Gene expression responses to Rice tungro spherical virus in susceptible and resistant near-isogenic rice plants

Virus Research 171 (2013) 111–120

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Gene expression responses to Rice tungro spherical virus in susceptible and resistant near-isogenic rice plants Kouji Satoh a , Hiroaki Kondoh a , Teresa B. De Leon b , Reena Jesusa A. Macalalad b , Rogelio C. Cabunagan b , Pepito Q. Cabauatan b , Ramil Mauleon c , Shoshi Kikuchi a,∗∗ , Il-Ryong Choi b,∗ a

Plant Genome Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan Plant Breeding, Genetics, and Biotechnology Division, DAPO Box 7777, Metro Manila, Philippines c T.T. Chang Genetic Resources Center, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines b

a r t i c l e

i n f o

Article history: Received 3 August 2012 Received in revised form 11 November 2012 Accepted 16 November 2012 Available online 23 November 2012 Keywords: Rice RTSV Transcriptome Microarray Resistance

a b s t r a c t Rice cultivar Taichung Native 1 (TN1) is susceptible to Rice tungro spherical virus (RTSV). TW16 is a backcross line developed between TN1 and RTSV-resistant cultivar Utri Merah. RTSV accumulation in TW16 was significantly lower than in TN1, although both TN1 and TW16 remained asymptomatic. We compared the gene expression profiles of TN1 and TW16 infected by RTSV to identify the gene expression patterns accompanying the accumulation and suppression of RTSV. About 11% and 12% of the genes in the entire genome were found differentially expressed by RTSV in TN1 and TW16, respectively. About 30% of the differentially expressed genes (DEGs) were detected commonly in both TN1 and TW16. DEGs related to development and stress response processes were significantly overrepresented in both TN1 and TW16. Evident differences in gene expression between TN1 and TW16 instigated by RTSV included (1) suppression of more genes for development-related transcription factors in TW16; (2) activation of more genes for development-related peptide hormone RALF in TN1; (3) TN1- and TW16-specific regulation of genes for jasmonate synthesis and pathway, and genes for stress-related transcription factors such as WRKY, SNAC, and AP2-EREBP; (4) activation of more genes for glutathione S-transferase in TW16; (5) activation of more heat shock protein genes in TN1; and (6) suppression of more genes for Golden2like transcription factors involved in plastid development in TN1. The results suggest that a significant number of defense and development-related genes are still regulated in asymptomatic plants even with a very low level of RTSV, and that the TN1- and TW16-specific gene regulations might be associated with regulation of RTSV accumulation in the plants. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Rice tungro disease (RTD) is a major constraint in the production of rice (Oryza sativa) in South and Southeast Asia. Rice plants affected by RTD show symptoms such as stunting and yelloworange discoloration of leaves (Hibino, 1983). RTD is caused by two viruses, Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). Both viruses are transmitted by green leafhoppers (GLH) such as Nephotettix virescens, but RTBV can be transmitted by GLH only in the presence of RTSV (Hibino, 1983). RTBV is mainly responsible for causing the disease symptoms, while RTSV plays the role of a helper virus for insect transmission of RTBV, and also enhances the disease symptoms caused by RTBV (Hibino, 1983).

∗ Corresponding author. Tel.: +63 2 580 5600, fax: +63 2 580 5699. ∗∗ Corresponding author. Tel.: +81 29 838 7007; fax: +81 29 838 7007. E-mail addresses: [email protected] (S. Kikuchi), [email protected] (I.-R. Choi). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.11.003

RTSV is the type member of the genus Waikavirus belonging to the Sequiviridae family (Choi, 2008). RTSV has a single-stranded polyadenylated plus-sense RNA genome of approximately 12 kb encapsidated in polyhedral particles (Hull, 1996; Shen et al., 1993). RTSV alone generally does not cause evident symptoms in rice, except mild stunting (Hibino, 1983). Indonesian rice cultivar Utri Merah is highly resistant to RTSV and RTBV (Encabo et al., 2009). RTSV resistance in Utri Merah is controlled by a single recessive locus (tsv1) mapped around 22.1 Mb of chromosome 7 (Lee et al., 2010). A survey of various rice cultivars for the allele types of a gene for translation initiation factor 4G (eIF4Gtsv1 ) located within tsv1 indicated an association of RTSV resistance with single nucleotide polymorphisms (SNP) in the eIF4Gtsv1 gene (Lee et al., 2010). Responses of plants at the gene expression level to various viruses were examined using microarrays to explore the molecular basis of symptom development and defense systems (AgudeloRomero et al., 2008; Babu et al., 2008; Catoni et al., 2009; Dardick, 2007; García-Marcos et al., 2009; Whitham et al., 2003, 2006).

