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The outcome of certain plant-virus interaction is symptom recovery, which is accompanied with the emergence of asymptomatic tissues in which the virus accumulation decreased dramatically. This phenomenon shows the potential to reveal critical molecular factors for controlling viral disease. MicroRNAs act as master regulators in plant growth, development, and immunity. However, the mechanism by which miRNA participates in regulating symptom recovery remains largely unknown. Here, we reported that miR172 was scavenged in the recovered tissue of tobacco mosaic virus (TMV)-infected Nicotiana tabacum plants. Overexpression of miR172 promoted TMV infection, whereas silencing of miR172 inhibited TMV infection. Then, TARGET OF EAT3 (TOE3), an APETALA2 transcription factor, was identified as a downstream target of miR172. Overexpression of NtTOE3 significantly improved plant resistance to TMV infection, while knockout of NtTOE3 facilitated virus infection. Furthermore, transcriptome analysis indicated that TOE3 promoted the expression of defense-related genes, such as KL1 and MLP43. Overexpression of these genes conferred resistance of plant against TMV infection. Importantly, results of dual-luciferase assay, chromatin immunoprecipitation-quantitative PCR, and electrophoretic mobility shift assay proved that TOE3 activated the transcription of KL1 and MLP43 by binding their promoters. Moreover, overexpression of rTOE3 (the miR172-resistant form of TOE3) significantly reduced TMV accumulation compared to the overexpression of TOE3 (the normal form of TOE3) in miR172 overexpressing Nicotiana benthamiana plants. Taken together, our study reveals the pivotal role of miR172/TOE3 module in regulating plant immunity and in the establishment of recovery in virus-infected tobacco plants, elucidating a regulatory mechanism integrating plant growth, development, and immune response., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Calcium is an important plant immune signal that is essential for activating host resistance, but how RNA viruses manipulate calcium signals to promote their infections remains largely unknown. Here, we demonstrated that tobacco mosaic virus (TMV) coat protein (CP)-interacting protein L (IP-L) associates with calmodulin-like protein 30 (NbCML30) in the cytoplasm and nucleus, and can suppress its expression at the nucleic acid and protein levels. NbCML30, which lacks the EF-hand conserved domain and cannot bind to Ca 2+ , was located in the cytoplasm and nucleus and was downregulated by TMV infection. NbCML30 silencing promoted TMV infection, while its overexpression inhibited TMV infection by activating Ca 2+ -dependent oxidative stress in plants. NbCML30-mediated resistance to TMV mainly depends on IP-L regulation as the facilitation of TMV infection by silencing NbCML30 was canceled by co-silencing NbCML30 and IP-L. Overall, these findings indicate that in the absence of any reported silencing suppressor activity, TMV CP manipulates IP-L to inhibit NbCML30, influencing its Ca 2+ -dependent role in the oxidative stress response. These results lay a theoretical foundation that will enable us to engineer tobacco (Nicotiana spp.) with improved TMV resistance in the future., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Viruses use and subvert host cell mechanisms to support their replication and spread between cells, tissues and organisms. Microtubules and associated motor proteins play important roles in these processes in animal systems, and may also play a role in plants. Although transport processes in plants are mostly actin based, studies, in particular with Tobacco mosaic virus (TMV) and its movement protein (MP), indicate direct or indirect roles of microtubules in the cell-to-cell spread of infection. Detailed observations suggest that microtubules participate in the cortical anchorage of viral replication complexes, in guiding their trafficking along the endoplasmic reticulum (ER)/actin network, and also in developing the complexes into virus factories. Microtubules also play a role in the plant-to-plant transmission of Cauliflower mosaic virus (CaMV) by assisting in the development of specific virus-induced inclusions that facilitate viral uptake by aphids. The involvement of microtubules in the formation of virus factories and of other virus-induced inclusions suggests the existence of aggresomal pathways by which plant cells recruit membranes and proteins into localized macromolecular assemblies. Although studies related to the involvement of microtubules in the interaction of viruses with plants focus on specific virus models, a number of observations with other virus species suggest that microtubules may have a widespread role in viral pathogenesis., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T-DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, alpha-tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus-end marker GFP-EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell-to-cell movement of TMV encoding GFP-tagged movement protein (MP-GFP) is reduced in ATER2 accompanied by a reduced association of MP-GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.

