Differential influence of molybdenum and tungsten on the growth of barley seedlings and the activity of aldehyde oxidase under salinity.

The influence of molybdenum, tungsten on germination and growth of barley Hordeum vulgare L. was studied. Results of this study revealed the differential effect of heavy metals on seedlings growth. Exogenous molybdenum treatment stimulated the growth of seedlings. The addition of the metal significantly stimulated root elongation. Contrastingly, the addition of tungsten resulted in increased seed germination and inhibits the growth of seedlings. The negative effect of tungsten on the growth of barley was more profound for roots of plants. In addition, the influence of metals on the growth of plants was also tested in saline conditions. It is shown that under salinity stress plant growth drastically decreased in presence of tungsten. Results of this study showed that activity of molybdenum-containing aldehyde oxidase (AO; EC 1.2.3.1) was also significantly affected by metals. The activity of AO in leaves and roots enhanced with increasing concentrations of molybdate, while tungstate treatment inhibited the enzyme activity. Perhaps, the differential influence of molybdenum and tungsten on the growth of barley is a direct effect of metals on aldehyde oxidase activity in plants. Moreover, the intense negative effect of tungsten treatment on barley growth under salinity conditions emphasizes an important role of aldehyde oxidase in plant resistance to stress factors.

The involvement of ROS producing aldehyde oxidase in plant response to Tombusvirus infection.

The influence of Tomato bushy stunt virus (TBSV) infection on the activity and isoformic composition of aldehyde oxidase and catalase in Nicotiana benthamiana plants was investigated. It was shown that the infection of plants with TBSV results in enhancement of leaf aldehyde oxidase (AO) isoforms AO2 and AO3. Significantly enhanced levels of superoxide radical producing activity of AO isoforms were also detected. This is the first demonstration of involvement of plant AO in defense mechanisms against viral infection. In addition, the infection caused an increased accumulation of hydrogen peroxide, compared to mock-inoculated plants. The virus infection resulted in increased activity of catalase (CAT) and superoxide dismutase (SOD) in roots and leaves of N. benthamiana. Moreover, activation of two additional CAT isoforms was observed in the leaves of plants after virus inoculation. Our findings indicate that the virus infection significantly affects enzymes responsible for the balance of ROS accumulation in plant tissue in response to pathogen attack.

An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity.

Infection of Nicotiana benthamiana plants with Tomato bushy stunt virus (TBSV) mutants compromised for silencing suppression induces formation of an antiviral RISC (vRISC) that can be isolated using chromatography procedures. The isolated vRISC sequence-specifically degrades TBSV RNA in vitro, its activity can be down-regulated by removing siRNAs, and re-stimulated by exogenous supply of siRNAs. vRISC is most effective at hydrolyzing the ~4.8kb genomic RNA, but less so for a ~2.2kb TBSV subgenomic mRNA (sgRNA1), while the 3' co-terminal sgRNA2 of ~0.9kb appears insensitive to vRISC cleavage. Moreover, experiments with in vitro generated 5' co-terminal viral transcripts show that RNAs of ~2.7kb are efficiently cleaved while those of ~1.1kb or shorter are unaffected. The isolated antiviral ribonuclease complex fails to degrade ~0.4kb defective interfering RNAs (DIs) in vitro, agreeing with findings that in plants DIs are not targeted by silencing.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

Differential requirements for Tombusvirus coat protein and P19 in plants following leaf versus root inoculation.

Traditional virus inoculation of plants involves mechanical rubbing of leaves, whereas in nature viruses like Tomato bushy stunt virus (TBSV) are often infected via the roots. A method was adapted to compare leaf versus root inoculation of Nicotiana benthamiana and tomato with transcripts of wild-type TBSV (wtTBSV), a capsid (Tcp) replacement construct expressing GFP (T-GFP), or mutants not expressing the silencing suppressor P19 (TBSVΔp19). In leaves, T-GFP remained restricted to the cells immediately adjacent to the site of inoculation, unless Tcp was expressed in trans from a Potato virus X vector; while T-GFP inoculation of roots gave green fluorescence in upper tissues in the absence of Tcp. Conversely, leaf inoculation with wtTBSV or TBSVΔp19 transcripts initiated systemic infections, while upon root inoculation this only occurred with wtTBSV, not with TBSVΔp19. Evidently the contribution of Tcp or P19 in establishing systemic infections depends on the point-of-entry of TBSV in the plants.

Biological chemistry of virus-encoded suppressors of RNA silencing: an overview.

