Directions from Nature: How to Halt the Tomato Brown Rugose Fruit Virus

Abstract

Tomato brown rugose fruit virus (ToBRFV) is a recently emerged serious viral threat to tomato production. The virus is named after its symptoms consisting of characteristic brown wrinkled (rugose) patches on the fruits of infected tomato plants. ToBRFV is a member of the genus Tobamovirus and a very stable mechanically transmitted virus. So far, most tomato cultivars are susceptible, enabling a swift spread of ToBRFV. In this review, we present strategies to halt devastating disease outbreaks of ToBRFV based on the collective research data of various tobamovirus–plant interactions. Viruses, like ToBRFV, are biotrophic pathogens with small genomes. Hence viral proliferation depends on various host factors, also termed susceptibility (S) genes. However, S genes often have an intrinsic function for the host plant. Thus, mutations in S genes may lead to pleiotropic phenotypes. Therefore, identifying mutant variants of S genes with no pleiotropic effects is essential for exploring impaired S genes in breeding tomatoes resistant to ToBRFV.

1. Introduction: Tomato Crop Model for Viral Immunity

Tomato (Solanum lycopersicum L.) has become one of the most important and extensively grown fruit/vegetable crops, with global production increasing by over 49 million tonnes between 2000 and 2019 [1]. Tomato belongs to the dicot Solanaceae family. Solanaceous plants share a high degree of sequence similarity, and this enables comparative genetic studies [2]. Other major crop plants within this family include potato, pepper, eggplant, tobacco, and petunia [3].

Tomato is a model crop for genetic research because the genome sequence is available, it can easily be transformed, and interspecific crosses can be generated between tomato cultivars and many wild relative Solanum species. Tomato is also a model plant for studying immunity because it is susceptible to a wide range of pathogens, including viruses. At least 312 viruses, satellite viruses, or viroid species in 22 families and 39 genera are associated with tomatoes [4]. This is likely the highest number of recorded viral and related species in a single crop. However, we should consider that this number could still increase because viral interactions are intensively studied and monitored in tomatoes. Moreover, before discussing in more detail different tomato factors that play a role during tomato–viral interactions, we will introduce tomato brown rugose fruit virus (ToBRFV) because, as stated by Johanna Westerdijk in 1917: ‘knowledge of a disease and the way to fight it, must be based on an understanding of the physiology of both the host plant and the parasite’. In this review, we discuss aspects of the tobamovirus–plant interaction that could potentially lead to ToBRFV-resistant tomato genotypes.

2. ToBRFV Is a Tobamovirus

A virus consists of one or a few small nucleic acid molecules, generally protected by a protein coat, that can only multiply within the living cells of a host. Moreover, since viruses are so small, it took decades before scientific consensus agreed they existed.

2.1. Tobamoviruses Are the First Described Viruses

Around 1900, viruses were identified by three pioneers in plant pathology [5,6]. Adolf Mayer described the tobacco mosaic disease and found that the disease could be transferred between plants. Dmitri Ivanoysky and Martinus Beijerinck independently showed that the infectious agent of tobacco mosaic disease is within the filtrate of infected plant sap. Beijerinck named the agent that could replicate and multiply in living plants as “contagium vivum fluidum” (contagious living fluid) and the new pathogen virus (liquid poison) to specify its non-bacterial nature [7]. In 1939 the first electron micrographs of a virus, tobacco mosaic virus (TMV), were produced by Gustav Kausche, Edgar Pfankuch, and Helmut Ruska (reviewed by [5]). In 1958 Rosalind Franklin speculated that TMV was not solid but hollow and carried a single-stranded RNA molecule [8]. Consequently, the discovery of viruses, pathogens with a hitherto unknown lifestyle, was made by three plant pathologists who wanted to halt the tobacco mosaic disease. They were assisted by fellow scientists who could visualize the viral particles and their content.

2.2. Pathogenic Tomato Viruses

The tomato crop is susceptible to many different plant viruses. Viruses can be classified into seven “Baltimore classes” (I-VII) depending on their genome and how messenger RNA is generated during viral genome replication. A viral genome can be double-stranded (ds)DNA (I), single-stranded (ss)DNA (II), dsRNA (III), positive ssRNA (IV), negative ssRNA (V), RNA reverse-transcribing viruses with positive RNA (VI), and DNA reverse-transcribing viruses with dsDNA or RNA-DNA (VII) [9]. Pathogenic tomato viruses belong to several classes. They are transmitted and spread in different ways. The economically most important viruses for tomato crops are transmitted by whiteflies (e.g., members of the genera Begomovirus, Crinivirus, Torradovirus), aphids (members of the genera Potyvirus, Cucumovirus, Alfamovirus), and thrips (Orthotospovirus) [10]. However, members of the genus Tobamovirus (Baltimore class IV), such as ToBRFV, tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV), are not transmitted by insect vectors but by contact and in low percentages via seed [11,12].

2.3. The Genome Organisation of ToBRFV

The ssRNA(+) genome of ToBRFV is about 6400 nucleotides in length containing four open reading frames (ORF) (Figure 1). ORF1 and ORF2 encode two replication (REP)-associated proteins of ~126 kDa and ~183 kDa. The latter protein is synthesized by readthrough of the ~126 kDa protein ORF. ORF3 encodes a movement protein (MP) of ~30 kDa and ORF4 a coat protein (CP) of ~17.5 kDa. ORF3 and ORF4 are translated from subgenomic RNAs. Since ToBRFV only encodes a limited number of proteins, it is expected that these viral proteins have multiple functions to establish viral proliferation.

Comparison of the tomato-infecting tobamoviruses TMV, ToMV, ToMMV, and ToBRFV genome sequences showed that their sequences are very similar, with approximately 90% nucleotide (nt) identity and the concatenated ORF amino acid sequence with approximately 80% amino acid (aa) identity (Figure 2). The relationships among the different tobamoviral species were determined by comparison of complete concatenated ORF amino acid sequences and shown in a phylogenetic tree (on the left in Figure 2) [13]. Our phylogenetic analysis of nucleotide and amino acid levels indicates that ToBRFV-IL is most similar to TMV (91.58% identity on nucleotide and 81.57% identity on amino acid levels).

