Defense signaling pathways in resistance to plant viruses: Crosstalk and finger pointing.

Resistance to infection by plant viruses involves proteins encoded by plant resistance (R) genes, viz., nucleotide-binding leucine-rich repeats (NLRs), immune receptors. These sensor NLRs are activated either directly or indirectly by viral protein effectors, in effector-triggered immunity, leading to induction of defense signaling pathways, resulting in the synthesis of numerous downstream plant effector molecules that inhibit different stages of the infection cycle, as well as the induction of cell death responses mediated by helper NLRs. Early events in this process involve recognition of the activation of the R gene response by various chaperones and the transport of these complexes to the sites of subsequent events. These events include activation of several kinase cascade pathways, and the syntheses of two master transcriptional regulators, EDS1 and NPR1, as well as the phytohormones salicylic acid, jasmonic acid, and ethylene. The phytohormones, which transit from a primed, resting states to active states, regulate the remainder of the defense signaling pathways, both directly and by crosstalk with each other. This regulation results in the turnover of various suppressors of downstream events and the synthesis of various transcription factors that cooperate and/or compete to induce or suppress transcription of either other regulatory proteins, or plant effector molecules. This network of interactions results in the production of defense effectors acting alone or together with cell death in the infected region, with or without the further activation of non-specific, long-distance resistance. Here, we review the current state of knowledge regarding these processes and the components of the local responses, their interactions, regulation, and crosstalk.

Critical points for the design and application of RNA silencing constructs for plant virus resistance.

Control of plant virus diseases is a big challenge in agriculture as is resistance in plant lines to infection by viruses. Recent progress using advanced technologies has provided fast and durable alternatives. One of the most promising techniques against plant viruses that is cost-effective and environmentally safe is RNA silencing or RNA interference (RNAi), a technology that could be used alone or along with other control methods. To achieve the goals of fast and durable resistance, the expressed and target RNAs have been examined in many studies, with regard to the variability in silencing efficiency, which is regulated by various factors such as target sequences, target accessibility, RNA secondary structures, sequence variation in matching positions, and other intrinsic characteristics of various small RNAs. Developing a comprehensive and applicable toolbox for the prediction and construction of RNAi helps researchers to achieve the acceptable performance level of silencing elements. Although the attainment of complete prediction of RNAi robustness is not possible, as it also depends on the cellular genetic background and the nature of the target sequences, some important critical points have been discerned. Thus, the efficiency and robustness of RNA silencing against viruses can be improved by considering the various parameters of the target sequence and the construct design. In this review, we provide a comprehensive treatise regarding past, present and future prospective developments toward designing and applying RNAi constructs for resistance to plant viruses.

Naturally occurring substitution of an amino acid in a plant virus gene-silencing suppressor enhances viral adaptation to increasing thermal stress.

Cereal yellow dwarf virus (CYDV-RPV) encodes a P0 protein that functions as a viral suppressor of RNA silencing (VSR). The strength of silencing suppression is highly variable among CYDV-RPV isolates. In this study, comparison of the P0 sequences of CYDV-RPV isolates and mutational analysis identified a single C-terminal amino acid that influenced P0 RNA-silencing suppressor activity. A serine at position 247 was associated with strong suppressor activity, whereas a proline at position 247 was associated with weak suppressor activity. Amino acid changes at position 247 did not affect the interaction of P0 with SKP1 proteins from Hordeum vulgare (barley) or Nicotiana benthamiana. Subsequent studies found P0 proteins containing a P247 residue were less stable than the P0 proteins containing an S247 residue. Higher temperatures contributed to the lower stability and in planta and the P247 P0 proteins were subject to degradation via the autophagy-mediated pathway. A P247S amino acid residue substitution in P0 increased CYDV-RPV replication after expression in agroinfiltrated plant leaves and increased viral pathogenicity of P0 generated from the heterologous Potato virus X expression vector system. Moreover, an S247 CYDV-RPV could outcompete the P247 CYDV-RPV in a mixed infection in natural host at higher temperature. These traits contributed to increased transmission by aphid vectors and could play a significant role in virus competition in warming climates. Our findings underscore the capacity of a plant RNA virus to adapt to climate warming through minor genetic changes in gene-silencing suppressor, resulting in the potential for disease persistence and prevalence.

