Research on Rx1 in Potato Has Illuminated the Initial Molecular Steps of Extreme Resistance
The initial processes of extreme resistance are best-characterized in the potato Rx1-PVX system. Also referred to as Rx, Rx1 is a coiled-coil NLR protein that confers extreme resistance to the Potexvirus, PVX, which can cause yield losses of up to 20% and is a common problem for potato growers (Adams et al., 1985). The Rx1 gene is located on chromosome XII and has been introgressed from the wild species of potato, Solanum andigena, into some commercial potato cultivars (e.g., cv. Cara, Atlantic; Ritter et al., 1991; Bendahmane et al., 1997). The Rx1 gene shares high sequence complementarity and is functionally redundant to another NLR, Rx2, which is found on potato chromosome V and was introgressed into potato from Solanum acuale (Ritter et al., 1991; Bendahmane et al., 1997). Despite similarities between the two genes, Rx1 has received a much greater research focus than Rx2.
Previous research on NLR protein function indicates that most, if not all, NLRs rely on chaperone proteins for stability and proper function (Kadota et al., 2010). A triad of highly conserved NLR chaperone proteins, SUPRESSOR OF G2 ALLELE OF SKP1 (SGT1), HEAT SHOCK PROTEIN 90 (HSP90), and REQUIRED FOR MLA12 RESISTANCE (RAR1) are essential for disease resistance. Many studies involve the expression of Rx1 in the genetically tractable model plant, Nicotiana benthamiana (Nb). Silencing of NbSGT1 suppressed Rx1-mediated extreme resistance to PVX in Nb, and indicated that the extreme resistance conferred by Rx1 relies on proteins similar to non-extreme resistance conferring NLRs (Botër et al., 2007; Figure 1). Despite no known nuclear or cytoplasmic localization signals, Rx1 must be distributed in both the cytoplasm and nucleus for an extreme resistance immune response to occur (Slootweg et al., 2010; Tameling et al., 2010). The Rx1 coiled-coil domain is necessary for accumulation in the nucleus, while the LRR domain is required for cytoplasmic localization (Slootweg et al., 2018). Binding between Rx1 and N. benthamiana RAN GTPase ACTIVATING PROTEIN (NbRG2) results in the retention of Rx1 in the cytoplasm, which is required for their dual roles in pathogen sensing and gene activation (Rairdan and Moffett, 2006; Sacco et al., 2007; Tameling et al., 2010).
The function of NbRG2 in the NbRG2-Rx1 complex is not clear, although a few possible roles are apparent (Slootweg et al., 2010; Hao et al., 2013). RanGAP proteins, like NbRG2, facilitate the GTPase activity of Ran GTPase proteins, which aid in the transport of protein complexes into and out of the nucleus (Dasso, 2002). There are no known transport activities of RanGAP proteins themselves and NbRG2 hydrolazing activity is not required for a successful resistance response (Tameling and Baulcombe, 2007). Many NLR proteins have evolved to monitor host proteins that are vulnerable targets of pathogen effector proteins (Collier and Moffett, 2009). Slootweg et al. (2010) suggest that NbRG2 may act as a decoy target of the PVX coat protein, in which Rx1 would be “monitoring” NbRG2 until bound by the PVX coat protein, after which Rx1 could initiate an immune response (Slootweg et al., 2010). This claim is buttressed by the findings that Gpa2, an NLR protein that shares a high amount of amino acid conservation with Rx1, guards NbRG2 and initiates an immune response only when NbRG2 is bound by effector proteins secreted by nematodes during infection (Sacco et al., 2009). Interactions between NbRG2 and the PVX coat protein could produce a conformational change in NbRG2, which could in turn allow Rx1 to reach an activated state (Hao et al., 2013). Many NLR proteins work in pairs with other NLRs or helper NLRs to effectively transmit immune signals (Adachi et al., 2019a). A study by Wu et al. (2017) found that silencing of three genes that encode helper NLR proteins, NLR REQUIRED FOR CELL DEATH 1 (NRC1), NRC2, NRC3, disabled Rx1-conferred extreme resistance, but only if all three genes were silenced simultaneously (Wu et al., 2017). These results indicate levels of redundancy and possible robustness to interference by pathogens within plant immune signaling.
