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. 2017 Feb 8;19(2):300–312. doi: 10.1111/mpp.12518

Differential expression of cucumber RNA‐dependent RNA polymerase 1 genes during antiviral defence and resistance

Diana Leibman 1, Michael Kravchik 1, Dalia Wolf 2, Sabrina Haviv 1, Mira Weissberg 3, Ron Ophir 3, Harry S Paris 4, Peter Palukaitis 5, Shou‐Wei Ding 6, Victor Gaba 1, Amit Gal‐On 1,✉
PMCID: PMC6637986  PMID: 27879040

Summary

RNA‐dependent RNA polymerase 1 (RDR1) plays a crucial role in plant defence against viruses. In this study, it was observed that cucumber, Cucumis sativus, uniquely encodes a small gene family of four RDR1 genes. The cucumber RDR1 genes (CsRDR1a, CsRDR1b and duplicated CsRDR1c1/c2) shared 55%–60% homology in their encoded amino acid sequences. In healthy cucumber plants, RDR1a and RDR1b transcripts were expressed at higher levels than transcripts of RDR1c1/c2, which were barely detectable. The expression of all four CsRDR1 genes was induced by virus infection, after which the expression level of CsRDR1b increased 10–20‐fold in several virus‐resistant cucumber cultivars and in a broad virus‐resistant transgenic cucumber line expressing a high level of transgene small RNAs, all without alteration in salicylic acid (SA) levels. By comparison, CsRDR1c1/c2 genes were highly induced (25–1300‐fold) in susceptible cucumber cultivars infected with RNA or DNA viruses. Inhibition of RDR1c1/c2 expression led to increased virus accumulation. Ectopic application of SA induced the expression of cucumber RDR1a, RDR1b and RDRc1/c2 genes. A constitutive high level of RDR1b gene expression independent of SA was found to be associated with broad virus resistance. These findings show that multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles.

Keywords: cucumber, gene expression, RDR1, silencing, virus resistance

Introduction

RNA silencing is a general eukaryotic cellular defence mechanism which regulates transcriptional or post‐transcriptional gene expression in a sequence‐specific manner by small interfering RNA (siRNA) molecules (Baulcombe, 2004; Dalmay et al., 2000; Ding and Voinnet, 2007; Meister and Tuschl, 2004; Waterhouse et al., 2001). In plants, the initial signals for the activation of gene silencing are exogenous (e.g. RNA virus) and endogenous (transposon) double‐stranded RNA (dsRNA) molecules (Agrawal et al., 2003; Hamilton and Baulcombe, 1999). RNA‐dependent RNA polymerases (RDRs) are key regulators of RNA and virus silencing via the synthesis of dsRNAs that activate gene silencing after processing by DICER‐like nucleases (DCLs) (Garcia‐Ruiz et al., 2010; Qi et al., 2009; Qu, 2010; Qu et al., 2008; Schiebel et al., 1993; Xie et al., 2001). Recently, it has been shown that the tomato genes for resistance against Tomato yellow leaf curl virus, designated Ty‐1 and Ty‐3, are RDR genes (Verlaan et al., 2013).

RDR1 is mainly associated with the antiviral RNA silencing pathway via the production and amplification of exogenous viral dsRNA in infected plants, which are subsequently digested by DCL‐4 and DCL‐2 into 21‐nucleotide siRNA duplexes (Diaz‐Pendon et al., 2007; Qi et al., 2009; Wang et al., 2010). RDR1, RDR6 and, to some extent, RDR2 are associated with defence against viral RNA accumulation (Donaire et al., 2008; Garcia‐Ruiz et al., 2010; Qi et al., 2009; Wang et al., 2010). RDR1 and RDR6 can be coordinated in defence against viruses, as demonstrated with Cucumber mosaic virus (CMV) in Arabidopsis, but seem to be antagonistic in Plum pox virus in transgenic Nicotiana benthamiana Domin expressing NtRDR1 (Ying et al., 2010).

The silencing of RDR1 enhances the infection of several viruses in Nicotiana tabacum L., Arabidopsis and rice (Alamillo et al., 2006; Rakhshandehroo et al., 2009; Wang et al., 2016; Xie et al., 2001; Yu et al., 2003). Furthermore, the absence of a functional RDR1 in N. benthamiana can explain the enhanced susceptibility of this species to many plant viruses (Yang et al., 2004). However, suppression of RDR1 in potato is not associated with increased virus susceptibility (Hunter et al., 2016). By contrast, the constitutive expression of RDR1, observed in a transgenic cucumber Line 823 (Cucumis sativus L.), causes broad potyvirus resistance (Leibman et al., 2011). The regulation of RDR1 following virus infection is not completely understood. Xie et al. (2001) demonstrated that either Tobacco mosaic virus infection or salicylic acid (SA) application induced RDR1 activity. Subsequently, this phenomenon was shown in different plant hosts for several viral pathogens and fungi, which implicates an important role for RDR1 in plant antiviral defence and against other biotic and abiotic stresses (Gilliland et al., 2003; Hunter et al., 2013; Liu et al., 2009; Yang et al., 2004; Yu et al., 2003). Several studies have shown the involvement of RDR1 in the regulation of plant defences, e.g. phytohormone biosynthesis genes that mediate herbivore protection (Pandey et al., 2008), cuticular wax deposition (Lam et al., 2012; Pandey et al., 2008) and several other defence‐related genes (Hunter et al., 2013; Rakhshandehroo et al., 2009). In addition, it has been shown that rice RDR1 plays a role in gene regulation, and the null mutant Osrdr1 shows alterations of small RNA (sRNA) accumulation and specific alterations of DNA methylation (Wang et al., 2014). RDR1 is associated with DNA methylation in tomato, rice and Arabidopsis (Stroud et al., 2013; Wang et al., 2014). Recently, it has been shown that plant viruses induce microRNA (miRNA) expression, which regulates RDR1 expression in rice via a molecular cascade (Wang et al., 2016). In addition, the biogenesis of viral‐activated siRNAs has been demonstrated from the coding region of many host genes by RDR1 in response to virus infection (Cao et al., 2014).

Here, we characterize a unique RDR1 family of four genes in cucumber and their responses to virus infection. We show that, in cucumber, RDR1b is constitutively expressed at a high level only in resistant plants, whereas RDR1c1 and RDR1c2 are barely expressed in healthy plants, but induced to very high levels by RNA and DNA virus infection. The inhibition of cucumber RDR1c1/2 leads to increased virus accumulation, demonstrating a role for these inducible genes in the control of virus infection. Multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles. These RDR1 genes may have either distinct or overlapping functions.

