Phylogenetic analysis of endogenous viral elements in the rice genome reveals local chromosomal evolution in Oryza AA-genome species.

Introduction: Rice genomes contain endogenous viral elements homologous to rice tungro bacilliform virus (RTBV) from the pararetrovirus family Caulimoviridae. These viral elements, known as endogenous RTBV-like sequences (eRTBVLs), comprise five subfamilies, eRTBVL-A, -B, -C, -D, and -X. Four subfamilies (A, B, C, and X) are present to a limited degree in the genomes of the Asian cultivated rice Oryza sativa (spp. japonica and indica) and the closely related wild species Oryza rufipogon. Methods: The eRTBVL-D sequences are widely distributed within these and other Oryza AA-genome species. Fifteen eRTBVL-D segments identified in the japonica (Nipponbare) genome occur mostly at orthologous chromosomal positions in other AA-genome species. The eRTBVL-D sequences were inserted into the genomes just before speciation of the AA-genome species. Results and discussion: Ten eRTBVL-D segments are located at six loci, which were used for our evolutionary analyses during the speciation of the AA-genome species. The degree of genetic differentiation varied among the eRTBVL-D segments. Of the six loci, three showed phylogenetic trees consistent with the standard speciation pattern (SSP) of the AA-genome species (Type A), and the other three represented phylogenies different from the SSP (Type B). The atypical phylogenetic trees for the Type B loci revealed chromosome region-specific evolution among the AA-genome species that is associated with phylogenetic incongruences: complex genome rearrangements between eRTBVL-D segments, an introgression between the distant species, and low genetic diversity of a shared eRTBVL-D segment. Using eRTBVL-D as an indicator, this study revealed the phylogenetic incongruence of local chromosomal regions with different topologies that developed during speciation.

Available cloned genes and markers for genetic improvement of biotic stress resistance in rice.

Biotic stress is one of the major threats to stable rice production. Climate change affects the shifting of pest outbreaks in time and space. Genetic improvement of biotic stress resistance in rice is a cost-effective and environment-friendly way to control diseases and pests compared to other methods such as chemical spraying. Fast deployment of the available and suitable genes/alleles in local elite varieties through marker-assisted selection (MAS) is crucial for stable high-yield rice production. In this review, we focused on consolidating all the available cloned genes/alleles conferring resistance against rice pathogens (virus, bacteria, and fungus) and insect pests, the corresponding donor materials, and the DNA markers linked to the identified genes. To date, 48 genes (independent loci) have been cloned for only major biotic stresses: seven genes for brown planthopper (BPH), 23 for blast, 13 for bacterial blight, and five for viruses. Physical locations of the 48 genes were graphically mapped on the 12 rice chromosomes so that breeders can easily find the locations of the target genes and distances among all the biotic stress resistance genes and any other target trait genes. For efficient use of the cloned genes, we collected all the publically available DNA markers (~500 markers) linked to the identified genes. In case of no available cloned genes yet for the other biotic stresses, we provided brief information such as donor germplasm, quantitative trait loci (QTLs), and the related papers. All the information described in this review can contribute to the fast genetic improvement of biotic stress resistance in rice for stable high-yield rice production.

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia.

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen's biological impact on rice tissue.

Reemerging Rice Orange Leaf Phytoplasma with Varying Symptoms Expressions and Its Transmission by a New Leafhopper Vector-Nephotettix virescens Distant.

Rice orange leaf phytoplasma (ROLP) belongs to the "Candidatus Phytoplasma asteris" 16SrI-B subgroup, which is solely transmitted by the zigzag-striped leafhopper (Recilia dorsalis Motchulsky) and the green leafhopper (Nephotettix cincticeps Uhler) (Hemiptera: Cicadellidae). Recently, rice plants showing orange leaf discoloration have become ubiquitous in several paddies of two provinces in the Philippines. In total of 98 symptomatic rice plants, 82% (Laguna) and 95% (Mindanao) were ROLP-positive by nested PCR detection. These plants showed more varying symptoms than previously reported. The vector insect R. dorsalis was scarcely present but green paddy leafhopper, N. virescens Distant (Hemiptera: Cicadellidae), was commonly observed in the paddies, thus the ability of N. virescens to transmit ROLP was thoroughly investigated. Newly emerged adult N. virescens, which fed on ROLD-source rice plants, were used to inoculate a susceptible rice seedling and was serially transferred into a new healthy seedling. Resultant positive transmission rates varied from 5.1% to 17.8%. The transmission ability of the insects was generally decreased over time. These findings suggest that N. virescens is an alternative vector of ROLP in the Philippines. Altogether, this study highlighted the increasing importance of ROLD-reemergence in Southeast and East Asia and proved the need for careful management of this alternative vector insect.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Infection with an asymptomatic virus in rice results in a delayed drought response.

