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.

GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean.

RAR1, SGT1, and HSP90 are important components of effector-triggered immunity (ETI) in diverse plants, where RAR1 and SGT1 are thought to serve as HSP90 co-chaperones. We show that ETI in soybean requires RAR1 and SGT1 but not HSP90. Rsv1-mediated extreme resistance to Soybean mosaic virus (SMV) and Rpg-1b-mediated resistance to Pseudomonas syringae were compromised in plants silenced for GmRAR1 and GmSGT1-2 but not GmHSP90. This suggests that RAR1- or SGT1-dependant signaling is not always associated with a dependence on HSP90. Unlike in Arabidopsis, SGT1 in soybean also mediates ETI against the bacterial pathogen P. syringae. Similar to Arabidopsis, soybean RAR1 and SGT1 proteins interact with each other and two related HSP90 proteins. Plants silenced for GmHSP90 genes or GmRAR1 exhibited altered morphology, suggesting that these proteins also contribute to developmental processes. Silencing GmRAR1 and GmSGT1-2 impaired resistance to virulent bacteria and systemic acquired resistance (SAR) in soybean as well. Because the Arabidopsis rar1 mutant also showed a defect in SAR, we conclude that RAR1 and SGT1 serve as a point of convergence for basal resistance, ETI, and SAR. We demonstrate that, although soybean defense signaling pathways recruit structurally conserved components, they have distinct requirements for specific proteins.

Multiple activities associated with the capsid protein of satellite panicum mosaic virus are controlled separately by the N- and C-terminal regions.

The 17-kDa capsid protein (CP) of satellite panicum mosaic virus (SPMV) contains a distinct N-terminal arginine-rich motif (N-ARM) which is required for SPMV virion assembly and the activity of SPMV CP to promote systemic accumulation of its cognate RNA. The present study indicates that SPMV CP also is involved in SPMV RNA accumulation in inoculated leaves and that this activity is also dependent on a functional N-ARM. In addition, deletions of a C-terminal region abolish virion assembly and impair SPMV RNA accumulation in both inoculated and systemic leaves. Unlike the N-ARM mutations, substantial deletions of the SPMV CP C-terminus do not affect SPMV RNA binding activity. Interestingly, SPMV CP also binds Panicum mosaic virus genomic RNA via N-ARM-mediated CP:RNA interactions. Mutations of the N-ARM and the C-terminal regions significantly reduce SPMV CP titers and result in symptom attenuation. In contrast, virions were not associated per se with symptom exacerbation or successful SPMV RNA accumulation. The results show the existence of a correlation between N- and C-termini-mediated contributions for CP accumulation, symptom induction, defective-interfering RNA accumulation, and temperature sensitivity of SPMV RNA maintenance. The data provide further evidence that SPMV CP has multiple roles during infection, which might involve the formation of nonvirion CP:RNA complexes whose stability is controlled in a biologically relevant manner by the N- and C-termini of the CP.

SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double-stranded RNA derived from transgenic or endogenous loci and formed by intra- or inter-molecular interactions of single-stranded RNAs or the action of RNA-dependent RNA polymerases (RDRs). Double-stranded RNA from various sources is processed by one of the four Dicer-like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans-acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper-susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat-induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double-stranded RNA from specific single-stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double-stranded form by RDR6.

Aspects of adaptive answering formation in virus-host plant pathosystem for different wheat cultivars under simulating microgravity condition.

Investigations of prolonged clinorotation effect on some morphological and physiological parameters under Wheat streak mosaic virus WSMW-infection of Apogee and Lada wheat cultivars were carried out. Experiments were held on universal clinostat CYCLE-2. Clinorotation caused changing of WSMV virions shape and reducing of the virus reproduction. Apogee wheat plants grown under two stress factors (infection and clinorotation) produced more kernels than stationary (motionless) plants, but the average weight of kernel was lower. Under clinorotation changes in host plant-virus system take place and adaptive reactions for simulated microgravity conditions form. These lead to reduction of potyvirus replication.

Characterization of an NBS-LRR resistance gene homologue from soybean.

