Cucumber Mosaic Virus

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THE VIRUS MOLECULAR BIOLOGY

Cucumber

Mosaic Virus Edited by

Peter Palukaitis and Fernando García-Arenal

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Host Responses: Resistance ●

Chikara Masuta Hokkaido University, Research Faculty of Agriculture, Sapporo, Japan

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Hideki Takahashi Tohoku University, Graduate School of Agricultural Science, Sendai, Miyagi, Japan

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INTRODUCTION

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RNA silencing and resistance (R) gene-mediated resistance are major defense systems against virus infection in host plants. The molecular mechanisms responsible for RNA silencing against several viruses, including cucumber mosaic virus (CMV), are well characterized (see reviews by Ding, 2010; Ding & Voinnet, 2007).

With regard to the resistance to viruses conferred by dominant R genes encoding nucleotide-binding and leucine-rich repeat (NB-LRR) class immune receptors, the function of the R protein in response to a limited number of viruses—such as N to tobacco mosaic virus, Rx to potato virus X, HRT to turnip crinkle virus, and RCY1 to CMV—has been the focus of most studies (Cournoyer & Dinesh-Kumar, 2011; Kachroo et al., 2006; Zvereva & Pooggin, 2012). Furthermore, a few non-NB-LRR class-dominant or recessive R genes controlling resistance to some viruses have been characterized for certain plant species (Ronde et al., 2014). Here, we will describe the state of knowledge of resistance responses to CMV.

R GENE-MEDIATED RESISTANCE: RCY1 AS A MODEL

R Gene-Induced HR as Programmed Cell Death NB-LRR class R gene-mediated resistance to viruses is generally associated with the induction of programmed cell death (PCD), which is visible as the formation of necrotic local lesions on the host at the primary infection site of the virus as a result of a hypersensitive response (HR). The restriction of virus spread is often concomitant with the induction of PCD; however, in some combinations of an avirulent strain of virus and a host plant carrying an NB-LRR class R gene, the restriction of virus spread is uncoupled from the development of a necrotic local lesion at the infection site (Bendahmane et al., 1999; Bhattacharjee et al., 2009; Kim et al., 2010; Sekine et al., 2006; Takahashi et al., 2012a). For example, overexpression of RESISTANCE TO CMV(Y) (RCY1), the NB-LRR class R gene to CMV, shifts the HR to extreme

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resistance in Arabidopsis thaliana (Sekine et al., 2008) (Fig. 5.1). While there have been several studies on the relationship between necrotic local lesion formation at the viral primary infection site and resistance to the virus, whether PCD by the HR has a role in NB-LRR class R gene-conferred resistance to viruses remains a matter of discussion.

RCY1/HRT/RPP8 Locus in Arabidopsis thaliana In the Arabidopsis genome, ~150 genes encode NBLRR class R proteins (Bakker et al., 2006; Meyers et al., 2003). Generally, the recognition of the NB-LRR class R proteins against pathogens is very specific. To recognize

FIG. 5.1. Response to CMV(Y) in Arabidopsis thaliana ecotype Col-0 transformed with RCY1 tagged with HA-epitope sequence. A, Necrotic local lesions on CMV(Y)-inoculated leaves of wild-type Col-0 that does not carry RCY1 and of two RCY1-HA transgenic lines, #12 and #13 (bright field optics). HR, hypersensitive reaction; ER, extreme resistance. B, Trypan blue staining of CMV(Y)-inoculated leaves of inoculated plants. C, Immunological detection of the virus coat protein in CMV(Y)-inoculated leaves of inoculated plants. D, Immunological detection of RCY1-HA protein of two RCY1-HA transgenic lines #12 and #13.

In transgenic line #12, the level of RCY1-HA expression is similar to wild-type C24. Transgenic line #13, containing 10 copies of RCY1-HA, accumulates more RCY1-HA than does line #12, which has one copy of the RCY1 transgene. (Courtesy H. Takahashi—© APS)

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a wide range of pathogens, several NB-LRR class R genes often are arranged in clusters generated by tandem duplications due to unequal crossing over and to gene conversion (Holub, 2001). Some of these NB-LRR class R genes are located singly on the plant genome. RCY1 against CMV(Y) is a singleton in the Arabidopsis genome.

