Suppression of cell wall-related genes associated with stunting of Oryza glaberrima infected with Rice tungro spherical virus.

Rice tungro disease is a complex disease caused by the interaction between Rice tungro bacilliform virus and Rice tungro spherical virus (RTSV). RTSV alone does not cause recognizable symptoms in most Asian rice (Oryza sativa) plants, whereas some African rice (O. glaberrima) plants were found to become stunted by RTSV. Stunting of rice plants by virus infections usually accompanies the suppression of various cell wall-related genes. The expression of cell wall-related genes was examined in O. glaberrima and O. sativa infected with RTSV to see the relationship between the severity of stunting and the suppression of cell wall-related genes by RTSV. The heights of four accessions of O. glaberrima were found to decline by 14-34% at 28 days post-inoculation (dpi) with RTSV, whereas the height reduction of O. sativa plants by RTSV was not significant. RTSV accumulated more in O. glaberrima plants than in O. sativa plants, but the level of RTSV accumulation was not correlated with the degree of height reduction among the four accessions of O. glaberrima. Examination for expression of genes for cellulose synthase A5 (CESA5) and A6 (CESA6), cellulose synthase-like A9 (CSLA9) and C7, and α-expansin 1 (expansin 1) and 15 precursors in O. glaberrima and O. sativa plants between 7 and 28 dpi with RTSV showed that the genes such as those for CESA5, CESA6, CSLA9, and expansin 1were more significantly suppressed in stunted plants of O. glaberrima at 14 dpi with RTSV than in O. sativa, suggesting that stunting of O. glaberrima might be associated with these cell wall-related genes suppressed by RTSV. Examination for expression of these genes in O. sativa plants infected with other rice viruses in previous studies indicated that the suppression of the expansin 1 gene is likely to be a signature response commonly associated with virus-induced stunting of Oryza species. These results suggest that stunting of O. glaberrima by RTSV infection might be associated with the suppression of these cell wall-related genes at the early stage of infection with RTSV.

Relationship between gene responses and symptoms induced by Rice grassy stunt virus.

Rice grassy stunt virus (RGSV) is a serious threat to rice production in Southeast Asia. RGSV is a member of the genus Tenuivirus, and it induces leaf yellowing, stunting, and excess tillering on rice plants. Here we examined gene responses of rice to RGSV infection to gain insight into the gene responses which might be associated with the disease symptoms. The results indicated that (1) many genes related to cell wall synthesis and chlorophyll synthesis were predominantly suppressed by RGSV infection; (2) RGSV infection induced genes associated with tillering process; (3) RGSV activated genes involved in inactivation of gibberellic acid and indole-3-acetic acid; and (4) the genes for strigolactone signaling were suppressed by RGSV. These results suggest that these gene responses to RGSV infection account for the excess tillering specific to RGSV infection as well as other symptoms by RGSV, such as stunting and leaf chlorosis.

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

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 Golden2-like 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.

Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress.

BACKGROUND: Plant roots are important organs to uptake soil water and nutrients, perceiving and transducing of soil water deficit signals to shoot. The current knowledge of drought stress transcriptomes in rice are mostly relying on comparative studies of diverse genetic background under drought. A more reliable approach is to use near-isogenic lines (NILs) with a common genetic background but contrasting levels of resistance to drought stress under initial exposure to water deficit. Here, we examined two pairs of NILs in IR64 background with contrasting drought tolerance. We obtained gene expression profile in roots of rice NILs under different levels of drought stress help to identify genes and mechanisms involved in drought stress. RESULTS: Global gene expression analysis showed that about 55% of genes differentially expressed in roots of rice in response to drought stress treatments. The number of differentially expressed genes (DEGs) increased in NILs as the level of water deficits, increased from mild to severe condition, suggesting that more genes were affected by increasing drought stress. Gene onthology (GO) test and biological pathway analysis indicated that activated genes in the drought tolerant NILs IR77298-14-1-2-B-10 and IR77298-5-6-B-18 were mostly involved in secondary metabolism, amino acid metabolism, response to stimulus, defence response, transcription and signal transduction, and down-regulated genes were involved in photosynthesis and cell wall growth. We also observed gibberellic acid (GA) and auxin crosstalk modulating lateral root formation in the tolerant NILs. CONCLUSIONS: Transcriptome analysis on two pairs of NILs with a common genetic background (~97%) showed distinctive differences in gene expression profiles and could be effective to unravel genes involved in drought tolerance. In comparison with the moderately tolerant NIL IR77298-5-6-B-18 and other susceptible NILs, the tolerant NIL IR77298-14-1-2-B-10 showed a greater number of DEGs for cell growth, hormone biosynthesis, cellular transports, amino acid metabolism, signalling, transcription factors and carbohydrate metabolism in response to drought stress treatments. Thus, different mechanisms are achieving tolerance in the two tolerant lines.

Relationship between symptoms and gene expression induced by the infection of three strains of Rice dwarf virus.

