Evaluation of potato virus X resistance in potato cultivars and identification of an innate immunity-independent resistance phenotype

Potato virus X (PVX) is a widely distributed viral pathogen that causes significant losses in potato production by co-infecting with potato virus Y or potato virus A. In this study, the resistance of 23 potato cultivars to PVX was dissected in detail using a PVX infectious clone containing a yellow fluorescent protein (YFP). Among them, four potato cultivars (Longshu-3, Eugene, Atlantic and Waiyin-2) were found to carry an Rx gene that confers extreme resistance to PVX; one cultivar (Waiyin-1) displayed partial resistance and was able to delay PVX infection by ~ 5 days; while the rest eighteen potato cultivars were susceptible to PVX. Moreover, we found that the replication but not cell-to-cell or long-distance movement of PVX was inhibited in Waiyin-1. Finally, we determined that the expression of pathogenesis-related (PR) genes in Waiyin-1 was not triggered by PVX infection at early infection stage, whereas they were triggered in the Rx-carrying cultivar Atlantic during this period of time. In conclusion, our results confirm that Rx is a major type of resistance gene in potato cultivars in the Northeast part of China. Furthermore, the possible mechanism underlying Waiyin-1 resistance to PVX is discussed.


Background
Potato (Solanum tuberosum L.) is the world's fourth most important food crop, with a global production of about 388 million tons per year according to the Food and Agriculture Organization of the United Nations (FAO) statistical databases (www. stati sta. com/ stati stics/ 192966/ us-potato-produ ction-since-2000). China is the largest potato producer, accounting for 26.3% of global production. Along with the transportation of seed potatoes, potato diseases, especially viral diseases are rapidly spreading across potato planting areas worldwide.
At present, potato production is being seriously threatened by virus infection, which not only causes yield and quality decline but also induces seed degradation (Solomon-Blackburn and Barker 2001a). Hitherto, more than 50 viruses have been identified in potato plants (Kreuze et al. 2020a).
Potato virus X (PVX), the type species of the genus Potexvirus in the family Alphaflexiviridae, is one of the major viruses infecting potato plants worldwide (Kreuze et al. 2020b). Although it does not induce severe symptoms on most potato cultivars, coinfection of PVX with other viruses, such as potato virus A (PVA) and potato virus Y (PVY), can cause serious yield and quality losses in potato production (Kreuze et al. 2020a). For example, coinfection of PVX with potato virus A (PVA) and potato virus Y (PVY) results in crinkle symptoms and even plant death (Nie and Singh 2013). The genome of PVX consists of a positive-sense single-stranded RNA (+ ssRNA) of about 6.4 kilobase pairs (kb) in length that is encapsulated in flexuous, filamentous and non-enveloped viral particles, and encodes five open reading frames (ORFs) (Kreuze et al. 2020b). The largest ORF (ORF1) on the 5´-end of the genome encodes the viral RNA-dependent RNA polymerase (RdRp), which is absolutely required for viral replication (Betten et al. 2012). The partially overlapping ORFs [2, 3 and 4] comprise a so-called triple-geneblock (TGB) module, encoding three viral movement proteins, namely TGBp1, TGBp2 and TGBp3 (Beck et al. 1991;Tilsner et al. 2012). ORF5 encodes the viral coat protein (CP) that is involved in viral genome encapsulation and cell-to-cell movement (Cruz et al. 1998).
During a long history of coevolution with pathogens, plants have evolved sophisticated resistance mechanisms to combat the invasion of all phytopathogens including viruses (Chisholm et al. 2006). Effector-triggered immunity (ETI) is one of the main antiviral machineries, which is triggered via the recognition of pathogen effectors by intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) that are historically termed resistance (R) proteins. To date, three R genes, namely R x , N x and N b , have been characterized in potato, which confer resistance to PVX (Cockerham 1970;Solomon-Blackburn and Barker 2001b). The R x gene encodes a coiled-coil NLR (CC-NLR) that specifically recognizes PVX CP and confers an extreme resistance (ER), a specific form of resistance without inducing visible local necrotic lesions (hypersensitive responses) (Bendahmane et al. 1995(Bendahmane et al. , 1997(Bendahmane et al. , 1999Moffett et al. 2002). N x and N b induce necrotic lesions on the infection sites by recognizing viral CP and TGBp1, respectively (Tommiska et al. 1998;Malcuit et al. 1999;Marano et al. 2002). In addition, several atypical dominant viral resistance proteins (ADVRPs) have been characterized. For instance, Jacalin-type lectin required potexvirus resistance 1 (JAX1) from Arabidopsis thaliana, putative methyltransferase 1 from Nicotiana benthamiana (PNbMTS1) and Remorins (REMs) from potato. Both JAX1 and PNbMTS1 interfere with virus replication by targeting viral RdRp, whereas REMs inhibit the function of TGBp1 in viral cell-to-cell movement (Cheng et al. 2009;Yamaji et al. 2012;Sugawara et al. 2013;Yoshida et al. 2019).
Identification of virus-resistant potato cultivars and dissection of their resistance mechanisms will not only facilitate sustainable potato production but also benefit potato resistance breeding. In this study, we evaluated the PVX resistance of twenty-three potato cultivars and identified a novel resistance phenotype to PVX in a potato cultivar, which will further deepen our understanding of potato resistance to PVX.