Interestingly, RCY1 is allelic to HRT, a resistance gene to turnip crinkle virus in ecotype Di-17, and to RPP8, a resistance gene to Hyaloperonospora arabidopsidis in ecotype Landsberg (Cooley et al., 2000; McDowell et al., 1998; Takahashi et al., 2002). Thus, the RCY1/HRT/RPP8 locus seems to have evolved from a certain ancient NBLRR class R gene and diversified among three different ecotypes, so that each specifically recognizes a different pathogen (Kuang et al., 2008).

CC-NB-LRR Class Resistance R Gene Against Y-CMV The most common structure of NB-LRR class R proteins consists of an N-terminal response domain involved in the activation of downstream signaling, an NB-ARC domain (a central molecular switch), and an LRR domain at the C-terminal end (Marone et al., 2013). The NB-LRR class R proteins are divided into two subclasses according to the N-terminal structure: a coiled-coil (CC) or a Toll and interleukin-1 receptor (TIR) domain (Soosaar et al., 2005). Major known NBLRR class R proteins, such as RCY1, belong to the CCNB-LRR subclass, whereas the TIR-NB-LRR subclass is a minor group (Bakker et al., 2006; Meyers et al., 2003).

A. thaliana is a host plant of CMV, but a limited number of ecotypes of A. thaliana are resistant to CMV (Takahashi et al., 1994). RCY1, which encodes a CC-NB-LRR subclass R protein in A. thaliana ecotype C24, confers resistance to a yellow strain of CMV (Y-CMV) (Takahashi et al., 1994, 2002). RCY1-mediated resistance to Y-CMV is accompanied by the development of necrotic local lesions at the primary infection sites (Fig. 5.1A and B), elevated expression of defense-related genes such as PATHOGENESIS-RELATED 1a (PR-1a), and accumulation of salicylic acid (SA) (Ishihara et al., 2008; Takahashi et al., 1994), but the RCY1 transcript levels do not increase in response to Y-CMV infection (Sato et al., 2014). Interestingly, the level of RCY1 protein regulates the strength of resistance to Y-CMV. Among a series of RCY1-transgenic lines of A. thaliana ecotype Col-0 lacking RCY1, a transgenic line in which RCY1 protein tagged with HA-epitope (RCY1-HA) accumulated at a level similar to that in wild-type C24 also exhibited an HR against Y-CMV infection. However, extreme

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resistance to Y-CMV was induced in a transgenic line that overaccumulated RCY1-HA (Fig. 5.1) (Sekine et al., 2008). This higher level of RCY1 protein conferring resistance to Y-CMV is regulated by the presence of the intron sequence in the genomic RCY1 gene, rather than by the induction of RCY1 gene expression in response to Y-CMV infection (Sato et al., 2014). Thus, intronmediated enhancement of RCY1 gene expression seems to play a key role in maintaining the level of RCY1 protein in Arabidopsis. On the other hand, when RCY1 was expressed transiently in Nicotiana benthamiana leaves after agroinfiltration, degradation of RCY1 coincided with the development of PCD in Y-CMV-inoculated leaves but not in mock-inoculated leaves (Takahashi et al., 2012b). RCY1 degradation may contribute to negative feedback and suppress developing systemic PCD. These findings indicate that precise control of the RCY1 protein level is critical for inducing resistance to Y-CMV; the virus is completely restricted to the primary infection site so that the virus cannot escape from the site to cause systemic necrosis.

Functional Analysis of RCY1 Chimeric constructs between the alleles of the RCY1/ HRT/RPP8 locus have been used to analyze the function of the domains in pathogen recognition specificity. A series of constructs was analyzed by exchanging domaincoding regions between RCY1 and its allelic RPP8 with differing pathogen recognition specificity. Expression of chimeric constructs sharing the LRR domain of RCY1 in transgenic A. thaliana ecotype Col-0 (Fig. 5.2A and B) induced PCD in Y-CMV-inoculated leaves (Fig. 5.2C), and systemic spread of virus was restricted. By contrast, individual expression of the CC-NB domains and LRR domain of RCY1 did not activate PCD in the agroinfiltrated regions in Y-CMV-inoculated leaves. Thus, the LRR domain of RCY1 is the primary determinant of Y-CMV recognition specificity, and the CC-NB domain of RCY1 is necessary but replaceable by that of RPP8 for inducing the HR resistance. The LRR domain is a structural motif characterized by a conserved pattern of hydrophobic leucine residues and a broad surface for proteins to interact (Kobe & Kajava, 2001). Variation in the amino acid sequences among RCY1, RPP8, and HRT proteins indicates that LRR domains undergo strong positive selection and possibly interact with pathogens (Cooley et al., 2000; McDowell et al., 1998; Takahashi et al., 2002). According to the commonly accepted models of the action of NB-LRR class R protein (Collier & Moffett, 2009), Y-CMV is recognized by the LRR

