Transcriptional Regulatory Networks Associate with Early Stages of Potato Virus X Infection of Solanum tuberosum.

Potato virus X (PVX) belongs to genus Potexvirus. This study characterizes the cellular transcriptome responses to PVX infection in Russet potato at 2 and 3 days post infection (dpi). Among the 1242 differentially expressed genes (DEGs), 268 genes were upregulated, and 37 genes were downregulated at 2 dpi while 677 genes were upregulated, and 265 genes were downregulated at 3 dpi. DEGs related to signal transduction, stress response, and redox processes. Key stress related transcription factors were identified. Twenty-five pathogen resistance gene analogs linked to effector triggered immunity or pathogen-associated molecular pattern (PAMP)-triggered immunity were identified. Comparative analysis with Arabidopsis unfolded protein response (UPR) induced DEGs revealed genes associated with UPR and plasmodesmata transport that are likely needed to establish infection. In conclusion, this study provides an insight on major transcriptional regulatory networked involved in early response to PVX infection and establishment.

Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > ß-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) < PP2A < GAPDH. For local infection by TMV, the most stable genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH < PP2A < UCE. Using two of the most stable and the two least stable validated reference genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus.

Inoculation of Nicotiana tabacum with recombinant potato virus X induces RNA interference in the solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

OBJECTIVES: The chitin synthase 1 (CHS1) gene in Phenacoccus solenopsis (PsCHS1) was evaluated as a potential target of RNA interference (RNAi) by using Potato virus X (PVX) as a vector (recombinant PVX) for expressing RNAi triggering elements in Nicotiana tabacum L. RESULTS: RT-PCR analysis confirmed the expression of PsCHS1 in N. tabacum inoculated with recombinant-PVX-PsCHS1 (treated). RT- and multiplex-PCR further showed a reduction in mRNA levels of the target gene in mealybugs feeding on treated plants. Mortality in parent adults and emerging nymphs (21 and 29%) exposed to the treated plants was significantly higher (P < 0.05) than those exposed to uninoculated (-ve control) or inoculated with non-recombinant PVX (PVX-control). The number of surviving adults and the combined number of adults and nymphs (47 and 60%) was significantly (P < 0.05) lower on the treated plants than the -ve (76%) or PVX (74%) control. The visual observations verified the physical deformities in mealybugs exposed to the treated plants. CONCLUSION: chitin synthase 1 is a potential RNAi target in P. solenopsis and the recombinant PVX can be used as a tool to evaluate candidate RNAi triggering elements in plants.

The P25 protein of potato virus X (PVX) is the main pathogenicity determinant responsible for systemic necrosis in PVX-associated synergisms.

UNLABELLED: Most plant viruses counter the RNA silencing-based antiviral defense by expressing viral suppressors of RNA silencing (VSRs). In this sense, VSRs may be regarded as virulence effectors that can be recognized by the host as avirulence (avr) factors to induce R-mediated resistance. We made use of Agrobacterium-mediated transient coexpression of VSRs in combination with Potato virus X (PVX) to recapitulate in local tissues the systemic necrosis (SN) caused by PVX-potyvirus synergistic infections in Nicotiana benthamiana. The hypersensitive response (HR)-like response was associated with an enhanced accumulation of PVX subgenomic RNAs. We further show that expression of P25, the VSR of PVX, in the presence of VSR from different viruses elicited an HR-like response in Nicotiana spp. Furthermore, the expression of P25 by a Plum pox virus (PPV) vector was sufficient to induce an increase of PPV pathogenicity that led to necrotic mottling. A frameshift mutation in the P25 open reading frame (ORF) of PVX did not lead to necrosis when coexpressed with VSRs. These findings indicate that P25 is the main PVX determinant involved in eliciting a systemic HR-like response in PVX-associated synergisms. Moreover, we show that silencing of SGT1 and RAR1 attenuated cell death in both PVX-potyvirus synergistic infection and the HR-like response elicited by P25. Our study underscores that P25 variants that have impaired ability to suppress RNA silencing cannot act as elicitors when synergized by the presence of other VSRs. These findings highlight the importance of RNA silencing suppression activity in the HR-like response elicited by VSRs in certain hosts. IMPORTANCE: The work presented here describes how the activity of the PVX suppressor P25 elicits an HR-like response in Nicotiana spp. when overexpressed with other VSR proteins. This finding suggests that the SN response caused by PVX-associated synergisms is a delayed immune response triggered by P25, once it reaches a threshold level by the action of other VSRs. Moreover, this work supports the contention that the silencing suppressor activity of PVX P25 protein is a prerequisite for HR elicitation. We propose that unidentified avr determinants could be involved in other cases of viral synergisms in which heterologous "helper" viruses encoding strong VSRs exacerbate the accumulation of the avr-encoding virus.