Results

The RDR1 gene family in cucumber

The RDR1 sequences of several plant families were used to identify Cucumis spp. RDR1 genes in the Cucurbit Genomics Database. We identified a number of putative RDR1 genes within the RDRα clade (Willmann et al., 2011) of Cucumis spp. (Fig. 1). In cucumber, four putative unique RDR1 family genes were identified in addition to CsRDR2 and CsRDR6 (Figs 1 and S1, Table S1, see Supporting Information). The CsRDR1a and CsRDR1b genes were positioned very close to each other (approximately 2.5 kbp apart) in a head‐to‐head orientation (http://www.icugi.org/cgi-bin/gb2/gbrowse/cucumber_v2/?name=gene:Csa5G239140). The CsRDR1c1 and CsRDR1c2 genes were within approximately 350 kbp of each other. The four exons of the CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes were similarly organized and, in each gene, similar exons were of comparable size (Fig. 1). This may indicate an ancestral RDR1 for all RDR1 genes in Cucumis spp.

Figure 1.

Figure 1

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Genome maps of CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes of cucumber (Cucumis sativus). Boxes represent exons and lines indicate introns. The genome maps were based on the cucumber Genomics Database (http://www.icugi.org). The numbers indicate the size of the exons (in nucleotides) and the start codon (ATG) is represented by an arrow. Exon numbers are marked in Roman numerals (I–IV).

The CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes were all expressed, but the expression of CsRDR1c1 and CsRD1c2 was scarcely detected in healthy plants under our conditions, compared with the high gene expression levels in virus‐infected plants (Figs 2, S1 and S2, see Supporting Information). The coding regions of the CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes of cucumber cv. ‘Shimshon’ [resistant to CMV, Cucumber green mottle mosaic virus (CGMMV) and Cucumber vein yellow virus (CVYV)] and susceptible cv. ‘Bet‐Alfa’ were cloned, sequenced and mapped to an RDR1 cluster based on sequence homology (Fig. S3, see Supporting Information). There were no sequence differences between the CsRDR1 genes of cv. ‘Shimshon’ and the corresponding genes in cv. ‘Bet‐Alfa’ (data not shown). This was also the case for the putative promoter regions (approximately 3 kbp upstream of the coding region) of the four RDR1 genes in cv. ‘Shimshon’ vs. the corresponding putative promoter regions in cv. ‘Bet‐Alfa’ (data not shown). However, within a cultivar, the putative promoter regions of the RDR1a, RDR1b and RDR1c genes did not exhibit sequence similarity with each other, but the putative promoter regions of RDR1c1 and RDR1c2 were identical (data not shown).

Figure 2.

Figure 2

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Expression levels of CsRDR1 genes (CsRDR1a, CsRDR1b and CsRDR1c (CsRDR1c1 + CsRDR1c2 together) in non‐inoculated cucumber plants. Gene expression analysis of CsRDR1a, CsRDR1b and CsRDR1c genes of virus‐susceptible ‘Bet‐Alfa’ and ‘Ilan’, Cucumber mosaic virus (CMV)‐, Cucumber green mottle mosaic virus (CGMMV)‐ and Cucumber vein yellow virus (CVYV)‐resistant ‘Shimshon’, transgenic cucumber line 823 [Zucchini yellow mosaic virus (ZYMV) (resistant), Watermelon mosaic virus (resistant) and Papaya ringspot virus (tolerant)] and line 887 (ZYMV resistant). Total RNAs were extracted from leaves of each genotype and the relative expression levels of each gene were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and were calculated using the ΔΔCt method normalized to Fbox gene expression levels. Each bar is the mean of three replicates, each of three plants. The error bars denote standard deviations, and the different letters above the bars indicate statistically significant differences between the investigated transgenic lines and cultivars (P < 0.01).

The coding regions of the four CsRDR1 genes from cucumber were compared with regard to their putative protein sequence (Fig. S3). The RNA binding domain and the catalytic motif ‘DLDGD’ were found in all RDR1 sequences (Fig. S3). The amino acid alignment of the putative cucumber RDR1a, RDR1b, RDR1c1 and RDR1c2 exhibited 55%–60% homology. RDR1a and RDR1b exhibited 60% homology, and both showed c. 55% homology to RDR1c1 and RDRc2. The latter pair shared 98% sequence homology to each other.

Phylogenetic analysis was performed on the four putative cucumber CsRDR1s and RDR1 sequences from other cucurbit species [Citrullus lanatus (Thunb.) Matsum. & Nakai (watermelon) and Cucumis melo (muskmelon) (XN_008467032)], together with RDRs from other selected species (Fig. 3). The three types of CsRDR1 clustered into separate groups. CsRDR1a clustered together with melon and watermelon RDR1a homologues. CsRDR1b and CsRDR1c clustered with their watermelon homologues, respectively, in two separate groups. The analysis showed that CsRDR1b was more related to CsRDR1a than to CsRDR1c1 or CsRDR1c2. CsRDR1b was closely related to watermelon ClRDR1b, whereas CsRDR1c1 and CsRDR1c2 clustered together with watermelon ClRDR1c. The latter were more closely related to the Hordeum vulgare L. and Solanaceae RDR1 (Solanum lycopersicum L. and Nicotiana tabacum) than to A. thaliana (L.) Heynh. AtRDR1 was more closely related to CsRDR1b and ClRDR1b than to any other RDR1 (Fig. 3).

Figure 3.

Figure 3

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Phylogenetic tree of RNA‐dependent RNA polymerase α (RDRα) clade members. The phylogenetic tree was constructed from 25 proteins of 10 species using the maximum likelihood method implemented in ‘phyml’. The analysis separated the genes into three distinct clades: RDR1, RDR2 and RDR6. Values left of the internal nodes are the percentage of bootstrap resampling replicates (out of 100) that support the tree topology. Only bootstrap values of ≥90% and calculated distances are shown. The accession numbers of the RDR genes are listed in Table S1.