The Secret Life of the Inhibitor of Virus Replication.

The inhibitor of virus replication (IVR) is an inducible protein that is not virus-target-specific and can be induced by several viruses. The GenBank was interrogated for sequences closely related to the tobacco IVR. Various RNA fragments from tobacco, tomato, and potato and their genomic DNA contained IVR-like sequences. However, IVRs were part of larger proteins encoded by these genomic DNA sequences, which were identified in Arabidopsis as being related to the cyclosome protein designated anaphase-promoting complex 7 (APC7). Sequence analysis of the putative APC7s of nine plant species showed proteins of 558-561 amino acids highly conserved in sequence containing at least six protein-binding elements of 34 amino acids called tetratricopeptide repeats (TPRs), which form helix-turn-helix structures. The structures of Arabidopsis APC7 and the tobacco IVR proteins were modeled using the AlphaFold program and superimposed, showing that IVR had the same structure as the C-terminal 34% of APC7, indicating that IVR was a product of the APC7 gene. Based on the presence of various transcription factor binding sites in the APC7 sequences upstream of the IVR coding sequences, we propose that IVR could be expressed by these APC7 gene sequences involving the transcription factor SHE1.

The Virus-Induced Transcription Factor SHE1 Interacts with and Regulates Expression of the Inhibitor of Virus Replication (IVR) in N Gene Tobacco.

The transcription factor SHE1 was induced by tobacco mosaic virus (TMV) infection in tobacco cv. Samsun NN (SNN) and SHE1 inhibited TMV accumulation when expressed constitutively. To better understand the role of SHE1 in virus infection, transgenic SNN tobacco plants generated to over-express SHE1 (OEx-SHE1) or silence expression of SHE1 (si-SHE1) were infected with TMV. OEx-SHE1 affected the local lesion resistance response to TMV, whereas si-SHE1 did not. However, si-SHE1 allowed a slow systemic infection to occur in SNN tobacco. An inhibitor of virus replication (IVR) was known to reduce the accumulation of TMV in SNN tobacco. Analysis of SHE1 and IVR mRNA levels in OEx-SHE1 plants showed constitutive expression of both mRNAs, whereas both mRNAs were less expressed in si-SHE1 plants, even after TMV infection, indicating that SHE1 and IVR were associated with a common signaling pathway. SHE1 and IVR interacted with each other in four different assay systems. The yeast two-hybrid assay also delimited sequences required for the interaction of these two proteins to the SHE1 central 58-79% region and the IVR C-terminal 50% of the protein sequences. This suggests that SHE is a transcription factor involved in the induction of IVR and that IVR binds to SHE1 to regulate its own synthesis.

Cucumber Mosaic Virus 1a Protein Interacts with the Tobacco SHE1 Transcription Factor and Partitions between the Nucleus and the Tonoplast Membrane.

The transcription factor SHE1 was identified as an interacting partner with the cucumber mosaic virus (CMV) 1a protein in the yeast two-hybrid system, by a pull-down assay, and via bimolecular fluorescent complementation. Using fluorescent-tagged proteins and confocal microscopy, the CMV 1a protein itself was found distributed predominantly between the nucleus and the tonoplast membrane, although it was also found in speckles in the cytoplasm. The SHE1 protein was localized in the nucleus, but in the presence of the CMV 1a protein was partitioned between the nucleus and the tonoplast membrane. SHE1 expression was induced by infection of tobacco with four tested viruses: CMV, tobacco mosaic virus, potato virus X and potato virus Y. Transgenic tobacco expressing the CMV 1a protein showed constitutive expression of SHE1, indicating that the CMV 1a protein may be responsible for its induction. However, previously, such plants also were shown to have less resistance to local and systemic movement of tobacco mosaic virus (TMV) expressing the green fluorescent protein, suggesting that the CMV 1a protein may act to prevent the function of the SHE1 protein. SHE1 is a member of the AP2/ERF class of transcription factors and is conserved in sequence in several Nicotiana species, although two clades of SHE1 could be discerned, including both different Nicotiana species and cultivars of tobacco, varying by the presence of particular insertions or deletions.

