Detection of a natural point mutation in Potato virus A that overcomes resistance to vascular movement in Nicandra physaloides., and studies on seed-transmissibility of the mutant virus

Isolate M of Potato virus A (PVA‐M; genus Potyvirus) is avirulent in Nicandra physaloides L. (family Solanaceae). The inoculated leaves are infected but no systemic infection is observed. Forty plants of ‘Black Pod’ (BP) and ‘Black Pod Alba’ (BPA), two variants of N. physaloides described in this study, were inoculated with PVA‐M. Two plants of BP and one plant of BPA were systemically infected. Mosaic, blistering and dark green islands developed on the systemically infected leaves, and flowers showed colour‐break symptoms. PVAprogeny were sequence‐characterised for the 6K2 protein and viral genome‐linked protein (VPg) encoding regions known to control the long distance movement of PVA in N. physaloides. All virus progeny (designated as PVA‐Mm) in the systemically infected leaves of the plants inoculated with PVA‐M contained only a single amino acid substitution (Vail 16Met) in the central part of VPg due to a nucleotide substitution G6033A, as compared to PVA‐M. Other PVA isolates that infected N. physaloides systemically also contained Metll6 in VPg. In a previous study using chimeric viruses, Metl16 in VPg was shown to be a major determinant for vascular movement of PVA in N. physaloides, and this study reveals that the mutation for Metl16 can occur in vivo during replication of the avirulent PVA‐M in infected plants. Immunolocalisation studies on BP and BPA plants showed that the pods (berries) and seed coat contained PVA‐Mm in the developing seeds, but no virus was detected in embryons. Up to 27% of the mature seeds contained PVA‐Mm but no transmission to seedlings was observed in a total of 450 seeds tested, and no test plants were infected following mechanical inoculation with extracts prepared from the seeds.     

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

Large quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double-antibody sandwich or plate-trapped antigen forms of enzyme-linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')₂ ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10^--⁴ but not 10^--⁵; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S_20,w, a single coat protein molecule of c . mol. wt 21 000, and a single-stranded RNA genome of c . mol. wt 2.0 ± 10⁶. Several dsRNA species, including the putative viral replicative form of c . mol. wt 4.1 ± 10⁶, were isolated from virus-infected cassava and N. benthamiana .     

Transient expression of HPV16 E7 peptide (aa 44-60) and HPV16 L2 peptide (aa 108-120) on chimeric potyvirus-like particles using Potato virus X-based vector

The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.     

Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.     

Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection.

A chimeric tomato golden mosaic virus (TGMV) A component DNA, which results from replacement of the coding region of the viral coat protein gene (CP) with the larger bacterial beta-glucuronidase coding sequence (GUS), can replicate in agroinoculated leaf discs but is unstable in systemically infected plants (1). We have made similar replacements of the TGMV CP gene with the GUS coding sequence in both the sense and antisense orientations. Both derivatives replicated in leaf discs inoculated via Agrobacterium. However, systemic movement of the GUS substituted vectors was not detected in agroinoculated Nicotiana benthamiana plants. The only TGMV A derivatives detected in systemically infected leaves of inoculated plants were similar in size to the wild type viral component. Sequence analysis of derivatives from six independently inoculated plants revealed that they did not result from internal deletions of the larger replicons detected in leaf discs but, instead, were generated by fusion events occuring within the original T-DNA insert. These results indicate that systemic movement of TGMV in N. benthamiana plants provides a strong selective pressure favoring viral derivatives similar in size to the wild type virus components.     

An alfalfa mosaic virus RNA 2 mutant,which does not induce a hypersensitive reaction in cowpea plants,is multiplied to a high concentration in cowpea protoplasts

A mutant of alfalfa mosaic virus (AMV), which in contrast to wild type (wt) can invade cowpea plants systemically, is replicated more efficiently in cowpea protoplasts than the wt. Mutant preparations isolated from infected cowpea protoplasts contained a higher amount of middle component (M, containing RNA 2) than wt preparations. Both in cowpea plants and in cowpea protoplasts a wt phenotype is obtained upon addition of wt M to this mutant, suggesting a correlation between the type of plant reaction evoked by the virus infection and the regulation of viral RNA synthesis.     

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.     

A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana.