BACKGROUND: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which often results in severe yield losses of rice in East Asian countries. The disease symptoms are stunted growth, chlorotic specks on leaves, and delayed and incomplete panicle exsertion. Three RDV strains, O, D84, and S, were reported. RDV-S causes the most severe symptoms, whereas RDV-O causes the mildest. Twenty amino acid substitutions were found in 10 of 12 virus proteins among three RDV strains. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the gene expression of rice in response to infection with the three RDV strains using a 60-mer oligonucleotide microarray to examine the relationship between symptom severity and gene responses. The number of differentially expressed genes (DEGs) upon the infection of RDV-O, -D84, and -S was 1985, 3782, and 6726, respectively, showing a correlation between the number of DEGs and symptom severity. Many DEGs were related to defense, stress response, and development and morphogenesis processes. For defense and stress response processes, gene silencing-related genes were activated by RDV infection and the degree of activation was similar among plants infected with the three RDV strains. Genes for hormone-regulated defense systems were also activated by RDV infection, and the degree of activation seemed to be correlated with the concentration of RDV in plants. Some development and morphogenesis processes were suppressed by RDV infection, but the degree of suppression was not correlated well with the RDV concentration. CONCLUSIONS/SIGNIFICANCE: Gene responses to RDV infection were regulated differently depending on the gene groups regulated and the strains infecting. It seems that symptom severity is associated with the degree of gene response in defense-related and development- and morphogenesis-related processes. The titer levels of RDV in plants and the amino acid substitutions in RDV proteins could be involved in regulating such gene responses.

Gene structures, classification and expression models of the AP2/EREBP transcription factor family in rice.

We identified 163 AP2/EREBP (APETALA2/ethylene-responsive element-binding protein) genes in rice. We analyzed gene structures, phylogenies, domain duplication, genome localizations and expression profiles. Conserved amino acid residues and phylogeny construction using the AP2/ERF conserved domain sequence suggest that in rice the OsAP2/EREBP gene family can be classified broadly into four subfamilies [AP2, RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein) and ERF (ethylene-responsive factor)]. The chromosomal localizations of the OsAP2/EREBP genes indicated 20 segmental duplication events involving 40 genes; 58 redundant OsAP2/EREBP genes were involved in tandem duplication events. There were fewer introns after segmental duplication. We investigated expression profiles of this gene family under biotic stresses [infection with rice viruses such as rice stripe virus (RSV), rice tungro spherical virus (RTSV) and rice dwarf virus (RDV, three virus strains S, O and D84)], and various abiotic stresses. Symptoms of virus infection were more severe in RSV infection than in RTSV and RDV infection. Responses to biotic stresses are novel findings and these stresses enhance the ability to identify the best candidate genes for further functional analysis. The genes of subgroup B-5 were not induced under abiotic treatments whereas they were activated by the three RDV strains. None of the genes of subgroups A-3 were differentially expressed by any of the biotic stresses. Our 44K and 22K microarray results suggest that 53 and 52 non-redundant genes in this family were up-regulated in response to biotic and abiotic stresses, respectively. We further examined the stress responsiveness of most genes by reverse transcription-PCR. The study results should be useful in selecting candidate genes from specific subgroups for functional analysis.

Genome-wide analysis of NAC transcription factor family in rice.

We investigated 151 non-redundant NAC genes in rice and 117 in Arabidopsis. A complete overview of this gene family in rice is presented, including gene structures, phylogenies, genome localizations, and expression profiles. We also performed a comparative analysis of these genes in rice and Arabidopsis. Conserved amino acid residues and phylogeny construction using the NAC conserved domain sequence suggest that OsNAC gene family was classified broadly into two major groups (A and B) and sixteen subgroups in rice. We presented more specific phylogenetic analysis of OsNAC proteins based on the DNA-binding domain and known gene function, respectively. Loss of introns was observed in the segmental duplication. Homologous, paralogous, and orthologous searches of rice and Arabidopsis revealed that the major functional diversification within the NAC gene family predated the divergence of monocots and dicots. The chromosomal localizations of OsNAC genes indicated nine segmental duplication events involving 18 genes; 32 non-redundant OsNAC genes were involved in tandem duplications. Expression levels of this gene family were checked under various abiotic stresses (cold, drought, submergence, laid-down submergence, osmotic, salinity and hormone) and biotic stresses [infection with rice viruses such as RSV (rice stripe virus) and RTSV (rice tungro spherical virus)]. Biotic stresses are novel work and increase the possibilities for finding the best candidate genes. A preliminary search based on our microarray (22K and 44K) data suggested that more than 45 and 26 non-redundant genes in this family were upregulated in response to abiotic and biotic stresses, respectively. All of the genes were further investigated for their stress responsiveness by RT-PCR analysis. Six genes showed preferential expression under both biotic RSV and RTSV stress. Eleven genes were upregulated by at least three abiotic treatments. Our study provides a very useful reference for cloning and functional analysis of members of this gene family in rice.

Single nucleotide polymorphisms in a gene for translation initiation factor (eIF4G) of rice (Oryza sativa) associated with resistance to Rice tungro spherical virus.