Resistance to Turnip Mosaic Virus in the Family Brassicaceae.

Resistance to diseases caused by turnip mosaic virus (TuMV) in crop species of the family Brassicaceae has been studied extensively, especially in members of the genus Brassica. The variation in response observed on resistant and susceptible plants inoculated with different isolates of TuMV is due to a combination of the variation in the plant resistome and the variation in the virus genome. Here, we review the breadth of this variation, both at the level of variation in TuMV sequences, with one eye towards the phylogeny and evolution of the virus, and another eye towards the nature of the various responses observed in susceptible vs. different types of resistance responses. The analyses of the viral genomes allowed comparisons of pathotyped viruses on particular indicator hosts to produce clusters of host types, while the inclusion of phylogeny data and geographic location allowed the formation of the host/geographic cluster groups, the derivation of both of which are presented here. Various studies on resistance determination in particular brassica crops sometimes led to further genetic studies, in many cases to include the mapping of genes, and in some cases to the actual identification of the genes. In addition to summarizing the results from such studies done in brassica crops, as well as in radish and Arabidopsis (the latter as a potential source of candidate genes for brassica and radish), we also summarize work done using nonconventional approaches to obtaining resistance to TuMV.

R gene mediated defense against viruses.

The relationship of Resistance (R) gene-mediated defense to other forms of resistance in plants is considered, and the natures of the products of dominant and recessive R genes are reviewed. Various factors involved in expressing R gene-mediated resistance are described. These include phytohormones and plant effector molecules: the former regulating different pathways for disease resistance and the latter having direct effects on viral genomes or encoded proteins. Finally, the status of our knowledge concerning the cell-death hypersensitive response and its relationship to the actual resistance response involved in inhibiting virus infection is examined.

A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation.

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

ICTV Virus Taxonomy Profile: Bromoviridae.

Bromoviridae is a family of plant viruses with tri-segmented, positive-sense, single-stranded RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain subgenomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and are transmitted between hosts mechanically, in/on the pollen and non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomato, cucurbits, bananas, fruit trees and alfalfa. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bromoviridae, which is available at www.ictv.global/report/bromoviridae.

Tomato geminivirus encoded RNAi suppressor protein, AC4 interacts with host AGO4 and precludes viral DNA methylation.

Plant RNA silencing systems are organized as a network, regulating plant developmental pathways and restraining invading viruses, by sharing cellular components with overlapping functions. Host regulatory networks operate either at the transcriptional level via RNA-directed DNA methylation, or at the post-transcriptional stage interfering with mRNA to restrict viral infection. However, viral-derived proteins, including suppressors of RNA silencing, favour virus establishment, and also affect plant developmental processes. In this investigation, we report that Tomato leaf curl New Delhi virus-derived AC4 protein suppresses RNA silencing activity and mutational analysis of AC4 showed that Asn-50 in the SKNT-51 motif, in the C-terminal region, is a critical determinant of its RNA silencing suppressor activity. AC4 showed interaction with host AGO4 but not with AGO1, aggregated around the nucleus, and influenced cytosine methylation of the viral genome. The possible molecular mechanism by which AC4 interferes in the RNA silencing network, helps virus establishment, and affects plant development is discussed.

Differential expression of cucumber RNA-dependent RNA polymerase 1 genes during antiviral defence and resistance.

