Eighteen accessions of O. glaberrima (International Rice Germplasm Collection (IRGC) accession numbers 86741, 96717, 96718, 96790, 96793, 96864, 96868, 100139, 100153, 102556, 102569, 103437, 103477, 104545, 104589, 104914, 112576, and 115633) were obtained from the T. T. Chang Genetic Resources Center, International Rice Research Institute (IRRI). O. sativa cultivar Taichung Native 1 (TN1) is susceptible to RTSV. TW16 is a backcross line (BC₅) resistant to RTSV, and was developed by serial backcrosses of RTSV-resistant cultivar Utri Merah (IRGC number 16682) with TN1 (Lee et al., 2010).

RTSV strain A (Cabauatan et al., 1995) maintained in TN1 was used as the source of inoculum. Five plants each of TN1 and TW16, and 18 O. glaberrima accessions were used per treatment (mock control or RTSV-inoculated) and sampling timing [7, 14, 21, 28, or 30 days post-inoculation (dpi)]. The plants were grown in a 12-cm-diameter pot with each pot containing five seedlings. GLH-mediated inoculation of RTSV to plants was done by the tube method as described by Cabauatan et al. (1995). GLH were given a 3-day acquisition access period to RTSV-infected plants and were allowed an inoculation access period of 24 h to 9-day-old plants at three insects per plant. Plants for mock control were prepared by feeding three virus-free insects per plant for 24 h. At Twenty Four hours after inoculation started, GLH were removed by insecticide. The inoculated plants were maintained in the greenhouse of IRRI.

The height of individual mock- and RTSV-inoculated plants was measured at 7, 14, 21, and 28 dpi) (n = 5 per treatment and time point in one experiment). Height reduction rates (%) due to RTSV infection were computed as 100 × [(height of mock-inoculated plant—height of RTSV-infected plant)/height of mock-inoculated plant]. After the height measurement, the second youngest fully expanded leaf was collected from each plant for RNA extraction (see below). The leaf samples were quickly frozen in liquid nitrogen and immediately kept at −80°C until RNA extraction. Subsequently, approximately 1/3 of the upper part of each plant was collected for evaluation of RTSV accumulation in the plant. One gram of plant samples collected were pulverized in liquid nitrogen, and homogenized with 10 ml of phosphate buffer saline containing Tween-20 to prepare 10 time-diluted sap samples. The diluted plant sap samples were used to estimate RTSV accumulation in plants by enzyme-linked immunosorbent assay (ELISA) using an antibody raised against purified RTSV as described by Shibata et al. (2007). The experiment for height measurement and that for estimation of RTSV accumulation were repeated three times. The differences between the heights of mock- and RTSV-inoculated plants were examined by the analysis of variance (ANOVA) and a t-test, and the differences in height reduction rate and RTSV accumulation (absorbance at 405 nm in ELISA) among O. glaberrima and O. sativa plants were examined by ANOVA and the least significant difference (LSD) test.

Cell wall-related genes investigated for expression in O. glaberrima (Table 1) were selected based on their expression patterns in O. sativa TN1 and TW16 after infection with RTSV (Satoh et al., 2013). Genes orthologous between O. sativa and O. glaberrima were identified by BLAST-Like Alignment Tool (BLAT) (Kent, 2002) using the O. sativa japonica gene sequences (http://rice.plantbiology.msu.edu) as query and the O. glaberrima genome assembly (www.genome.arizona.edu) as the database. Genes were considered to be orthologous if the length of the BLAT match along the O. glaberrima scaffold is more than 90% of the length of the O. sativa japonica gene with minimal mismatches.

Total RNA was isolated individually from the second fully expanded leaves of single plants using TRIzol (Invitrogen, USA) according to the manufacturer's instructions. The equal amounts of the RNA samples from five independent plants of the same treatment were pooled prior to cDNA synthesis. First-strand cDNA molecules were synthesized from 3 μg of the pooled RNA samples using SuperScript III (Invitrogen, USA) and an oligo(dT) primer (Invitrogen, USA) in 20 μl of reaction mix according to the manufacturer's instruction. Primers specific to six cell wall-related genes were designed to target the 3′ untranslated regions of the corresponding orthologs in both O. sativa and O. glaberrima, and to avoid amplification from the gene family of selected genes (Table 1). Real-time PCR was performed with the gene-specific primers using the Light Cycler 480 SYBR Green I Master Mix (Roche, USA) in a Light Cycler 480 thermal cycler (Roche, USA) according to the manufacturer's instructions. Three replicates were performed for each sample. A threshold cycle (C_T) value was obtained for each reaction. Fold changes in cell wall-related gene expression between mock-inoculated and RTSV-inoculated plants were computed using the comparative C_T method (Schmittgen et al., 2000) with an actin gene (LOC_Os03g50885 in O. sativa and Oglab03_unplaced058 in O. glaberrima) as the internal reference control (Table 1). The differences in fold changes in expression of the genes among O. sativa and O. glaberrima plants were examined by ANOVA and LSD test.