3. ToBRFV Spread, Symptoms, and Host Range

Tobamoviruses spread mechanically, and viral particles enter the plant cells after wounding the epidermal cells. Therefore, the virus will spread rapidly with all plant handling, and a gentle touch is already sufficient as virus concentrations are very high even in trichomes [14]. Bumblebees can transmit the virus, but they are not considered vectors as the mode of transmission is mechanical [15]. The presence of ToBRFV in tomatoes was first reported in the fall of 2014 in southern Israel and during the spring of 2015 in Jordan. Both virus isolates were similar and termed corresponding to the disease symptoms “tomato brown rugose fruit virus” (ToBRFV) [16,17]. Subsequently, ToBRFV was detected and reported (between 2018 and 2023) on tomatoes and other plants in more than 25 countries all over the world [18].

3.1. ToBRFV Disease Symptoms in Tomato

The ToBRFV-caused symptoms on tomatoes are similar to those caused by TMV, ToMV, and ToMMV. They are not easily distinguished from other viral tomato symptoms, e.g., cucumber mosaic virus (CMV), or even herbicide effects [19,20]. ToBRFV disease symptoms in tomatoes can be severe. The most obvious ones are curling, malformation of the leaves (shoestring or fern leaf), and stunting of the whole plant (Figure 3). Additional symptoms on the leaves are mosaic discoloration, mottling, and necrosis. Deformation and necrosis can also occur on the stem. In Figure 3, the bottom left panel shows deformation and anthocyanin accumulation on the stem. Additionally, ToBRFV infection reduces fruit number due to early flower abortion and reduced fruit size. Figure 3, bottom right panel, shows the reduced fruit number of Micro-Tom plants. Mock-treated Micro-Tom plants yielded an average of 55 fruits per plant, while ToBRFV-infected plants yielded 22.4.

Although the disease is termed tomato brown rugose fruit virus, neither discoloration (marbling) nor brown wrinkled (rugose) patches could be detected on Micro-Tom or Moneymaker fruits, whether ToBRFV inoculation was performed on 10-day-old seedlings or mature plants just before fruit set under greenhouse conditions. However, others detected fruits with marbling, yellow patches, brown rugosity, and deformation symptoms on tomato cultivars Piccolo, Kivu, and Moneymaker [21]. Symptom development can vary due to environmental growth conditions (temperature), age during viral infection initiation, and the genetic background of the tomato plant [22]. In addition, as expected, when ToBRFV is present in a greenhouse compartment, the virus swiftly spreads. Two ToBRFV-contaminated tomato plants, corresponding to <0.5% of the total crop arsenal, could contaminate the entire tomato crop (98.96%) under normal cultivation procedures within 9 months [23].

3.2. Additional Host Plants of ToBRFV

In addition to tomatoes, ToBRFV has been reported in sweet pepper plants in Jordan and Sicily (Italy) [16,24]. The host range of ToBRFV was evaluated for 31 plant species from seven families, and 20 plant species (encompassing four families) showed local symptoms or the presence of ToBRFV in systemic leaves [25]. The various host plants include petunia, pepper, tobacco, and tomato (from the Solanaceae), globe amaranth, quinoa and lamb’s quarters (from Amaranthaceae), rosy periwinkle (from Apocynaceae), lilac tassel flower and crown daisy (from Asteraceae) [25]. Probably these 20 plant species and many of the thus far unidentified host plants will never be tested for the presence of ToBRFV either because ToBRFV infection causes only mild symptoms or because the host plant has no economic impact. However, the various ornamental species and weeds can still facilitate ToBRFV viral spread.

3.3. Preventing ToBRFV Distribution

To better understand the worldwide distribution of ToBRFV, all countries should investigate and report the absence or presence of ToBRFV in all known host plants, not only tomatoes. Obviously, this is not a realistic task, and eliminating ToBRFV by eradicating all the infected host plants is impossible. The current geographical overview of ToBRFV expansion solely based on reported occurrences does not reflect the true global spreading of the virus. Possibly, measuring ToBRFV in waste water (all over the world), similar to the detection of the COVID-19 virus, and indicating positive locations on the world map would give a more realistic overview.

To halt the spreading of ToBRFV and the possibility of new ToBRFV variants evolving, tomato cultivars with durable ToBRFV resistance are required. Although ToBRFV is a recently emerged virus species, its high genomic similarity to the three tobamoviruses TMV, ToMV, and ToMMV (Figure 2) is helpful in deducing the biological function of the viral proteins and how they can establish disease. Furthermore, the extensive knowledge and comparable life style among the different tomato tobamoviruses can help us to combat the tomato brown rugose fruit disease in tomato. Four strategies that can be utilized by the plant as a defense against viral disease are given in the next paragraph.

4. Viral Disease Resistance Strategies in Plants

Four well-known strategies that plants use to become resistant against plant viruses are (1) RNA silencing mediated by the plant, (2) the presence of dominant plant resistance proteins (R-proteins), (3) plant hormone-mediated resistance, and (4) absence of a functional plant susceptibility factors (S-factors). Resistance strategies can differ in expected durability, and a strategy with high durability (+++) is preferred. Simultaneously, a specific strategy can have a negative impact on plant physiology and lead to pleiotropy, and a strategy with a low pleiotropic likelihood (−) is preferred. The durability of the pleiotropic effect on the plant as a result of the chosen resistance strategy is based on current knowledge and discussed for each strategy.

4.1. ToBRFV Resistance Conferred by Host-Based Viral RNA Silencing

RNA silencing encompasses the plant’s first layer of viral defense (Figure 4, box 1). Viral RNA is converted into viral double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase encoded by the plant. Similar to Pathogen Associated Molecular Patterns (PAMPS), viral dsRNA can be considered a Virus-Associated Molecular Pattern (VAMP). The viral dsRNA is recognized by Dicer-like (DCL) plant enzymes, and DCLs process viral dsRNA into virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are incorporated in the RNA silencing complex (RISC), where single vsiRNA strands act as RNA guides for viral RNA silencing (reviewed by [26]). Mutations in genes that play a role in the plant RNA silencing mechanism often have a significant impact on plant physiology because the plant RNA silencing mechanism plays a key role in plant development [27], indicated by a single + for the likelihood of pleiotropy in Figure 4 beside box 1. In addition, successful plant viruses express viral suppressors of RNA silencing (VSRs) that attenuate or block this defense. VSRs are produced from the first ORF of tobamoviruses and are part of the 126-kDa replicase (REP) protein (top horizontal red line in Figure 1) [28]. The 126-kDa protein (P126) consists of four parts, methyltransferase, helicase, and two non-conserved regions, I and II (Figure 1). The methyltransferase, helicase, and non-conserved region II each possess RNA silencing-suppression activity [29].