Infection of viruses in plants often modifies plant responses to biotic and abiotic stresses. In the present study we examined the effects of Rice tungro spherical virus (RTSV) infection on drought response in rice. RTSV infection delayed the onset of leaf rolling by 1-2 days. During the delay in drought response, plants infected with RTSV showed higher stomatal conductance and less negative leaf water potential under drought than those of uninfected plants, indicating that RTSV-infected leaves were more hydrated. Other growth and physiological traits of plants under drought were not altered by infection with RTSV. An expression analysis of genes for drought response-related transcription factors showed that the expression of OsNAC6 and OsDREB2a was less activated by drought in RTSV-infected plants than in uninfected plants, further suggesting improved water status of the plants due to RTSV infection. RTSV accumulated more in plants under drought than in well-watered plants, indicating the increased susceptibility of rice plants to RTSV infection by drought. Collectively, these results indicated that infection with RTSV can transiently mitigate the influence of drought stress on rice plants by increasing leaf hydration, while drought increased the susceptibility of rice plants to RTSV.

Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Bunyavirales: second update 2018.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Ancient Endogenous Pararetroviruses in Oryza Genomes Provide Insights into the Heterogeneity of Viral Gene Macroevolution.

Endogenous viral sequences in eukaryotic genomes, such as those derived from plant pararetroviruses (PRVs), can serve as genomic fossils to study viral macroevolution. Many aspects of viral evolutionary rates are heterogeneous, including substitution rate differences between genes. However, the evolutionary dynamics of this viral gene rate heterogeneity (GRH) have been rarely examined. Characterizing such GRH may help to elucidate viral adaptive evolution. In this study, based on robust phylogenetic analysis, we determined an ancient endogenous PRV group in Oryza genomes in the range of being 2.41-15.00 Myr old. We subsequently used this ancient endogenous PRV group and three younger groups to estimate the GRH of PRVs. Long-term substitution rates for the most conserved gene and a divergent gene were 2.69 × 10-8 to 8.07 × 10-8 and 4.72 × 10-8 to 1.42 × 10-7 substitutions/site/year, respectively. On the basis of a direct comparison, a long-term GRH of 1.83-fold was identified between these two genes, which is unexpectedly low and lower than the short-term GRH (>3.40-fold) of PRVs calculated using published data. The lower long-term GRH of PRVs was due to the slightly faster rate decay of divergent genes than of conserved genes during evolution. To the best of our knowledge, we quantified for the first time the long-term GRH of viral genes using paleovirological analyses, and proposed that the GRH of PRVs might be heterogeneous on time scales (time-dependent GRH). Our findings provide special insights into viral gene macroevolution and should encourage a more detailed examination of the viral GRH.

Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus.

Rice tungro disease (RTD) is a serious constraint in rice production across tropical Asia. RTD is caused by the interaction between Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. RTSV resistance found in traditional cultivars has contributed to a reduction in the incidence of RTD in the field. Natural RTSV resistance is a recessive trait controlled by the translation initiation factor 4 gamma gene (eIF4G). The Y1059 V1060 V1061 residues of eIF4G are known to be associated with the reactions to RTSV. To develop new sources of resistance to RTD, mutations in eIF4G were generated using the CRISPR/Cas9 system in the RTSV-susceptible variety IR64, widely grown across tropical Asia. The mutation rates ranged from 36.0% to 86.6%, depending on the target site, and the mutations were successfully transmitted to the next generations. Among various mutated eIF4G alleles examined, only those resulting in in-frame mutations in SVLFPNLAGKS residues (mainly NL), adjacent to the YVV residues, conferred resistance. Furthermore, our data suggest that eIF4G is essential for normal development, as alleles resulting in truncated eIF4G could not be maintained in homozygous state. The final products with RTSV resistance and enhanced yield under glasshouse conditions were found to no longer contain the Cas9 sequence. Hence, the RTSV-resistant plants with the novel eIF4G alleles represent a valuable material to develop more diverse RTSV-resistant varieties.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Loss-of-Function Alleles of Heading date 1 (Hd1) Are Associated With Adaptation of Temperate Japonica Rice Plants to the Tropical Region.

