Molecular and biological characterization of a putative new sobemovirus infecting Physalis peruviana.

Physalis peruviana is a perennial solanaceous plant that has recently been established as a commercial crop in Brazil. This work reports the near-complete genome sequence, particle morphology, and plant host responses to a putative new sobemovirus, named "physalis rugose mosaic virus". The virus, characterized by isometric particles of ca. 30 nm in diameter, causes foliar symptoms of mosaic, malformation and blistering, accompanied by stunting. The near-complete genome sequence comprises 4175 nucleotides and contains five open reading frames that are similar to those of other sobemoviruses. In addition to P. peruviana, the new virus systemically infected Capsicum annuum, Nicotiana tabacum and Solanum lycopersicum by mechanical inoculation. Thus, this virus may cause disease in these crops in the field.

Characterization of the genome of a novel ilarvirus naturally infecting Cape gooseberry (Physalis peruviana).

As part of an initiative to characterize viruses infecting Cape gooseberry in the province of Antioquia (Colombia), we report the genome sequence of a new member of the genus Ilarvirus (family Bromoviridae). This virus was identified in a Cape gooseberry plot in the municipality of Marinilla in a mixed infection with potato virus Y (PVY) as part of high-throughput sequencing initiative. Results were confirmed by nested RT-PCR and DAS-ELISA. Phylogenetic analysis suggested that the Cape gooseberry ilarvirus is a new member of subgroup 1 and it is most closely related to ageratum latent virus (AgLV). The name "Cape gooseberry ilarvirus 1" (CGIV-1) is proposed for this new ilarvirus.

Potato virus Y (PVY) Isolates from Physalis peruviana are Unable to Systemically Infect Potato or Pepper and Form a Distinct New Lineage Within the PVYC Strain Group.

Poha, or cape gooseberry (Physalis peruviana L.), is a plant species cultivated in Hawaii for fresh fruit production. In 2015, an outbreak of virus symptoms occurred on poha farms in the South Kohala District of the island of Hawaii. The plants displayed mosaic, stunting, and leaf deformation, and produced poor fruit. Initial testing found the problem associated with Potato virus Y (PVY) infection. Six individual PVY isolates, named Poha1 to Poha6, were collected from field-grown poha plants and subjected to biological and molecular characterization. All six isolates induced mosaic and vein clearing in tobacco, and three of them exhibited O-serotype while the other three reacted only with polyclonal antibodies and had no identifiable serotype. Until now, PVY isolates have been broadly divided into pepper or potato adapted; however, these six PVY isolates from poha were unable to establish systemic infection in pepper and in four tested potato cultivars. Whole-genome sequences for the six isolates were determined, and no evidence of recombination was found in any of them. Phylogenetic analysis placed poha PVY isolates in a distinct, monophyletic "Poha" clade within the PVYC lineage, suggesting that they represented a novel, biologically and evolutionarily unique group. The genetic diversity within this poha PVYC clade was unusually high, suggesting a long association of PVYC with this solanaceous host or a prolonged geographical separation of PVYC in poha in Hawaii.

Complete genome sequence of an isolate of Potato virus X (PVX) infecting Cape gooseberry (Physalis peruviana) in Colombia.

Transcriptome analysis of a Cape gooseberry (Physalis peruviana) plant with leaf symptoms of a mild yellow mosaic typical of a viral disease revealed an infection with Potato virus X (PVX). The genome sequence of the PVX-Physalis isolate comprises 6435 nt and exhibits higher sequence similarity to members of the Eurasian group of PVX (~95 %) than to the American group (~77 %). Genome organization is similar to other PVX isolates with five open reading frames coding for proteins RdRp, TGBp1, TGBp2, TGBp3, and CP. 5' and 3' untranslated regions revealed all regulatory motifs typically found in PVX isolates. The PVX-Physalis genome is the only complete sequence available for a Potexvirus in Colombia and is a new addition to the restricted number of available sequences of PVX isolates infecting plant species different to potato.

Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.

Differential pathogenicity of two different recombinant PVY(NTN) isolates in Physalis floridana is likely determined by the coat protein gene.

A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVY(NTN)) in China and sequenced the complete genome of the variant PVY(NTN)-HN2. In this study, the complete genome of isolate PVY(NTN)-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVY(NTN)-HN1, like other typical European-PVY(NTN) isolates such as PVY(NTN)-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVY(NTN)-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVY(O), PVY(N), PVY(N:O), PVY(NTN_-HN1 and PVY(NTN)-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVY(N) and PVY(NTN)-HN1 induced mild symptoms (mainly mild mottling) whereas PVY(O), PVY(N:O) and PVY(NTN)-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana.

Stability and competitiveness of interviral recombinant RNAs derived from a chimeric cucumovirus.

We previously described interviral recombinant RNAs derived from a chimeric virus having RNAs 1 and 2 of cucumber mosaic virus (CMV) with RNA 3 from the related tomato aspermy virus (TAV) and the 2b gene from either TAV or another strain of CMV. Here, we show that these interviral recombinant RNAs 3 were stable in the infected plants and could co-exist with their wild-type parental viral RNAs in the same plants, but their de novo generations were inhibited in the presence of the wild-type parental viral RNAs. The recombinant viral genomes did not prevent the replication of other viral RNAs or vice versa, but one of the interviral recombinant viruses induced different symptoms in Physalis floridana from those induced by the parental chimeric virus without the interviral RNA 3 recombinant. Factors such as the nature of the 2b gene and/or the presence or absence of competing wild-type parental RNAs influenced the generation of the recombinant RNAs described. Our data provide additional mechanistic insight into generation, stabilization and competition of recombinant viral RNA in infected host plants.

Co-infection by two criniviruses alters accumulation of each virus in a host-specific manner and influences efficiency of virus transmission.

Tomato chlorosis virus (ToCV), and Tomato infectious chlorosis virus (TICV), family Closteroviridae, genus Crinivirus, cause interveinal chlorosis, leaf brittleness, and limited necrotic flecking or bronzing on tomato leaves. Both viruses cause a decline in plant vigor and reduce fruit yield, and are emerging as serious production problems for field and greenhouse tomato growers in many parts of the world. The viruses have been found together in tomato, indicating that infection by one Crinivirus sp. does not prevent infection by a second. Transmission efficiency and virus persistence in the vector varies significantly among the four different whitefly vectors of ToCV; Bemisia tabaci biotypes A and B, Trialeurodes abutilonea, and T. vaporariorum. Only T. vaporariorum can transmit TICV. In order to elucidate the effects of co-infection on Crinivirus sp. accumulation and transmission efficiency, we established Physalis wrightii and Nicotiana benthamiana source plants, containing either TICV or ToCV alone or both viruses together. Vectors were allowed to feed separately on all virus sources, as well as virus-free plants, then were transferred to young plants of both host species. Plants were tested by quantitative reverse-transcription polymerase chain reaction, and results indicated host-specific differences in accumulation by TICV and ToCV and alteration of accumulation patterns during co-infection compared with single infection. In N. benthamiana, TICV titers increased during co-infection compared with levels in single infection, while ToCV titers decreased. However, in P. wrightii, titers of both TICV and ToCV decreased during mixed infection compared with single infection, although to different degrees. Vector transmission efficiency of both viruses corresponded with virus concentration in the host in both single and mixed infections. This illustrates that Crinivirus epidemiology is impacted not only by vector transmission specificity and incidence of hosts but also by interactions between viruses and efficiency of accumulation in host plants.

The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains.