Binding between Rx1 and PVX coat protein occurs in the cytoplasm (Bendahmane et al., 1995). Recognition of PVX coat protein by Rx1 and subsequent binding causes the release of NbRG2 from Rx1, allowing Rx1 to translocate to the nucleus through a yet unknown process, as there are no detectable nuclear localization signals within Rx1 (Tameling and Baulcombe, 2007). However, nuclear localization signals are notoriously difficult to predict and can be hidden within secondary structure. Although many studies have focused on NLR functionality in the cytoplasm, there are other examples of NLR nuclear localization and function (Burch-Smith et al., 2007; Shen et al., 2007). The barley NLR, MLA10, interferes with repression of defense genes by binding to WRKY transcription factors in the nucleus (Chang et al., 2013). Experiments in which nuclear exclusion signals or nuclear localization signals were added to an Rx1-GFP fusion protein indicated that the DNA binding capabilities of Rx1 are contingent upon Rx1 recognition of the PVX coat protein in the cytoplasm, which is followed by movement of Rx1 to the nucleus. Resistance, but not cell death responses were compromised in experiments in which Rx1 was localized predominantly to the nucleus or the cytoplasm (Bendahmane et al., 1999; Knip et al., 2019; Richard et al., 2021). These results indicate that Rx1 must be activated (ATP-bound) in order to successfully bind DNA and likely must be able to translocate from the cytoplasm to the nucleus in order to initiate the extreme resistance response. Activation of Rx1 and other NLRs occurs after binding effector proteins, in this case the PVX coat protein. In vitro binding assays indicate that the DNA binding activity of Rx1 is inhibited, while in an inactivated state (ADP-bound), while the activated (ATP-bound) Rx1 can bind DNA (Fenyk et al., 2015). Further, recognition of the PVX coat protein likely results in a perturbed binding between the LRR and ARC2 (Apaf-1, R proteins, and CED-4) domains of Rx1, a process which may play a role in initiation of resistance pathways. Rairdan and Moffett (2006) suggest that the LRR domain can repeatedly dissociate and reassociate with the ARC2 domain after recognition of the PVX coat protein, and that this iterative process may serve to amplify the resistance signal and could play a key role in the extreme resistance response (Rairdan and Moffett, 2006).
Upon entering the nucleus, the activated nucleotide-binding domain of Rx1 allows for binding and melting of double-stranded DNA in a non-sequence specific manner, but with a higher affinity for DNA topologies similar to transcription start site bubbles (Finzi and Dunlap, 2010; Tang et al., 2011; Townsend et al., 2018). The DNA binding activities of Rx1 likely become sequence-specific when in complex with NbGLK1 (a Golden 2-like transcription factor), although this remains to be definitively proven (Townsend et al., 2018). The activation state of Rx1 likely determines the DNA binding activity of NbGLK1, as inactivated Rx1 in complex with NbGLK1 does not bind DNA in planta (Townsend et al., 2018). Golden 2-like transcription factors preferentially bind DNA sequences with GLK-like motifs and are known to regulate the transcription of genes involved in abscisic acid (ABA) signaling (Ahmad et al., 2019). Although the genes that NbGLK1 regulates in response to PVX infection are not known, GLK-like transcription factors play a role in resistance to cucumber mosaic virus and fungal pathogens in Arabidopsis (Savitch et al., 2007; Murmu et al., 2014; Han et al., 2016).