Rice tungro disease (RTD) is a serious constraint to rice production in South and Southeast Asia. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Rice cv. Utri Merah is resistant to RTSV. To identify the gene or genes involved in RTSV resistance, the association of genotypic and phenotypic variations for RTSV resistance was examined in backcross populations derived from Utri Merah and rice germplasm with known RTSV resistance. Genetic analysis revealed that resistance to RTSV in Utri Merah was controlled by a single recessive gene (tsv1) mapped within an approximately 200-kb region between 22.05 and 22.25 Mb of chromosome 7. A gene for putative translation initiation factor 4G (eIF4G(tsv1)) was found in the tsv1 region. Comparison of eIF4G(tsv1) gene sequences among susceptible and resistant plants suggested the association of RTSV resistance with one of the single nucleotide polymorphism (SNP) sites found in exon 9 of the gene. Examination of the SNP site in the eIF4G(tsv1) gene among various rice plants resistant and susceptible to RTSV corroborated the association of SNP or deletions in codons for Val(1060-1061) of the predicted eIF4G(tsv1) with RTSV resistance in rice.

Selective modification of rice (Oryza sativa) gene expression by rice stripe virus infection.

Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in east Asia. Rice plants infected with RSV usually show symptoms such as chlorosis, weakness, necrosis in newly emerged leaves and stunting. To reveal rice cellular systems influenced by RSV infection, temporal changes in the transcriptome of RSV-infected plants were monitored by a customized rice oligoarray system. The transcriptome changes in RSV-infected plants indicated that protein-synthesis machineries and energy production in the mitochondrion were activated by RSV infection, whereas energy production in the chloroplast and synthesis of cell-structure components were suppressed. The transcription of genes related to host-defence systems under hormone signals and those for gene silencing were not activated at the early infection phase. Together with concurrent observation of virus concentration and symptom development, such transcriptome changes in RSV-infected plants suggest that different sets of various host genes are regulated depending on the development of disease symptoms and the accumulation of RSV.

Suppression of two tungro viruses in rice by separable traits originating from cultivar Utri Merah.

Rice tungro disease (RTD) is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV) transmitted by green leafhoppers. Rice cv. Utri Merah is highly resistant to RTD. To define the RTD resistance of Utri Merah, near-isogenic lines (NIL, BC(5) or BC(6)) developed from Utri Merah and susceptible cv. Taichung Native 1 (TN1) were evaluated for reactions to RTSV and RTBV. TW16 is an NIL (BC(5)) resistant to RTD. RTBV was able to infect both TN1 and TW16 but the levels of RTBV were usually significantly lower in TW16 than in TN1. Infection of RTSV was confirmed in TN1 by a serological test but not in TW16. However, the global gene-expression pattern in an RTSV-resistant NIL (BC(6)), TW16-69, inoculated with RTSV indicated that RTSV can also infect the resistant NIL. Infection of RTSV in TW16 was later confirmed by reverse-transcription polymerase chain reaction but the level of RTSV was considerably lower in TW16 than in TN1. Examination for virus accumulation in another NIL (BC(6)), TW16-1029, indicated that all plants of TW16-1029 were resistant to RTSV, whereas the resistance to RTBV and symptom severity were segregating among the individual plants of TW16-1029. Collectively, these results suggest that RTD resistance of Utri Merah involves suppression of interacting RTSV and RTBV but the suppression trait for RTSV and for RTBV is inherited separately.

Phase 1 trial of 13-valent pneumococcal conjugate vaccine in Japanese adults.

BACKGROUND: A 7-valent pneumococcal conjugate vaccine (7vPnC) has markedly reduced invasive pneumococcal disease (IPD) in routine use in the USA and is in clinical development in Japan. But a 13-valent pneumococcal conjugate vaccine (13vPnC) would cover even more serotypes. Because vaccines are administered to children by s.c. injection in Japan but by i.m. injection in the USA, a phase I study of s.c. injected 13vPnC in healthy Japanese adults was appropriate before commencing trials in Japanese children and older adults. METHODS: This was a randomized comparison in healthy Japanese adults of s.c. administered 13-valent pneumococcal conjugate vaccine and s.c. administered 23-valent plain polysaccharide pneumococcal vaccine (23vPn). Local and systemic reactions were recorded in a daily diary for 14 days after injection. IgG antibodies to serotype-specific capsular polysaccharide were measured on enzyme-linked immunosorbent assay on samples taken before and approximately 1 month after immunization. RESULTS: A total of 15 subjects were evaluable for safety review in each treatment group. There was a trend towards more local reactions in the 13vPnC group, which may be associated with s.c. administration of aluminum-containing vaccines as used routinely in Japan; but the local reactogenicity was mostly mild or moderate. Both 13vPnC and 23vPn were immunogenic for all types, with the exception of 6A, which is not included in 23vPn and for which only 13vPnC was immunogenic. CONCLUSIONS: Overall, immunogenicity and tolerance was adequate to lead to studies of 13vPnC in both infants and older adults in Japan, using the s.c. route if appropriate.