In healthy plants extracellular ATP (eATP) regulates the balance between cell viability and death. Here we show an unexpected critical regulatory role of eATP in disease resistance and defensive signalling. In tobacco, enzymatic depletion of eATP or competition with non-hydrolysable ATP analogues induced pathogenesis-related (PR) gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci. Artificially increasing eATP concentrations triggered a drop in levels of the important defensive signal chemical salicylic acid (SA) and compromised basal resistance to viral and bacterial infection. Inoculating tobacco leaf tissues with bacterial pathogens capable of activating PR gene expression triggered a rapid decline in eATP. Conversely, inoculations with mutant bacteria unable to induce defence gene expression failed to deplete eATP. Furthermore, a collapse in eATP concentration immediately preceded PR gene induction by SA. Our study reveals a previously unsuspected role for eATP as a negative regulator of defensive signal transduction and demonstrates its importance as a key signal integrating defence and cell viability in plants.

Active resistance to viruses is afforded by plant disease resistance (R) genes, which encode proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. Upon recognition of pathogen-derived elicitors, these NB-LRR proteins are thought to initiate a number of signaling pathways that lead to pathogen restriction. However, little is known about the molecular mechanisms that ultimately curtail virus accumulation. Here, we show that the co-expression of a plant NB-LRR protein with its cognate elicitor results in an antiviral response that inhibits the translation of virus-encoded proteins in Nicotiana benthamiana. This antiviral response is dependent on viral cis elements, and, upon activation of the NB-LRR protein, viral transcripts accumulate but do not associate with ribosomes. The induced inhibition of viral transcript translation and NB-LRR-mediated virus resistance were compromised by the downregulation of Argonaute4-like genes. Argonaute proteins have been implicated in small RNA-mediated RNA degradation, and in degradation-independent translational control. Our results suggest that the engagement of Argonaute proteins in the specific translational control of viral transcripts is a key factor in virus resistance mediated by NB-LRR proteins.

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.

Plant mitogen-activated protein kinases (MAPKs) represented by tobacco wounding-induced protein kinase (WIPK) have unique regulation at the level of transcription in response to stresses. By using transcriptional and translational inhibitors, it has been shown previously that WIPK gene expression and de novo protein synthesis are required for the high-level activity of WIPK in cells treated with elicitins from Phytophthora spp. However, regulation of WIPK expression and the role(s) of WIPK in plant disease resistance are unknown. In this report, we demonstrate that WIPK gene transcription is regulated by phosphorylation and de-phosphorylation events. Interestingly, salicylic acid-induced protein kinase (SIPK) was identified as the kinase involved in regulating WIPK gene expression based on both gain-of-function and loss-of-function analyses. This finding revealed an additional level of interaction between SIPK and WIPK, which share an upstream MAPKK, NtMEK2. Depending on whether WIPK shares its downstream targets with SIPK, it could either function as a positive feed-forward regulator of SIPK or initiate a new pathway. Consistent with the first scenario, co-expression of WIPK with the active mutant of NtMEK2 leads to accelerated hypersensitive response (HR)-like cell death in which SIPK also plays a role. Mutagenesis analysis revealed that the conserved common docking domain in WIPK is required for its function. Together with prior reports that (i) WIPK is activated in NN tobacco infected with tobacco mosaic virus, and (ii) PVX virus-induced gene silencing of WIPK attenuated N gene-mediated resistance, we concluded that WIPK plays a positive role in plant disease resistance, possibly through accelerating the pathogen-induced HR cell death.

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.

Virus expression vectors based on the tobacco mosaic virus (TMV) genome are powerful tools for foreign gene expression in plants. However, the inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. To improve these properties of TMV vectors, the gene encoding the 30-kDa movement protein was subjected to mutagenesis and DNA shuffling. A vector that expresses the green fluorescent protein was used to allow simple visual discrimination of mutants with enhanced movement phenotypes. An initial round of mutagenesis produced 53 clones with a faster local movement phenotype. Two subsequent rounds of DNA shuffling produced additional clones that showed further increased rates of cell-to-cell movement and degrees of systemic invasion in restrictive hosts. Surprisingly, sequence analysis of the best performing shuffled genes revealed alterations resulting in coding and silent changes in the movement protein gene. Separation of these coding and silent alterations into distinct gene backgrounds revealed that each contributes to improved movement protein function to differing degrees. The resulting vectors demonstrate that the complex activities of the movement protein genes of viruses can be evolved to have improved movement phenotypes, as evidenced by cell-to-cell and systemic invasion. The experiments produced improved vectors that will be of use both for in planta functional screening and for therapeutic protein production and demonstrated the power of shuffling for plant virus vector improvement.

