Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.     

Overexpression of Rice Black-Streaked Dwarf Virus P7-1 in Arabidopsis Results in Male Sterility Due to Non-Dehiscent Anthers

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, is propagatively transmitted by the small brown planthopper (Laodelphax striatellus Fallén). RBSDV causes rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses in crops in China. Although several RBSDV proteins have been studied in detail, the functions of the nonstructural protein P7-1 are still largely unknown. To investigate the role of the P7-1 protein in virus pathogenicity, transgenic Arabidopsis thaliana plants were generated in which the P7-1 gene was expressed under the control of the 35S promoter. The RBSDV P7-1-transgenic Arabidopsis plants (named P7-1-OE) were male sterility. Flowers and pollen from P7-1-transgenic plants were of normal size and shape, and anthers developed to the normal size but failed to dehisce. The non-dehiscent anthers observed in P7-1-OE were attributed to decreased lignin content in the anthers. Furthermore, the reactive oxygen species levels were quite low in the transgenic plants compared with the wild type. These results indicate that ectopic expression of the RBSDV P7-1 protein in A. thaliana causes male sterility, possibly through the disruption of the lignin biosynthesis and H₂O₂-dependent polymerization pathways.     

Dissemination and detection of peanut clump virus in groundnut seed

Experiments were done to assess the role of seed-transmission in the dissemination of peanut clump virus (PCV) in groundnut (Arachis hypogea L.), and the usefulness of enzyme-linked immunosorbent assay (ELISA) for detecting the virus in infected groundnut seed. The virus was present in 7.5% of seedling progeny from infected plants and could be detected in 16.5% of the seeds by ELISA. When groundnut seeds were grown in a field contaminated by the virus, it was shown that by roguing the infected plants, only 0.1% of the seeds from the remaining plants contained the virus. It was also established that the level of contamination of seeds by the virus was inversely proportional to the seed size.     

The TGB1 movement protein of Potato virus X reorganizes actin and endomembranes into the X-body, a viral replication factory

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX "X-body," a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus "factory." TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection.     

Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms

The 23 kDa protein (p23) coded by the 3'-terminal gene of Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, is an RNA-binding protein that contains a motif rich in cysteine and histidine residues in the core of a putative zinc-finger domain. On this basis, a regulatory role for CTV replication or gene expression has been suggested for p23. To explore whether over-expression of this protein in transgenic plants could affect the normal CTV infection process, transgenic Mexican lime plants were generated carrying the p23 transgene, or a truncated version thereof, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constitutive expression of p23 induced phenotypic aberrations that resembled symptoms incited by CTV in non-transgenic lime plants, whereas transgenic plants expressing the p23 truncated version were normal. The onset of CTV-like symptoms in p23 -transgenic plants was associated with the expression of p23, and its accumulation level paralleled the intensity of the symptoms. This demonstrates that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis. This is the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host. This finding also delimits a small region of the large CTV genome for the future mapping of specific pathogenic determinants.     

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.     

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene     

Further properties of peanut clump virus and studies on its natural transmission

Purified preparations of particles of peanut clump virus (PCV) had A₂₆₀/A₂₈₀ values (corrected for light scattering) of 1.00. They contained rod-shaped particles with sedimentation coefficients of 183 S and 224 S, and a density in CsCl of 1.32 g/ml. PCV infected 36 species in 8 plant families. No serological relationship was detected between PCV and barley stripe mosaic, beet necrotic yellow vein, Nicotiana velutina mosaic and tobacco mosaic viruses. PCV was seed-borne for two generations in groundnut (Arachis hypogaea) but was not seed-borne in great millet (Sorghum arundinaceum), Phaseolus mungo or Nicotiana benthamiana. Seedlings of groundnut, great millet and wheat (Triticum aestivum) became infected when grown in soil from groundnut fields with outbreaks of clump disease, and the infectivity of soil survived air-drying at 25°C for 3 months. Groundnut seedlings became infected when grown in sterilised soil contaminated with washed roots of naturally-infected S. arundinaceum but not in soil to which roots of naturally infected groundnut or shoots of infected groundnut were added, or in which mechanically inoculated groundnut seedlings were grown at the same time. The patchy distribution of PCV in a crop was related to the infectivity of the soil for groundnut and to the presence of Polymyxa graminis resting spores which could be detected in the roots of S. arundinaceum bait seedlings, but not in those of groundnut. The results indicate that PCV is transmitted by a vector that is resistant to air-drying and closely associated with S. arundinaceum roots. For these reasons P. graminis is thought to be the vector of PCV.     

Studies on transmission of Indian peanut clump virus disease by Polymyxa graminis*

The plasmodiophoromycete fungus, Polymyxa graminis was observed in the roots of Sorghum bicolor, S. sudanense, Pennisetum glaucum, Triticum aestivum, Cyperus rotundus, Eleucine coracana, Zea mays, Tridax procumbens and Arachis hypogaea collected from Indian peanut clump virus (IPCV)-infested fields. Examination of roots of IPCV-infected S. bicolor, S. sudanense, P. glaucum and T. aestivum grown in previously air dried field soil also showed the presence of cystosori of P. graminis. IPCV-infested soil stored at room temperature for 3 years transmitted the virus to A. hypogaea, T. aestivum and S. bicolor. Roots extracted from IPCV-infected P. glaucum and S. bicolor containing cystosori, and dried root fragments incorporated into sterile soil, transmitted the virus to A. hypogaea and T. aestivum. The root extracts contained primary zoospores of the fungus, presumably arising from cystosori. Utilising root fragments of S. sudanense containing cystosori as inoculum P. graminis was shown to infect both monocotyledonous and dicotyledonous plants. Profuse cystosorus production in rootlets only occurred in monocotyledonous plants. In dicotyledonous plants, in general, only few rootlets showed cystosori. Indian isolates of P. graminis appear to differ from isolates from temperate soils in that they can infect dicotyledonous plants and have a much wider host range.     

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars ‘SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP–SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP–SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.     

The occurrence of Indian peanut clump,a soil-borne virus disease of groundnuts (Arachis hypogaea) in India*

A disease characterised by severely stunted plants with small dark green leaves was found in groundnut (Arachis hypogaea) in sandy soils in Punjab State, India. The disease occurred in patches in the field and reappeared in the same positions in succeeding groundnut crops. Plants infected early did not produce mature pods. Seeds sown in soil collected from infected fields produced plants with typical disease symptoms. Phaseolus vulgaris cv. Local and Chenopodium quinoa were found to be good diagnostic hosts. The disease was shown to be caused by a rod-shaped virus c. 24 nm in diameter with predominant particle lengths of c. 249 and 184 nm when stained in uranyl acetate. The virus, named Indian peanut clump virus (IPCV), resembled peanut clump virus (PCV) reported from W. Africa in symptomatology on groundnuts, particle morphology and soil-borne nature. However, it is not serologically related to two W. African PCV isolates tested, or to tobacco rattle (PRN and CAM strains) or pea early browning virus (Dutch isolate) in microprecipitin, enzyme linked immunosorbent assay and immunosorbent electron microscopy tests.