Participation of CsRDR1 genes in virus resistance

We have shown previously that there is an association between multiple virus resistance and increased CsRDR1 gene expression (Leibman et al., 2011). Homozygous transgenic cucumber lines 823 and 887, expressing a Zucchini yellow mosaic virus (ZYMV) dsRNA fragment of the HCPro coding region in cv. ‘Ilan’, exhibited ‘immunity’ to ZYMV infection. Interestingly, only line 823 showed broad potyvirus resistance to ZYMV, Watermelon mosaic virus (immunity) and Papaya ringspot virus (tolerance), associated with a significant increase in CsRDR1 gene expression (Leibman et al., 2011). At the time of the study by Leibman et al. (2011), the existence of the cucumber RDR1 gene family was unknown. To determine which of the four CsRDR1 genes was associated with this resistance in line 823, but not line 887, we examined the expression of all CsRDR1s in the same cultivars and homozygous transgenic lines (Fig. 2).

The expression level of CsRDR1a in healthy cucumber leaves was higher in the resistant transgenic lines 823 and 887 and in cv. ‘Shimshon’ (about 2.3–4.1‐fold) than in the virus‐susceptible cvs. ‘Ilan’ and ‘Bet‐Alfa’ (Fig. 2). Substantial differences (approximately 10–14‐fold) were observed in the CsRDR1b expression level in the healthy plants of resistant cv. ‘Shimshon’ and transgenic line 823 compared with healthy plants of the susceptible cvs. ‘Bet‐Alfa’ and ‘Ilan’ and transgenic line 887 (Fig. 2). The constitutive CsRDR1b expression levels in healthy virus‐resistant cultivars indicate a possible association of the CsRDR1b expression level with broad virus resistance – ‘like a resistance gene’. The expression levels of CsRDR1c1 and CsRDR1c2 (combined in Fig. 2 as RDR1c) remained very low in leaves and roots, and no significant differences could be seen between the resistant and susceptible cultivars or the transgenic lines (Figs 2, S2 and S4, see Supporting Information). We compared the expression level of CsRDR1c1 and CsRDR1c2 in ZYMV‐ and CMV‐infected leaves of the resistant cv. ‘Shimshon’ and susceptible cv. ‘Bet‐Alfa’ cucumbers (Fig. S2). In both cultivars, the expression of CsRDR1c1 and CsRDR1c2 was highly induced compared with the barely detectable mRNA levels in healthy plants (Figs S1, S2 vs. Fig. 2). These data indicated that the expression levels of CsRDR1c1 and CsRDR1c2 had similar kinetics and response to virus infection, and therefore, in the following experiments, the expression level of CsRDR1c1 + CsRDR1c2 was measured together, and is given as CsRDR1c.

To determine whether virus accumulation was affected by higher basal levels of expression of the CsRDR1 gene family, resistance was determined for three RNA viruses of different families (Fig. 4): CMV, a member of the genus Cucumovirus, family Bromoviridae; CGMMV, a member of the genus Tobamovirus, family Virgaviridae; and CVYV, a member of the genus Ipomovirus, family Potyviridae. These results were compared with the responses to infection by ZYMV, a member of the genus Potyvirus, family Potyviridae (Leibman et al., 2011). Resistance was determined by symptom expression and viral RNA accumulation was measured by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) at various days post‐inoculation (dpi) (Fig. 4). Infection of cv. ‘Shimshon’ and transgenic line 823 with CMV and CVYV did not induce symptoms, whereas CGMMV induced very mild symptoms at 14 dpi. However, CMV and CGMMV infection caused severe symptom development in cvs. ‘Bet‐Alfa’, ‘Ilan’ and transgenic line 887 (Fig. 4). CVYV infection caused severe symptoms in cv. ‘Bet‐Alfa’ and mild chlorotic lesions in cv. ‘Ilan’ and transgenic line 887 (Fig. 4). The asymptomatic phenotype in cv. ‘Shimshon’ and line 823 was correlated with the low viral RNA accumulation (Fig. 4), i.e. tolerance. At 7 dpi, CMV RNAs accumulated to approximately 10‐fold greater levels in cv. ‘Bet‐Alfa’ relative to cv. ‘Ilan’ and line 887, but were undetectable in cv. ‘Shimshon’ and line 823. Significantly higher levels of CMV RNAs accumulated at 21 dpi in cvs. ‘Bet‐Alfa’, ‘Ilan’ and line 887 compared with cv. ‘Shimshon’ and line 823 (Fig. 4). Similarly, significantly higher levels of accumulation of CGMMV and CVYV RNAs, at 7 and 14 dpi, respectively, were observed in cv. ‘Bet‐Alfa’ and transgenic line 887 compared with ‘Shimshon’ and transgenic line 823 (Fig. 4). Differences in RNA accumulation following inoculation with CVYV were much more pronounced between susceptible (cvs. ‘Ilan’, ‘Bet‐Alfa’ and Line 887) and resistant (cv. ‘Shimshon’ and Line 823) plants at 14 dpi (Fig. 4). These results reinforce our previous observations with ZYMV (Leibman et al., 2011), in which the resistance of transgenic line 823 was independent of the transgene sequence and was probably associated with the level of expression of RDR1.

Figure 4.

Figure 4

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Analysis of virus resistance of cucumber cultivars and transgenic lines to three virus families. The cultivars ‘Bet‐Alfa’ (BA), ‘Shimshon’ (Shim) and ‘Ilan’, and transgenic lines 887 and 823, were sap inoculated with Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Cucumber vein yellow virus (CVYV), and symptoms were recorded at 14–18 days post‐inoculation (dpi) (right panels). Relative viral RNA accumulation was measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) (left panels) at different dpi, and the levels of each virus were calculated using the ΔΔCt method normalized to Fbox gene expression levels. Each bar is the mean of three replicates, each of three plants. The error bars denote standard deviations, and the different letters above the bars indicate statistically significant differences between transgenic lines and cultivars (P < 0.01, except for P < 0.05 for CMV at 21 dpi).