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Comparison of results from the previous studies indicated that, although many genes in various plant species respond specifically to different viruses, there is commonality in responses among different plant–virus interactions. For example, rice plants infected with Rice stripe virus (RSV; Satoh et al., 2010) and those with Rice dwarf virus (RDV; Satoh et al., 2011; Shimizu et al., 2007) exhibit leaf chlorosis and stunted growth. Such commonality in symptom development appears to be reflected in gene expression patterns in plants infected with RDV and RSV. The expression of genes related to cell wall formation and nuclear-encoded chloroplasttargeted genes decreased significantly in plants infected with either virus. RTSV synergistically enhances symptoms caused by RTBV in rice, although RTSV itself rarely causes any visible symptoms (Encabo et al., 2009). The expression of about 100 genes was changed more than twofold in an RTSV-susceptible rice plant by RTSV infection, while 150 genes were found significantly regulated by RTSV infection in an RTSV-resistant plant in which RTSV accumulated at a level much lower than in an RTSV-susceptible plant (Encabo et al., 2009). This observation prompted us to speculate that, despite lacking the ability to cause visible symptoms, RTSV may affect host gene expression potentially linked to the development of RTD. In this study, we examined the host gene expression responses to RTSV infection in two near-isogenic rice plants (a backcrossed plant and its recurrent parental plant) that share an extensive common genetic background but are clearly different in the trait linked to virus accumulation, to reveal the gene expression responses associated with the difference in RTSV accumulation between resistant and susceptible plants. Our results suggested that the genes related to defense and development processes were actively responding to RTSV in asymptomatic plants even without significant accumulation of RTSV, and that the gene expression responses specific to the resistant or susceptible plants might be associated with various defense mechanisms contributing to the regulation of RTSV accumulation in the plants.

2. Materials and methods 2.1. Plant materials TW16 is a backcross line (BC5 F7–8 ) developed from donor cultivar Utri Merah (International Rice Germplasm Collection accession number 16682) and recurrent parent Taichung Native 1 (TN1) (Lee et al., 2010). TN1 is susceptible to both RTSV and RTBV, whereas TW16 is resistant to both viruses (Encabo et al., 2009). Genetic similarity between TW16 and TN1 was evaluated by a kinship analysis based on data obtained from the 1536 rice SNP platform (Zhao et al., 2010).

2.2. Inoculation of virus and estimation of virus accumulation RTSV strain A (Cabauatan et al., 1995) maintained in TN1 was used as the source of inoculum. Insect 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 10-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. Five seedlings per pot (12 cm in diameter) were grown in a temperature-controlled greenhouse (27–29 ◦ C). Collection of plant samples for microarray (6, 9, and 15 days postinoculation (dpi)) and estimation for RTSV accumulation (0, 3, 6, 9, 12, 15, 18, and 21 dpi) were repeated three times (biological replications). The relative RTSV accumulation in plants was estimated