Resistance of different potato cultivars to PVX
Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the tissue-culture plantlets of all 23 potato cultivars were free of PVX (Fig. 1a). These plantlets were then infiltrated with agrobacteria harboring PVX infectious clone pGR107. At 12 days post-inoculation (dpi), Kexin-18, Shepody and Netherlands-15 showed mild yellowing and/or mosaic symptoms, while other cultivars remained symptomless (Fig. 1b). Systemic leaves were analyzed by DAS-ELISA at 12 dpi. The results showed that the optical density values at 405 nm (OD405) of five cultivars, namely Longshu 3, Eugene, Atlantic, Waiyin-1 and Waiyin-2, were close to that of the negative control (healthy Zhongshu-2), whereas those of other 18 varieties were close to that of the positive control (Table 1). These data suggest that Longshu 3, Eugene, Atlantic, Waiyin-1 and Waiyin-2 are probably resistant to PVX infection. We also investigated the presence of PVX genomic RNA in the systemic leaves by RT-PCR at 15 dpi. Consistent with the DAS-ELISA results, an amplicon of the expected size of 1.5 kb was observed in all 18 ELISA-positive cultivars, whereas no band was amplified from Longshu 3, Eugene, Atlantic and Waiyin-2 (Fig. 1c). Unexpectedly, a 1.5-kb band was amplified in Waiyin-1 at 15 dpi (Fig. 1c). To further confirm the infection of PVX in Waiyin-1, we re-examined PVX genomic RNA in the systemic leaves of Waiyin-1 by RT-PCR at 9, 12 and 15 dpi. The results showed that the 1.5-kb band could only be amplified in the systemic leaves of Waiyin-1 at 15 dpi but not at 9 or 12 dpi (Fig. 1d). All amplicons were cloned and Sanger sequenced. The results confirmed that amplicons from the 18 ELISA-positive cultivars and Waiyin-1 were the genomic sequence of PVX. These results suggest that Longshu-3, Eugene, Atlantic and Waiyin-2 are fully resistant to PVX, Waiyin-1 is partial resistant to PVX, while the other 18 cultivars are susceptible to PVX.
To further confirm the resistance of Waiyin-1 to PVX, we constructed a PVX infectious clone that expresses a yellow fluorescent protein (pGR107-YFP) to facilitate the direct visualization of virus infection (Fig. 2a). Infection of Nicotiana benthamiana seedlings with pGR107-YFP caused typical symptoms of PVX such as mosaic and leaf scroll-down on systemic leaves at 7 dpi (Fig. 2b,left panel). Under ultra-violet (UV) light, bright yellow fluorescence was observed on symptomatic leaves (Fig. 2b, right panel). Potato seedlings of Longshu 3, Eugene, Atlantic, Waiyin-1, Waiyin-2 and Kexin-1 were then agroinfiltrated with pGR107-YFP. It was shown that yellow fluorescence was only observed on systemic leaves of Kexin-1 at 12 dpi, which was used as a susceptible control ( Fig. 2c, left panel). Interestingly, yellow fluorescence also appeared on systemic leaves of Waiyin-1 at 17 dpi ( Fig. 2c, right panel), whereas no visible fluorescence signal was observed on the other 4 cultivars throughout the experimental period (data not shown). Together, these results confirm that Longshu 3, Eugene, Atlantic and Waiyin-2 are fully resistant to PVX, whereas Waiyin-1 has a partial resistance.