Conserved motifs such as the nucleotide-binding site (NBS) were found in many characterized plant disease resistance genes. Based on the NBS domain, resistance gene analogs have been isolated in our previous study and were used as probes to screen a soybean (Glycine max) cDNA library. A full-length cDNA, KR4, was isolated by screening the library and rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA was 3818 bp in length and the open reading frame coded for a polypeptide of 1211 amino acids with an NBS and five leucine-rich repeats domains, which were identified by Pfam protein analysis. Sequence alignment showed that KR4 was similar to 12 protein of tomato. Southern analysis indicated that the KR4 gene had low copies in soybean genome and it was mapped on the molecular linkage group E. Its expression was also investigated and it was found that KR4 was induced by exogenous salicylic acid and responded upon infection of soybean mosaic virus strain N3.

Soilborne wheat mosaic virus movement protein and RNA and wheat spindle streak mosaic virus coat protein accumulate inside resting spores of their vector, Polymyxa graminis.

To study virus-vector interactions between Soilborne wheat mosaic virus (SBWMV) or Wheat spindle streak mosaic virus (WSSMV) and Polymyxa graminis Ledingham, P. graminis was propagated in plants grown hydroponically. P. graminis accumulated to high levels in several barley cultivars tested. Multiple developmental stages of P. graminis could be identified in infected barley roots. Accumulation of SBWMV and WSSMV inside P. graminis sporosori in the roots of soil-grown winter wheat and hydroponically grown barley was compared to determine if data obtained from plants naturally infected plants and plants infected by manual inoculation were similar. WSSMV coat protein (CP), SBWMV RNAs, SBWMV movement protein but not SBWMV CP were detected in both soil-grown winter wheat and hydroponically grown barley roots. These data are the first direct evidence that SBWMV and WSSMV are internalized by P. graminis.

Salicylic acid-, but not cytokinin-induced, resistance to WClMV is associated with increased expression of SA-dependent resistance genes in Phaseolus vulgaris.

Two-week-old Phaseolus vulgaris plants, wick-fed with 1 mmol/L salicylic acid (SA) or 50 nmol/L dihydrozeatin (DHZ), showed partial inhibition of the accumulation of white clover mosaic virus (WClMV) in infected primary leaves. This inhibition was measured as a decrease in the accumulation of both viral mRNA and viral coat protein, especially at the early stages of infection. Salicylic acid treatment resulted in moderately increased expression of phenylalanine ammonia lyase (PAL), NPR1, PR1 and HSP70 genes that participate in resistance to pathogens in plants. In contrast, DHZ treatments did not induce significant changes in expression of these genes. The expression of the P. vulgaris alternative oxidase (AOX) gene homolog, an enzyme implicated in plant resistance to viruses, showed low constitutive expression during the first 11 days post-infection and was not affected by either SA or DHZ. It appears that, while SA induced the NPR1-PR1 pathogen defense pathway genes, both SA and DHZ may use a different pathway to induce resistance to WClMV infection in P. vulgaris plants.

Biological and molecular characterization of lettuce mosaic virus from Tehran province in Iran.

In this study, lettuce samples having LMV infection symptoms were collected from Tehran fields during 2003. Samples tested for LMV infection by immuno printing. Three positive samples in immuno printing collected and their characteristics were determined. In mechanical inoculation, these Isolates produced symptoms on Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana benthamiana, Lactuca sativa cv. Mantilia and cv. Terocadero (which contains the mol1 resistance gene and susceptible respectively), but not cv. Salinas 88 (which contains the mol2 resistance gene). LMV was purified and LMV polyclonal antiserum was produced in rabbit by a series of intravenous and intramuscular injections, the precipitin titre of this antiserum was 1:1024. Gel double diffusion test (GDDT) was performed, and precipitin bands appeared. SDS-PAGE and western blotting showed the presence of coat protein 29 kDa. In IC-RT-PCR with on LMV specific primer pair, an approximately 1300 bp fragment was amplified.

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2(2) from Lycopersicon esculentum.

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2(2) resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2(2) resistance gene and the susceptible allele, tm-2. The Tm-2(20 gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2(2) gene survive. The Tm-2(2) locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2(2) sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2(2) allele. Between the tm-2 and the Tm-2(2) polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2(2) gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2(2) resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2(2)-encoded resistance is aimed at the Achilles' heel of the virus.

[Virus resistance in transgenic watermelon plants containing a WMV-2 coat protein gene].