Eighteen accessions of O. glaberrima were inoculated with RTSV to see whether RTSV induces stunting in O. glaberrima accessions. Most accessions of O. glaberrima infected with RTSV appeared to be evidently stunted at 30 dpi (Supplementary Material 1), and the number of tillers usually decreased in the plants stunted with RTSV. However, many plants of O. glaberrima accessions died or grew poorly during the initial evaluation. This was probably due to a loss of vigor after long-term storage. Therefore, among the 18 accessions of O. glaberrima, we selected and propagated four (96790, 96793, 102569, and 104545) for further evaluation to confirm their reactions to RTSV. They were selected because O. glaberrima-96793 and -102569 appeared to be severely stunted by RTSV infection in the preliminary evaluation, whereas O. glaberrima-96790 and -104545 seemed not stunted or stunted only slightly.

The height reduction due to RTSV infection in the four accessions of O. glaberrima and two genotypes of O. sativa, TN1 susceptible to RTSV and TW16 resistant to RTSV, was monitored until 28 dpi to characterize the stunting phenotypes of the plants. The heights of O. sativa TN1 and TW16 were not reduced significantly after infection with RTSV, whereas the heights of the four accessions of O. glaberrima were significantly reduced after RTSV infection (Table 2).

The height reduction rates of the O. sativa and O. glaberrima plants were compared to examine whether the severity of stunting by RTSV infection was different among the plants. The height reduction rates of O. glaberrima-102569 were significantly higher than those of O. sativa TN1 and TW16 at 14 dpi and onwards, and they were also significantly higher than those of the other three O. glaberrima accessions at 21 and 28 dpi (Figure 1A, Supplementary Material 2). The reduction rates of O. glaberrima-96790, -96793, and -104545 were significantly higher than that of O. sativa TN1 only at 21 dpi. (Figure 1A, Supplementary Material 2). The height reduction rates of the four accessions of O. glaberrima were between approximately 8 and 14% at 7 dpi (Figure 1A, Supplementary Material 2). The height reduction rates for the four accessions of O. glaberrima increased to approximately 18–21% at 14 dpi, indicating that stunting of plants became more evident at 14 dpi. Stunting of O. glaberrima-102569 became more severe after 14 dpi, showing height reduction rates of approximately 31% at 21 dpi and 34% at 28 dpi (Figures 1A,B, Supplementary Material 2). In contrast, the height reduction rates of three other O. glaberrima accessions (96790, 96793, and 104545) decreased from approximately 18% at 14 dpi to approximately 14–15% at 28 dpi, indicating that stunting of these three O. glaberrima accessions became milder (Figures 1A,B, Supplementary Material 2). The height reduction rates for O. sativa TN1 and TW16 were between approximately −0.3 and 5% during the observation (Figure 1A, Supplementary Material 2), but the differences in height between the mock-inoculated O. sativa plants and the corresponding RTSV-infected plants were not statistically significant throughout the observation (Table 2). These results suggested that, unlike O. sativa, a majority of O. glaberrima accessions are vulnerable to RTSV, but the severity of stunting varies significantly among O. glaberrima accessions.

The accumulation of RTSV in the O. glaberrima and O. sativa plants was examined to see whether it is correlated with the height reduction rate. The levels of RTSV accumulation determined by ELISA in the four accessions of O. glaberrima and O. sativa increased rapidly until 14 dpi and then were maintained at similar levels or decreased slightly until 28 dpi (Figure 2, Supplementary Material 3). The accumulation of RTSV in O. glaberrima-96790 (OD₄₀₅ of 2.40 ± 0.25) was significantly higher than that in the other three O. glaberrima accessions (1.48 ± 0.27–1.75 ± 0.21) at 14 dpi. The levels of RTSV accumulated in O. glaberrima-96793, -102569, and -104545 at a time point were not significantly different from one another throughout the observation. The levels of RTSV accumulated in O. sativa TN1 were significantly lower than those in at least one of the O. glaberrima accessions-96790, -96793, and 104545 at 14 dpi and onwards. The levels of RTSV accumulated in O. glaberrima-102569 were not significantly different from those in O. sativa TN1 throughout the observation, despite the fact that their height reduction rates were significantly different at 14 dpi and onwards. Accumulation of RTSV in O. sativa TW16 was not detected by ELISA. However, as in the previous studies by Encabo et al. (2009) and Satoh et al. (2013) showing detection of a very low level of RTSV in TW16 by RT-PCR, we assumed that most O. sativa TW16 plants inoculated with RTSV were infected since most O. sativa TN1 plants (susceptible control) inoculated at the same time were found to be infected with RTSV. The comparison between the height reduction rates (Figure 1, Supplementary Material 2) and the RTSV accumulation (Figure 2, Supplementary Material 3) indicates that the level of RTSV accumulation in plants is not correlated with the severity of stunting.