by enzyme-linked immunosorbent assay (ELISA) as described by Shibata et al. (2007) 2.3. Microarray Total RNA samples were extracted from shoot samples pooled from five independent plants of the same treatment using the RNeasy Maxi kit (Qiagen). Cyanine 3(Cy3)- or Cyanine 5 (Cy5)labeled complementary RNA (cRNA) samples were synthesized from 850 ng of the total RNA using the low-input RNA labeling kit (Agilent Technologies) according to the manufacturer’s instructions. Transcriptome profiles specific to RTSV-infected plants were examined by direct comparison of transcription activities between RTSV-infected and mock (RTSV-free GLH)-inoculated plants on the customized oligoarray (Satoh et al., 2010). Hybridization solution was prepared with 825 ng each of Cy3- and Cy5-labeled cRNA preparations using the in situ Hybridization Kit Plus (Agilent Technologies). After washing, the slide image files were produced by a DNA microarray scanner (G2505B; Agilent Technologies). Examination by microarray was repeated three times with independent samples. 2.4. Data analysis Signal intensities of Cy3 and Cy5 were extracted from the image files and normalized to remove the dye-effect in signal intensity by the rank consistency and LOWESS methods processed by Feature Extraction version 9.5 (Agilent Technologies). Signal intensities of all samples were transformed into log2 -based numbers and normalized according to the quantile method for standardization among array slides by EXPANDER ver. 4.1 (Ulitsky et al., 2010). A gene was declared “expressed” if the average signal intensity of the gene was higher than the median intensity of the complete intensity data at least in one condition; otherwise, the gene was considered not expressed. A differentially expressed gene (DEG) was defined as an expressed gene with (1) a log2 -based ratio (RTSVinoculated sample/mock-inoculated sample) higher than 0.585 (1.5-fold) or lower than −0.585 (0.667-fold) and (2) significant of changes in gene expression between two plants at P ≤ 0.05 by a paired t-test (permutation: all, FDR correction: adjusted Bonferroni method). Data processing was performed using Mev version 4.4 (Saeed et al., 2006). The outputs of microarray analysis used in this study (series number GSE 16142) are available at NCBI-GEO (Barrett et al., 2009). 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) for rice and virus genes cDNA fragments for transcripts of selected rice genes or the RTSV genome were synthesized using 1000 ng of the corresponding RNA with 50 pmol of oligo(dT)20 by SuperScript III reverse transcriptase (Invitrogen, USA). The 4-fold diluted cDNA reaction mixtures were used for PCR. To examine the expression of rice genes, PCR was performed with 4 ␮l of diluted cDNA reaction mixture in a final volume of 20 ␮l using Taq DNA polymerase (New England Biolabs). An actin gene (LOC Os11g06390), whose expression remained nearly constant under all experimental conditions, was used for the control of gene expression analysis by RT-PCR. PCR for RTSV RNA was performed with 4 ␮l of 4-fold diluted cDNA reaction mixture, and primers 5 -GAAGAAGCCTATCATGTTCGCGT3 and 5 -CCTCCACGATATTGTACGAGG-3 targeted at a region of the coat protein cistron in a final volume of 20 ␮l using Taq polymerase. The cycling program was initial denaturation for 1 min at 94 ◦ C, followed by 25–40 cycles of 15 s at 94 ◦ C (denaturation), 15 s at 60 ◦ C (annealing), and 45 s at 72 ◦ C (extension), and then a final extension of 1 min at 72 ◦ C.

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2007). For some ontology categories, the ratios of the number of DEGs to the number of expressed genes were significantly (P < 0.01 by a chi-square test) higher than the ratios of the total number of DEGs to the total number of expressed genes in the corresponding conditions (4.6%) (1097 DEGs/23,899 expressed genes in TN1 at 9 dpi) to 6.8% (1630 DEGs/23,899 expressed genes in TW16 at 6 dpi) (Table 1). Especially, the categories “Response to abiotic stimulus,” “Response to biotic stimulus,” and “Multicellular organismal development” were significantly overrepresented in many conditions (Table 1). We hypothesized that the overrepresented functional categories are more likely to be the ones either influenced by or influencing RTSV accumulation in TN1 and TW16. Therefore, the expression patterns of the individual genes comprising the overrepresented biological processes were more closely examined to assess the relationship between the modified biological processes and the levels of RTSV infection. 3.2. Gene expression profiles related to defense responses

Fig. 1. Accumulation of RTSV in TN1 and TW16. (A) RTSV accumulation in TN1 (open circle) and TW16 (closed circle) estimated by enzyme-linked immunosorbent assay. Data for mock-inoculated TN1 and TW16 are not shown to avoid confusion with the data for RTSV-inoculated TW16. Vertical lines: standard errors. (B) Detection of RTSV RNA in TN1 and TW16 by RT-PCR.