RNA interference (RNAi) plays multiple biological roles in eukaryotic organisms to regulate gene expression. RNAi also operates as a conserved adaptive molecular immune mechanism against invading viruses. The antiviral RNAi pathway is initiated with the generation of virus-derived short-interfering RNAs (siRNAs) that are used for subsequent sequence-specific recognition and degradation of the cognate viral RNA molecules. As an efficient counter-defensive strategy, most plant viruses evolved the ability to encode specific proteins capable of interfering with RNAi, and this process is commonly known as RNA silencing suppression. Virus-encoded suppressors of RNAi (VSRs) operate at different steps in the RNAi pathway and display distinct biochemical properties that enable these proteins to efficiently interfere with the host-defense system. Recent molecular and biochemical studies of several VSRs significantly expanded our understanding of the complex nature of silencing suppression, and also remarkably advanced our overall knowledge on complex host-virus interactions. In this review, we describe the current knowledge on activities and biochemical mechanisms of selected VSRs with regard to their biological role of suppressing RNAi in plants.

An antiviral RISC isolated from Tobacco rattle virus-infected plants.

The RNAi model predicts that during antiviral defense a RNA-induced silencing complex (RISC) is programmed with viral short-interfering RNAs (siRNAs) to target the cognate viral RNA for degradation. We show that infection of Nicotiana benthamiana with Tobacco rattle virus (TRV) activates an antiviral nuclease that specifically cleaves TRV RNA in vitro. In agreement with known RISC properties, the nuclease activity was inhibited by NaCl and EDTA and stimulated by divalent metal cations; a novel property was its preferential targeting of elongated RNA molecules. Intriguingly, the specificity of the TRV RISC could be reprogrammed by exogenous addition of RNA (containing siRNAs) from plants infected with an unrelated virus, resulting in a newly acquired ability of RISC to target this heterologous genome in vitro. Evidently the virus-specific nuclease complex from N. benthamiana represents a genuine RISC that functions as a readily employable and reprogrammable antiviral defense unit.

Diverse and newly recognized effects associated with short interfering RNA binding site modifications on the Tomato bushy stunt virus p19 silencing suppressor.

The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.

The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection.

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport.

RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex.

Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNA-induced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete approximately 500-kDa protein complex that contained approximately 21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequence-preferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus-host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus.

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.

Activity and protein level of AO isoforms in pea plants (Pisum sativum L.) during vegetative development and in response to stress conditions.

Among three AO isoforms detected in pea plants, the activity of PAO-1 was dominant in leaves of seedlings and young leaves of mature plants, while PAO-3 revealed the highest band intensity in old leaves and roots. PAO-1 and PAO-3 are homodimers consisting of 145 kDa and 140 kDa subunits, respectively, while PAO-2 is a heterodimer of one 145 kDa and one 140 kDa subunit. In leaves, the activity of PAO-1 disappeared gradually with leaf ageing, while in roots it was present only in seedlings but not in mature pea plants. PAO-3 could oxidize abscisic aldehyde, a precursor of abscisic acid, indicating the possible involvement of this isoform in ABA synthesis in pea. The ability of PAO-3 to oxidize abscisic aldehyde was higher in old leaves than in young ones and increased significantly both in roots and leaves of plants exposed to salinity and ammonium treatments. A marked increase of the AO protein level was observed after ammonium application but not under salinity. Interestingly, the activity of PAO isoforms may be transcriptionally and post-transcriptionally regulated during vegetative growth and in response to stress conditions, and such a regulation might be particularly important to adjust ABA levels to the recent requirements of the plant. The observations suggest that the AO isoforms have different metabolic roles and that the activity and protein level of each isoform is regulated not only by environmental conditions but also through plant developmental stages.

Host-specific generation and maintenance of Tomato bushy stunt virus defective interfering RNAs.

The accumulation of Tomato bushy stunt virus (TBSV) defective interfering RNAs (DIs) has been observed in several species of plants, but the involvement of host-specific processes and the functional role of DIs are still poorly understood. In this study, the accumulation of DIs was compared after several passages of TBSV through Nicotiana benthamiana and pepper (Capsicum annuum). As anticipated, passages of wild-type TBSV through N. benthamiana resulted in the accumulation of significant levels of TBSV DIs, which caused symptom attenuation and prevented the plants from lethal necrosis. On the contrary, TBSV infection of pepper plants caused severe local and systemic chlorosis, but continuous virus passages did not result in detectable levels of DIs accumulation. In addition, the inoculation of pepper plants with a mixture of helper virus and DI either from in vitro generated transcripts or from infected N. benthamiana did not yield DI in upper pepper leaves. Our cumulative results suggest that complex host-specific determinants play an important role in TBSV DI generation and their subsequent maintenance and accumulation.