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form ß-strands, namely ß1 and ß2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the ß1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within ß1 without preventing the membrane association or movement function of P4.

Viral nanoparticles for in vivo tumor imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. (1) and (2) for further information on VNPs). From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies. In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.

Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9.

The immunogenic properties of chimeric potato virus X (PVX) particles engineered to display the synthetic R9 peptide have been evaluated. The R9 peptide is a consensus sequence derived from diverse variants of the hypervariable region 1 from the hepatitis C virus (HCV) envelope protein E2. Two different constructs were designed, with the R9 peptide expressed either as an indirect fusion via the ribosomal skip 2A (PVX(R9-2A)CP) sequence or as a direct PVX coat protein fusion (PVX(R9)CP). Systemic infection of Nicotiana benthamiana plants was only achieved with PVX(R9-2A)CP constructs, and the presence of the R9 peptide was detected in extracts from these plants by ELISA, Western blot and electron microscopy using specific anti-R9 antibodies. The virus particles were recovered at yields of up to 125mg/kg from leaf material. BALB/c mice immunized with purified PVX(R9-2A)CP particles developed specific anti-R9 IgG titers of up to 1:50,000. Monoclonal anti-R9 antibodies were obtained from the spleen of a mouse immunized with PVX(R9-2A)CP particles and characterized by Western blot and electron microscopy. Sera from patients infected chronically with HCV were found to react specifically with PVX(R9-2A)CP particles in 35% of cases.

Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1.

Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 degrees C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 degrees C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the 'core' Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P(1110),R(1121),K(1255)]+TGB1(L(88))] always induced systemic necrosis at 15 degrees C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1(P(88)) significantly reducing silencing suppression. The difference in silencing suppression between TGB1(P(88)) and TGB1(L(88)) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1(P(88)) and TGB1(L(88)) resulted in interference in the suppression of silencing by TGB1(L(88)). Yeast two-hybrid analysis confirmed that TGB1(P(88)) and TGB1(L(88)) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.

Mixed infections of Pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus.

Pepino mosaic virus (PepMV) is an emerging pathogen that causes severe economic losses in tomato crops (Solanum lycopersicum L.) in the Northern hemisphere, despite persistent attempts of control. In fact, it is considered one of the most significant viral diseases for tomato production worldwide, and it may constitute a good model for the analysis of virus emergence in crops. We have combined a population genetics approach with an analysis of in planta properties of virus strains to explain an observed epidemiological pattern. Hybridization analysis showed that PepMV populations are composed of isolates of two types (PepMV-CH2 and PepMV-EU) that cocirculate. The CH2 type isolates are predominant; however, EU isolates have not been displaced but persist mainly in mixed infections. Two molecularly cloned isolates belonging to each type have been used to examine the dynamics of in planta single infections and coinfection, revealing that the CH2 type has a higher fitness than the EU type. Coinfections expand the range of susceptible hosts, and coinfected plants remain symptomless several weeks after infection, so a potentially important problem for disease prevention and management. These results provide an explanation of the observed epidemiological pattern in terms of genetic and ecological interactions among the different viral strains. Thus, mixed infections appear to be contributing to shaping the genetic structure and dynamics of PepMV populations.

The role of viral infection in inducing variability in virus-free progeny in tomato.

The effect of virus-host interactions on subsequent generations is poorly understood. The evaluation of the effects of viral infection on inheritance of quantitative traits in the progeny of infected plants and elucidation of a possible relationship between chiasma frequency in the infected plants and variability of traits in the progeny were investigated. The current study involved genotypes of four intraspecific hybrids of tomato (Solanum lycopersicum L.), their parental forms and two additional cultivars. Used as infection were the tobacco mosaic virus (TMV) and potato virus X (PVX). The consequences of the effect of viral infection were evaluated based on chromosome pairing in diakinesis and/or by examining quantitative and qualitative traits in the progeny of the infected tomato plants. Tomato plants infected with TMV + PVX were found to differ in chiasma frequency per pollen mother cell or per bivalent. Deviations have been observed for genotypes of both F(1) hybrids and cultivars. At the same time, differences in mean values of the traits under study have only been found for progeny populations (F(2)-F(4)) derived from virus-infected F(1) hybrids, but not in the case of progeny of the infected cultivars. The rate of recombinants combining traits of both parents increased significantly (2.22-8.24 times) in progeny populations of hybrids infected with TMV + PVX. The above suggests that the observed effects could be the result of modification of recombination frequencies that can be manifested in heterozygous hybrids and make small contributions to variability in cases of 'homozygous' tomato genotypes (i.e. cultivars).