3. Results and discussion 3.1. Cellular processes affected by RTSV infection at the transcriptome level A kinship analysis between TN1 and TW16 by the 1536 rice SNP platform (Zhao et al., 2010) confirmed their extensive common genetic background (Supplementary material Fig. S1). Any evident disease symptoms were not observed in TN1 and TW16 by infection with RTSV during our observation (28 dpi). In TN1, the accumulation of RTSV coat protein (CP) was first detected at 6 dpi, and accumulation increased until 18 dpi. RTSV CP was not detected in TW16 by ELISA (Fig. 1A), but we were able to confirm the presence of RTSV RNA in TW16 by RT-PCR for the CP gene (Fig. 1B). These results showed that RTSV accumulates at a much lower level in TW16 than in TN1. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2012.11.003. The number of expressed genes in plants infected with RTSV was 23,899. The numbers of genes detected as a DEG by RTSV infection at least at one of the time points (6, 9, and 15 dpi) were 2874 (12.0% of total expressed genes) and 2597 (10.9%) in TW16 and TN1, respectively (Supplementary material Table S1). The proportion of common DEGs detected in both TN1 and TW16 was about 30%. The total number of DEGs by RTSV infection detected in either TN1 or TW16 was 4474 (Supplementary material Table S1). To assess the accuracy of microarray data, we selected eight DEGs belonging to ontology categories apparently affected by RTSV infection (see below) and examined the similarity between gene responses observed by microarray and those by RT-PCR. Most cases of activation or suppression of gene expression detected by microarray were also observed by RT-PCR (Supplementary material Fig. S2). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2012.11.003. To discern the biological processes that might have been affected by RTSV infection, genes examined by microarray were classified based on the biological process categories according to the rice genome annotation database Osa1 version 5 (Ouyang et al.,

Plant hormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene are involved in controlling responses to diseases (Karban and Chen, 2007; Sánchez et al., 2010; Takahashi et al., 2004). The expression of many genes encoding enzymes involved in JA synthesis was found regulated in TN1 and TW16 by RTSV (Fig. 2). Five lipoxygenase genes were found regulated by RTSV infection. Among them, two genes (LOC Os12g37260 and LOC Os12g37350) were specifically suppressed in TW16. Four genes encoding 12oxo-phytodienoic acid (OPDA) reductase were found activated in both TN1 and TW16, but, at 6 dpi, the OPDA reductase genes were more evidently activated in TW16 than in TN1. Three genes encoding enzymes involved in ␤-oxidation of oxopentenylcyclopentane-octanoyl-CoA (LOC Os05g07090, LOC Os05g29880, and LOC Os11g39220) and a gene encoding phospholipase A1 (LOC Os09g33820) were more significantly suppressed in TN1, especially at 6 dpi, than in TW16 (Fig. 2). JA is usually associated with a defense mechanism against necrotrophic pathogens (reviewed in Bari and Jones, 2009). Endogenous JA and OPDA were increased by infection with Potato virus Y especially in resistant potato plants (Kovaˇc et al., 2009). Thus, activation of genes for OPDA reductase by RTSV in TW16 may be a typical response of plants resistant to virus infection. Suppression of two lipoxygenase genes in TW16 (Fig. 2) may be associated with the suppression of TCP genes in TW16 by RTSV infection (see below). Tify and JAMyb are transcription factors involved in JA-mediated signaling pathways (Lee et al., 2001; Ye et al., 2009). Genes for Tify and JAMyb were found more evidently activated in TW16 by RTSV infection than in TN1 (Fig. 2), suggesting that the JA-mediated defense system is more actively operating in TW16 after RTSV infection than in TN1. WRKY transcription factors are often involved in responses to biotic stresses in plants, including responses to virus infection (Babu et al., 2008; Catoni et al., 2009; Dardick, 2007; Satoh et al., 2011; Shimizu et al., 2007). The expression of 23 WRKY genes was found regulated by RTSV infection (Supplementary material Fig. S3). In particular, the expression patterns of WRKY genes LOC Os01g18584 (OsWRKY9), LOC Os03g21710 (OsWRKY45), LOC Os05g04640 (OsWRKY5), LOC Os06g44010 (OsWRKY28), and LOC Os07g02060 (OsWRKY29) were different between TN1 and TW16. More WRKY genes were suppressed in TW16 than in TN1, while WRKY genes were predominantly activated in TN1 at 9 and 15 dpi. OsWRKY28 (LOC Os06g44010, Supplementary material Fig. S3) was activated especially in TW16, suggesting the involvement of OsWRKY28 as a positive regulator of defense response against RTSV. On the contrary, OsWRKY28 reportedly acted as a negative regulator of basal resistance to Magnaporthe grisea (Delteil et al., 2012). Therefore, OsWRKY28 may direct antagonistic