Kinetics of expression of CsRDR1 genes during virus infection

As RDR1 gene expression is involved in resistance against plant viruses (Xie et al., 2001; Yu et al., 2003), mediated by RNA silencing (Cao et al., 2014; Garcia‐Ruiz et al., 2010; Wang et al., 2010), we assessed the expression levels of the RDR1 genes in virus‐infected plants. The expression levels of CsRDR1a–c in ZYMV‐infected leaves of cucumber cv. ‘Shimshon’ and cvs. ‘Bet‐Alfa’ and ‘Ilan’ (all susceptible to infection by ZYMV) (Fig. 5A) were compared. In all cultivars, significantly increased levels of CsRDR1a were observed at 11 dpi compared with healthy plants. However, earlier (4 and 7 dpi), no differences could be seen in the responses of cvs. ‘Shimshon’ and ‘Bet‐Alfa’, although an increased level of CsRDR1a (approximately two‐fold) was measured in cv. ‘Ilan’ at 7 dpi (Fig. 5A). CsRDR1b expression levels increased as a result of ZYMV infection at 7 and 11 dpi in cvs. ‘Ilan’ and ‘Bet‐Alfa’ and at 11 dpi in cv. ‘Shimshon’ (Fig. 5A). It is important to note that the approximately eight‐fold increase in the level of CsRDR1b expression in cv. ‘Shimshon’ at 11 dpi was in addition to the high CsRDR1b expression level observed in healthy plants of cv. ‘Shimshon’ (Fig. 5A).

Figure 5.

Figure 5

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The expression levels of the RDR1 gene family in cucumber are affected by virus infection, and CsRDR1c is highly induced with RNA and DNA viruses. (A) CsRDR1a, CsRDR1b and CsRDR1c expression was analysed in healthy (white bars) and Zucchini yellow mosaic virus (ZYMV)‐infected (black bars) cucumber leaves from ‘Ilan’, ‘Shimshon’ and ‘Bet‐Alfa’. The relative levels of ZYMV in infected cucumber are presented (grey bars). Total RNAs were extracted from plants at 4, 7 and 11 days post‐inoculation (dpi) with ZYMV. (B) Relative expression levels of CsRDR1c and virus nucleic acids in cucumber ‘Shimshon’ (as a reference) and ‘Bet‐Alfa’, healthy and infected with Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), Cucumber vein yellow virus (CVYV) (14 dpi) and Squash leaf curl virus (SLCV). Total RNA was extracted from virus‐infected (black bars) and healthy (white bars) cucumber plants at different days after sap inoculation. First‐strand cDNAs were prepared with oligo‐dT and specific CGMMV and CMV primers. Quantitative polymerase chain reaction (qPCR) was performed with appropriate primers for RDR1 mRNA for the coat protein genes of the different viruses and for the host Fbox gene for normalization. The relative expression level of each gene was calculated using the ΔΔCt method normalized to the Fbox gene expression level. DNA was extracted from infected (with SLCV) and healthy cucumber at 9 and 15 days after whitefly inoculation and qPCR was performed on 15 dpi samples (not infected as a reference). Each bar is the mean of three replicates of three plants. The error bars denote standard deviations, and the different letters above the bars indicate statistically significant differences between infected and non‐infected cultivars (P < 0.01, except P < 0.05 for the level of CsRDR1a in Bet‐Alfa infected with CMV).

The level of CsRDR1c gene expression increased dramatically in all cucumber cultivars after ZYMV infection (Fig. 5A): in cv. ‘Bet‐Alfa’, the CsRDR1c level increased about four‐fold at 4 dpi and by more than 400‐fold at 7 and 11 dpi; in susceptible cv. ‘Ilan’, a >300‐fold increase in CsRDR1c expression was observed at 7 and 11 dpi; and, in cv. ‘Shimshon’, a 122‐fold increase in RDR1c expression was observed at 11 dpi. These data indicated that CsRDR1c expression was highly induced via ZYMV infection and was almost undetectable in healthy cucumber. An important question is whether the induction of CsRDR1c expression is a general phenomenon for cucumber infected with RNA and/or DNA viruses.

To examine this, the expression level of CsRDR1c was determined in cvs. ‘Shimshon’ and ‘Bet‐Alfa’ at 14 dpi for RNA viruses (CMV, CGMMV and CVYV) and at 15 dpi for a DNA virus, the geminivirus Squash leaf curl virus (SLCV) (Fig. 5B). The expression of RDR1c was highly induced to different extents by all three RNA viruses and the geminivirus SLCV. A higher level of RDR1c induction was observed in cv. ‘Bet‐Alfa’ infected with CVYV and CMV (approximately 600–700‐fold). However, in cv. ‘Shimshon’ following infection by the same viruses (CVYV and CMV; Fig. 5B), the induction of RDR1c was rather less (3–90‐fold) as a result of the resistance of cv. ‘Shimshon’ to these viruses (Figs 4 and 5B).

The increase in expression level of RDR1c in CGMMV‐infected plants was lower (about 20‐fold) when compared with infection with CMV, CVYV (Fig. 5B) and ZYMV (Fig. 5A). It has been shown that geminiviruses are subject to RNA silencing (Vanitharani et al., 2003). Therefore, we tested the CsRDR1c expression level in cv. ‘Bet‐Alfa’ infected with SLCV. The expression of CsRDR1c was induced about 20‐fold in leaves infected with SLCV (Fig. 5B). Interestingly, SLCV‐infected cv. ‘Bet‐Alfa’ plants showed mild symptoms at 2 weeks post‐infection, from which the plants recovered.

Inhibition of CsRDR1c gene expression enhances virus accumulation

To determine whether CsRDR1c has a specific effect on virus systemic infection, we used ZYMV‐based virus‐induced gene silencing (VIGS) technology (Shoresh et al., 2006) to suppress the accumulation of CsRDR1c mRNA. A 250‐bp CsRDR1c sequence was inserted into two engineered ZYMV strains: ZYMVFRNK (severe) and ZYMVFINK (symptomless) (Gal‐On, 2007). ZYMV constructs with a CsRDR1c fragment (ZYMV‐Rc and ZYMV‐Ic) were infectious on cucumber and caused symptoms similar to those of the parental strains. Cucumber plants infected with recombinant viruses (ZYMV‐Rc, ZYMV‐Ic and ZYMV‐wt) expressed similar levels of CsRDR1a and CsRDR1b (Fig. 6). However, the expression level of CsRDR1c was significantly decreased in cucumber leaves infected with ZYMV‐Rc or ZYMV‐Ic compared with infection with ZYMV‐wt (Fig. 6). These data show that the CsRDR1c transcript level was partially silenced by the ZYMV‐VIGS system (Fig. 6). The decrease in CsRDR1c expression levels accompanied a significant increase in the level of both recombinant viruses (5.2‐fold with ZYMV‐Rc and 3.9‐fold with ZYMV‐Ic), compared with ZYMV‐wt, indicating the involvement of CsRDR1c in defence.