VSRs target various factors of the silencing pathway. P126 of TMV interferes with HEN1-mediated methylation and accumulation of novel miRNAs, while P130 of ToMV blocks the siRNA accumulation. Crucifer-infecting-TMV P122 binds to siRNAs and miRNAs, thereby preventing their incorporation into RISC, and also enhances AGO1 downregulation via miR168 upregulation [28,30]. The production of P126 as the first viral protein with a combined function of replication and VSR is a perfect viral survival strategy since the viral genome is most exposed to silencing-mediated RNA degradation during its replication. Therefore, due to the production of VSRs, the strategy of RNA silencing mediated by the plant is expected not to be highly durable (indicated by a single + beside box 1 in Figure 4).

4.2. ToBRFV Resistance Conferred by Host Resistance (R) Proteins

Viral proliferation can also be inhibited by a host R protein-mediated defense response, the second strategy (Figure 4). R genes are usually dominant genes that provide full or partial resistance to pathogens [31]. Most plant R proteins are encoded by nucleotide-binding leucine-rich repeat receptor (NLR) genes. More than 200 NLR genes identified from different plant species can prevent viral proliferation [32]. Dominant R proteins directly or, in most cases, indirectly sense and interact with a viral protein, also termed effector [32] (Figure 4, box 2). The sensing or recognition between NLRs and their cognate viral protein usually triggers rapid localized cell death, also termed the hypersensitive response (HR), indicated by the red star in Figure 4, box 2. Two NLR encoding R proteins for resistance against tobamoviruses were identified, the N protein from tobacco yielding resistance to TMV and Tm-2/Tm-2² from tomato yielding resistance to ToMV and TMV [33,34]. However, not all viral dominant R proteins code for NLRs. The tomato Tm-1 R protein against TMV has a triosephosphate isomerase (TIM)-barrel-like domain and unknown function [35]; for several R proteins against viruses, the sensed or interacting viral protein that leads to resistance is known. The Tm-2/Tm-2² protein detects the MP of TMV and ToMV. Detection or sensing of the helicase domain from the TMV REP protein by the host N and/or Tm-1 protein results in resistance (reviewed by [32]). Although R proteins are successfully used to halt viral proliferation and prevent disease, the achieved resistance is mostly only effective against one viral species. In addition, viral genomes have a high mutation frequency due to their rapid replication rate and generation of large populations [36]. Viruses with RNA genomes, such as ToBRFV, are expected to have the highest mutation frequency since RNA polymerases lack proofreading activity [36]. Another source of genetic variation is caused by recombination between two viral species, which is known to occur when multiple viral species are present in a single host plant [36]. The high genetic variability of tobamoviruses potentially leads to changes in viral proteins that overcome the R-protein-based resistance due to the inability to sense the altered viral protein. For example, initially, the two allelic R proteins Tm-2 and Tm-2² were successfully exploited to halt ToMV viral disease in tomatoes. But in 1993, two ToMV isolates (from Japan and Europe) broke the Tm-2² resistance [37]. These ToMV isolates had three amino acid changes in their MP [37,38]. Furthermore, currently, none of the three dominant resistance genes Tm-1, Tm-2/Tm-2² used in tomato cultivars provide complete resistance to ToBRFV disease [39].

4.3. Search for Additional ToBRFV Host Resistance (R) Proteins

Currently, wild tomato accessions are explored to identify additional/novel R proteins against ToBRFV. Multiple ToBRFV resistance traits are described in patents by various breeding companies. A dominant ToBRFV resistance trait conferred by a CC-NBS-LRR gene was found on chromosome 8 of the S. habrochaites LYC4943 accession [40]. A recessive ToBRFV resistance trait was found on chromosome 11 from S. pimpinellifolium accession PI79532 (LA2348) [41]. Patent WO2019110130 describes a polygenic ToBRFV resistance trait based on three loci (located on chromosomes 6, 11, and 12) from three different S. pimpinellifolium accessions [42]. Interestingly, some identified novel ToBRFV resistance traits involve alleles and loci of the previously ‘broken’ Tm genes. Zinger et al. (2021) reported a tomato genotype resistant to ToBRFV. The ToBRFV resistance of this accession depends on a locus on chromosome 2 that includes the Tm-1 gene, in combination with a locus on chromosome 11 (associated with tolerance to ToBRFV) [43]. In addition, the presence of the Tm-1 locus with at least one of the two recessive loci on chromosome 9 (fruit tolerance) and chromosome 11 (foliar tolerance) can lead to resistant plants [44]. Finally, a recent patent describes a gain-of-function in resistance to ToBRFV and ToMV by amino acid changes in the protein sequence of Tm-2² [45]. The recent claims and findings on novel ToBRFV-resistant sources indicate the great desire to halt this pathogen in tomatoes.

Although the novel identified R genes lead to ToBRFV-resistant tomato cultivars, the occurrence of resistance-breaking viral isolates and newly evolving species, as seen in the past, is likely to occur (also illustrated by a single plus (+) for durability in Figure 4). One benefit of this strategy is that the R proteins-based resistance is widely used and rarely leads to pleiotropy (indicated by a minus (−) for pleiotropy in Figure 4 beside box 2). Therefore, a good approach would be to stack multiple R genes within a single tomato cultivar. The application of multiple R proteins will increase the durability of the disease resistance significantly. The disadvantage of this strategy is that the introgression of multiple R genes in each cultivar requires a lot of time and effort.

