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

Abstract

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: RDR1; cucumber; gene expression; silencing; virus resistance.

Figure 1

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).

Figure 2

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).

Figure 3

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.

Figure 4

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).

Figure 5

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).

Figure 6

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).

Figure 7

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.

Figure 8

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.