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Table 1 Numbers of differentially expressed genes (DEGs) grouped based on gene ontology categories in TN1 and TW16 infected with RTSV.

regulation defense modes to different types of pathogens. WRKY6 activates the expression of genes from a PR1 gene promoter in Arabidopsis, indicating that WRKY6 is a positive regulator of defense mechanisms (Robatzek and Somssich, 2002). OsWRKY1.V2 (LOC Os01g14440) is an ortholog of WRKY6. The expression of OsWRKY1.V2 was activated in both TN1 and TW16 (Supplementary material Fig. S3), suggesting that OsWRKY1.V2 also acts as a positive regulator of defense mechanisms against RTSV in rice. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2012.11.003. AP2/EREBP (APETALA 2/ETHYLENE RESPONSIVE ELEMENT BINDING PROTEIN) family genes encode transcription regulators functioning in the responses to abiotic and biotic stresses (Catoni et al., 2009; Dubouzet et al., 2003; Fischer and DrögeLaser, 2004; Satoh et al., 2010, 2011). Expression of 44 genes for AP2/EREBP was found regulated by RTSV infection in TN1 and TW16 (Supplementary material Fig. S3). More AP2/EREBP genes were suppressed than activated in both TN1 and TW16 at 6 and 15 dpi, whereas more genes were activated than suppressed in both plants at 9 dpi. The expression patterns of AP2/EREBP genes such as LOC Os01g54890, LOC Os05g49700 (at 6 dpi), LOC Os06g03670 (at 6 dpi), and LOC Os07g13170 were especially different between TN1 and TW16. NAC (named from “NAM,” “ATAF1,” and “CUC2”) transcription factors are involved in the regulation of development and stress

responses (Fang et al., 2008). Five genes for NAC related to stress responses (SNAC) were found regulated by RTSV infection. At 6 dpi, the SNAC genes were predominantly suppressed in TN1, whereas they were activated in TW16 (Fig. 3). The role of SNAC during defense response is not clear yet, but SNAC may act as a positive regulator of defense mechanisms against pathogens since rice plants overexpressing one of the SNAC (OsNAC6) gene exhibited enhanced tolerance to M. grisea (Nakashima et al., 2007). Meanwhile, a NAC transcription factor was found to interact with TMV replicase in Arabidopsis (Wang et al., 2009b). The interaction between the NAC and the TMV replicase resulted in suppression of host basal defense, indicating that the interaction serves as a means to promote systemic infection of TMV (Wang et al., 2009b). Among various pathogenesis-related (PR) protein genes encoded in the rice genome, the expression of genes for PR2, 5, 7, 15, and 16 was found more significantly affected by RTSV infection than other PR protein genes (Supplementary material Fig. S4). It appeared that more PR2 genes encoding ␤-1,3-glucosidases (van Loon et al., 2006) were suppressed than activated in TN1 and TW16 by RTSV infection. More PR5 genes encoding thaumatinlike proteins (Shatters et al., 2006) and more PR7 genes encoding subtilisin-like serine proteases (Golldack et al., 2003) were activated than suppressed in both TN1 and TW16 by RTSV infection. More PR15/16 genes encoding germin-like proteins (Banerjee and Maiti, 2010) were activated in TW16, but they were largely suppressed in TN1 by RTSV infection.