4.4. Hormone-Based ToBRFV Resistance

Another complex viral resistance strategy is mediated by hormone-based resistance (box 3 in Figure 4). The complexity is caused by the crosstalk between different hormone signaling pathways [46]. Crosstalk refers to the phenomenon that different hormones regulate each other; e.g., the accumulation of salicylic acid (SA) can cause a reduction in jasmonic acid (JA). Additionally, various hormones are affected by the viral presence, as viruses hijack host components to deregulate the plant hormone production in order to proliferate. Zhao and Li [39] composed a graphical summary of six different viral outcomes/effects (replication, accumulation, symptom development, virus movement, host resistance, and insect vector relationship) by eight different hormones, including SA, JA, ethylene (ET), abscisic acid (ABA), gibberellic acid (GA), auxin, cytokinin and brassinosteroids (BRs) for 19 plant viruses. Their main conclusion is that changes in hormone levels are tightly coordinated with viral movement, replication, symptom development, and defense responses and directly affect viral disease outcomes. ABA accumulation has a negative effect on TMV accumulation, movement, and symptom development. The induction of ABA signaling causes a down-regulation of callose-degrading enzymes, resulting in callose accumulation at the plasmodesmata (PD), which leads to restricted viral cell-to-cell movement [47]. ET accumulation has a negative effect on TMV accumulation and symptom development but a positive effect on the accumulation of viral crucifer-infecting tobacco mosaic virus (TMV-cg) [46]. SA accumulation has a negative effect on TMV and ToMV accumulation [46]. In addition, exogenous application of SA, JA, or a combination resulted in reduced levels of TMV in N. benthamiana plants. Therefore enhanced SA and JA levels possibly activate systemically induced defense in tobacco leaves against TMV [48]. Exogenous application of BR in tobacco also enhanced resistance to TMV, independent of SA accumulation [49]. BRs modulate plant–pathogen interactions, but depending on the involved plant species and the pathogen’s lifestyle, BRs induce resistance or susceptibility. The presence of BR induces resistance to most biotrophic pathogens and susceptibility to necrotrophic and hemibiotrophic pathogens. Importantly, BRs do not always enhance plant viral defense, as there is evidence that BRs induce viral susceptibility [50].

Overall, resistance based on altered hormone levels can lead to durable virus resistance, and this strategy is marked by ++ behind box 3 in Figure 4. Simultaneously, hormones, especially the balance between hormones, influence all stages of the tomato composite leaf development [51]. Therefore, the predicted effect on pleiotropy is indicated with ++ beside box 3 in Figure 4. Remarkably, the shoestring/fern leaf symptoms on the ToBRFV-infected tomato leaves (Figure 3) resemble a phenotype caused by hormonal imbalance in non-infected tomato plants.

4.5. ToBRFV Resistance Based on Dysfunctional Host Susceptibility (S) Proteins

Viral proliferation depends on the presence of a considerable number of host susceptibility (S-) factors. Therefore, the fourth resistance strategy (box 4 in Figure 4) depends on the absence of crucial S-factor(s). The absence of some host S-factors can prevent or inhibit viral proliferation. Therefore, this type of resistance is expected to be highly durable, illustrated by +++ in Figure 4 beside box 4. However, this may be accompanied by slight to severe pleiotropic effects on plant morphology and fitness, marked by ++ in Figure 4. In the next section of this review, we will discuss why we consider the S-factor-based viral resistance strategy as the preferred strategy to achieve durable ToBRFV resistance.

Until now, at least 115 plant host factors that could affect viral proliferation for at least 52 different viral species have been identified (based on current literature studies). Thirty-eight S-factors that could or are proven to affect tobamoviral proliferation, including references, are presented in Supplemental Table S1. In the next section, we discuss the molecular role of 11 plant host factors (indicated in bold in Supplemental Table S1) in more detail and in what manner they are instrumental for tobamovirus viral proliferation (also illustrated in Figure 5). Obviously, plant host factors that are nonessential for the fitness of the host are preferred; these would be scored as—for pleiotropy.

5. Molecular Factors of Plant Host and Tobamoviruses That Are Instrumental for Viral Proliferation

To make maximal use of their limited genomes, viruses exploit the machinery and metabolism of a living host cell for their life cycle. The viral proliferation of tobamoviruses can be divided into three stages: (1) entrance, (2) replication and translation, and (3) movement and spread through the plant vascular tissue (Figure 5) [52].

For each stage, viral and plant proteins are expected to collectively contribute to viral proliferation. As the life cycle of ToBRFV conceivably resembles that of TMV, based on the genome structure and sequence similarity (Figure 1 and Figure 2), it is likely that ToBRFV exploits similar host cell components as TMV (or ToMV and ToMMV) to cause disease. Therefore, in this review, we propose the host factors required for TMV and/or ToMV proliferation as the host factors for ToBRFV proliferation [52]. Figure 5 illustrates the three stages of the ToBRFV life cycle in the plant and how virus and plant proteins together could contribute to enhancing tobamovirus viral proliferation. Due to the mechanical transmission of tobamoviruses, most host factors are utilized by the virus during the second and third stages. Plant proteins that associate with the viral REP play a key role during the second stage, and those that associate with viral MP or CP are more relevant during the third stage. Obviously, as research continues, the depicted interactions and locations are not set in stone, and additional viral and plant proteins that take part in the ToBRFV proliferation remain to be discovered.

5.1. Stage 1: Viral Entrance

Plant cells have cell walls that act as the first barrier against the invasion of pathogens. To overcome this, many plant viruses have to take advantage of different biological vectors or physical damage (wounding) of the plant tissue caused by environmental stresses to enter plant cells. For example, ToBRFV enters the host cell through mechanical wounding of the plant tissue, which either transiently opens the plasma membrane (PM) or allows pinocytosis (endocytosis of fluids) [52]. Therefore, no host proteins are likely required for tobamoviral entrance or are not identified yet (only hypothetical proteins are illustrated in Figure 5). A putative role for the viral CP that interacts with a plant protein could be envisioned, but thus far entrance of tobamoviruses is known to rely only on a damaged cell.

5.2. Stage 2: Viral Replication and Translation

For the second stage, replication and translation, the REP proteins, REP-126-130 kDa and REP-180-183 kDa, play a central role. The viral REP proteins (indicated by red and orange circles in Figure 5) can interact directly or indirectly with host proteins to build the tobamovirus replication complex. Figure 5 depicts a set of plant proteins that directly interact and/or are part of the replication complex.

Details for each plant protein and how it might function in the replication complex and enhance viral replication and translation are provided below.

ARL8 (ADP-ribosylation factor-like 8, blue crescent shape in Figure 5) is a GTPase highly conserved in eukaryotic cells from animals to plants. The 180-kDa REP protein of ToMV interacts with ARL8 from Nicotiana tabacum, and the absence of the ARL8 protein in Arabidopsis inhibits intracellular TMV and ToMV virus multiplication [53]. Arabidopsis has two ARL8 homologs, and only a double knock-out of both AtARL8 genes led to the inhibition of TMV and ToMV viral proliferation [53]. Tomato has four ARL8 homologs, and solely knocking out SlARL8a was insufficient to reduce ToBRFV viral proliferation [54]. How the absence of ARL8 could lead to viral resistance is still unknown. Research in human cells showed that ARL8b regulates lysosomal motility and the fusion of lysosomes with late endosomes [55,56,57]. Lysosomes are organelles needed for the clearing and recycling of cellular components, also known as autophagy. Depending on the plant-virus pathogenesis system, autophagy can function as an antiviral mechanism or promote viral infection [58,59]. A putative role of autophagy in viral proliferation and whether viral S genes take part in the molecular mechanism of autophagy will be further discussed in Section 5.4.