The Potato virus Y (PVY) cDNA full-length clone created by Jakab et al. [Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J. Gen. Virol. 78, 3141-3145] was stabilized by inserting three introns into putatively toxic genes. Using this clone, hybrid viruses were constructed by in vitro recombination. The PVY-N/NTN and PVY-N/O chimeras carried the 3' end of NIb, the whole CP and 3'UTR region of PVY(NTN) and PVY(O), respectively, in a PVY(N) genetic background. The clones proved to be stable after several passages by re-sequencing the exchanged region. Both hybrid viruses showed reduced infectivity in particle bombardment experiments, but they were suitable for further mechanical plant inoculation. In five of the six host plant species, inoculated with the two chimeras and three parental strains, the chimeras produced similar symptoms to those of PVY(N). By contrast, Physalis floridana reacted with different pattern of symptoms. In this species, the symptoms caused by the N/O hybrid were similar to those of the 3'NIb-CP-donating PVY(O) strain, and not to those of the background (PVY(N)). The results suggest that symptom determinants may be different even between strains of the same virus species in a particular host.

The role of inter-subunit ionic interactions in the assembly of Physalis mottle tymovirus.

Physalis mottle tymovirus (PhMV) is a small spherical plant virus with its RNA genome encapsidated in a protein shell made of 180 identical coat protein (CP) subunits. The amino acid residues involved in two interfacial salt bridges, Asp-83/Arg-159 and Arg-68/Asp-150 and Lys-153, were targeted for mutagenesis with a view to delineate the role of interfacial ionic interactions in the subunit folding and assembly of the virus. R159A and D83A-R159A recombinant CP (rCP) mutants formed stable T = 3 capsids, indicating that the D83-R159 interfacial salt bridge is dispensable for the folding and assembly of PhMV. However, D150A and R68Q-D150A mutant rCPs were present in the insoluble fraction, suggesting that the R68-D150 interfacial salt bridge is crucial for subunit folding and assembly. Similarly, K153Q, D83A-K153Q, and H69A-K153Q mutant rCPs were present in the insoluble fraction. Interestingly, the R68Q-D150A, D83A-K153Q, and H69A-K153Q double mutant rCPs could be refolded into partially folded soluble heterogeneous aggregates of 14-16 S. The results further confirm our earlier observation that subunit folding and assembly are concerted events in PhMV.

Influence of the Potato leafroll virus and virus-infected plants on the arrestment of the aphid, Myzus persicae.

A series of experiments was conducted using membrane sachets containing MP148 diet or phosphate-buffered sucrose with and without purified Potato leafroll virus to determine if direct encounter with the virus would arrest the aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae). In only two out of 36 tests were there significantly more aphids settled on sachets containing the virus. In all other tests, there were either significantly fewer aphids on sachets containing virus or there were no differences between virus treatments and control sachets without virus. In an experiment using excised Physalis floridana leaves, twice as many M. persicae settled on virus-infected leaves as on noninfected control leaves. Taken together, the results indicate that arrestment of M. persicae on potato leaf roll virus-infected plants may be due to enhanced nutritional qualities resulting from disease, but not from direct encounter with or detection of the virus.

Mutation of interfacial residues disrupts subunit folding and particle assembly of Physalis mottle tymovirus.

Virus-like particles (VLPs) serve as excellent model systems to identify the pathways of virus assembly. To gain insights into the assembly mechanisms of the Physalis mottle tymovirus (PhMV), six interfacial residues, identified based on the crystal structure of the native and recombinant capsids, were targeted for mutagenesis. The Q37E, Y67A, R68Q, D83A, I123A, and S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble. However, except for the S145A mutant, which assembled into VLPs similar to that of wild type rCP capsids, all the other mutants failed to assemble into VLPs. Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies. The rCP mutants locked into such conformations probably lack the structural signals/features that would allow them to assemble into capsids. Thus, the mutation of residues involved in inter-subunit interactions in PhMV disrupts both subunit folding and particle assembly.

Host-dependent requirement for the Potato leafroll virus 17-kda protein in virus movement.

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.