Virus disease is a major cause that affects the quality and output of watermelon which is an important fruit in summer. So it is really urgent to develop disease resistance plants. But it takes a long time to breed such plants in conventional ways, and it is very difficult to get ideal result. With the development of plant genetic engineering, new ways have been found to breed plants with disease resistance. By using plant transgenic technique, much progress was been made in plant improvement. There are many successful cases of transgenic plants against corresponding virus disease through transferring coat protein gene. This paper reports the results of inheritance, segregation, expression of WMV-2 coat protein gene in inbred transgenic watermelon and its resistance to virus. Through PCR analysis of inbred plants, we found WMV-2 coat protein gene in the genome of progeny R1 separated with 3:1. After successive selection and identification of 4 generations, 8 transgenic pure lines with almost the same agronomic traits were obtained from 3 independent transformants of T7, T11 and T32. The result of Western blotting shows all 3 different transgenic lines of R4T7-1, R4T11-3 and R4T32-7 can produce coat protein. Disease resistance experiment on transgenic plants with WMV-2 shows that, compared with the control groups, transgenic plants can delay the disease infection and reduce the incidence and the symptoms of virus disease. And the transgenic line R4T32-7 expressed high resistance to infection by WMV-2, which lays a foundation for breeding of disease resistant varieties through plant transgenic technique.

Evolution of Wheat streak mosaic virus: dynamics of population growth within plants may explain limited variation.

Like many other plant RNA viruses, Wheat streak mosaic virus (WSMV) sequence diversity within and among infected plants is low given the large number of virions produced. This may be explained by considering aspects of plant virus life history. Intracellular replication of RNA viruses is predominately linear, not exponential, which means that the rate at which mutations accumulate also is linear. Bottlenecks during systemic movement further limit diversity. Analysis of mixed infections with two WSMV isolates suggests that about four viral genomes participate in systemic invasion of each tiller. Low effective population size increases the role of stochastic processes on dynamics of plant virus population genetics and evolution. Despite low pair-wise diversity among isolates, the number of polymorphic sites within the U.S. population is about the same as between divergent strains or a sister species. Characteristics of polymorphism in the WSMV coat protein gene suggest that most variation appears neutral.

High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles.

The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs.

The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses.

An Arabidopsis thaliana line bearing a transposon insertion in the gene coding for the isozyme form of the plant-specific cap-binding protein, eukaryotic initiation factor (iso) 4E (eIF (iso) 4E), has been isolated. This mutant line completely lacks both eIF(iso)4E mRNA and protein, but was found to have a phenotype and fertility indistinguishable from wild-type plants under standard laboratory conditions. In contrast, the amount of the related eIF4E protein was found to increase in seedling extracts. Furthermore, polysome analysis shows that the mRNA encoding eIF4E was being translated at increased levels. Given the known interaction between cap-binding proteins and potyviral genome-linked proteins (VPg), this plant line was challenged with two potyviruses, Turnip mosaic virus (TuMV) and Lettuce mosaic virus (LMV) and was found resistant to both, but not to the Nepovirus, Tomato black ring virus (TBRV) and the Cucumovirus, Cucumber mosaic virus (CMV). Together with previous data showing that the VPg-eIF4E interaction is necessary for virus infectivity and upregulates genome amplification, this shows that the eIF4E proteins are specifically recruited for the replication cycle of potyviruses.

Production of patchouli mild mosaic virus resistant patchouli plants by genetic engineering of coat protein precursor gene.

Patchouli (Pogostemon cablin (Blanco) Benth), an aromatic crop which yields an essential oil, is widely cultivated in South-east Asia. Patchouli mild mosaic virus (PaMMV) infects patchouli plants and causes decrease in leaf biomass and essential oil yield. Transgenic patchouli plants with PaMMV coat protein precursor (CP-P) gene have been produced by Agrobacterium-mediated transformation. PaMMV CP-P gene integration into the patchouli genome was confirmed by the PCR method and by Southern blot analysis. The transformants were estimated to contain one to three copy genes using Southern blot analysis. The transformant with three copy genes was tested for the resistance to PaMMV by artificially inoculating plants grown in an environmentally controlled cabinet, and this transformant was found to be highly resistant to PaMMV. The transgenic patchouli plant with PaMMV CP-P gene should provide valuable material for protecting against PaMMV.

Barley stripe mosaic virus-induced gene silencing in a monocot plant.

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.