PAP85 (Pokeweed antiviral protein, green rhombus shape in Figure 5) was originally identified in field beans (Vicia faba L.) as vicilin, a 7S seed globulin representing approximately 30% of the storage protein in mature seeds [60]. Remarkably, vicilins are mostly reported as defensive proteins against fungi and insects, probably due to their capacity to bind chitin [61,62]. In Arabidopsis, PAP85 is involved in membrane modification for transportation, and its transcript accumulates during the last stage of silique development [63]. Arabidopsis PAP85 interacts with the 126-kDa REP protein of TMV at the endoplasmic reticulum (ER) [64]. The interaction between PAP85 and P126 results in ER transition, formation, and transfer of small vesicles from ER to Golgi early in the infection process [64]. In general, newly synthesized proteins and lipids are transported from the ER to the PM via the Golgi apparatus. Viruses are known to hijack the secretory pathway that leads to membrane rearrangements for their life cycle (reviewed by [65]). Presumably, tobamoviruses exploit PAP85 for this function.

SYP22 and SYP23 (Figure 5, blue pentagon arrow shape) are also important for vesicle trafficking and interact with the methyltransferase domain of the 126-kDa REP TMV protein. SYP proteins are soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) [66]. The presence of SYP22 and/or SYP23 is necessary for TMV accumulation [67]. SNAREs associate with membranes and mediate the fusion of vesicle membranes with target membranes. SNARE proteins form highly stable protein–protein interactions, a SNARE complex. SNAREs can be classified according to their subcellular localization, t (target)- and v (vesicle)-SNAREs, or to their structure, with the presence of a conserved glutamine (Q) or arginine (R) residue in the center of the SNARE domain [68]. t-SNAREs have a Q residue and are termed Q-SNARE, and v-SNAREs have an R and are termed R-SNARE. A functional SNARE complex consists of one set of Qa-, Qb-, Qc-, and R-SNARE proteins with a four-helical bundle assembled by four SNARE motifs. The three types of Q-SNAREs, Qa, Qb, and Qc, are determined by their position in the SNARE complex [68]. The SYP2 SNARE subfamily (SYP21, SYP22, and SYP23) belongs to the Qa-SNAREs. In general, a Qa-SNARE is the core SNARE protein that regulates the formation of a SNARE complex at the target membrane. In Arabidopsis, the functions of SYP21, SYP22, and SYP23 are expected to be redundant and interchangeable [69,70]. Vesicle transport mediated by SYP22 is important for shoot gravitropism and morphogenesis. The predicted localization sites of SYP22 and SYP23 are cytosol, vacuole, or pre-vacuole compartment. SYP22 forms a SNARE complex with a v-SNARE, e.g., VAMP711, and the complex is localized to tonoplasts and the pre-vacuolar compartment [70]. SYP22 also interacts with R-SNARE VAMP727, and both bind to the PM-bound receptor BRASSINOSTEROID INSENSITIVE1 (BRI1), resulting in BR accumulation. BRs are plant-specific steroidal compounds essential for normal growth and development [50]. Accumulation of BR enhanced plant defense, e.g., against root-knot nematodes [71,72]. Possibly, the absence of a functional SYP2 protein may lead to an imbalance of hormones leading to viral resistance.

Arabidopsis TOM1 (Tobamovirus multiplication 1, green star shape in Figure 5) and TOM3 (a paralog of TOM1) have been shown to interact with the tobamovirus helicase domain from the 130-kDa/180-kDa REP proteins [73,74]. The Arabidopsis TOM1/TOM3 double mutant completely suppressed TMV replication [73]. TOM1 and TOM3 are predicted to be seven-pass transmembrane proteins. Therefore TOM1/3 could form a link between the host membrane and the tobamovirus replication proteins [52,74,75]. The effectivity of TOM1 and TOM3 as S proteins for TMV has been shown for various plant species besides Arabidopsis, including C. annuum, N. benthamiana, and tomato [76,77]. In tomato, both paralogs TOM1 and TOM3 have two homologs. Recent data showed that a quadruple knock-out mutant of all four homologs in tomatoes displayed resistance to four tobamoviruses: TMV, ToMV, YMV, and ToBRFV [78]. Presumably, based on the phylogenetic tree (Figure 4), ToMMV accumulation will also be inhibited in the quadruple tomato mutant. However, when only three of the four homologs were mutated, the virus could still multiply. Moreover, in the triple knock-out tomato plants, mutant viruses emerged that were multiplying more efficiently than the wild-type ToBRFV [78].

Arabidopsis TOM2a (orange cross shape in Figure 5) interacts with itself and the TOM1 integral membrane protein [79]. Both the Arabidopsis tom2-1 mutant that lacks TOM2a, and the tobacco genotype TI203 containing a natural TOM2a variant, showed reduced tobamovirus viral accumulation [79,80]. In addition, tomato CRISPR-Cas9 knock-out mutants of TOM2a displayed enhanced resistance against TMV [80]. TOM2a encodes a 280-amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, i.e., CaaX motif, that undergoes farnesylation [79]. Farnesylation is a post-translational protein modification by which an isoprenyl group is added to a cysteine residue. Farnesylation mediates protein–protein interactions and protein–membrane interactions [81]. RNA viruses such as ToBRFV lack post-translational protein modification enzymes. Instead, they probably use the host to modify their proteins and/or post-transcriptionally modified host proteins (e.g., the TOM1–TOM2a complex) to allocate to the membrane to promote viral proliferation.