Analysis of temperature modulation of plant defense against biotrophic microbes.

Plant-pathogen interactions are known to be affected by environmental factors including temperature; however, the temperature effects have not been systematically studied in plant disease resistance. Here, we characterized the effects of a moderate increase in temperature on resistance to bacterial pathogen Pseudomonas syringae and two viral elicitors in Arabidopsis thaliana and Nicotiana benthamiana. Both the basal and the resistance (R) gene-mediated defense responses to Pseudomonas syringae are found to be inhibited by a moderately high temperature, and hypersensitive responses induced by R genes against two viruses are also reduced by an increase of temperature. These indicate that temperature modulation of defense responses to biotrophic and hemibiotrophic pathogens might be a general phenomenon. We further investigated the roles of two small signaling molecules, salicylic acid and jasmonic acid, as well as two defense regulators, EDS1 and PAD4, in this temperature modulation. These components, though modulated by temperature or involved in temperature regulation or both, are not themselves determinants of temperature sensitivity in the defense responses analyzed. The inhibition of plant defense response by a moderately high temperature may thus be mediated by other defense signaling components or a combination of multiple factors.

The Rx gene confers resistance to a range of potexviruses in transgenic Nicotiana plants.

Rx-mediated resistance was analyzed in Rx-expressing transgenic Nicotiana plants. The infection outcome of nine Potato virus X isolates mutated at amino acid positions 121 and 127 of the coat protein (CP) confirmed the key role of these amino acids but provided a more complex picture than previously reported. In particular, in Rx-expressing Nicotiana spp., eliciting activity modulated by amino acid 121 was conditioned by the nature of amino acid 127. These results suggest that the specificity of recognition might be modulated by host factors that are somehow subtly modified between Rx-expressing potato and Rx-expressing transgenic Nicotiana plants. Moreover, the CP of three Potexviruses, Narcissus mosaic virus (NMV), White clover mosaic virus (WClMV), and Cymbidium mosaic virus (CymMV), are all recognized by the Rx-based machinery and able to trigger an Rx-dependant hypersensitive response. A smaller elicitor of 90 amino acids was identified in the CP of NMV and WClMV, which contains the previously identified key positions 121 and 127. This elicitor is only weakly conserved (approximately 40% identity) among the CP of the various recognized viruses, suggesting that the Rx molecular machinery targets a conserved structural element of the Potexvirus CP rather than a conserved amino acid motif.

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants.

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.

The coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein function in pathogen recognition and signaling.

Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure-function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase-activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.

The efficiency of interference of Potato virus X infection depends on the target gene.

RNA silencing is a natural defense response against viral infection. This phenomenon has been used to interfere with viral infections by exploiting fragments of viral genomes as sources of RNA silencing. Agrobacterium-mediated transient expression of a hairpin RNA derived from the TGBp1 gene of Potato virus X (PVX) induced RNA silencing of the TGBp1 gene and resulted in interference of PVX infection. The interference was induced in the infiltrated leaves but not in the upper non-infiltrated leaves. Transient expression of a CP hairpin RNA also induced interference of PVX. The TGBp1 hairpin RNA showed more efficient interference of PVX infection than the CP hairpin RNA.

Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14alpha-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L.

Obtusifoliol-14alpha-demethylase (CYP51) is implicated in plant sterol biosynthesis. An Arabidopsis expressed sequence tag encoding a CYP51 was used as a probe to isolate Nicotiana tabacum L. cDNAs. Two types of cDNA clones were identified. Nt CYP51-1 and Nt CYP51-2 shared 97% identity together and around 75% with other plant CYP51s. The function of the encoded enzyme has been demonstrated in planta by manipulating the sterol biosynthetic pathway at the gene level. The endogenous CYP51 of Nicotiana benthamiana was silenced upon inoculation of the plantlets with POTATO VIRUS X::Nt CYP51-1 transcripts. This resulted in the accumulation of obtusifoliol, the substrate of CYP51, and other 14alpha-methyl sterols, with a concomitant growth reduction phenotype. Virus-induced gene silencing was also applied to another steroidogenic enzyme, the Delta7-sterol-C5(6)-desaturase, and this resulted in the accumulation of Delta7-sterols in infected plants instead of the pathway end-products Delta5-sterols.

Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco.

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.

Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense.

RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a tobamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.

Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato.

Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.