There are likely other proteins that interact with Rx1 and NbGLK1 in the nucleus. Sukarta et al. (2020) recently described a direct interaction between Rx1 and a DNA-binding bromodomain containing protein, NbDBCP (Sukarta et al., 2020). The precise role(s) of NbDBCP remains unclear, though it likely acts as a repressor of Rx1-mediated resistance signaling. Silencing of NbDBCP as well as co-expression of non-functional NbDBCP decreased PVX coat protein accumulation during Potato virus X infection in plants expressing Rx1, indicating that NbDBCP may negatively regulate extreme resistance responses. Binding of DNA by NbDBCP occurs in situ, but not when co-expressed with Rx1 or during PVX infection. Size exclusion chromatography results indicate that Rx1, NbDBCP, and NbGLK1 may form a transient complex; however, this idea remains theoretical and untested. These results conservatively indicate a negative regulatory role of NbDBCP on Rx1-mediated extreme resistance, although its exact role(s) require more research (Sukarta et al., 2020).
Intriguingly, overexpression of NbGLK1 in N. benthamiana confers immunity to PVX even in the absence of Rx1, and this immunity does not result in HR (Townsend et al., 2018). These results may signal that NbGLK plays a role in controlling gene expression that is important for extreme resistance, but that role is likely independent or upstream of HR/cell death. Additionally, NbDBCP overexpression, in the presence of Rx1 and during PVX infection, resulted in increased cell death. Expression of a non-functional NbDBCP resulted in decreased cell death, lending credence to the idea that (1) NbDBCP negatively regulates the extreme resistance pathway and (2) extreme resistance and HR/programmed cell death are largely separate or sequentially activated pathways (Sukarta et al., 2020). Similar separation of cell death and resistance has been reported in N. benthamiana plants expressing the barley NLR, MLA10, with a nuclear localization tag (Bai et al., 2012). The MLA10 gene provides resistance to the barley powdery mildew fungus, indicating that nuclear functions of NLRs may not be specific to virus resistance.
Extreme resistance conferred by Rx1 does not limit viral spread through the phloem. Grafting experiments revealed that PVX moved from a susceptible rootstock through the phloem of a middle, resistant scion and into another upper, susceptible scion and caused infection (Bendahmane et al., 1999). A reaction similar to Rx1-mediated extreme resistance can occur in protoplasts, while HR does not (Otsuki et al., 1972; Baulcombe et al., 1984; Goulden et al., 1993; Kohm et al., 1993; Bendahmane et al., 1995). These results may indicate that intercellular signaling components or cell wall components may not be necessary for Rx1-conferred extreme resistance responses, whereas they are for HR responses. It is likely that the mechanisms controlling extreme resistance occur rapidly in the cell, as extreme resistance prevents viral replication and spread beyond the initial point of inoculation.
Overexpression of Rx1 in N. benthamiana results in HR, regardless of whether its elicitor, the PVX coat protein, is present or not. Transformation of Rx1 under its native promoter into N. benthamiana and Nicotiana tabacum results in a typical, symptomless extreme resistance to PVX, indicating that functionality and possible downstream interacting elements are conserved between species (Bendahmane et al., 1999). Overexpression of PVX coat protein in N. benthamiana expressing Rx1 under its native promoter results in HR. Bendahmane et al. (1999) suggest that the continued production of the PVX coat protein after the initial recognition event and extreme resistance activation may signal to the cell that extreme resistance has been overcome and that further immune action may be warranted, hence the subsequent HR (Bendahmane et al., 1999). This hypothesis is supported by the finding that extreme resistance conferred by Rx1 is epistatic to HR, as plants expressing both Rx1 and N, an NLR that provides resistance with an HR phenotype to Tobacco mosaic virus, were resistant to Tobacco mosaic virus infection but did not display HR when the virus was engineered to express both the PVX coat protein and the protein elicitor of N during infection. The addition of a nuclear localization signal to NbRG2 caused Rx1 to accumulate almost solely in the nucleus and prevented HR from occurring, even when auto-active Rx1 mutants were overexpressed (Slootweg et al., 2010). Thus, it is likely that Rx1 must be located in the cytoplasm in order for HR and concurrent signaling to occur. This conclusion is congruent with cytosolic location of PVX replication and PVX coat protein detection by Rx1 (Slootweg et al., 2010).