Figure 6.

Figure 6

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Inhibition of CsRDR1c mRNA accumulation increases virus accumulation. The expression levels of CsRDR1a, CsRDR1b and CsRDR1c were examined in cucumber ‘Bet‐Alfa’ infected with recombinant viruses ZYMV‐Ic (symptomless) and ZYMV‐Rc (severe) and with ZYMV‐wt (severe). Total RNAs were extracted from plants at 7 days post‐inoculation (dpi) and from healthy plants (H). First‐strand cDNAs were prepared with oligo‐dT and quantitative polymerase chain reaction (qPCR) was performed with the appropriate primers for CsRDR1a, CsRDR1b, CsRDR1c and the Zucchini yellow mosaic virus (ZYMV) coat protein (CP) gene. The relative expression level of each CsRDR1 gene was calculated using the ΔΔCt method normalized to Fbox gene expression. The relative virus titre is indicated in the bottom panel. Each bar is the mean of three replicates of three plants. The error bars denote standard deviations, and the different letters above the bars indicate statistically significant differences in CsRDR1a, CsRDR1b and CsRDR1c between infected plants and between the ZYMV titres (P < 0.01).

Signal transduction in CsRDR1c1 gene expression induced by virus infection

To examine whether a signal causes the induction of CsRDR1 expression in a leaf prior to the appearance of virus from an inoculated lower leaf, a time course analysis of CsRDR1b and CsRDR1c gene expression was performed in a systemic leaf, following cotyledon inoculation of cucumber cv. ‘Bet‐Alfa’ with CMV (Fig. S5, see Supporting Information). CMV systemic infection could not be detected at 24 and 48 h post‐infection (hpi) in the first true leaf above the inoculated cotyledon. Low levels of CMV accumulation were detected in the first true leaf at 65 hpi, and subsequently very high levels of CMV could be measured in the systemically infected leaf at 5 and 7 dpi (Fig. S5). The expression levels of CsRDR1b increased by approximately two‐ and five‐fold in virus‐free, true leaves of inoculated plants at 24 and 48 hpi, respectively, and by approximately 3.5‐ and nine‐fold in systemically infected leaves at 5 and 7 dpi, respectively (Fig. S5). CsRDR1c expression levels increased by about four‐fold at 48 hpi in the virus‐free true leaves of inoculated plants. However, CsRDR1c levels in the first systemically infected leaves increased by more than 1000‐fold at 5 and 7 dpi. These data indicate that CMV infection induces a systemic signal that increases the expression of CsRDR1b and CsRDR1c prior to virus infection and that, in infected tissues, CsRDR1c expression is induced to very high levels.

Exogenous SA induces RDR expression in different species (Carr et al., 2010). We therefore tested the effect of SA on the expression levels of CsRDR1a–c in resistant cv. ‘Shimshon’ and susceptible cv. ‘Bet‐Alfa’ cucumber. Ectopic application of SA for 6 days induced the expression of CsRDR1a–c in both cultivars (Fig. 7A). CsRDR1a and CsRDR1b levels in cv. ‘Bet‐Alfa’ (six‐fold increase for RDR1a and 15‐fold increase for RDR1b) were approximately double those observed in cv. ‘Shimshon’ (three‐fold for RDR1a and seven‐fold for RDR1b), but a much higher induction was observed with CsRDR1c in both cultivars (27‐fold in ‘Bet‐Alfa’ and 33‐fold in ‘Shimshon’) (Fig. 7A). CsRDR1a, CsRDR1b and CsRDR1c expression by SA treatment decreased to initial levels at 7 days after spraying (Fig. 7B), which may indicate a transient effect of SA on the induction of CsRDR1 gene expression (Fig. 7A).

Figure 7.

Figure 7

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The expression of CsRDR1a, CsRDR1b and CsRDR1c was induced by salicylic acid (SA). Expression levels of CsRDR1a, CsRDR1b and CsRDR1c in healthy cucumber (‘Shimshon’, ‘Bet‐Alfa’) sprayed daily with a 1% solution of SA (black bars) and water control (white bars). Total RNAs were extracted from plants after spraying for 6 days (A) and following a 7‐day interval after SA application (B). (C) SA content in cucumber ‘Ilan’, ‘Bet‐Alfa’ and ‘Shimshon’, and transgenic lines 823 and 887. SA was extracted from three plants per cultivar and from the two transgenic lines at 14 days post‐germination, and statistically significant differences between treatments were calculated by the MassLynx range test (P < 0.05). (D) Relative expression levels of SID2, EDS5, WIN3 and WRKY22 genes in healthy cv. Ilan and transgenic line 823. First‐strand cDNAs were prepared from isolated mRNAs using oligo‐dT, followed by quantitative polymerase chain reaction (qPCR) performed with appropriate primers for CsRDR1a, CsRDR1b, CsRDR1c, SID2, EDS5, WIN3 and WRKY22 genes, as well as for the Fbox gene used for normalization. The relative expression level of each gene was calculated using the ΔΔCt method normalized to Fbox gene expression levels. Each bar is the mean of three replicates of three plants. The error bars denote standard deviations, and the different letters above the bars indicate statistically significant differences between SA treatments (P < 0.01). The statistical analysis in (A) was made separately for Bet‐Alfa and Shimshon.

We tested the expression of other genes in the SA signalling pathway in transgenic line 823 (with high endogenous CsRDR1b expression levels) and non‐transgenic cv. ‘Ilan’ in the absence of added SA. Here, similar levels of expression were measured for the genes SID2 and EDS5 upstream of SA synthesis, and for the genes WIN3 and WRKY22 downstream of SA synthesis (Carr et al., 2010; Kumar and Klessig, 2000) (Fig. 7D). Similar accumulation levels of SA were measured in cvs. ‘Bet‐Alfa’ and ‘Shimshon’, and lines 887 and 823 (Fig. 7C). These data indicate that the high endogenous level of expression of CsRDR1b is not caused by increased endogenous levels of SA or of various genes in the SA signalling pathway. Nevertheless, exogenous SA can increase the levels of expression of CsRDR1b still further (Fig. 7A).