Identification and molecular tagging of two complementary dominant resistance genes to maize dwarf mosaic virus.

Maize dwarf mosaic is one of the devastating and widespread viral diseases in the world. So far, only a few genes were identified and mapped in the resistant materials. A new resistant elite inbred line Siyi was identified with resistance to maize dwarf mosaic virus strain B at early and adult stage. Two complementary dominant genes conditioned the resistance, with a new genetic model, of the maize inbred line were found at adult stage by the genetic analysis based on parents, F1, F2 and backcrosses in two years. The microsatellite analysis of a F2 population from the cross between Siyi and Mo17 was used to identify the two resistance genes on chromosome 3 and 6 respectively by 87 pairs of microsatellite markers. The linkage distance between phi029 and the one resistance gene on chromosome 3 is 14.5 cM, and phi126 to the other on chromosome 6 is 7.2 cM.

Intracellular ingestion and salivation by aphids may cause the acquisition and inoculation of non-persistently transmitted plant viruses.

Transmission of non-persistent plant viruses is related to aphid behaviour during superficial brief probes. A widely accepted hypothesis postulates that virus acquisition occurs during ingestion of plant cell contents, and inoculation during egestion or regurgitation of previously ingested sap. Although conceptually attractive, this ingestion-egestion hypothesis has not been clearly demonstrated. Furthermore, it overlooks the anatomy of the tips of the stylets (mouthparts) and, consequently, the potential role of salivation in the inoculation process. Here, we used the electrical penetration graph (EPG) technique to investigate aphid-stylet activities associated with uptake (acquisition) and release (inoculation) of two non-persistently transmitted viruses. Our results show that acquisition occurs primarily during the last sub-phase (II-3) of intracellular stylet punctures, whereas inoculation is achieved during the first sub-phase (II-1). An alternative mechanism to the ingestion-egestion hypothesis is proposed on the basis of our findings.

Virion formation is required for the long-distance movement of red clover necrotic mosaic virus in movement protein transgenic plants.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species. The polycistronic RNA-1 encodes the viral RNA polymerase and capsid protein (CP) and the monocistronic RNA-2 encodes the 35 kDa cell-to-cell movement protein (MP). Nicotiana benthamiana plants transformed with the RCNMV MP gene were generated. When inoculated onto the MP transgenic plants, cell-to-cell movement of RNA-1 occurred at a rate similar to wild-type virus. However, long-distance (leaf-to-leaf) movement of RNA-1 was not observed. Neither CP nor virions were detected in the inoculated leaves of the MP transgenic plants. When RNA-1 was coinoculated with RNA-2 mutants, which do not express a functional MP, onto MP transgenic plants, CP and virions were readily detected and a systemic infection resulted. These results demonstrate that both RNA-1 and RNA-2 are necessary for the accumulation of both CP and virions. Furthermore, CP accumulation was found to be required for long-distance movement of RCNMV. Therefore, these data provide evidence that CP, in the form of virions, is necessary for the long-distance movement of RCNMV.

Replication of the satellite RNA of pea enation mosaic virus is controlled by RNA 2-encoded functions.

The helper virus mediating replication of the satellite RNA (RNA 3) of pea enation mosaic virus (PEMV) consists of two autonomously replicating, taxonomically unrelated viral RNAs with ties to the luteovirus (RNA 1) and the newly proposed umbravirus (RNA 2) genera. The following study dissects the relative contribution of each of the genomic RNAs of PEMV to the subsistence and dissemination of this satellite RNA. Infectivity assays in a pea protoplast system demonstrate that RNA 2 alone is responsible for the replication of RNA 3, an observation that is supported in part by shared regions of sequence homology at the 5' and 3' termini of both RNAs. In pea seedlings, infectivity assays also demonstrated that the presence of RNA 2 alone is necessary for the systemic invasion of RNA 3. In contrast, the luteovirus-like phase of PEMV (RNA 1) is solely responsible for the encapsidation and aphid transmission of both RNA 2 and the satellite RNA. In a manner comparable to several other virus-satellite systems, the satellite of PEMV also displays a differential response in its capacity to attenuate symptom expression in selected host species. Thus, the satellite RNA of PEMV exists in a trilateral arrangement with its host and two viral RNAs, comparable in many respects to the satellite-virus-host interaction occurring with groundnut rosette disease.