The possible interconnectedness between HR and extreme resistance responses underscores the need for more sensitive resistance assays. To better understand extreme resistance, it is paramount that researchers try to replicate the native expression levels of Rx1 (and other genes that confer extreme resistance) when experimenting outside of its native potato system. Slootweg et al. (2010) expressed Rx1 from a vector with a second start codon inserted upstream and out of frame of the Rx1 start codon. The resulting “leaky” expression of Rx1 led to protein levels in N. benthamiana that were 5–10x lower than expression driven by a typical CaMV35S promoter, and a much more sensitive assay (Slootweg et al., 2010). Studies in A. thaliana also indicate the expression level of NLR proteins may in part determine the phenotype of the resistance response. For example, resistance to the yellow strain of Cucumber mosaic virus is conferred by RCY1, a coiled-coil NLR, in Arabidopsis ecotype C24. Resistance to Cucumber mosaic virus (Y) via RCY1 is normally accompanied by a hypersensitive response. Transgenic Arabidopsis lines that over-expressed RCY1 at high levels (i.e., ~100x greater than its native promoter) exhibited the extreme resistance phenotype. Transgenic plants that expressed RCY1 at moderately elevate levels (i.e., ~20x greater than native expression) exhibited enhanced resistance with very small areas of cell death (“micro-HR”; Sekine et al., 2008). The hypersensitive response was observed in transgenic lines with levels of RCY1 expression similar to native expression. None of the RCY1 transformed Arabidopsis lines became systemically infected with Cucumber mosaic virus (Y). These results indicate that RCY1 expression levels, at least in part, govern virus-resistance phenotypes, possibly by determining the type of the subsequent immune response.
Other publications studying the NLR, HRT, which confers resistance to Turnip crinkle virus, have noted similar results (Cooley et al., 2000). However, levels of resistance protein expression are likely not the only factor governing immune responses. Overexpression of the Turnip crinkle virus coat protein, which is the binding target of HRT, resulted in severe HR, similar to the reaction that occurs after overexpression of the PVX coat protein plants expressing Rx1 (Cooley et al., 2000). Expression and activity levels of NLR proteins in plants are regulated in many ways (e.g., transcriptionally, post-transcriptionally, post-translationally, etc.; Borrelli et al., 2018). It is possible that increased expression of HRT or RCY1 is sufficient to overcome some negative regulation, resulting in faster immune responses.
The mechanistic details of Rx1 conferred resistance restricting PVX viral replication and spread are not yet known, however, experiments indicate that the translational arrest of the PVX transcripts is likely a major component of these resistance processes (Richard et al., 2021). By employing an inducible effector protein expression system and nuclear- and cytoplasm-localized Rx1 expression, Richard et al. (2021) demonstrate that Rx1-conferred extreme resistance likely relies on PVX transcript-specific translational arrest and that this response occurs within a few hours after infection (Meteignier et al., 2016; Richard et al., 2021). These data also demonstrate that nuclear- or cytoplasm-localized Rx1 expressed individually or together, results in HR or trailing necrosis (i.e., HR that trails viral spread throughout the plant) after 4 hours of the induction of PVX coat protein transcription, but does not induce extreme resistance. These results further support that upon recognition of the PVX coat protein in the cytoplasm, Rx1 must translocate to the nucleus in order to initiate the extreme resistance response. Translational arrest is a common host antiviral strategy (Machado et al., 2017). Rx1-expressing, PVX-infected potato protoplasts did not support replication of either Tobacco mosaic virus or Cauliflower mosaic virus, indicating that the Rx1-mediated antiviral response was a general antiviral response (Goulden et al., 1993; Bendahmane et al., 1995). Further, Rx1 may not be unique in this regard, as recognition of the resistance elicitor, Tobacco mosaic virus p50, by the tobacco NLR gene, N, can initiate an immune response that prevents translation of PVX transcripts in N. benthamiana, but only in the presence of RNA containing the PVX coat protein coding sequence (Bhattacharjee et al., 2009).