PVX infection is repressed in Waiyin-1
To understand the resistance mechanism of Waiyin-1 to PVX, we sap-inoculated Waiyin-1 and Kexin-1 plantlets with inoculum of pGR107-YFP-infected N. benthamiana leaf tissues. Fluorescent infection foci were observed in inoculated leaves of Kexin-1 under UV light at 10 dpi, while no infection spot was found in leaves of Waiyin-1 at the same time point (Fig. 4a). A time course analysis was subsequently performed to monitor PVX infection in the inoculated leaves using RT-PCR from 24 to 96 h post-inoculation (hpi). It was found that the genomic RNA of PVX in Kexin-1 showed a significant increasing trend from 48 hpi onwards, while in Waiyin-1 it just started to increase from 96 hpi onwards (Fig. 4b), suggesting that the early stage of PVX infection could be inhibited in Waiyin-1. To further confirm this hypothesis, we infiltrated Waiyin-1 and Kexin-1 leaves with agrobacteria harboring pGR107-YFP at a low concentration (an OD600 value of 0.0005), so that isolated cells became infected. The inoculated leaves were then observed with a confocal fluorescence microscope from 48 to 72 hpi at a 12-h interval. At 48 hpi, YFP-expressing cells were only observed in the inoculated leaves of Kexin-1, suggesting that PVX replication had occurred in Kexin-1 at this time point (Fig. 4c). Most fluorescent foci in Kexin-1 were expanded into more than two cells at 60 hpi, while only very faint YFP-signal was observed in isolated cells in Waiyin-1 at the same time point (Fig. 4c). At 72 hpi, cell patches with strong fluorescence were observed in Kexin-1, whereas the fluorescence in Waiyin-1 remained in isolated cells (Fig. 4c). Moreover, the fluorescence intensity in Waiyin-1 was significantly lower than that in Kexin-1 at both 60 and 72 hpi (Fig. 4d). Thus, PVX replication occurs before 48 h and starts cell-to-cell movement at about 60 hpi in Kexin-1, while the replication of PVX takes place at about 60 hpi and no cell-to-cell movement was observed even at 72 hpi in Waiyin-1.
To rule out the influence of cell-to-cell movement, a movement-defective PVX infectious clone (pGR107-TGBp2 ∆52-60 -YFP) was constructed, in which a nineamino-acid motif (GGAYRDGTK) between the two transmembrane motifs of TGBp2 was deleted (Fig. 5a). Our previous study showed that this motif plays a critical role in PVX cell-to-cell movement ). Three-week-old N. benthamiana seedlings were infiltrated with agrobacteria harboring pGR107-YFP or pGR107-TGBp2 ∆52-60 -YFP. Yellow fluorescence was observed in the local leaves of these agroinfiltrated seedlings under UV light from 2 dpi onwards. The fluorescence of pGR107-TGBp2 ∆52-60 -YFP was restricted only in the infiltration area, whereas that of pGR107-YFP spread to upper systemic leaves at 6 dpi ( Fig. 5b), suggesting that pGR107-TGBp2 ∆52-60 -YFP is defective in cell-to-cell and long-distance movement. We then sap-inoculated seedlings of Waiyin-1, Kexin-1 and Atlantic using inoculum of pGR107-TGBp2 ∆52-60 -YFP-infected N. benthamiana leaf tissues. Kexin-1 and Atlantic were used as susceptible and resistant controls, respectively. The accumulation of PVX genomic RNA in the local inoculated leaves was evaluated by RT-qPCR at 48 and 72 hpi. It was shown that PVX genomic RNA was increased about 1.75 times from 48 to 72 hpi in Kexin-1, whereas it was decreased in Atlantic in the same time period. Interestingly, the relative level of viral genomic RNA was hardly increased in Waiyin-1 from 48 to 72 hpi (Fig. 5c). Together, these results suggest that the replication of PVX is inhibited in Waiyin-1.