Discussion

Organization and expression of cucumber RDR1 genes

Our studies reveal a unique small gene family of four CsRDR1s in cucumber, in addition to CsRDR2 and CsRDR6. Four cucumber RDR1 genes were identified (Figs 1 and 3). The cucumber RDR1 genes can be separated into 1a, 1b and 1c (1c1 + 1c2) based on sequence similarity and gene expression (Figs 2, S2 and S3). CsRDR1c1 and CsRDR1c2 are very similar (essentially copies), expressed to similar levels in different virus‐infected cucumber cultivars (Fig. S2). Such a RDR1 gene family (RDR1a–c), with members varying by 30%–40%, has not been demonstrated in other plant species. However, RDR1 gene duplication (such as RDR1c1 and RDR1c2, with members 98% identical) seems to be a common evolutionary phenomenon (Zong et al., 2009), observed in both monocots (barley) (Madsen et al., 2009) and other dicots (potato) (Hunter et al., 2016). The unique RDR1 family was also found in other Cucurbitaceae: Citrullus (watermelon; Cucurbit Genomics Database) (Fig. 3) and Cucurbita (squash, pumpkin; http://www.icugi.org/cgi-bin/ICuGI/index.cgi).

No differences were observed between each of the four CsRDR1 genes plus their putative promoters (3 kb sequence upstream) in virus‐susceptible cv. ‘Bet‐Alfa’ compared with the same genes and putative promoters in multiple virus‐resistant cv. ‘Shimshon’. Thus, differences in RDR1a–c expression between these cultivars (Leibman et al., 2011; Wang et al., 2003) may be caused by modifications of enhancer elements, specific transcription factors that bind to these elements, epigenetic effects or regulators of RDR1 gene expression, as demonstrated recently for the rice RDR1 (Wang et al., 2016). By contrast, the putative promoter sequences for CsRDR1a–c were different from each other (but with that of RDR1c1 being the same as RDR1c2), indicating that regulation could differ at the promoter level, as demonstrated in Arabidopsis (Xu et al., 2013).

Our gene cloning study enabled corrections of a situation which was previously unclear for this gene family. Previous interrogation of CsRDR in the Cucurbit Genomics Database identified five RDR1 genes, labelled CsRDR1a–e (Gan et al., 2016). In that in silico study, no RDR1 genes were cloned and sequenced, in contrast with our study. Thus, CsRDR1a and CsRDR1b are the same in both studies, although CsRDR1a in the database contains a 100‐bp deletion. In addition, CsRDR1c of Gan et al. (2016) has the same sequence as the 5′ half of CsRDR1b here, whereas CsRDR1d of Gan et al. (2016) has the same sequence as the 3′ half of our CsRDR1c1, and their CsRDR1e is the same as our CsRDR1c2.

Close phylogenetic relationships are notable between the RDR1a gene products of cucumber, melon and watermelon, as well as between the RDR1b and RDR1c gene products of cucumber and watermelon (Fig. 3). Such similarities suggest that the duplications leading to the accumulation of four RDR1 genes, in close proximity on an arm of chromosome 5 of cucumber (Cucurbit Genomics Database), occurred before the separation of the ancestral cucurbits into modern taxa. In addition, the great diversity between the CsRDR1a, CsRDR1b and CsRDR1c genes (homology levels of 55%–60%), whilst retaining a much higher homology among the same genes from cucurbit species (Fig. 3), indicates that the genes evolved earlier to provide separate, although possibly overlapping, defence functions.

Kinetics of expression of RDR1 genes during virus infection

CsRDR1a and CsRDR1b only showed moderate increases in gene expression as a result of ZYMV infection (2.5 to six‐fold) in cvs. ‘Ilan’, ‘Bet‐Alfa’ and ‘Shimshon’ (Fig. 5A). Notably, the increase in CsRDR1b levels in cv. ‘Shimshon’ was on top of the 13‐fold higher constitutive level of expression found in healthy plants (Fig. 5A vs. Fig. 2). However, CsRDR1c showed a much greater increase in expression after ZYMV infection, by several hundred‐fold or more (11 dpi) in cvs. ‘Ilan’, ‘Bet‐Alfa’ and ‘Shimshon’ (Fig. 5A).

A higher level of expression of CsRDR1c was also observed after infection by other viruses in cv. ‘Bet‐Alfa’: 20‐fold for CGMMV, 600‐fold for CMV, 700‐fold for CVYV and 20‐fold for SLCV (Fig. 5B). Interestingly, in cv. ‘Shimshon’, which has a high endogenous CsRDR1b expression level, expression of CsRDR1c was only stimulated six‐fold, three‐fold and 90‐fold following infection by CGMMV, CMV and CVYV, respectively (Fig. 5B). Cultivar ‘Shimshon’ shows good resistance to virus accumulation against all three viruses, but only moderate resistance against symptoms induced by CGMMV vs. good resistance to symptom development induced by CMV or CVYV (Figs 4 and 5B). Thus, the higher endogenous level of CsRDR1b may act as a new resistance gene, so that the reduced virus titre may obviate the need for strong CsRDR1c induction (Fig. 8). CsRDR1c plays a role in decreasing the virus titre, as demonstrated by the silencing of CsRDR1c expression using a ZYMV‐VIGS vector (Fig. 6). Similarly, an rdr1 rice mutant showed higher virus titre (Wang et al., 2016). Unfortunately, as ZYMV‐mediated VIGS of the CsRDR1a and CsRDR1b genes was unsuccessful, we could not establish the effect of each gene on the expression of the other, or the effect of the silencing of each gene on the infection by different viruses.

Figure 8.

Figure 8

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A model describing the effects of virus infection on the expression of CsRDR1 genes in susceptible and resistant plants. The model shows induced expression of the four CsRDR1 genes (short, thick, green arrows) by salicylic acid (SA) (long thin red arrows) and possibly other phytohormones [jasmonic acid (JA) or abscisic acid (ABA)]. Virus‐induced CsRDR1 expression occurs via SA induction (red broken arrow). However, virus infection (thick red arrow) induces a much higher level of CsRDR1c1/c2 (long, thick, green arrow) than does SA alone, leading to virus silencing. The high constitutively expressed CsRDR1b (thick green arrow) causes broad virus resistance. A high level of RDR1b expression is possibly associated with the production of viral‐activated small interfering RNAs (vasiRNAs) (Cao et al., 2014), which activate broad‐spectrum antiviral activity via widespread silencing of host genes.