These results collectively suggest that a conserved characteristic of viral RNAs, possibly secondary structure, may be specifically targeted by NLR-mediated translational inhibition responses and that this mechanism may play a key role in the extreme resistance response. Although the factor(s) governing translational arrest are not known, it is interesting to note that virus-induced gene silencing of ARGONAUTE 4 (AGO4) disabled symptomless resistance responses, and in turn allowed systemic PVX infection in N. benthamiana (Bhattacharjee et al., 2009). Similarly, in N. benthamiana plants in which the RNA interference (RNAi) suppressor proteins from Beet western yellows virus and Turnip crinkle virus, P0 and P38, were expressed, the antiviral response was also disabled. The Turnip crinkle virus P0 protein targets and induces degradation of Argonaute proteins (Baumberger et al., 2007; Bortolamiol et al., 2007). The expression of other suppressors of RNAi, including potyviral Helper-component protease (HC-Pro), did not prevent an antiviral response from occurring, although HC-Pro disables RNAi through sequestration of virus-derived small RNAs, not through the degradation of Argonaute (Mallory et al., 2002). Another RNA virus, Tobacco rattle virus (TRV), expresses a suppressor of RNAi silencing protein, 16k, which binds AGO4 and ago4 mutant N. benthamiana plants are more susceptible to infection (Ma et al., 2015; Fernández-Calvino et al., 2016).
ARGONAUTE 4 is well known for its roles in transcriptional gene silencing and the RNA-directed DNA methylation pathway, as well as methylation-based antiviral defense against plant viruses with DNA genomes (Zilberman et al., 2003; Li et al., 2006; Raja et al., 2008, 2014; Gao et al., 2010; Greenberg et al., 2011; Dowen et al., 2012; Wierzbicki et al., 2012; Ye et al., 2012). These well-characterized roles of AGO4 all occur in the nucleus. Interestingly, cytoplasm-localized AGO4 is necessary for resistance to the potexvirus virus, Plantago asiatica mosaic virus, in Arabidopsis. This resistance does not involve other protein components of the RNA-directed DNA methylation pathway (e.g., DICER-LIKE 3, RNA POLYMERASE IV, and RNA POLYMERASE 5), indicating that AGO4 antiviral activity in this case is likely independent of its DNA methylation activity (Brosseau et al., 2016). The importance of AGO-4 non-methylation-based defense is not limited to antiviral responses, as silencing of AGO4 in Arabidopsis plants without functional RNA-directed DNA methylation pathways increased susceptibility to Pseudomonas syringae (Agorio and Vera, 2007).
Research on Rx1-conferred extreme resistance has illuminated the potential nuclear functions of Rx1 and laid a framework for future studies. In particular, gaining an understanding of the DNA sequences targeted by the Rx1-NbGLK1 complex and the possible transcriptional changes that occur after recognition of the PVX coat protein will aid in the identification of other genes and mechanisms involved in Rx1-conferred extreme resistance. Future experiments employing RNA sequencing, chromatin-immunoprecipitation sequencing, and translatome analysis would increase understanding of the regulation of this defense system. Additional studies are required to determine the role(s) of bromodomain containing proteins, to identify the DNA sequences that are targeted by the Rx1-GLK complex, and if targeted gene(s) are responsible for the next stages of the Rx1 conferred extreme resistance response. Further, studies that dissect the antiviral translational repression response and possible antiviral roles of AGO4 would provide a greater understanding of NLR-mediated immunity.