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domain of RCY1, leading to an intermolecular interaction between the LRR domain and other domains in the N-terminal region of RCY1. This results in a conformational change in the LRR domain that is required to induce RCY1-conferred resistance to Y-CMV (Takahashi et al., 2012b).

More recently, microRNA (miRNA)-mediated negative regulation of NB-LRR “microRNA” class R genes against viruses has been reported (Li et al., 2012; Shivaprasad et al., 2012; Zhai et al., 2011). Because the well-characterized viral RNA silencing suppressor (RSS) protein, CMV 2b, has a strong affinity to bind small RNAs (Goto et al., 2007), RSS may lead to the suppression of the miRNA-mediated silencing of NB-LRR class R genes and induce effector-triggered immunity. However, whether this mode of interplay between viral RSS and NB-LRR class R gene expression is conserved is still unknown.

Avirulence (Avr) Factor of Y-CMV The Avr of Y-CMV has been analyzed using reassortant CMV RNA between strains Y-CMV and virulent B2-CMV. The induction of RCY1-mediated resistance in C24 leaves inoculated with a series of reassortant virus indicates that the coat protein (CP) of Y-CMV acts as the Avr to RCY1 (Takahashi et al., 2001). Furthermore, when cDNAs of the various reassortant Y-CMV RNAs were expressed transiently under the control of the CaMV 35S promoter after agroinfiltration in RCY1transformed N. benthamiana leaves, PCD was induced in the infiltrated regions of the leaves only by the transient expression of cDNA to Y-CMV RNA 4, encoding the virus CP, but not to Y-CMV RNA 1, RNA 2, RNA 4A, or RNA 3. Therefore, the CP of Y-CMV acts as an Avr independently of other viral proteins. Moreover,

FIG. 5.2. Response of Arabidopsis thaliana ecotype Col-0 transformed with chimeric constructs with an LRR domain exchange between RCY1 and RPP8 cDNAs. A, Schematic structure of the chimeric constructs. CYCYRP-HA encodes CC-NB domains of RCY1 (CCRCY1 and NBRCY1) and LRR domain of RPP8 (LRR RPP8) tagged with the HA-epitope sequence. RPRPCY-HA composed of the coding sequences for the CC-NB domains of RPP8 (CCRPP8 and NBRPP8) and LRR domain of RCY1 (LRR RCY1), which are reciprocal in construction to CYCYRP-HA. B, Immunological detection of the RCY1/RPP8 chimeric protein in CYCYRP-HA- or RPRPCY-HA-transformed Col-0 plants. C, Necrotic local lesion formation on cucumber mosaic virus CMV(Y)-inoculated leaves of CYCYRP-HA- or RPRPCY-HA-transformed Col-0. (Courtesy H. Takahashi—© APS)

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transient expression of chimeric constructs between Y-CMV and B2-CMV CP genes in RCY1-transformed N. benthamiana leaves suggests that one amino acid at the N-terminal region of the CP determines the induction of RCY1-mediated resistance to Y-CMV (S. Miyashita, Y. Ando, and H. Takahashi, unpublished results).

Downstream Signaling of RCY1 In a survey of RCY1-interacting host proteins using the yeast two-hybrid system, the WRKY70 transcription factor was identified (Ando et al., 2014). WRKY70 can bind to the CC-NB domains of RCY1 but not to the full-length RCY1. In a Y-CMV-inoculated knockout Arabidopsis mutant wrky70 carrying the transgene RCY1-HA, restriction of the virus to the inoculated leaves was partially compromised. Furthermore, analysis of RCY1 downstream signaling in several Arabidopsis mutants impaired in hormonal signal transduction pathways indicates that SA- and ethylene-dependent signaling, which antagonistically cross-talk with jasmonate-dependent signaling, are indispensable for RCY1-mediated resistance, whereas an unknown novel signaling pathway seems to be required for complete resistance to Y-CMV (Takahashi et al., 2004).