The role of CsRDR1a in defence against virus infection is less clear, as it was induced only moderately, in line with the induction levels described for RDR1 from plants that contain only a single RDR1 (Alamillo et al., 2006; Rakhshandehroo et al., 2009; Xie et al., 2001; Yu et al., 2003), or two almost identical RDR1s (e.g. potato and barley) (Hunter et al., 2016; Madsen et al., 2009).

Signalling responses in RDR1 gene expression

Before virus could be detected in upper leaves, an increase in transcription of CsRDR1b and CsRDR1c genes was perceived (Fig. S5), suggesting that a signal, possibly SA, anticipated systemic virus appearance (Carr et al., 2010; Mayers et al., 2005; Zhu et al., 2014). Ectopic application of SA to cucumber leaves induced the expression of all four CsRDR1 genes, especially CsRDR1c (Fig. 7A), as shown previously for NtRDR1 and AtRDR1 (Xie et al., 2001; Yu et al., 2003). However, a high endogenous level of CsRDR1b gene expression in broad virus‐resistant lines (line 823 and cv. ‘Shimshon’) was not caused either by a higher basal level of SA (Fig. 7C) or increased activity of SA‐mediated defence pathway genes (Fig. 7D). The cause of the association of a constitutively high RDR1b level and broad virus resistance remains unclear. Potential effects of other phytohormones or transcriptional regulator(s) on CsRDR1b gene expression cannot be dismissed. In addition, we assume that, in transgenic line 823, unlike in cv. ‘Shimshon’, the high level of CsRDR1b expression probably depends on the very high level of transgene‐dsRNA/transgene‐siRNA accumulation (Leibman et al., 2011)

Virus resistance and susceptibility in cucumber

The combined data on the effects of virus and signalling responses on the expression of CsRDR1 genes are summarized in a model (Fig. 8) as follows: virus infection results in a weak induction of CsRDR1a and CsRDR1b (probably mediated by SA), but a strong induction of CsRDR1c. The strong induction of CsRDR1c by virus infection is much greater than that observed by application of SA alone (Figs 5, S2 and S5 vs. Fig. 7), and thus viruses may induce CsRDR1c by another pathway, possibly mediated by other phytohormones to which RDR1 genes have been shown to respond (Hunter et al., 2013; Liu et al., 2009; Pandey and Baldwin, 2007; Xu et al., 2013). In addition, RDR1c induction could be mediated by either miRNA (Wang et al., 2016) or transcription factors via virus‐associated siRNA (Cao et al., 2014). The higher level of induced CsRDR1c had an effect on virus accumulation, as demonstrated by gene silencing. In one cucumber cultivar (‘Shimson’) and a transgenic line (823), CsRDR1b was expressed constitutively at a high level (Figs 2 and 7). These plants are either tolerant or highly resistant to virus infection (Figs 4 and 5B). The constitutively high CsRDR1b transcript level strongly associated with virus resistance is independent of virus infection and endogenous SA level (Figs 2 and 7).

Overall, our study showed that multiple RDR1 genes are involved in virus resistance in cucumber, are regulated in a coordinated fashion with different expression profiles, and may have either distinct or overlapping functions.

Experimental Procedures

Plants, pathogens and inoculations

Three accessions of Bet‐Alfa‐type cucumbers, Cucumis sativus, were used for the biological and molecular analyses. These were the original ‘Bet‐Alfa’ (Paris et al., 2012), as well as ‘Shimshon’, ‘Ilan’ (Zera‘im Gedera Co., Gedera, Israel) and the homozygotic transgenic lines 823 and 887 (Leibman et al., 2011). Seeds were planted in a soil mixture and grown in a growth chamber under continuous white fluorescent light at 25 °C. Mechanical inoculation of cucumber seedlings with CMV, ZYMV, CGMMV (Table S2, see Supporting Information) and recombinant viruses ZYMV‐Ic and ZYMV‐Rc was performed according to Leibman et al. (2011). Inoculation of cucumber with CVYV and SLCV was performed by viruliferous whiteflies (Sufrin‐Ringwald and Lapidot, 2011).

All experiments were sampled in the same manner for molecular analysis of RNA, DNA, endogenous genes and virus accumulation. Nine plants in groups of three (i.e. three replicates) were used per sample. Two 8‐mm leaf discs were sampled per plant. Therefore, each repeat was a pool of six leaf discs from three plants.

Phylogenetic analysis

Protein sequences derived from three RDRα clade members (RDR1, RDR2 and RDR6) from nine different species were aligned with the MAFFT program. Multiple sequence alignment (MSA) was performed globally for all pairs with maximum iterations of 1000. The robustness of the MSA was estimated with guidance2 (Sela et al., 2015) to be 0.971, which was slightly more robust than the local pair option. Furthermore, one MSA site with a confidence value of <0.25 was removed for downstream analysis. The phylogenetic tree was constructed using the likelihood method by the ‘phyml’ program (Guindon and Gascuel, 2003; Shimodaira and Hasegawa, 2001) with the JTT substitution model. The robustness and confidence of tree nodes were estimated as a percentage of 1000 bootstrap resampling replicates

DNA and RNA extraction and PCR and qRT‐PCR analysis

Total genomic DNA was extracted from cucumber leaves (approximately 40 mg) (two leaf discs per plant) by the method of Dellaporta et al. (1983). Diluted DNA (1 : 25) was used for PCR, with the appropriate primers for RDR1 (RDR1a, RDR1b, RDR1c1 and RDR1c2), RDR2, RDR6 and F‐box genes (Table S3, see Supporting Information). PCR conditions were the same as those described in Leibman et al. (2011).

RNA expression of endogenous genes and virus accumulation were determined by RT‐PCR and qRT‐PCR according to Leibman et al. (2011). RNA samples were collected from the second and third leaves of cucumber, and from whole roots. Total RNAs were extracted by a TRI‐REAGENT kit (Molecular Research Center, Inc., Cincinnati, OH, USA) and adjusted to the same concentration prior to RT‐PCR using a NanoDrop ND1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). First‐strand cDNA was synthesized from 2 µg of total RNA using the Verso™ cDNA Kit (Thermo Fisher Scientific, Epsom, UK) with oligo(dT) primer (100 pmol) and specific virus reverse primers for CMV and CGMMV analysis. The cDNA was used for PCR and qPCR as described above. qPCRs were performed according to Leibman et al. (2011).