Adaptation of temperate japonica rice varieties to tropical regions is impeded by extremely early flowering probably due to photoperiod change from long to short. However, constant breeding efforts led to development of temperate japonica varieties adapted to tropical/subtropical regions, but the genetic factor underlying this is still elusive. We analyzed the 45 diverse rice accessions and 12 tropical-adapted temperate japonica lines for the allele types of seven major flowering genes Hd1, OsPPR37, DTH8, Ghd7, Ehd1, RFT1, and Hd3a and flowering time under three different field conditions in temperate and tropical locations. The accessions originated from the tropical/subtropical regions preferred the non-functional alleles of Hd1 and not other flowering genes. The genetic effect analysis of each gene showed that only the functional Hd1 caused early flowering in the tropical location. All 12 temperate japonica breeding lines adapted to the tropics possessed the loss-of-function alleles of Hd1 with no change of other flowering genes compared to common Korean temperate japonica varieties. A phylogenetic analysis using 2,918 SNP data points revealed that the genome status of the 12 breeding lines were very similar to Korean temperate japonica varieties. These results indicate that the functional Hd1 alleles of temperate japonica varieties induced extremely early flowering in the tropics and the non-functional hd1 alleles brought about the adaptation of temperate japonica rice to tropical regions.

Allele mining and enhanced genetic recombination for rice breeding.

Traditional rice varieties harbour a large store of genetic diversity with potential to accelerate rice improvement. For a long time, this diversity maintained in the International Rice Genebank has not been fully used because of a lack of genome information. The publication of the first reference genome of Nipponbare by the International Rice Genome Sequencing Project (IRGSP) marked the beginning of a systematic exploration and use of rice diversity for genetic research and breeding. Since then, the Nipponbare genome has served as the reference for the assembly of many additional genomes. The recently completed 3000 Rice Genomes Project together with the public database (SNP-Seek) provides a new genomic and data resource that enables the identification of useful accessions for breeding. Using disease resistance traits as case studies, we demonstrated the power of allele mining in the 3,000 genomes for extracting accessions from the GeneBank for targeted phenotyping. Although potentially useful landraces can now be identified, their use in breeding is often hindered by unfavourable linkages. Efficient breeding designs are much needed to transfer the useful diversity to breeding. Multi-parent Advanced Generation InterCross (MAGIC) is a breeding design to produce highly recombined populations. The MAGIC approach can be used to generate pre-breeding populations with increased genotypic diversity and reduced linkage drag. Allele mining combined with a multi-parent breeding design can help convert useful diversity into breeding-ready genetic resources.

Rice tungro spherical virus resistance into photoperiod-insensitive japonica rice by marker-assisted selection.

Rice tungro disease (RTD) is one of the destructive and prevalent diseases in the tropical region. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Cultivation of japonica rice (Oryza sativa L. ssp japonica) in tropical Asia has often been restricted because most japonica cultivars are sensitive to short photoperiod, which is characteristic of tropical conditions. Japonica1, a rice variety bred for tropical conditions, is photoperiod-insensitive, has a high yield potential, but is susceptible to RTD and has poor grain quality. To transfer RTD resistance into Japonica1, we made two backcrosses (BC) and 8 three-way crosses (3-WC) among Japonica1 and RTSV-resistant cultivars. Among 8,876 BC1F2 and 3-WCF2 plants, 342 were selected for photoperiod-insensitivity and good grain quality. Photoperiod-insensitive progenies were evaluated for RTSV resistance by a bioassay and marker-assisted selection (MAS), and 22 BC1F7 and 3-WCF7 lines were selected based on the results of an observational yield trial. The results demonstrated that conventional selection for photoperiod-insensitivity and MAS for RTSV resistance can greatly facilitate the development of japonica rice that is suitable for cultivation in tropical Asia.

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.