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Fig. 2. Responses to RTSV infection of genes related to jasmonate (JA) synthesis and signaling. (A) Metabolic pathway of JA synthesis and the responses of genes encoding enzymes involved in the pathway. 13(S)-HpOTrE: (9Z,11E,14Z)-(13S)-hydroperoxyoctadeca-(9,11,14)-trienoate, 12,13-EOTrE: (9Z)-(13S)-12,13-epoxyoctadeca-9,11,15trienoate, OPC8-CoA: 8-[(1R,2R)-3-oxo-2-{(Z)-pent-2-enyl}cyclopentyl]octanoate CoA, JA-CoA: (+)-7-isojasmonic acid CoA. (B) Responses of genes related to jasmonate (JA) synthesis and signaling. The log2 -based differential expression ratios (signal intensity in RTSV-infected plant/signal intensity in mock-inoculated plant) of a gene in TN1 and TW16 at the respective time points (6–15 dpi) are indicated by a row of green (suppressed) and red (activated) colors of various intensities. Only the ratios of significant differential gene expression (P < 0.05) at least at one time point are shown.

Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2012.11.003. Other groups of stress-related genes that were noticeably responding to RTSV infection were those for glutathione Stransferases (GST) (Jain et al., 2010), glutaredoxins (Rouhier et al., 2006), and heat shock proteins (HSP). A majority of these genes were activated by RTSV infection (Fig. 3). Many genes for GST were activated in TW16 (Fig. 3), suggesting that GST proteins are actively involved in defense mechanisms against RTSV, especially in TW16. In contrast, many genes for HSP were activated more prominently in TN1 (Fig. 3). This observation is consistent with previous studies indicating that activation of HSP genes such as those for HSP70, which may serve as a chaperone, is a general phenomenon resulting from virus infection (Chen et al., 2008) or simply the outcome of protein accumulation in cytosol (Aparricio et al., 2005). However, specific interactions of HSP and viral proteins may also be important for virus replication. For example, the interaction of replicases

of tomato bushy stunt virus and cucumber necrosis virus with HSP70 is crucial for the assembly and localization of virus replicases (Pogany et al., 2008; Serva and Nagy, 2006; Wang et al., 2009a). Activation of HSP in TN1 may reflect such an interaction between RTSV replicase and HSP, which may have occurred, especially in TN1, to support the active replication of RTSV in TN1. 3.3. Gene expression profiles related to development processes The homeobox gene family is associated with the development and morphogenesis of plants (Jain et al., 2008). Among 77 homeobox genes examined in the microarray, the expression of 23 genes was changed significantly by RTSV infection (Fig. 4). More homeobox genes were suppressed in TW16 than in TN1. Growing evidence revealed that homeobox transcription factors are actually involved in response to biotic stresses. Homeobox-like transcription factor OCP3 in Arabidopsis is involved in JA-mediated

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Fig. 3. Responses to RTSV infection of genes related to stress response, and defense processes. See Fig. 2 for details.

defense pathway (Coege et al., 2005; Ramirez et al., 2010). The OCP3 gene is constitutively expressed in healthy plants, but repressed in response to infection with necrotrophs. An ocp3 mutant constitutively expressed genes for GST and JA-inducible PDF1.2. These observations are consistent with the gene expression patterns observed in TN1 and TW16 infected with RTSV; the expression of many homeobox genes was suppressed, especially in TW16, by RTSV (Fig. 4). Many genes for GST were activated, especially in TW16, by RTSV (Fig. 3), and many genes associated with synthesis of, and response to, JA were activated in TN1 and TW16 by RTSV (Fig. 2). Homeobox gene SAB23 in rice also appeared to be a negative regulator of resistance to Xanthomonas oryzae (Seo et al., 2011). Several other homeobox genes were also found to be involved in the regulation of JA-mediated response to biotic stresses (Dezar et al., 2011; Manavella et al., 2008; Ramirez et al., 2010; Yoon et al., 2009). Meanwhile, homeobox gene NTH201 encoding KNOTTED1 was found to facilitate cell-to-cell movement of Tobacco mosaic virus (TMV) (Yoshii et al., 2008). The results in these previous

Fig. 4. Responses to RTSV infection of genes for transcription factors related to development and morphogenesis processes. See Fig. 2 for details.

studies imply that the suppression of many homeobox genes in TW16 contributes to resistance to RTSV. More genes of other development-related transcription factors were also suppressed in TW16 than in TN1. Such suppressed transcription factor genes were those encoding C2C2-CO (CONSTANS)-like (Kim et al., 2008), C2C2-DOF (DNA-binding with one finger) (Shigyo et al., 2007), SBP (SQUAMOSA promoter-binding proteins) (Shikata et al., 2009), and TCP (named from “TEOSINTE BRANCHED 1,” “CYCLOIDEA,” and “PROLIFERATING CELL FACTORS 1 and 2”) (Martín-Trillo and Cubas, 2010) (Fig. 4). TCP transcription factors are known to control biosynthesis of JA by regulating the expression of genes encoding lipoxygenase (Schommer et al., 2008). Thus, the suppression of two lipoxygenase genes in TW16 (Fig. 2) may be associated with the predominant suppression of TCP genes in TW16, by RTSV (Fig. 4).