5.3. Stage 3: Viral Movement and Spread

For the third stage, viral movement and spread, plant viruses move from cell to cell via plasmodesmata (PD), indicated as short distance, and via the phloem (sieve elements), indicated as long distance (Figure 5). PD are unique membrane-lined cytoplasmic nanobridges that connect plant cells for cell-to-cell movement [82]. Due to studies on the role of viral MP in cell-to-cell movement, it was discovered that plant proteins could also move between cells via PD [83]. Microinjection of fluorescently-labeled viral MP revealed that MP proteins could increase the size exclusion limit of PD to facilitate the movement of viral RNA, MP, and other proteins to surrounding cells [83]. Six host factors that facilitate viral movement were selected and are depicted in Figure 5. Although most of the host proteins that facilitate the viral movement and spreading interact with the tobamoviral MP or CP protein, the REP from TMV was also shown to have a role in viral movement [84,85]. Most of the discussed proteins in this review interact directly with the viral MP (ANK, BAM1, PME, and SYTA), while LeT12 co-localizes with MP, and IP-L interacts with CP. The depicted proteins in Figure 5 are ANK (pink rectangle shape), BAM1 (blue semicircle shape), IP-L (orange pentagon shape), LeT12 (green crescent shape), PME (purple rhombus shape), and SYTA (oval red shape).

The plant ankyrin repeat-containing protein ANK probably promotes short-distance viral spread. The binding of the viral MP protein to the plant ANK protein results in the reduction of callose levels at the plasmodesmata, also termed callose sphincters. This causes the relaxation of the PD, which promotes viral cell-to-cell movement through the PD [86]. The ANK repeat domain-containing proteins comprise one of the largest known protein superfamilies in plants [87]. ANK proteins can mediate protein–protein interactions, and most plant ANK proteins play crucial roles in defense responses [87]. Still, ANKs’ role in viral proliferation is not well understood.

BAM1 (Barley any meristem 1) is a receptor-like kinase (RLK) from Arabidopsis that interacts with MP from TMV. Viral movement through PD by BAM1 is independent of its kinase activity [88]. Arabidopsis mutants bam1-3 and bam2-3 both showed reduced levels of TMV RNA accumulation when compared to wild-type plants at 6 days post-inoculation [88]. Interestingly, BAM1 and BAM2 were previously identified to interact with a small protein (C4) of tomato yellow leaf curl virus (TYLCV) at the PM and PD. TYLCV contains a DNA genome and is unrelated to tobamoviruses. BAM1 is required for the cell-to-cell spread of RNA silencing. In addition, in this case, the kinase activity of BAM1 does not seem to be required [89]—the targeting of BAM1 by C4 of TYLCV results in the suppression of intercellular spreading of RNA silencing. In C4-overexpressing transgenic tomato plants, four RLK genes (including BAM1 and BAM2) transcript levels were reduced. Therefore, the TYLCV C4 could compromise the RLK-mediated plant defense system. For tobamoviruses, it is not yet known whether the absence of BAM1 also causes reduced viral spread due to the reduction of cell-to-cell spread of RNA silencing or RLK-mediated plant defense.

The tobacco-interacting protein-L (IP-L) is a host protein interacting with the ToMV CP. IP-L was previously identified as an ‘elicitor-responsive protein’ gene [90]. For the protein–protein interaction, the N-terminal helical region of IP-L (155 aa) and two α-helical domains of ToMV CP are essential [91]. Both proteins, IP-L and ToMV CP, co-localize in the chloroplast thylakoid membranes [91]. This localization could explain the occurrence of chlorosis during viral infection because the interaction of ToMV CP with IP-L may affect chloroplast function and stability. Multiple studies of chloroplast protein–viral protein interactions have shown that the chloroplast is a common target of plant viruses for viral pathogenesis or propagation [92]. TMV infection induces the transcript levels of IP-L, and the reduction of IP-L transcripts in N. benthamiana resulted in delayed systemic ToMV symptoms at 7 days post-inoculation [90,93]. How IP-L supports viral proliferation is still unknown. For several viruses, the role of chloroplasts and associated proteins during viral proliferation has been described [94]. For example, the MP of ToMV and TMV interact with the small subunit of Rubisco (RbCS). This interaction takes place in the cytoplasm prior to the re-localization of RbCS to the chloroplasts of N. benthamiana. Silencing of NbRbCS in susceptible N. benthamiana plants enhanced local virus infectivity but delayed the development of systemic viral symptoms [95]. Additionally, NbRbCS silencing compromises Tm-2²-dependent resistance in transgenic Tm-2² N. benthamiana (ToMV-resistant) plants [95]. Thus, hijacking light-dependent or light-induced chloroplast factors by tobamovirus in plants can enhance local viral proliferation and/or prevent R protein-based viral defense.

LeT12 (Lycopersicon esculentum clone 12) is a class II knotted-like homeodomain protein (KNOX2) isolated from a tomato cDNA library [96]. The tomato LeT12 protein is a host factor in stages 2 and 3. The closest homolog of tomato LeT12 in tobacco is NTH201. NTH201 regulates the formation of VRC and the accumulation of TMV MP [97]. Silencing of the NTH201 gene transcript caused a delay in viral RNA accumulation and viral spread in TMV-infected tobacco plants [97]. Although NTH201 was observed to migrate from the nucleus to cytoplasm and PD, where NTH201 colocalized with MP, no direct interaction between NTH201 and TMV MP was detected [97]. The closest Arabidopsis homolog of LeT12 is Knotted1-like homeobox gene 4 (KNAT4). KNAT4 is one of the four Arabidopsis KNOX2 genes, together with KNAT3, KNAT5, and KNAT7, expressed in the inflorescence stems [98]. KNAT3 and KNAT7 may play a significant role in secondary cell wall formation as detected by mutation analysis [98]. Single gene mutations of KNAT4 and KNAT5 did not result in obvious morphological cell wall phenotypes in Arabidopsis, indicating possible redundancy in gene function, or KNAT4 and KNAT5 have another role, and absence does not lead to direct cell wall malformation. Still, modification of the plant cell wall by tobamoviruses is a likely strategy to improve viral spreading.

The tomato and citrus pectin methylesterase (PME) interacts with the MP from TMV, Turnip vein-clearing virus (TVCV, crucifer-infecting RNA tobamovirus), or Cauliflower mosaic virus (CaMV, DNA pararetrovirus) [99]. PMEs catalyze the de-methylesterification of pectin resulting in the release of protons and methanol. Methanol emission triggers the expression of methanol-induced genes, including β-1,3-glucanases. β-1,3-glucanases degrade callose locally deposited at the cell wall-embedded neck region of PD to restrict cell-to-cell communication and viral spreading. PME-dependent methanol emission triggers PD dilation, and the accumulated protons in the apoplast lead to acidification of the cell wall. This results in cell wall loosening due to the activation of several cell wall-degrading enzymes [100]. Dorokhov and coworkers identified an additional or contradicting role of PME, in which PME suppresses TMV RNA accumulation. They showed that PME levels increase due to the presence of TMV (or other pathogens), and this resulted in rapid siRNA accumulation indicative of induced RNA silencing [101,102]. In Figure 5, we have only depicted the PD dilation by PME, enhancing the viral spread, with the PD outward dashed arrow. Directed mutagenesis of PME will elucidate whether tobamovirus movement is inhibited by a non-functional or reduced functional PME.