Temporal changes in expression of six cell wall-related genes were examined in O. glaberrima-96793 and -102569 and O. sativa TN1 to find cell wall-related genes associated with stunting of O. glaberrima caused by RTSV. The three accessions were selected since the severity of stunting by RTSV was significantly different from one another at 21 dpi (Figure 1, Supplementary Material 2). A genome-wide gene expression analysis in O. sativa TN1 and TW16 had been performed previously by Satoh et al. (2013). Genes for CESA5 and cellulose synthase A6 (CESA6), CSLA9 and cellulose synthase-like C7 (CSLC7), expansin 1, and α-expansin 15 precursor (expansin 15) were selected based on their expression patterns in O. sativa TN1 infected with RTSV. The previous microarray analysis by Satoh et al. (2013) showed that the expression 1) was activated for CESA5 and CSLC7 genes, 2) was suppressed for CSLA9 and expansin 15 genes, and 3) was not significantly changed for CESA6 and expansin 1 genes by RTSV infection between 6 and 15 dpi (Table 3).

At 14 dpi, the relative expression levels (fold changes) of four (CESA5, CESA6, CSLA9, and expansin 1genes) out of the six genes examined were significantly higher in O. sativa TN1 than in at least either O. glaberrima-96793 or -102569 (Figure 3). The expression of the CESA5 gene was up-regulated more than 4-fold by RTSV infection in O. sativa TN1 at 14 dpi, whereas the expression of the gene was found to be down-regulated slightly (approximately 0.7- to 0.8-fold) by RTSV infection in O. glaberrima-96793 and -102569 (Figure 3A). The expression of the expansin 1 gene was also up-regulated approximately 1.6-fold by RTSV infection in O. sativa TN1 at 14 dpi, whereas the gene was down-regulated to approximately 0.2- to 0.4-fold in both O. glaberrima accessions (Figure 3E). The expression of the genes for CESA6 and CSLA9 was down-regulated more significantly by RTSV infection in O. glaberrima-96793 and -10569 than in O. sativa TN1 at 14 dpi (Figures 3B,C).

At 21 dpi, the relative expression levels of five genes (CESA5, CESA6, CSLA9, CSLC7, and expansin 15 genes) were significantly higher in O. glaberrima-96793 than in O. sativa TN1 (Figure 3). The expression of all six genes was down-regulated to approximately 0.2- to 0.8-fold in O. sativa TN1 at 21 dpi (Figure 3). The expression of the genes for CESA5 and CESA6 was up-regulated slightly (approximately 1.1-to 1.2-fold) in O. glaberrima-102569 at 21 dpi (Figures 3A,B).

At 28 dpi, the relative expression levels of five genes (CESA5, CSLA9, CSLC7, expansin 1, and expansin 15 genes) were significantly higher in O. glaberrima-102569 than in O. sativa TN1 (Figure 3). The relative expression levels of four genes (CSLA9, CSLC7, expansin 1, and expansin 15 genes) were significantly higher in O. glaberrima-102569 than in O. glaberrima-97693 (Figure 3). The expression of all six genes was down-regulated in O. sativa TN1 (approximately 0.02- to 0.95-fold), and also in O. glaberrima-96793 (approximately 0.03- to 0.92-fold) at 28 dpi (Figure 3).

Overall, at 14 dpi, the relative expression levels of a majority of the six cell wall-related genes were significantly higher in O. sativa TN1 than in the two accessions of O. glaberrima which were stunted by RTSV infection. In contrast, at 28 dpi, the relative expression levels of a majority of the six genes were significantly higher in most severely stunted O. glaberrima-102569 than in O. sativa TN1 and O. glaberrima-96793.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.