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NATURALLY OCCURRING RESISTANCE TO CMV IN CROPS

A class of distinct recessive R genes, encoding eukaryotic translation initiation factors eIF4E, eIF(iso)4E, eIF4G, and eIF(iso)4G, has been reported largely for resistance to potyviruses, as well as to bymoviruses, melon necrotic spot virus, rice yellow mottle virus, and CMV (Truniger & Aranda, 2009). In the case of CMV, two genes, cum1 and cum2, both from Arabidopsis, encoded translation factors eIF4E and eIF4G, respectively (Yoshii et al., 2004). However, resistance to CMV in crops largely involves two or more genes with complex inheritance (Kang et al., 2005). CMV resistance sources in various crops, wild species, and Arabidopsis have been well summarized by Palukaitis and GarciaArénal (2003) and Jacquemond (2012).

Although considerable efforts have been made to produce CMV-resistant crops in the world, such distinct resistance as R gene-mediated resistance in Arabidopsis ecotype C24 (see previous discussion of RCY1) has not

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been discovered in many important crops. Here, we describe several studies on naturally occurring resistance genes to CMV in some important crops. Among them, a CMV resistance system that does not activate defense reactions has been reported recently in melon (Guiu-Aragonés et al., 2015). The proposed mechanism differs completely from the one for RCY1 (Sato et al., 2014; Takahashi et al., 2002), suggesting that plants actually have a wide range of strategies to resist CMV depending on their evolutionary history and breeding genealogy.

In the study of CMV resistance in melon lines, Pitrat and Lecoq (1980) found that line PI161375 had complete resistance to CMV when the virus was inoculated through Aphis gossypii aphids and that the resistance was controlled by a single dominant gene, Vat (Boissot et al., 2016). The Vat gene was found to be unique in conferring resistance to both A. gossypii and the aphidtransmissible viruses. In this context, the Vat gene can be used in many other crops for breeding programs of viral resistance.

Cucurbita and Cucumis Species Cucurbita species generally vary greatly in their CMV tolerance and resistance (Lebeda & K ř ístková, 1996). In cucumber (Cucumis sativus), Wang et al.

(2004) showed that the cultivar Delila had resistance at the single-cell level that restricted viral accumulation to a low level. They also discovered that the resistance was broken by coinfection with zucchini yellow mosaic virus. A similar resistance to CMV was discovered in the cucumber cultivar Shimshon, which correlated with overexpression of the RNA-DEPENDENT RNA POLYMERASE 1b gene (Leibman et al., 2018). A single recessive gene, cmv1 of melon (Cucumis melo), which confers complete resistance to CMV Subgroup II strains, has recently been analyzed in detail (Guiu-Aragonés et al., 2014, 2015). In a comparative study between CMV Subgroup I and Subgroup II strains, Guiu-Aragonés and colleagues revealed that the CMV 3a movement protein was associated with cmv1 resistance. Guiu-Aragonés et al. (2016) most recently proposed an elegant model of the molecular mechanism for cmv1 resistance that is based on the interactions between CMV 3a protein and the cmv1 product at plasmodesmata (Fig. 5.3). For practical breeding, the CMV resistance gene designated Cmv-2 was isolated from Cucurbita moschata var. Seminole pumpkin and transferred to other Cucurbita species, including squash (Nicolet et al., 2007). Cmv-2 is a single dominant gene and confers a high level of stable resistance.

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FIG. 5.3. Model of cmv1 resistance to CMV. PS is the plant containing the dominant CMV1; SC12-1-99 has the recessive gene cmv1. CMV-FNY can infect the cmv1-containing plant. A, The CMV1-encoded protein can interact with the CMV movement protein (MP), resulting in either viral transport to plasmodesmata (PDs) or the opening of PDs. B, CMV-LS MP cannot interact with the cmv1 protein and thus CMV cannot move through PDs. C, CMV-FNY MP has the ability to interact with the cmv1 protein, and the interaction then allows viral movement as in panel A. (Reproduced by permission of the publisher, from Guiu-Aragonés, C., et al. 2016. cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon. Molecular Plant Pathology 17:973-984. © BSPP and John Wiley & Sons Ltd. DOI: 10.1111/mpp.12351)

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Pepper (Capsicum annuum) The nature of resistance of pepper to CMV has been reported to be associated mostly with limited systemic movement of CMV, thus a non-HR type of resistance (Garcia-Ruiz & Murphy, 2001). Although many quantitative trait loci (QTLs) conferring CMV resistance have been reported, the resistance phenotypes were largely dependent on the particular combination of pepper cultivar and local CMV strain tested (Caranta et al., 2002; Yao et al., 2013). Kang et al.