The final host factor is synaptotagmin A (SYTA). SYTA is a protein that localizes to endosomes in plant cells and is essential to form ER-PM contact sites (EPCSs). The MP from TMV (and viruses from other genera) interact with SYTA to allocate the MPs to PD and to alter the PDs. The combination of MP allocation to and altering of the PD can facilitate tobamovirus cell-to-cell movement [103,104,105]. In Arabidopsis, the absence of SYTA/SYT1 affects the formation of the ER immobile tubules [106]. Two SYT1 interacting proteins, SYT5 and SYT7, were identified and shown to contribute together with SYT1 to enhance PD permeability for tobamoviral MP [107]. Because SYTA is suggested to assist viral movement through the PD, this protein is indicated near the PD (Figure 5).

5.4. Identification of Additional Host Factors Based on Host Processes That Are Manipulated by Tobamoviruses

Most identified host proteins reside in the cytosol/lysosomes or at the cell membrane. Host factors that function in the mitochondria or vacuole are, to our knowledge, not (yet) identified (depicted with the grey cloud shapes and a question mark in Figure 5). The number of host (susceptibility) factors currently identified is limited due to the identification method, co-purification with viral proteins. As a result, susceptibility-related host factors that do not directly interact or associate with a viral protein will be overlooked. Some S factors were identified via a forward genetics approach, e.g., TOM1, TOM2a, and TOM3 [73,74,75,79,108]. If existing, forward genetic screens of mutant plant populations will allow the identification of additional and non-viral protein-interacting host proteins. The drawback of forward genetic screens is that these will only disclose single proteins that result in a clear reduction of viral titer. This method will not reveal proteins that facilitate the virus proliferation if multiple or redundant proteins are required in concert. In addition, mutations that cause strong pleiotropic or lethal effects will be eliminated from the screen, and the corresponding host factors will not be identified.

In this review, we have focused on the utilization of host protein factors that interact with viral proteins. The absence of these specific host factors can lead to resistance. However, the identification of tobamoviral resistance can also be based on exploring which mechanisms are manipulated by the virus since these factors could also lead to novel and durable resistance. For example, viral resistance can be based on altered hormonal balance, membrane composition, apoptosis pathway, vesicle trafficking, or RNAi silencing mechanism of the host plant. For each mechanism, examples of S factors have been found.

S factors that alter the hormonal balance are discussed in Section 4.4. Altered membrane composition by plant cell fortification (discussed in Section 5.3), caused by PME-based callose deposition or overexpression of PME inhibitors (PMEIs) in tobacco and Arabidopsis plants, limits viral movement and reduces susceptibility to TMV and TVCV [100]. Autophagy can inhibit viral proliferation. Some S factors are negative regulators of autophagy, and their absence will trigger autophagy [58,59]. Five of the six host proteins in stage 2 (discussed in Section 5.2) prevent autophagy. However, autophagy can also have a pro-viral role, and several viruses have evolved strategies to use autophagy to their benefit. Therefore, only the autophagy-related genes (ATGs) with a pro-viral function should be further explored as S factors to combat viral infections of plants [58,59,109]. Importantly, the induction of autophagy and resistance depends on which plant proteins interact/bind with the viral helicase domain. The binding of the viral helicase to the N and/or Tm-1 R proteins can enhance autophagy resulting in resistance. In contrast, binding the viral helicase to the S proteins could prevent autophagy, enhancing susceptibility. For example, ARL8 binding to the REP protein could promote viral proliferation. ARL8 also interacts with TOM1 [53]. TOM1/3, TOM2a and ARL8 are essential to the tobamovirus replication complex. In human cells, the knockdown of ARL8b resulted in an increased fusion of autophagosomes with lysosomes [110]. The presence of ARL8 could promote viral infection by preventing the fusion of autophagosomes with lysosomes, as such inhibiting autophagy. In addition, SNARE motif-containing proteins, such as SYP23, are also required for autophagosome-lysosome fusion. Thus, we could postulate that host targets are incorporated into the VRC complex to prevent autophagy.

Another mechanism that is linked to the above S factors is membrane trafficking. SYP23 contains a SNARE motif, and TOM1/3 and TOM2a are putative transmembrane proteins. The TOM1/3 and TOM2a proteins could function in cell or vesicle membranes. A major role was found for endomembrane deformation during virus replication of ssRNA(+) viruses such as ToBRFV (reviewed by [111]). Additional host proteins that associate with VRC and are part of the intracellular membrane are interesting S factor candidates to explore.

Finally, studying the RNAi silencing mechanism and factors involved in this pathway can also lead to the identification of potential S factors. Plants produce small noncoding RNAs (sRNAs) that can alter viral proliferation (reviewed by [112]), including miRNAs. miRNAs are derived and excised from primary nonprotein-coding MIR transcripts that form stem-loop structures [113]. Three main modes of miRNA antiviral defense response could be anticipated. A plant miRNA could (1) directly target and silence viral RNA, (2) trigger the biogenesis of siRNA resulting in RNA silencing, or (3) target the mRNA of an S gene resulting in the absence of the viral S factor from the host. An example of the first mode by miRNAs is from two miRNAs from cotton (Gossypium arboreum), Ga-miR398 and Ga-miR2950. Both miRNAs can target multiple ORFs of the Cotton leaf curl Multan virus (CLCuMuV), leading to enhanced viral immunity [114]. Interestingly, for TMV, an opposite role of miRNAs in viral proliferation has been found. The presence of TMV in tobacco (N. benthamiana) causes the accumulation of two miRNAs, Nb-miR6019 and Nb-miR6020, which results in the cleavage and silencing of the mRNA of the resistance gene N, leading to enhanced susceptibility [115]. It would be interesting to identify and utilize the promotor of the two miRNAs to produce viral dsRNA fragments that halt viral proliferation.