Innate immunity is not involved in early antiviral responses in Waiyin-1
To investigate whether the resistance of Waiyin-1 to PVX involves plant innate immunity, the expression of pathogenesis-related (PR) genes, e.g., PR1, PR2, PR3 and PR4, was evaluated during PVX infection using RT-qPCR. The expression levels of PR1, PR2, PR3 and PR4 were almost unaltered after PVX infection in Kexin-1, whereas in Atlantic, they were peaked at 48 hpi and decreased at 72 hpi ( Fig. 6a-d). These results suggest that the innate immunity triggered by the Rx gene typically peaked at 48 hpi. Intriguingly, the expression of PR1 and PR4 was not changed in Waiyin-1 at 48 hpi, but significantly increased at later infection stage, e.g., 72 hpi (Fig. 6a, d), while the expression of PR2 and PR3 was gradually increased from 48 to 72 hpi in Waiyin-1 (Fig. 6b, c). We further investigated the expression of WRKY1, a member of the WRKY transcription factor family involved in disease resistance, abiotic stress and some developmental processes, e.g., stomatal movement, light and nitrogen signaling pathways (Qiao et al. 2016;Heerah et al. 2019), during PVX infection. The results showed that the expression of WRKY1 was slightly increased at 48 hpi and decreased at 72 hpi in Atlantic, while no significant changes occurred in Keyin-1. Interestingly, the expression of WRKY1 was also significantly increased in Waiyin-1 at 72 hpi but not altered at 48 hpi (Fig. 6e). Together, these results suggest that although the PVX resistance in Waiyin-1 requires the expression of PR genes, it is apparently differed from the Rx-triggered innate immunity.

Discussion
In this study, we evaluated the PVX resistance of 23 potato cultivars widely cultivated in China. The results suggest that most potato varieties we tested are susceptible to PVX infection and only 4 cultivars (Longshu-3, Eugene, Atlantic and Waiyin-2) are completely resistant to PVX (Table 1 and Fig. 1c). Our results further reveal that the resistance of the 4 cultivars to PVX is conferred by the Rx gene, since the deduced amino acid sequences of Rx identified in these cultivars share high identities with Rx1 and Rx2, and all of them were clustered in the same phylogenetic branch (Fig. 3b, c). Noticeably, our bioinformatics analyses indicate that these Rx genes share considerable sequence diversity; especially, the deduced amino acid sequence of the Rx from Waiyin-2 lacks 32 aa in the LRR domain. It has been reported that mutations in the LRR domain are capable of altering the specificity of disease resistance conferred by the Rx gene (Farnham and Baulcombe 2006;Harris et al. 2013). Therefore, whether these Rx varieties have the same resistance spectrum to different PVX isolates is unknown since only the PVX UK3 isolate was evaluated in this study. Nevertheless, our results suggest that the genetic resources of PVX resistance in current potato varieties are considerably narrow, although it is believed that potato cultivars are abundant in genetic variability (Hardigan et al. 2017). As a consequence of the lack of proofreading activity in RNA-dependent RNA polymerases (RdRp), RNA viruses including PVX have a very high mutation rate (Steinhauer et al. 1992;Duffy et al. 2008), which may allow PVX to easily escape Rx-mediated resistance. Therefore, it is necessary to introduce new PVX resistance genes in future breeding projects to avoid any potential damage caused by resistance-breaking PVX isolates.
The most interesting finding in this study is the identification of a partial PVX resistance phenotype in Waiyin-1. In comparison to susceptible Kexin-1, the infection of PVX in Waiyin-1 was delayed by about 5 days. Replication and cell-to-cell movement assays showed that the replication but not movement of PVX was inhibited in Waiyin-1. Moreover, our results reveal that the expression profiles of PR genes in Waiyin-1 are apparently different from the Rx-triggered transcriptome reprogramming in which PR genes, e.g., PR1, PR2, PR3 and PR4, are upregulated at early stage of PVX infection (Knip et al. 2019). The infection of PVX in Waiyin-1 activates the expression of the four PR genes and stress-related WRKY1 at a later stage. These results suggest that the resistance of Waiyin-1 is not likely associated with a typical R protein. The resistances of most known ADVRPs, such as JAX1, REMs, PNbMTS1, h-type thioredoxin (ZmTrxh) and Tm-1, do not induce the expression of PR genes (Ishibashi et al. 2007;Cheng et al. 2009;Yamaji et al. 2012;Sugawara et al. 2013;Yoshida et al. 2019). Recently, a novel class of ADVRP that is able to confer a broad resistance to both positive-and negative-stranded RNA viruses was discovered. This class of ADVRP encodes a sulfotransferase to catalyze the conversion of salicylic acid (SA) into sulphonated SA (SSA), which serves as a signal to trigger SA biosynthesis, resulting in higher expression of PR genes (Bake et al. 2010;Wang et al. 2014;Zhao et al. 2021). Whether the resistance of Waiyin-1 is also conferred by a sulfotransferase needs to be investigated in future studies. In conclusion, our results provide new understanding on the resistance to PVX in potato plants.