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial role in plant defence against viruses. In this study, it was observed that cucumber, Cucumis sativus, uniquely encodes a small gene family of four RDR1 genes. The cucumber RDR1 genes (CsRDR1a, CsRDR1b and duplicated CsRDR1c1/c2) shared 55%-60% homology in their encoded amino acid sequences. In healthy cucumber plants, RDR1a and RDR1b transcripts were expressed at higher levels than transcripts of RDR1c1/c2, which were barely detectable. The expression of all four CsRDR1 genes was induced by virus infection, after which the expression level of CsRDR1b increased 10-20-fold in several virus-resistant cucumber cultivars and in a broad virus-resistant transgenic cucumber line expressing a high level of transgene small RNAs, all without alteration in salicylic acid (SA) levels. By comparison, CsRDR1c1/c2 genes were highly induced (25-1300-fold) in susceptible cucumber cultivars infected with RNA or DNA viruses. Inhibition of RDR1c1/c2 expression led to increased virus accumulation. Ectopic application of SA induced the expression of cucumber RDR1a, RDR1b and RDRc1/c2 genes. A constitutive high level of RDR1b gene expression independent of SA was found to be associated with broad virus resistance. These findings show that multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles.

The Use of a Tobacco mosaic virus-Based Expression Vector System in Chrysanthemum.

Chrysanthemums (Chrysanthemum morifolium) are susceptible to tobacco mosaic virus (TMV). TMV-based expression vectors have been used in high-throughput experiments for production of foreign protein in plants and also expressing green fluorescent protein (GFP) to allow visualization of TMV movement. Here, we used TMV expressing the GFP to examine the infection of chrysanthemum by a TMV-based expression vector. Viral replication, movement and GFP expression by TMV-GFP were verified in upper leaves of chrysanthemums up to 73 days post inoculation (dpi) by RT-PCR. Neither wild-type TMV nor TMV-GFP induced symptoms. GFP fluorescence was seen in the larger veins of the inoculated leaf, in the stem above the inoculation site and in petioles of upper leaves, although there was no consistent detection of GFP fluorescence in the lamina of upper leaves under UV. Thus, a TMV-based expression vector can infect chrysanthemum and can be used for the in vivo study of gene functions.

Manipulation of induced resistance to viruses.

Induced resistance against plant viruses has been studied for many years. However, with the exception of RNA silencing, induced resistance to viruses remains mechanistically less well understood than for other plant pathogens. In contrast, the induction processes involved in induced resistance, comprising basal resistance signaling, effector-triggered immunity, and phytohormone pathways, have been increasingly well characterized in recent years. This has allowed induced resistance to viruses to be placed in a broader conceptual framework linking it to other defense systems, which we discuss in this review. We also discuss the range of agents, including chemicals and beneficial microorganisms and application methods that can be used to induce resistance to viruses.

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.

Silencing the tobacco gene for RNA-dependent RNA polymerase 1 and infection by potato virus Y cause remodeling of cellular organelles.

RNA-dependent RNA polymerase 1 (RDR1) has been shown to be involved in DNA methylation, RNA silencing and regulating expression of other genes. RDR1 gene expression is stimulated by infection with potato virus Y° (PVY). Transgenic Nicotiana tabacum plants silenced for RDR1 gene expression showed morphological changes in mesophyll cells, associated with remodeling of the nuclei, chloroplasts and mitochondria. RDR1 silencing led to decreased nuclear size, increased heterochromatin content and aggregation, decreased numbers of chloroplasts, plus changes in shape, internal structures and integrity of chloroplasts and mitochondria. RDR1-silenced transgenic plants showed increased PVY accumulation and ultrastructural remodeling was intensified in both chloroplasts and mitochondria of PVY-infected, RDR1-silenced plants. By contrast, heterochromatin condensation was reduced by PVY infection, and in non-transgenic plants the nuclei were translucent and lacked morphology after PVY infection. Thus, RDR1 regulates gene expression leading to remodeling of chromosomes, and PVY infection counteracts these effects on chromosomal remodeling.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSsS189R) and the other in a non-Trp/GH3 motif (NSsL172R). Tobacco rattle virus (TRV) expressing NSsS189R enhanced the PCD response, but not TRV-NSsL172R, while RNA silencing suppression activity was lost in TRV-NSsL172R, but not in TRV-NSsS189R. Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.

Corrigendum: Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants.

[This corrects the article on p. 903 in vol. 6, PMID: 26579153.].