Brome mosaic virus (BMV) is a positive-strand RNA virus with a multipartite genome that causes symptomless infection in Nicotiana benthamiana. We have isolated and characterized a strain of BMV that produced uniform vein chlorosis in systemically infected N. benthamiana. Analysis of pseudorecombinants constructed by exchanging RNA 1 and 2 and RNA 3 components between wild-type (non-symptom-inducing) and vein chlorosis-inducing strains of BMV indicated that the genetic determinant for the induction of the chlorotic phenotype is located on RNA 3. Sequence analysis of progeny RNA 3 recovered from symptomatic N. benthamiana plants revealed that vein chlorosis is due to the single nucleotide transition 887G-->887A, which changes the codon for Val-266 to Ile-266 in the movement protein gene. The mutation had no detectable effect on the accumulation of virus in either inoculated or systematically infected leaves of N. benthamiana. The vein chlorosis phenotype is the manifestation of the substitution of Ile-266 for Val-266 in the movement protein gene, since additional alterations in this region (a silent mutation, i.e., 887GUU889-->GUC, and an alteration of valine to phenylalanine, i.e., 887GUU889-->887UUU889) resulted in symptomless infections on N. benthamiana. The modulation of the symptom phenotype by the substitution of Ile-266 for Val-266 is specific for N. benthamiana, since neither movement nor the symptom phenotype in barley plants was affected.     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.     

The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation

The interaction between viral capsid protein (CP) and its cognate viral RNA modulates many steps in the virus infection cycle, such as replication, translation and assembly. The N-terminal 50 amino acids of the Red clover necrotic mosaic virus (RCNMV) CP are rich in basic residues (especially lysine) and are essential for the core functions of the CP, namely RNA binding and virion assembly. To further elucidate additional biological roles for these basic residues, a series of alanine substitution mutations was introduced into infectious clones of RCNMV RNA-1 and assayed for symptomatology, virion formation and systemic infection. Infectivity assays conducted in Nicotiana benthamiana revealed that all nine alanine substitution mutants (ASMs) were competent for systemic infection. Two ASMs (K4A and K7A/K8A) induced severe symptoms and delayed the systemic spread of viral genomes when compared with wild-type RCNMV. However, these ASMs were still competent for virion formation. Three other ASMs (K25A, K33A and K38A) displayed milder symptoms and significant reductions in virion accumulation when compared with wild-type RCNMV, but retained the ability to spread systemically. Evidence from these last three ASMs, as well as a CP null mutant, showed that RCNMV is able to move systemically in N. benthamiana as a nonvirion form. These observations reaffirm the necessity of the N-terminal lysine-rich residues of the RCNMV CP for efficient virion accumulation. They also reveal additional roles for the CP in the modulation of host symptomatology, independent of its role in virion assembly and the rate of systemic viral movement in N. benthamiana.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Replication and movement of a coat protein mutant of cymbidium ringspot tombusvirus.

The spread of cymbidium ringspot tombusvirus (CyRSV) in host tissue was studied by using a coat protein gene mutant with a six-nucleotide deletion; the deletion removes two amino acids from the shell domain (S) of the capsid protein. Mutated protein subunits were synthesized in infected cells but could not assemble into virus particles. Virions were formed, however, with inoculation of mutated RNA in transgenic plants expressing normal CyRSV coat protein. The mutant is restricted in long-distance movement in Nicotiana clevelandii, whereas it spreads systemically in N. benthamiana. These results suggest that tombusviruses may spread either as complete virions or in a nonvirion form, depending on the host plant species.     

Induction of plant cell division by beet curly top virus gene C4

Beet curly top virus (BCTV) is a small DNA virus that causes tumorigenic growths (enations) in infected plants by inducing division of phloem parenchyma cells (hyperplasia). It has previously been shown that BCTV C4 plays an important role in symptom development in sugarbeet and Nicotiana benthamiana , and it has been suggested that this gene is responsible for the induction of hyperplasia. Using in situ hybridization, we show that BCTV infection is closely associated with the vascular system in these hosts, although hyperplastic cells associated with wild-type virus infection frequently do not contain detectable levels of viral DNA. Extensive hyperplasia was not observed in plants infected with a C4 mutant, demonstrating a role for C4 in virus-induced cell proliferation. Ectopic expression of C4 in transgenic N. benthamiana resulted in abnormal plant development and the production of tumorigenic growths, confirming that this gene alone is sufficient to initiate cell division in permissive cells when removed from the context of the viral genome.