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Fig. 5. Responses to RTSV infection of genes related to plant hormone synthesis and signaling. See Fig. 2 for details.

Plant hormone auxin is involved in the regulation of various development processes (Kieffer et al., 2010). Auxin was also found to be associated with biotic stress responses (Bari and Jones, 2009; Ghanashyam and Jain, 2009). The expression of many genes related to synthesis and signaling of a major auxin, indole acetic acid (IAA), such as genes for ARF (auxin response factor) and the Aux/IAA family (Guilfoyle and Hagen, 2007), was predominantly suppressed in both TN1 and TW16 by RTSV infection (Fig. 5). It was shown that TMV reprograms the auxin response pathway and controls disease development through interaction between TMV replicase protein and Aux/IAA proteins (Padmanabhan et al., 2005, 2008). Therefore, the response patterns of many genes related to IAA synthesis and signaling in TN1 and TW16 by RTSV infection (Fig. 5) may be related to susceptibility or resistance to RTSV.

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Two auxin-responding genes for indole-3-acetic acid-amido synthetase (GH3) (Jain et al., 2006) were significantly more activated by RTSV infection in TN1 than in TW16 (Supplementary material Fig. S5). The GH3 genes prevent accumulation of free IAA. IAA induces expression of genes for expansin, which are involved in loosening of cell wall. Thus, excess IAA may make plants more vulnerable to pathogens (Ding et al., 2008). The activation of GH3 genes may result in a reduction in IAA in TN1 and TW16, which, in turn, may have led to the suppression of genes for expansin in the plants (Supplementary material Fig. S5) (see below). Therefore, the activation of the GH3 genes in rice plants by RTSV infection may be a defense response to RTSV. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2012.11.003. Regulation patterns of genes for SAUR in TN1 and TW16 after RTSV infection were variable (Supplementary material Fig. S5). A SAUR gene was activated by infection with beet curly top virus in Arabidopsis, and the induction of the SAUR gene was correlated with symptom development (Park et al., 2004). A TCP transcription factor in Arabidopsis was found to activate an auxin response gene and a SAUR gene in Arabidopsis (Koyama et al., 2010). Many TCP and SAUR genes were suppressed concomitantly in TN1 and TW16 by RTSV. The result suggests that a regulation mechanism similar to that of the SAUR gene by TCP in Arabidopsis may also operate in rice. Gibberellic acid (GA) is another plant hormone regulating development and morphogenesis (Schwechheimer and Willige, 2009). Four genes related to GA synthesis were found suppressed in TN1 and TW16 by RTSV infection (Fig. 5). The P2 protein of RDV interacts with ent-kaurene oxidase required for GA synthesis (Zhu et al., 2005). This interaction results in a reduction in GA synthesis and may make plants more competent for virus replication. Thus, the suppression of genes for GA synthesis by RTSV may have resulted from the interaction of RTSV proteins with genes associated with GA synthesis. Among 14 genes for the GA receptor family (Itoh et al., 2008) whose expression was changed by RTSV infection, eight genes were suppressed in either TN1 or TW16. Genes belonging to the GRAS (named from “GIBBERELLIC-ACID INSENSITIVE,” “REPRESSOR of GAI,” and “SCARECROW”) family encode negative regulators of GA signaling (Hirsch and Oldroyd, 2009). More GRAS genes were found regulated in TW16 by RTSV infection than in TN1. Overall, the GA signaling process was seemingly more drastically affected in TW16 by RTSV infection than in TN1. The involvement of GA in defense mechanisms is not understood well, but a GA–GA receptor-GRAS interaction mechanism may enable plants to prioritize resource allocation to divert resources away from growth in favor of defense against pathogens (Harberd et al., 2009). DELLA proteins, a subfamily of GRAS proteins (Itoh et al., 2008), promote resistance to necrotrophs by activating JA/ET (ethylene)-dependent defense responses (Navarro et al., 2008). Activation of GRAS genes in TN1 and TW16 by RTSV infection (Fig. 5) may therefore be associated with the activation of a JA-mediated defense pathway by RTSV infection (Fig. 2). Rapid alkalinization factors (RALF) are peptide hormones involved in the regulation of plant development (Germain et al., 2005), Genes for RALF were more significantly activated in TN1 than in TW16 (Fig. 5). Gupta et al. (2010) hypothesized that RALF is induced by pathogens to exploit it as a decoy to decrease pathogenic load without adding pathogen fitness or activating defense signals in plants, so that the pathogens can propagate actively in the hosts. Thus, evident activation of the RALF genes observed in TN1 after RTSV infection may be associated with active propagation of RTSV in TN1. Cell wall formation is one of the important processes in plant morphogenesis. The expression of four cellulose synthase (-like)