(2010) characterized the single dominant gene from cultivar Bukang, Cmr1, which inhibits systemic movement of CMV, suggesting that major genes controlling CMV resistance can be isolated from pepper (Kang et al., 2010). Suzuki et al. (2003) found a distinct HRtype response to CMV in Capsicum baccatum, as well as a general non-HR-type resistance in C. annuum (Fig. 5.4); thus, an R gene-mediated resistance to CMV may be present in pepper. In addition, Grube et al.

(2000) reported that Capsicum frutescens ‘BG2814-6’

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was resistant to several CMV isolates and that the resistance is conferred by at least two major recessive genes.

Tomato (Solanum lycopersicum) Cultivated tomato varieties have provided few genetic resources against CMV infection. Although some wild tomato species are resistant (tolerant) to CMV, introducing this trait into commercial cultivars was not successful before the introgression of the single dominant gene, Cmr, from Solanum chilense into tomato (Stamova & Chetelat, 2000). In tomato, the CMV satellite RNAs, referred to as “necrogenic satellite RNAs,” induced a lethal necrosis that devastated the tomato industry in several countries (Jordá et al., 1992). Cillo et al. (2007) tested wild tomato species for tolerance to lethal necrosis induced by CMV containing necrogenic satellite RNAs and found that Solanum habrochaites had some tolerance that is controlled by complex QTLs.

FIG. 5.4. Hypersensitive response to CMV in Capsicum baccatum (PI 439381-1-1) and non-HR resistance in C. annuum (cultivars Sapporo- and Nanbu-oonaga) after inoculation with CMV. (Courtesy K. Suzuki— © APS)

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Potato (Solanum tuberosum) CMV is often detected in potato plants diagnosed with viral infections, but CMV infection is not important in cultured potatoes because potatoes per se are thought to have some resistance to CMV (Valkonen et al., 1995).

No genes (not even QTLs) responsible for the resistance have been identified. To confirm this resistance, Celebi et al. (1998) inoculated 32 potato genotypes (mostly tetraploid) with an ordinary strain of CMV (Fny-CMV) and found that CMV was restricted at the infection sites in two-thirds of the inoculated plants, suggesting that potatoes indeed have a mechanism to block the longdistance movement of CMV. Valkonen and Watanabe (1999) then conducted crosses using different diploid genotypes to identify the resistance gene(s) and found that the gene restricting CMV long-distance movement was controlled by a single major trait. They also found that the resistance was easily overcome at a higher temperature (>28°C). Celebi-Toprak et al. (2003) further revealed that potato resistance was specific to some CMV strains, suggesting that CMV resistance in potato is not necessarily absolute.

Soybean (Glycine max) Soybean cultivars have various resistance responses to CMV infection, but absolute resistance to a broad range of CMV soybean strains has not been reported.

In soybean breeding for CMV resistance, the resistance responses seem to have developed specifically against the local CMV soybean isolates (Hong et al., 2003).

Ohnishi et al. (2011) found that the resistance to the SC-CMV strain in soybean cultivar Harosoy operates at the level of viral long-distance movement. In addition,

by QTL analysis, they further demonstrated that at least three QTLs were involved in the systemic infection by SC-CMV. However, since then, little progress has been made toward identifying soybean resistance genes.