Another method that is currently successfully applied to halt viruses is the application of topical dsRNA delivery. Topical dsRNA application is sometimes referred to as a ‘plant vaccine’. However, technically it is not a vaccine because the dsRNA only works temporarily (10–20 days) when the pathogen is present [116]. The use of dsRNA production in-planta driven by a promotor induced in the presence of a virus could lead to a more durable solution. However, as the application of genetically modified plants is restricted in the European Union, other approaches need to be considered.

5.5. Antisense Oligonucleotide Therapeutics: Targeting the Secondary RNA Structures from ToBRFV for Durable Resistance

Although the discovery of the first viruses occurred in plants by plant pathologists, new insights from the fast-evolving human virology field may lead to solutions for plant breeding. For example, research on the human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of Coronavirus Disease 2019 (COVID-19), could lead to new insights or solutions for durable plant virus resistance. SARS-CoV-2 virus, like ToBRFV, contains a single-stranded positive-sense RNA genome. The SARS-CoV-2 genome is ~30 kb RNA, and the secondary RNA structure of the entire SARS-CoV-2 virus genome was recently described [117].

The secondary RNA structure of ToBRFV is probably vital to evade plant-based RNA silencing, the first layer of viral defense by the host plant. For TMV, secondary RNA structures for the 5′ and 3′ parts of the genome have been described, but a genome-wide predicted RNA structure is still missing [118]. Secondary RNA structures from multiple animal viruses can be targeted by small molecules that affect interactions, structural stability, or conformational changes and thereby block processes that are essential for viral replication [119]. Can the secondary RNA structure of ToBRFV also be targeted by small molecular inhibitors? Comparison of human (SARS-CoV2), Pengolin, Porcine, and Bat coronaviruses resulted in the identification of conserved sites with persistent single-stranded sequences in the SARS-CoV2 and other coronavirus genomes in vivo [117]. These regions might represent ideal targets for the design of antisense oligonucleotide therapeutics, already proven to represent a promising approach for the treatment of infections by other RNA viruses [117].

The ToBRFV RNA molecule is only ~6.4 kb. It may be interesting to investigate whether a full genome comparison of secondary RNA structures from several tobamoviruses would also result in the identification of conserved sites with persistent single-stranded sequences. Antisense oligonucleotide therapeutics in tomatoes could also cause conformational changes or instability of secondary ToBRFV RNA structures and, as such, inhibit viral replication leading to durable ToBRFV resistance.

6. Conclusion and Future Directions towards Durable ToBRFV Tomato Resistance

Mutant S gene alleles can provide viral-resistant plants, and the abovementioned host factors for tobamoviruses are excellent candidates for which mutant alleles should be identified. Mutant alleles can be obtained by non-targeted chemical mutagenesis (e.g., EMS) or targeted mutagenesis using, e.g., CRISPR/Cas9 technology. Genome editing by CRISPR/Cas9 is well established in tomatoes and is the ideal method to explore and generate S gene-based resistance against ToBRFV. However, caution is required, as was shown by a CRISPR/Cas9 knock-out study on the well-known eIF4E and eIF4G S genes for potyviruses [120]. Knocking out eIF4E1 in Arabidopsis resulted in clover yellow vein virus (ClYVV) resistance but simultaneously increased turnip mosaic virus (TuMV) susceptibility. This is probably because potyviruses, such as both CIYVV and TuMV, hijack different eIF4E factors, and the strategy of developing resistance by eIF4E loss-of-function could be jeopardized by existing or emerging viruses that are able to recruit the remaining eIF4 paralogs in the plant [120].

As in the European Union, the application of CRISPR/Cas9 technology to generate mutations is restricted, and alternative methods to generate durable ToBRFV resistance have to be explored. A search for natural variants of a gene impacting the function of the encoded protein may be considered, especially if the complete absence of a functional S protein causes undesired pleiotropic effects.

6.1. The Use of Allelic Polymorphisms of Susceptibility Genes for Durable Resistance

Previously, some well-known S genes were identified from naturally occurring polymorphic alleles. These include eIF4E and eIF4G for resistance to potyviruses in Arabidopsis and pepper [121,122] and pelota for resistance against geminiviruses in tomato and pepper [123,124]. Thus, allelic variants of susceptibility genes (S genes) can be utilized to achieve recessively inherited disease resistance. The identification of natural variation in R proteins from wild tomato relatives is already a valuable source in tomato breeding. Wild relatives genetically related to cultivated species (Solanum lycopersicum), allowing compatible crosses, present a great opportunity to increase the genetic diversity in tomato breeding programs for disease resistance. Furthermore, such allelic S gene variants can be introgressed into cultivated tomatoes to obtain durable ToBRFV resistance. Currently, with the large list of identified host S genes, allelic S gene variants that could potentially contribute to ToBRFV resistance can be readily identified in the available genome sequences of various crop and wild tomato species. For example, ToBRFV-resistant tomato could be obtained using allelic variants of some of our discussed S genes since the absence of TOM1/3 led to ToMV or TMV resistance in tomato, pepper (Capsicum annuum) and tobacco (N. benthamiana) [76,125,126]. In addition, recently, allelic variants of TOM2a were claimed to be the main cause of ToBRFV resistance in wild tomato accessions [127].

The combination of available literature on tobamoviruses S gene candidates, wild tomato resources, genome sequences of the various wild species, single nucleotide polymorphism (SNP) detection methods, molecular techniques, and breeding methods make this reverse genetics approach to obtain durable resistant ToBRFV tomatoes accessible.

6.2. The Best Strategy for Durable Resistance Is the Absence of Susceptibility Genes

In this review, we highlighted four strategies to achieve durable ToBRFV-resistance in tomatoes (Figure 4). Although the second strategy (host R proteins) does not show a high risk of pleiotropy, viral genomes can adjust fast and break the resistance. The best strategy for durable resistance is an absence of functional susceptibility genes because viruses fully depend on host proteins for their proliferation due to the small number of viral proteins. As illustrated in the previous sections, most viral proteins interact with S proteins. Therefore, we need to investigate which amino acids from the S protein are relevant for viral protein binding and responsible for the S factor function. Simultaneously, we should identify which amino acid variants of the S protein can prevent viral protein binding and proliferation while maintaining the intrinsic protein function for the host plant. Deployment of S protein variants that lost their viral “helper” ability but kept their intrinsic in planta function is expected to result in durable virus resistance.