Hypersensitive reactions to plant diseases are typically mediated by R genes. Many R genes that have been cloned only confer resistance to a particular pathogen. However, Chenopodium spp. have multivirus hypersensitive resistance, thus making the understanding of this broad-spectrum resistance mechanism attractive. Using tobacco mosaic virus (TMV) tagged with green fluorescent protein to follow infection over time, cDNA-AFLP to find genes up-regulated during virus infection in C. amaranticolor and quantitative RT-PCR to accurately measure gene expression at different time points, the first dissection of this significant defense response pathway is presented. The detected disease-expressed sequences in C. amaranticolor (DESCA) are similar to those that encode p450 monooxegenases, hypersensitivity-related genes, cellulases, ABC transporters, receptor-like kinases, serine/threonine kinases, phosphoribosylanthranilate transferases and hypothetical R genes, many of which are associated with pathogen defense in other plants. The expressions of these DESCA genes are also induced by infection with the taxonomically distinct tobacco rattle virus (TRV) in C. amaranticolor. In particular, DESCA1, one of the gene fragments from C. amaranticolor that lacks similarity to any other sequence in the GenBank database, is induced at least 200 fold 4 d after infection (dai) by both TMV and TRV. The potential role of DESCA genes in a C. amaranticolor multivirus defense response with regard to their levels and time of gene expression is discussed.

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.

Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA.

A clone encoding a putative soluble epoxide hydrolase (EH-1), an enzyme which converts epoxides to diols, was isolated by differential screening of a cDNA library prepared from tobacco mosaic virus (TMV)-infected tobacco leaves. To confirm that EH-1 encodes an epoxide hydrolase, the recombinant EH-1 protein produced in bacteria was shown to have high epoxide hydrolase activity in vitro. Infection of resistant but not susceptible tobacco cultivars induced the accumulation of EH-1 transcripts in both the inoculated and uninoculated, systemic leaves. EH-1 expression was also induced in the inoculated and systemic tissues of TMV-infected NahG plants, which are unable to accumulate salicylic acid (SA). However, EH-1 expression in the inoculated leaves of NahG plants was delayed, whilst in the systemic leaves the induction was both later and weaker, compared to that observed in wild-type plants. Furthermore, exogenously applied SA or its functional analog 2,6-dichloroisonicotinic acid (INA) caused a rapid and transient accumulation of EH-1 transcripts, whereas an inactive SA analog did not. Thus, the induction of EH-1 gene expression appears to be regulated by both SA-independent and SA-dependent pathways. Since EH-1 was expressed only in TMV-resistant tobacco after infection, and the encoded enzyme is thought to help metabolize toxic compounds, we propose that EH-1 may play a role in protection from oxidative damage associated with defense responses. It may also play a role in generating signals for activation of certain defense responses.

To investigate the role of salicylic acid (SA) in the hypersensitive response (HR) its accumulation was compromised during different phases of lesion development by differential expression of a salicylate hydroxylase gene (SH-L). Constitutive suppression of SA accumulation was achieved by expression of a gene fusion between the CaMV35S promoter (35S) and SH-L. Using the H2O2-responsive AoPR1 promoter to drive SH-L SA accumulation could be compromised at an early stage, on lesion formation and possibly prior to visible necrosis, whilst use of the salicylate-responsive PR1a promoter reduced SA accumulation at a later stage as lesions expand. TMV infection of 35S-SH-L and AoPR1-SH-L, but not PR1a-SH-L, tobacco resulted in significantly greater rates of lesion growth than in wild-type tobacco. TMV was detected in asymptomatic tissue surrounding lesions only in 35S-SH-L and AoPR1-SH-L lines; subsequently these transgenic lines exhibited a 'spreading-necrosis' originating from the lesion which entered the stem and eventually other leaves, a phenotype which could be correlated with the presence of TMV particles. Analysis of TMV-infected and 'temperature-shifted' tobacco indicated that both 35S-SH-L and AoPR1-SH-L, but not PR1a-SH-L, transgenics exhibited delayed cell-death compared to wild-type infections. We propose that the SH-L phenotypes indicate that early SA accumulation is a major factor in preventing viral escape, via mechanism(s) which may include influencing the rate of host-cell death and, possibly, an effect on viral function.

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a nonvirion form of the coat protein.

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.