Cowpea (Vigna unguiculata) Most CMV strains induce an HR on the inoculated leaves of cowpea plants, while some strains, designated “CMV legume strains,” systemically infect legume species. Therefore, most cowpea cultivars are actually resistant to CMV infection; cowpea has been well utilized as an indicator plant to identify CMV infection (Palukaitis et al., 1992). Nasu et al. (1996) demonstrated that an R gene is responsible for the resistance and designated the locus as the single dominant gene, Cry. The HR to CMV is conferred by a combination of the single

dominant R gene Cry of cowpea and the CMV 2a gene product as the Avr (Chida et al., 2000; Hu et al., 2012; Karasawa et al., 1999; Kim & Palukaitis, 1997). Based on the results of a comparative study between an HRinducing strain and a resistance-breaking strain, Kim and Palukaitis (1997) identified two amino acids in the 2a protein that are responsible for the HR induction.

They also revealed that two host genes are probably involved in the resistance; one can restrict viral spread from the infection site, and the other induces HR. However, in two other reports (Hu et al. 2012; Karasawa et al., 1999), only one amino acid change is required for the HR determination. This difference could be due to different genetic backgrounds between the cowpea lines used. In spite of these extensive studies on Cry early in the research on CMV resistance, the gene still has not been identified. For CMV legume strains, sources of resistance have been screened because CMV infection causes significant losses in cowpea production, and some resistant/tolerant genotypes have been reported (Gillaspie, 2006).

Common Bean (Phaseolus vulgaris) The RT4-4 gene, encoding a TIR-NBS-LRR class protein, has been isolated from the bean cultivar Othello.

The expression of RT4-4 in N. benthamiana actually activated a resistance-like response (systemic necrosis) to CMV with the coexpression of the CMV 2a gene product (Seo et al., 2006). Recently, Azizi and Shams-bakhsh (2014) reported that common bean also has a non-HR type of resistance and tolerance. However, in spite of such efforts to find CMV resistance traits in common bean, no successful commercial application has been reported.

Lettuce (Lactuca sativa) Because CMV is important in lettuce production, it is a target of breeding programs for resistance. The CMVresistant line PI261653 was established from a related species, L. saligna. However, a resistance-breaking strain, CMV-LsS, has been isolated from the resistant lines; CMV RNAs 2 and 3 were found to be responsible for the resistance breaking (Edwards et al., 1983).

Lupin (Lupinus sp.)

Narrow-leafed lupin (Lupinus angustifolius) is very susceptible to CMV, which is seed transmitted. In Australia, breeding programs for L. angustifolius to prevent CMV seed transmission were conducted extensively

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from 1987 to 1992. Although highly resistant lines were not obtained, at least moderately resistant lines (1–6% seed transmission) were found (Jones & Cowling, 1995).

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CMV TOLERANCE

In a tolerant plant, unlike a resistant one, viruses accumulate at a certain level (even a high level) but do not induce severe symptoms (Lapidot et al., 1997). For CMV tolerance, there are few reports dissecting its molecular basis. Cucumber cultivars have been reported to have a variety of levels of tolerance or resistance to CMV. To be accurate, some so-called resistance should be regarded as tolerance, especially when CMV can replicate to some extent at the single-cell level and accumulate at a relatively low level in systemic leaves without producing severe symptoms (Maule et al., 1980; Wang et al., 2004). Symptomless, CMV-tolerant plants may serve as viral reservoirs, which would allow viral transmission to other plants. We should also note that coinfection with a potyvirus can greatly intensify CMV accumulation in CMV-infected tolerant plants through synergistic interactions (Wang et al., 2004). As to the host (cucumber) genes involved in CMV tolerance, Leibman et al. (2018) recently showed that the high expression levels of an RNA-DEPENDENT RNA POLYMERASE 1 gene were associated with resistance in an SA-independent manner. This resistance may be regarded as tolerance, because CMV is reduced to a very low level but spreads systemically without distinct symptoms. In addition, a brassinosteroid plant hormone has been demonstrated to be important for CMV tolerance in both Arabidopsis and cucumber (Xia et al., 2009; Zhang et al., 2015).

Although elevated H2O2 in brassinosteroid-treated cucumber was suggested as a secondary mediator (Xia et al., 2009), at this time, we do not know much about the signal transduction pathway for CMV tolerance.

CONCLUDING REMARKS There seem to be many genetic resources for CMV resistance, including R genes, and we understand some of the molecular mechanisms for CMV resistance. Even so, we have yet to produce crops with strong CMV resistance, because several divergent CMV strains can break the resistance that is effective against other strains. In the future, we may be able to overcome this problem by combining such resistant traits in one crop.

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THE PATHOLOGY

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