Phloem connectivity and transport are not involved in mature plant resistance (MPR) to Potato Virus Y in different potato cultivars, and MPR does not protect tubers from recombinant strains of the virus.

The aims of this study were: i) to investigate mature plant resistance (MPR) against four strains of Potato virus Y (PVYO, PVYN, PVYNTN and PVYN-Wi) in potato cultivars that differ in maturity (e.g. early or maincrop) at different developmental stages, and ii) to determine whether phloem translocation of photoassimilates at different stages including the source-sink transition influences MPR. The data showed that MPR was functional by the flowering stage in all cultivars, and that the host-pathogen interaction is highly complex, with all three variables (potato cultivar, virus strain and developmental stage of infection) having a significant effect on the outcome. However, virus strain was the most important factor, and MPR was less effective in protecting tubers from recombinant virus strains (PVYNTN and PVYN-Wi). Development of MPR was unrelated to foliar phloem connectivity, which was observed at all developmental stages, but a switch from symplastic to apoplastic phloem unloading early in tuber development may be involved in the prevention of tuber infections with PVYO. Recombinant virus strains were more infectious than parental strains and PVYNTN has a more effective silencing suppressor than PVYO, another factor that may contribute to the efficiency of MPR. The resistance conferred by MPR against PVYO or PVYN may be associated with or enhanced by the presence of the corresponding strain-specific HR resistance gene in the cultivar.

Natural resistance to Potato virus Y in Solanum tuberosum Group Phureja.

KEY MESSAGE: Novel major gene resistance against Potato virus Y in diploid populations of Solanum tuberosum Groups Phureja and Tuberosum was biologically and genetically characterised. Named Ry(o)phu, it mapped to chromosome 9. A new source of genetic resistance derived from Solanum tuberosum Group Phureja against Potato virus Y (PVY) was identified and genetically characterised in three diploid biparental potato populations. Segregation data for two populations (05H1 and 08H1) suggested the presence of a single dominant gene for resistance to PVY which, following DaRT analysis of the 08H1 cross, was mapped to chromosome 9. More detailed genetic analysis of resistance utilised a well-characterised SNP-linkage map for the 06H1 population, together with newly generated marker data. In these plants, which have both S. tuberosum Group Phureja and S. tuberosum Group Tuberosum in their pedigree, the resistance was shown to map to chromosome 9 at a locus not previously associated with PVY resistance, although there is evidence for at least one other genetic factor controlling PVY infection. The resistance factor location on chromosome 9 (named as Ry(o)phu) suggests a potential role of NB-LRR genes in this resistance. Phenotypic analysis using a GUS-tagged virus revealed that a small amount of PVY replication occurred in occasional groups of epidermal cells in inoculated leaves of resistant plants, without inducing any visible hypersensitive response. However, the virus did not enter the vascular system and systemic spread was completely prevented.

Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.

The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids.

The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1-2 µm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

Unusual features of pomoviral RNA movement.

Potato mop-top pomovirus (PMTV) is one of a few viruses that can move systemically in plants in the absence of the capsid protein (CP). Pomoviruses encode the triple gene block genetic module of movement proteins (TGB 1, 2, and 3) and recent research suggests that PMTV RNA is transported either as ribonucleoprotein (RNP) complexes containing TGB1 or encapsidated in virions containing TGB1. Furthermore, there are different requirements for local or systemic (long-distance) movement. Research suggests that nucleolar passage of TGB1 may be important for the long-distance movement of both RNP and virions. Moreover, and uniquely, the long-distance movement of the CP-encoding RNA requires expression of both major and minor CP subunits and is inhibited when only the major CP sub unit is expressed. This paper reviews pomovirus research and presents a current model for RNA movement.

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement.

The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.

Unusual long-distance movement strategies of Potato mop-top virus RNAs in Nicotiana benthamiana.

The Potato mop-top virus (PMTV) genome encodes replicase, movement, and capsid proteins on three different RNA species that are encapsidated within tubular rod-shaped particles. Previously, we showed that the protein produced on translational readthrough (RT) of the coat protein (CP) gene, CP-RT, is associated with one extremity of the virus particles, and that the two RNAs encoding replicase and movement proteins can move long distance in the absence of the third RNA (RNA-CP) that encodes the capsid proteins, CP and CP-RT. Here, we examined the roles of the CP and CP-RT proteins on RNA movement using infectious clones carrying mutations in the CP and CP-RT coding domains. The results showed that, in infections established with mutant RNA-CP expressing CP together with truncated CP-RT, systemic movement of the mutant RNA-CP was inhibited but not the movement of the other two RNAs. Furthermore, RNA-CP long-distance movement was inhibited in a mutant clone expressing only CP in the absence of the CP-RT polypeptide. CP-RT was not necessary for particle assembly because virions were observed in leaf extracts infected with the CP-RT deletion mutants. RNA-CP moved long distance when protein expression was suppressed completely or when CP expression was suppressed so that only CP-RT or truncated CP-RT was expressed. CP-RT but not CP interacted with the movement protein TGB1 in the yeast two-hybrid system. CP-RT and TGB1 were detected by enzyme-linked immunosorbent assay in virus particles and the long-distance movement of RNA-CP was correlated with expression of CP-RT that interacted with TGB1; mutant RNA-CP expressing truncated CP-RT proteins that did not interact with TGB1 formed virions but did not move to upper noninoculated leaves. The results indicate that PMTV RNA-CP can move long distance in two distinct forms: either as a viral ribonucleoprotein complex or as particles that are most likely associated with CP-RT and TGB1.

Two plant-viral movement proteins traffic in the endocytic recycling pathway.

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.

A naturally occurring deleted form of RNA 2 of Potato mop-top virus.

A spontaneous deletion in RNA 2 of Potato mop-top virus (PMTV) was identified by RT-PCR. The deletion occurred reproducibly during manual passage of two isolates of PMTV and during fungal inoculation of plants with viruliferous soil. The borders of the deletion were conserved in all instances and sequence analyses showed that a contiguous segment of 2113 nucleotides was deleted internally from the genomic RNA 2, leaving the 5'- and 3'-terminal sequences. RT-PCR experiments also showed that the deletion was present in preparations of PMTV particles.