Two TOBAMOVIRUS MULTIPLICATION 2A homologs in tobacco control asymptomatic response to tobacco mosaic virus

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom—in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1–TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Tobacco TOBAMOVIRUS MULTIPLICATION 2A homologs control the asymptomatic response to tobacco mosaic virus and have highly conserved biological functions related to virus multiplication.     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes

The TOM1 gene of Arabidopsis thaliana encodes a putative multipass transmembrane protein which is necessary for the efficient multiplication of tobamoviruses. We have previously shown that mutations severely destructive to the TOM1 gene reduce tobamovirus multiplication to low levels but do not impair it completely. In this report, we subjected one of the tom1 mutants (tom1-1) to another round of mutagenesis and isolated a new mutant which did not permit a detectable level of tobamovirus multiplication. In addition to tom1-1, this mutant carried a mutation referred to as tom3-1. Positional cloning showed that TOM3 was one of two TOM1-like genes in Arabidopsis. Based on the similarity between the amino acid sequences of TOM1 and TOM3, together with the results of a Sos recruitment assay suggesting that both TOM1 and TOM3 bind tobamovirus-encoded replication proteins, we propose that TOM1 and TOM3 play parallel and essential roles in the replication of tobamoviruses.     

Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1

The tom2-1 mutation of Arabidopsis thaliana reduces the efficiency of intracellular multiplication of tobamoviruses. The tom2-1 mutant was derived from fast-neutron-irradiated seeds, and the original mutant line also carries ttm1, a dominant modifier that increases tobamovirus multiplication efficiency in a tobamovirus-strain-specific manner in the tom2-1 genetic background. Here, we show that the tom2-1 mutation involved a deletion of approximately 20 kb in the nuclear genome. The deleted region included two genes named TOM2A and TOM2B that were both associated with the tom2-1 phenotype, whereas ttm1 corresponded to the translocation of part of the deleted region that included intact TOM2B but not TOM2A. TOM2A encodes a 280 amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, while TOM2B encodes a 122 amino acid basic protein. The split-ubiquitin assay demonstrated an interaction of TOM2A both with itself and with TOM1, an integral membrane protein of A.thaliana presumed to be an essential constituent of tobamovirus replication complex. The data presented here suggest that TOM2A is also an integral part of the tobamovirus replication complex.     

Alpha-momorcharin enhances Nicotiana benthamiana resistance to tobacco mosaic virus infection through modulation of reactive oxygen species

Alpha-momorcharin (α-MMC), a member of the plant ribosomal inactivating proteins (RIPs) family, has been proven to exhibit important biological properties in animals, including antiviral, antimicrobial, and antitumour activities. However, the mechanism by which α-MMC increases plant resistance to viral infections remains unclear. To study the effect of α-MMC on plant viral defence and how α-MMC increases plant resistance to viruses, recombinant DNA and transgenic technologies were employed to investigate the role of α-MMC in Nicotiana benthamiana resistance to tobacco mosaic virus (TMV) infection. Treatment with α-MMC produced through DNA recombinant technology or overexpression of α-MMC mediated by transgenic technology alleviated TMV-induced oxidative damage and reduced the accumulation of reactive oxygen species (ROS) during TMV-green fluorescent protein infection of N. benthamiana. There was a significant decrease in TMV replication in the upper leaves following local α-MMC treatment and in α-MMC-overexpressing plants relative to control plants. These results suggest that application or overexpression of α-MMC in N. benthamiana increases resistance to TMV infection. Finally, our results showed that overexpression of α-MMC up-regulated the expression of ROS scavenging-related genes. α-MMC confers resistance to TMV infection by means of modulating ROS homeostasis through controlling the expression of antioxidant enzyme-encoding genes. Overall, our study revealed a new crosstalk mechanism between α-MMC and ROS during resistance to viral infection and provides a framework to understand the molecular mechanisms of α-MMC in plant defence against viral pathogens.     

Primitive Defence: The MiAMP1 Antimicrobial Peptide Family

The founder of the MiAMP1 protein family was originally isolated from Macadamia integrifolia and had antimicrobial activity in vitro. MiAMP1 was the first plant protein with a structure containing a βγ-crystallin precursor fold, a structural superfamily associated with antimicrobial proteins in other kingdoms. In recent times, expanding plant genomics information has revealed that genes encoding homologues of MiAMP1 are conserved across the plant kingdom from lycophytes, gymnosperms to early angiosperms (e.g. Amborella, Papaver) and some monocots (e.g. Zantedeschia, Zea, Sorghum). Many studies of plant–pathogen interactions in gymnosperms have demonstrated a potential role for MiAMP1 family members in defence against fungal pathogens. This commentary describes the discovery and diversity of this protein family and considers current evidence supporting, and future opportunities for substantiating, a role in defence in primitive plants, and why this role may have diminished in higher plants.     

Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.     

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.     

Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-22 resistance gene

Tomato cultivars containing the Tm-2² resistance gene have been widely known to resist tobacco mosaic virus (TMV) and tomato mosaic virus. Tomato brown rugose fruit virus (ToBRFV), a new emerging tobamovirus, can infect tomato plants carrying the Tm-2² gene. However, the virulence determinant of ToBRFV that overcomes the resistance conferred by the Tm-2² gene remains unclear. In this study, we substituted the movement protein (MP) encoding sequences between ToBRFV and TMV infectious clones and conducted infectivity assays. The results showed that MP was the virulence determinant for ToBRFV to infect Tm-2² transgenic Nicotiana benthamiana plants and Tm-2²-carrying tomato plants. A TMV MP chimera with amino acid residues 60–186 of ToBRFV MP failed to induce hypersensitive cell death in the leaves of Tm-2² transgenic N. benthamiana plants. Chimeric TMV containing residues 60–186 of ToBRFV MP could, but chimeric ToBRFV containing 61–187 residues of TMV MP failed to infect Tm-2² transgenic N. benthamiana plants, indicating that 60–186 residues of MP were important for ToBRFV to overcome Tm-2² gene-mediated resistance. Further analysis showed that six amino acid residues, H⁶⁷, N¹²⁵, K¹²⁹, A¹³⁴, I¹⁴⁷, and I¹⁶⁸ of ToBRFV MP, were critical in overcoming Tm-2²-mediated resistance in transgenic N. benthamiana plants and tomato plants. These results increase our understanding of the mechanism by which ToBRFV overcomes Tm-2²-mediated resistance.     

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant‐pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface‐localized receptor Ve1 confers race‐specific resistance against race 1 strains of the soil‐borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.     

Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component

Virus‐induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite‐like and single‐stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication‐associated protein open reading frame and the A‐rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co‐agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co‐agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene‐silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.     

Phytophthora capsici CBM1-containing protein CBP3 is an apoplastic effector with plant immunity-inducing activity

Carbohydrate-binding module family 1 (CBM1) is a cellulose-binding domain that is almost exclusively found in fungi and oomycetes. CBM1-containing proteins (CBPs) have diverse domain architectures and play pivotal roles in the plant–microbe interaction. However, only a few CBPs have been functionally investigated. In this study, we identified PcCBP3 in an oomycete pathogen, Phytophthora capsici. PcCBP3 contains two tandem CBM1 domains and its orthologs from other Phytophthora species exhibit diversity including gene loss, pseudogenization, variations in sequences, and domain structures. PcCBP3 is upregulated during infection and knockout of PcCBP3 results in significantly decreased virulence. Moreover, PcCBP3 requires signal peptide to induce BAK1-dependent cell death in Nicotiana benthamiana. Further studies indicate that PcCBP3-triggered cell death and plant immunity require its N-terminal region, which is conserved in CBM1-containing proteins and other small, secreted, cysteine-rich protein from oomycetes. These results suggest that PcCBP3 is an apoplastic effector and could be perceived by the plant immune system.     

Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-2² and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

Editing of host susceptibility genes in tomato confers strong resistance against an emerging virus capable of overcoming currently available resistance genes.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.     

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE‐containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.     

Activation of Tm-22 resistance is mediated by a conserved cysteine essential for tobacco mosaic virus movement

The tomato Tm-2² gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm-2², damaging tomato production worldwide. Tm-2² encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP^ToBRFV) enabled the virus to overcome Tm-2²-mediated resistance. Yet, it was unknown how Tm-2² remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MP^TMV) plays a dual role in Tm-2² activation and viral movement. Substitution of MP^ToBRFV amino acid H67 with the corresponding amino acid in MP^TMV (C68) activated Tm-2²-mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-2² immune activation could explain how TMV was unable to overcome this resistance for such a long period.     

Glutathione contributes to resistance responses to TMV through a differential modulation of salicylic acid and reactive oxygen species

Systemic acquired resistance (SAR) is induced by pathogens and confers protection against a broad range of pathogens. Several SAR signals have been characterized, but the nature of the other unknown signalling by small metabolites in SAR remains unclear. Glutathione (GSH) has long been implicated in the defence reaction against biotic stress. However, the mechanism that GSH increases plant tolerance against virus infection is not entirely known. Here, a combination of a chemical, virus-induced gene-silencing-based genetics approach, and transgenic technology was undertaken to investigate the role of GSH in plant viral resistance in Nicotiana benthamiana. Tobacco mosaic virus (TMV) infection results in increasing the expression of GSH biosynthesis genes NbECS and NbGS, and GSH content. Silencing of NbECS or NbGS accelerated oxidative damage, increased accumulation of reactive oxygen species (ROS), compromised plant resistance to TMV, and suppressed the salicylic acid (SA)-mediated signalling pathway. Application of GSH or l -2-oxothiazolidine-4-carboxylic acid (a GSH activator) alleviated oxidative damage, decreased accumulation of ROS, elevated plant local and systemic resistance, enhanced the SA-mediated signalling pathway, and increased the expression of ROS scavenging-related genes. However, treatment with buthionine sulfoximine (a GSH inhibitor) accelerated oxidative damage, elevated ROS accumulation, compromised plant systemic resistance, suppressed the SA-mediated signalling pathway, and reduced the expression of ROS-regulating genes. Overexpression of NbECS reduced oxidative damage, decreased accumulation of ROS, increased resistance to TMV, activated the SA-mediated signalling pathway, and increased the expression of the ROS scavenging-related genes. We present molecular evidence suggesting GSH is essential for both local and systemic resistance of N. benthamiana to TMV through a differential modulation of SA and ROS.