Conclusions
In short, four potato cultivars (Longshu-3, Eugene, Atlantic and Waiyin-2) were found to contain the Rx gene, which confers the extreme resistance to PVX. The partial resistance of Waiyin-1 to PVX is associated with the suppression of viral replication and expression of pathogenesis-related genes. The rest eighteen potato cultivars are susceptible to PVX infection.

Virus resource, plant materials and growth condition
The infectious clone of PVX UK3 isolate (pGR107) has been described previously (Lu et al. 2003). Potato cultivars were kindly provided by Heilongjiang Academy of Agricultural Sciences. Virus-free potato seedlings were cultivated in fertile soil at 23℃, 60% humidity in a plant growth chamber with a light/dark cycle of 16 h/8 h. N. benthamiana plants were grown in pots in a plant growth chamber at 23℃ with a light/dark cycle of 16 h/8 h.

Virus inoculation
For agroinfiltration assay, Agrobacterium tumefaciens strain GV3101 harboring pSoup helper plasmid was used. PVX infectious clones were transformed by electroporation. Agrobacteria harboring proper plasmid were collected by centrifuge, washed twice with infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl 2 , 100 µM acetosyringone), and resuspended in infiltration buffer at room temperature. After keeping at room temperature for 1 h, agrobacteria were infiltrated into N. benthamiana or potato leaves at an optical density at 600 nm (OD 600 ) of 0.4 or as indicated in the context using 1-mL needleless syringes.
For sap-inoculation, potato and N. benthamiana leaves were pre-dusted with 600-mesh carborundum powder, and then were rubbed with crude sap of virus-infected N. benthamiana leaves in 10 mM of sodium phosphate buffer (pH 7.0). At 2 min post-inoculation, the inoculated leaves were washed with distilled water and covered with a pre-wetted tissue napkin, and were subsequently incubated in a growth chamber at 23℃ with a light/dark cycle of 16 h/8 h for symptom development.

Enzyme-linked immunosorbent assay (ELISA)
ELISA assay was conducted using the ELISA Reagent Set for potato virus X (Agdia, Elkhart, IN, USA) according to the provided protocol. The detailed procedure is as follows: potato leaf tissues were homogenized in general extract buffer (GEB) (0.1 g per 1 mL), and resulting leaf extracts were dispensed in capture antibodies-pretreated ELISA plates (100 µL per well). After incubating at room temperature for 2 h in a humid box, the plate was washed 7 times with 1 × PBST. Alkaline phosphatase-conjugated secondary antibody diluted in ECI buffer was then added to each well at 100 µL per well. The plate was washed 8 times with 1 × PBST after incubating at room temperature for 2 h in a humid box. Each well was added with PNP solution (1 mg/mL in PNP substrate buffer) and incubated for 60 min in the dark. Absorbance values at 405 nm were read using a Microplate reader (Thermo Scientific). All samples were repeated triply.

DNA sequencing and sequence analyses
DNA sequences obtained via Sanger sequencing were pretreated using the SeqBuilder program to remove the vector backbone and primer sequences and then assembled using the SeqMan program in the Lasergene 7.1 software package (DNASTAR Inc.) (Burland 2000). For multiple sequence alignment, sequences were aligned by Clustal Omega with default parameters and displayed by ESPript 3.0 (https:// espri pt. ibcp. fr/ ESPri pt/ ESPri pt/ index. php) (Robert and Gouet 2014;Sievers and Higgins 2018). Maximum Likelihood (ML) phylogenetic tree was constructed using the MEGA X software (Kumar et al. 2018). In detail, sequences were aligned using the Clustal W with default parameters (Chenna 2003), and then rendered for phylogeny analysis using the ML method. The node significance was evaluated with 1,000 bootstrap replicates.

Microscopic observation
Microscopic observations were performed as described previously (Wu et al. 2018). In brief, the fluorescence signal in the infiltrated leaf area was observed with a TCS SP8 LIGHTNING Confocal Microscope (Leica, Germany). YFP was excited at 496 nm and collected between 520 and 535 nm.