Genes Conferring Extreme Resistance to Potato Virus Y Rely on Conserved Proteins That Are Also Necessary for HR
The Ry genes in potato (e.g., Rysto, Ryfsto, and Ryadg) provide resistance to particular strains of PVX and PVY. The Rysto gene (Resistance to PVY from S. stoloniferum), which conveys resistance to a broad spectrum of strains of PVY and Potato virus A (PVA) in potato and tobacco, is the only one of the genes controlling extreme resistance that has been isolated from the Ry loci (Cockerham, 1970; Barker, 1996; Grech-Baran et al., 2020). Global potato production is reliant on pathogen-free seed tubers, which are vulnerable to generational buildup and spread of pathogens, particularly viruses. Various PVY strains (including PVYNTN and PVYN-Wi) are the most economically harmful viral pathogens involved in potato production and genetic resistance to PVY is a major focus of breeding programs (Karasev and Gray, 2013). Wild potato varieties and landraces are sources of PVY-specific NLR resistance genes that can be introgressed into commercial potato cultivars. Loci conferring extreme resistance to PVY have been mapped in Solanum chacoense (Rychc), S. tuberosum group Andigena (Ryadg), and S. stoloniferum (Ry
sto; Herrera et al., 2018). Grech-Baran et al. (2020) employed resistance enrichment sequencing (RenSeq) to isolate the gene conferring Rysto-mediated extreme resistance from the commercial potato cultivar, Alicja. Introgressed from S. stoloniferum, Rysto is a Toll-interleukin receptor (TIR) NLR protein, similar to other potato virus resistance genes (e.g., N, Pvr4, Y-1, etc.). The broad-spectrum resistance conferred by Rysto lends it an attractive trait for breeders of Solanaceous plants. The Rysto gene is present in various commercial potato cultivars, including American cultivars Payette Russet and Castle Russet and European cultivars Alicja, White Lady, and Pirola. The Rysto protein either directly or indirectly recognizes or binds the coat protein of PVY and PVA to elicit the extreme resistance response (Grech-Baran et al., 2020).
The Rysto gene has been cloned and expressed in PVY-susceptible Solanaceous plants. Challenge of transgenic plants expressing Rysto under its native promoter with PVY usually results in an extreme resistance response (i.e., no infection, no symptoms), but can cause veinal necrosis or HR in response to some isolates of PVYO (Hinrichs-Berger et al., 1999). Co-expression of Rysto and the PVY coat protein under control of a CaMV35S promoter results in HR in potato and N. benthamiana. Expression of Rysto in tobacco and subsequent challenge with PVY produced some localized necrosis in inoculated leaves. Grech-Baran et al. (2020) suggest that establishment of either extreme resistance or HR depends on at least three variables: expression level of the resistance gene; abundance of the cognate effector protein; and the genetic background of the host. Extreme resistance conferred by Rysto relies on at least two other genes for successful immune activation: the lipase-like ENHANCED DISEASE SUSCEPTIBILTY (EDS1) and the CC-NLR, N REQUIREMENT GENE 1 (NRG1). These dependencies corroborate studies on other TIR-NLRs (Grech-Baran et al., 2020).
Similar to the Rx1-PVX system, the genes controlling Rysto-mediated extreme resistance downstream of virus recognition are not known, although some results have hinted at the involvement of proteins that interact with plasmodesmata. β-1,3-glucanase proteins aid in plant virus infection, likely by hydrolyzing callose and increasing the size exclusion limit of plasmodesmata, thus allowing for cell to cell spread of the virus (Iglesias and Meins, 2000). Callose is a polysaccharide that influences the size exclusion limit of the plasmodesmata and also serves as a deposition site for defense compounds (Zavaliev et al., 2011). Overexpression experiments of β-1,3-glucanase (class III) proteins were carried out in potato cultivars Santé (which displays extreme resistance to PVY) and Désirée (PVY-susceptible) within the context of PVYNTN infection. Resistant Santé plants overexpressing β-1,3-glucanase exhibited modest, transient increases in PVYNTN that dissipated within days of infection and the virus did not spread beyond the inoculated leaf. Susceptible Désirée plants that overexpressed β-1,3-glucanase may have exhibited slightly faster systemic infection of PVYNTN, although a relatively small sample size precluded more definitive conclusions (Dobnik et al., 2013).