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in either TN1 or TW16 (Supplementary material Fig. S5) is significantly less than the number of genes affected by RSV infection (30 genes; Satoh et al., 2010). Such differences in gene expression and the symptoms between plants infected with RSV and those with RTSV may suggest that the host gene expression responses to RTSV infection in TN1 are not drastic enough to cause evident symptoms. 3.4. Gene expression profiles related to photosynthesis Genes associated with photosynthesis and chlorophyll synthesis were predominantly activated in both TN1 and TW16, especially at 9 dpi (Fig. 6), whereas genes for ribosomal proteins were predominantly suppressed in both TN1 and TW16. Many Golden2-like genes involved in plastid development (Bravo-Garcia et al., 2009) were suppressed by RTSV infection more noticeably in TW16 than in TN1. Savitch et al. (2007) reported that overexpression of the Golden2-like (GLK1) gene in Arabidopsis resulted in a high constitutive expression of genes encoding disease defense-related proteins, suggesting a positive relationship between GLK1 and the defense response. Thus, the predominant suppression of the Golden2-like genes after RTSV infection in TN1 (Fig. 6) implies its weakened defense response against RTSV. In conclusion, the global expression profiles of two genetically similar rice plants differing in permissiveness to RTSV propagation revealed that the expression of various genes was affected by RTSV infection not only in a plant susceptible to RTSV (TN1) but also in a plant resistant to RTSV (TW16). The host responses involving genes associated with biological systems such as defense and development processes were significantly different between susceptible and resistant plants. Such dissimilarity may have resulted from or in the difference in accumulation of RTSV between the two plants. Further studies such as examination for (1) RTSV accumulation in rice mutants for genes whose expression was drastically changed by RTSV, and (2) host gene expression in plants transformed with the individual genes of RTSV should follow to unveil the basis of interaction between rice and RTSV, which leads to the differential gene expression responses between susceptible and resistant plants. Acknowledgments Fig. 6. Responses to RTSV infection of genes for photosynthesis-related processes. See Fig. 2 for details.

genes was activated, while three of these genes were suppressed in TN1 and TW16 by RTSV infection (Supplementary material Fig. S5). Expansin is involved in pH-dependent loosening of the cell wall, while xyloglucan endotransglucosylase/hydrolase (XET) is involved in the restructuring of xyloglucan-backbones (Cosgrove, 2005). The expression of a few genes for expansin and XET was also changed by RTSV infection. The suppression of expansin genes by RTSV infection was more evident in TW16. Genes related to cell wall formation were also suppressed in rice plants infected with either RDV or RSV (Satoh et al., 2010, 2011; Shimizu et al., 2007). The expression of many cellulose synthase (-like) and expansin family genes in plants stunted by RDV or RSV infections decreased significantly, often to less than 50% of the levels in healthy plants, suggesting an association between the suppression of cell wall-related genes and stunting (Satoh et al., 2010, 2011; Shimizu et al., 2007). The expression of some cell wall-related genes was also suppressed by RTSV infection, but their expression in RTSV-infected plants was mostly not less than 50% of the level in healthy plants. The number of cell wall-related genes whose expression was evidently suppressed by RTSV infection (10 genes)

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