Construction of the ZYMV‐VIGS vector

ZYMVFRNK and ZYMVFINK are potyvirus‐based vectors for the expression of foreign genes and gene silencing in cucurbits (Arazi et al., 2001; Shoresh et al., 2006). To silence RDR1c, two ZYMV constructs were made which contained an RDR1c fragment: ZYMV‐Rc and ZYMV‐Ic. The RDR1c 225‐bp fragment was amplified from CsRDR1c cDNA clones by PCR (Arazi et al., 2001; Table S3).

Cloning and sequencing

RDR1a, RDR1b, RDR1c1 and RDR1c2 were cloned from cucumbers ‘Bet‐Alfa’ and ‘Shimshon’ using the Cucurbit Genomics Database (http://www.icugi.org/). The intact RDR1 mRNAs of cucumber were cloned with primers corresponding to the coding regions (Table S3). RT‐PCR was performed as above and PCRs were performed using Phusion high‐fidelity DNA polymerase and the manufacturer's protocol (New England BioLabs, Hitchin, UK). Fragments were cloned into pJet1.2/blunt Vector (Thermo Scientific) and sequenced twice.

Endogenous cucumber genes (homologous to Arabidopsis WRKY22, EDS5, WIN3 and SID3) were cloned based on the Cucurbit Genomics Database and using the primers listed in Table S3. F‐box genes were used for gene expression controls in cucumber (Leibman et al., 2011). The expression of the endogenous genes SID2, EDS5, WIN3 and WRKY22 was analysed using specific primers (Table S3) from the Cucurbit Genomics Database.

SA sample preparation and analysis

Samples for SA analysis were extracted from three separate plants of each accession: ‘Bet‐Alfa’, ‘Shimshon’ and transgenic lines 887 and 823. Leaf samples (250 mg per leaf) were taken from plants at 14 days post‐germination, frozen in liquid N2 and ground to a powder with a mortar and pestle. The powder was mixed with 750 µL MeOH–H2O–HOAc (90 : 9 : 1, v/v/v) and centrifuged for 1 min at 9,600 g. Extraction of SA from the mixture was performed according to Segarra et al. (2006). Liquid chromatography‐mass spectrometry (LC‐MS) analyses were conducted using a UPLC‐Triple Quadrupole‐MS (Waters Xevo TQ, Milford, MS, USA). Samples were filtered through a Millex‐HV Durapore (Bio‐Rad laboratories, Hercules, CA, USA) [poly(vinylidene difluoride), PVDF] membrane (0.22 µm) before injection into the LC‐MS instrument. Separation was performed on a 2.1 × 100 mm2, 1.7‐µm UPLC BEH C18 column. Chromatographic and MS parameters were as follows: the mobile phase consisted of water (phase A) and 0.1% formic acid in acetonitrile (phase B). The linear gradient program was as follows: 75% to 25% A over 0.1 min, 75% to 0% A over 5 min, held at 0% A over 1 min, and then returned to the initial conditions (75% A) over 1 min and held at 75% A for 6 min. The flow rate was 0.3 mL/min and the column temperature was kept at 35 °C. All the analyses were performed using the Electrospray ionization (ESI) source in negative ion mode with the following settings: capillary voltage, 3.2 kV; cone voltage, 30 V; desolvation temperature, 350 °C; desolvation gas flow, 650 L/h; source temperature, 150 °C. Quantification was performed using multiple reaction monitoring (MRM) acquisition by monitoring the 137/65 and 137/93 (Retention time (RT) = 2.05; dwell time of 61 ms for each transition) peaks for SA, and 141/97 (RT = 2.05; dwell time of 61 ms) peak for d4‐SA (used as internal standard).

Statistical analysis

Results are expressed as means ± standard deviation. Statistical analysis was performed using JMP 5 software (SAS Institute Inc., 2002, Cary, NC, USA). Data were subjected to one‐way analysis of variance (ANOVA) and Tukey honestly significant difference for comparison of means.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website.

Fig. S1 RDR1 gene expression in cucumber leaves.

Fig. S2 Expression of CsRDR1c1 and CsRDR1c2 was induced to similar levels by virus infection in cucumbers ‘Shimshon’ and ‘Bet‐Alfa’.

Fig. S3 Amino acid sequence alignment of putative proteins encoded by the CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes of cucumber ‘Bet‐Alfa’ and the AtRDR1a gene of Arabidopsis thaliana.

Fig. S4 RDR1 gene expression in cucumber roots.

Fig. S5 Time course analysis of CsRDR1b and CsRDR1c1 expression following Cucumber mosaic virus (CMV) infection in cucumber.

Table S1 List of RNA‐dependent RNA polymerase (RDR) accession numbers.

Table S2 List of viruses tested.

Table S3 Primers used in this study.

Click here for additional data file. (1.9MB, docx)

Acknowledgements

This work was supported in part by grants from the Chief Scientist's Office, Ministry of Agriculture (132165015 to A.G.‐O.), the United States–Israel Binational Agricultural Research and Development Fund (BARD) (Grant‐IS‐4513‐12 to A.G.‐O. and S.‐W.D.), the United States–Israel Binational Science Foundation (BSF‐2011302 to A.G.‐O. and S.‐W.D.), the Korean National Research Foundation (grant no. NRF‐2013R1A2A2A01016282 to P.P.) and the Korean Rural Development Administration (grant no. PJ011309 to P.P.). Contribution from the Agricultural Research Organization, The Volcani Center, Bet‐Dagan, Israel, number 559/16.

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Associated Data

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Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's website.

Fig. S1 RDR1 gene expression in cucumber leaves.

Fig. S2 Expression of CsRDR1c1 and CsRDR1c2 was induced to similar levels by virus infection in cucumbers ‘Shimshon’ and ‘Bet‐Alfa’.

Fig. S3 Amino acid sequence alignment of putative proteins encoded by the CsRDR1a, CsRDR1b, CsRDR1c1 and CsRDR1c2 genes of cucumber ‘Bet‐Alfa’ and the AtRDR1a gene of Arabidopsis thaliana.

Fig. S4 RDR1 gene expression in cucumber roots.

Fig. S5 Time course analysis of CsRDR1b and CsRDR1c1 expression following Cucumber mosaic virus (CMV) infection in cucumber.

Table S1 List of RNA‐dependent RNA polymerase (RDR) accession numbers.

Table S2 List of viruses tested.

Table S3 Primers used in this study.

Click here for additional data file. (1.9MB, docx)

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