Callose deposition is also targeted by PVY during infection, as PVY-encoded HC-Pro suppresses callose deposition during PVYO infection through an unknown mechanism (Chowdhury et al., 2020). Callose deposition also occurs in cells surrounding HR activity during PVY infection and those cells can harbor viable PVY, thus further indicating that cell death is not the primary driver of resistance during HR and that callose deposition alone is not effective at arresting viral spread (Lukan et al., 2018). Cells undergoing HR/cell death processes may release signals to surrounding cells to initiate immune responses or that may act as defense compounds themselves (Lamb and Dixon, 1997). These signals may include reactive oxygen species (ROS), which are a common component of plant immune responses (Qi et al., 2017). Whether or not reactive oxygen signaling acts a component of extreme resistance is not known, although there are examples of symptomless resistance to Tobacco mosaic virus in tobacco plants induced by application of ROS (Kuenstler et al., 2016). The timing of foliar treatments was key to inducing resistance to Tobacco mosaic virus, as resistance did not occur in plants that were treated with reactive oxygen species 3 days after virus inoculation, but an HR-like reaction and cell death did.
The NLR resistance gene, Ny-1, also provides resistance to PVY, but the immune response is accompanied by HR. Extreme resistance conferred by Rysto is epistatic to Ny-1-mediated HR, as plants expressing both Ny-1 and Rysto exhibit an extreme resistance phenotype but lack HR when challenged with a PVYNTN isolate that is recognized by both Rysto and Ny-1 (Grech-Baran et al., 2020). For many TIR-NLR proteins, including Ny-1, resistance breaks down at high or low temperatures, while Rysto function is not limited by high ambient temperatures. Modulation of defense responses by temperature is likely controlled by NLR proteins, as point mutations in NLR genes can decrease nuclear accumulation of NLR proteins at higher temperatures and reduce NLR immune function (Zhu et al., 2010). At higher temperatures plants may preferentially activate pattern-triggered immunity (PTI), rather than NLR-dependent immunity (Cheng et al., 2013). Since NLR proteins confer resistance to plant pathogens that disable host pattern-triggered immune responses, a greater understanding of NLR thermosensitivity is needed as global temperatures continue to rise (Trebicki, 2020).
The expression levels of both the genes conferring extreme resistance and the viral elicitor protein seems to be a critical factor if HR or extreme resistance occurs in both Rx1-PVX and Rysto-PVY systems. The cellular distribution of Rysto both before and during resistance responses is not known but should be the focus of further research given the potential nuclear functions of Rx1. The advent and expanded use of RenSeq, which allows for the expedited identification of NLR genes conferring specific resistance phenotypes, and CRISPR technologies, which allow for precise genome editing, should facilitate faster identification and breeding of resistance genes (Witek et al., 2016; Zhang et al., 2019). As extreme resistance is largely characterized by a lack of infection symptoms, there may be a pool of genes that confer extreme resistance to viruses that are yet to be discovered within landraces or wild Solanaceous species. For example, the PVR4 gene, which encodes an NLR protein that originated in a landrace of hot pepper (Capsicum annum), confers extreme resistance to multiple potyviruses, including many PVY strains, Pepper mottle virus, and Pepper severe mosaic virus (Kim et al., 2015). The PVR4 protein recognizes the potyviral RNA-dependent RNA polymerase (NIb) to elicit the extreme resistance response (Kim et al., 2015). Global potato production relies on labor-intensive seed tuber certification programs to prevent pathogen accumulation, particularly viruses, with a large focus on PVY strains. Given that many NLR genes can be shuttled between Solanaceous species without a loss of function, transferring multiple, broad-spectrum NLR genes that target different potyviral components (i.e., Rysto, PVR4) to potato could provide durable and sustainable immunity to potyviruses (Rivero et al., 2012; Zhu et al., 2012; Jo et al., 2014).