemic gene silencing.

Dark green islands in plant virus infection are the result of posttranscriptional gene silencing.

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.

Seed Transmission of the High Plains virus in Sweet Corn.

The High Plains virus (HPV), which infects corn and other cereals, was first found in 1993 in the United States. Research was initiated in 1995 to investigate the potential for seed transmission of HPV. Sweet corn seeds of various cultivars harvested in 1994 to 1996 from 13 fields and research plots in southwestern Idaho, Colorado, and Nebraska were seeded in potting mix in the greenhouse. Leaf samples collected at the three- to six-leaf stage from both symptomatic and asymptomatic plants were tested by enzyme-linked immunosorbent assay (ELISA). Of the 46,600 seeds planted, 38,473 seedlings emerged, and three tested positive by ELISA, exhibited mosaic symptoms, and had the presence of HPV confirmed by an additional test. One of the positive plants was used for successful acquisition and transmission of HPV by the wheat curl mite to Westford barley. The other two plants were used to successfully transfer HPV to other corn plants by vascular puncture inoculation of seed. These results indicate that HPV can be seed transmitted at a very low frequency in sweet corn.

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.

Distribution and diversity of New Zealand isolates of ryegrass mosaic virus.

Sequences from the coat protein cistron of five ryegrass mosaic virus (RgMV) isolates indicated the presence of two distinct strains in New Zealand. The nucleotide differences between the strains, and their distribution, suggested that both strains were introduced recently, either as a mixed infection or as two independent introductions. The relationship between these New Zealand strains and other strains and isolates of RgMV, and their potential severity is discussed.

Phormium Yellow Leaf Phytoplasma Is Associated with Strawberry Lethal Yellows Disease in New Zealand.

A yellows disease of strawberry plants was identified in propagation beds in New Zealand. Affected plants were flatter to the ground, showed purpling of older leaves, reduced leaf size, yellowing of younger leaves, and sometimes plant death. A phytoplasma was observed in the phloem of affected plants. The 16S rRNA gene of the phytoplasma was amplified by polymerase chain reaction from symptomatic plants and from one asymptomatic plant, but not from 36 other asymptomatic plants. Nucleotide sequence analysis of the 16S rRNA gene showed that the phytoplasma is closely related or identical to the phytoplasma associated with the yellow leaf disease of New Zealand flax (Phormium tenax).

Biological, serological, and molecular differences among isolates of potato a potyvirus.

ABSTRACT Sequences of the coat protein (CP) and 3'-end nontranslated region (3'NTR) of 13 isolates and the helper component proteinase (HC) of nine isolates of potato A potyvirus (PVA) were determined and compared with the eight previously determined PVA CP and 3'NTR sequences and one HC sequence. CP amino acid (aa), 3'NTR nucleotide, and HC aa sequence identities were 92.9, 93.4, and 94.8%, respectively. Sequence data, serological tests, and the necrotic local lesions induced in the leaves of the potato hybrid 'A6' confirmed that tamarillo mosaic virus is a strain of PVA. The aa substitutions A6T and G7S in the CP N-terminus were correlated with loss of aphid transmissibility. Development of necrotic lesions or nonnecrotic symptoms in the systemically infected leaves or lack of systemic spread in potato cv. King Edward were used to place the PVA isolates into four strain groups, but this grouping was not correlated with any differences in CP, HC, or 3'NTR. Recognition of CP by three monoclonal antibodies was used to place the PVA isolates into three groups different from the four groups above. The epitopes of two mono-clonal antibodies were mapped by site-directed mutagenesis to the same lysine residue at the CP aa 34.

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene.

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v f are also placed on the map. The map around v f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.

Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.

White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.

Characterization of NTPase, RNA-binding and RNA-helicase activities of the cytoplasmic inclusion protein of tamarillo mosaic potyvirus.

The 66-kDa cytoplasmic inclusion protein of tamarillo mosaic potyvirus was purified to near homogeneity using organic solvent clarification, differential centrifugation and sucrose density gradient centrifugation. ATPase and GTPase activities were shown to co-purify with the 66-kDa protein. ATPase activity was stimulated up to fivefold in the presence of 20 microM poly(A). The Km value for ATP hydrolysis (18 microM), was minimally affected upon addition of poly(A). In contrast, the Vmax value for ATP hydrolysis was increased fivefold by the addition of poly(A). Binding of RNA by the cytoplasmic inclusion protein was demonstrated by gel electrophoresis of ultraviolet cross-linked enzyme-RNA complexes. In the absence of added NTP, complexes between the cytoplasmic inclusion protein and single-stranded RNA species formed rapidly in the pH range 3-7, but not at pH 8 or 9. Binding to single-stranded RNA was markedly decreased by the addition of NaCl (10 mM), suggesting a weak association between RNA and enzyme. The cytoplasmic inclusion protein bound single-stranded RNA or partially double-stranded RNA duplexes with single-stranded overhangs of 35 bases and 81 bases, respectively, but did not bind 16-bp blunt-ended double-stranded RNA. RNA binding occurred in the absence of NTP (ATP, GTP, CTP or UTP), whereas dissociation of bound RNA occurred only in the presence of NTP. RNA duplex unwinding (helicase) activity of the enzyme was demonstrated in the presence of any of the above four NTPs using partially double-stranded RNA duplexes with 3' single-stranded overhangs. We propose that the cytoplasmic inclusion protein of tamarillo mosaic virus is an RNA helicase, which translocates in the 3' to 5' direction in an energy-dependent manner, unwinding double-stranded regions.

Nucleotide sequence of the coat protein genes of strawberry latent ringspot virus: lack of homology to the nepoviruses and comoviruses.

The sequence of the 3'-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3' untranslated region of 552 nucleotides and a 3'-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M(r) of 43K is located 5' of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and comoviruses than has been considered previously.