Eiko Nakazono-Nagaoka

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein.

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants.

Cross-Protection Against Bean yellow mosaic virus (BYMV) and Clover yellow vein virus by Attenuated BYMV Isolate M11

Attenuated isolate M11 of Bean yellow mosaic virus (BYMV), obtained after exposing BYMV-infected plants to low temperature, and its efficacy in cross-protecting against infection by BYMV isolates from gladiolus, broad bean (Vicia faba) and white clover (Trifolium repens) was assessed with western blotting and reverse transcription-polymerase chain reaction. The level of cross-protection varied depending on the challenge virus isolates. Cross-protection was complete against BYMV isolates from gladiolus, but incomplete against BYMV isolates from other hosts. M11 also partially cross-protected against an isolate of Clover yellow vein virus. A comparison of the nucleotide sequence of M11 and those of BYMV isolates from gladiolus and from other hosts showed higher homology among gladiolus isolates than the homology between gladiolus isolates and nongladiolus isolates. In the phylogenetic trees, constructed using the nucleotide sequences of an overall polyprotein of the genomes, five gladiolus isolates clustered together, completely separated from the three BYMV isolates from other hosts. A comparison of the amino acid sequences between M11 and its parental isolate IbG, and analysis of recombinant infectious clones between M11 and IbG revealed that an amino acid at position 314 was involved in the attenuation of BYMV.

Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

Transgenic strategies to confer resistance against viruses in rice plants

Rice (Oryza sativa L.) is cultivated in more than 100 countries and supports nearly half of the world’s population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. Many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA interference (RNAi), also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral “Achilles’ heel” gene to target for RNAi attack when engineering plants.

Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants.

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.

Evaluation of cross-protection with an attenuated isolate of Bean yellow mosaic virus by differential detection of virus isolates using RT-PCR

Cross-protection by an attenuated severe isolate of Bean yellow mosaic virus (BYMV), a member of genus Potyvirus, was evaluated. The attenuated isolate, M11, produced no or extremely mild symptoms on broad bean and Nicotiana benthamiana. To differentiate between M11 and severe isolates, we prepared specific primers for analysis by the reverse transcription-polymerase chain reaction (RT-PCR) based on a difference in the nucleotide sequence at the 1676th position in the HC-Pro (helper component proteinase) gene. M11 had a strong ability to cross-protect plants from infection by a severe isolate as shown by RT-PCR. Plants that were first inoculated with a severe isolate and then challenge-inoculated with M11 were dually infected by these viruses, and the symptoms on upper leaves were significantly milder than those on plants infected only with the severe isolate.

RT-PCR-RFLP analysis for evaluating cross protection by an attenuated isolate of Cucumber mosaic virus

CM95 is a good attenuated isolate of Cucumber mosaic virus (CMV) for practical use in cross protection. To assess the protective ability of CM95 in the field, reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) was developed to differentiate CM95 from severe CMV isolates in a short time. In cross-protection tests, protection by CM95 as evaluated by RT-PCR-RFLP on challenge-inoculated leaves 14 days after inoculation agreed with the results obtained in a concomitant experiment to assess symptom appearance on upper leaves 1 month after inoculation. It demonstrated that the RT-PCR-RFLP technique is a reliable method for evaluating the cross-protective ability of CM95 in a short time, in contrast to the month needed for symptoms to appear on upper leaves during the standard evaluation.

A single amino acid substitution in the 126-kDa protein of pepper mild mottle virus controls replication and systemic movement into upper non-inoculated leaves of bell pepper plants

Previously, we generated attenuated variants of pepper mild mottle virus by replacing residue 649 in the 126-kDa replicase protein with various amino acids. Here, we examined the biological properties of the 16 variants that caused either mild mosaic or no mosaic. All but one (A649N) of the mild-mosaic-inducing strains replicated at higher levels in pepper plants and systemically moved at higher rates into the upper non-inoculated leaves than the no-mosaic strains. C1421, previously selected for practical use, not only caused mild symptoms but also had an especially high replication rate in pepper plants and spread more efficiently into the upper non-inoculated leaves.

Nucleotide sequences of Japanese isolates of citrus vein enation virus.

The genomic sequences of five Japanese isolates of citrus vein enation virus (CVEV) isolates that induce vein enation were determined and compared with that of the Spanish isolate VE-1. The nucleotide sequences of all Japanese isolates were 5,983 nt in length. The genomic RNA of Japanese isolates had five potential open reading frames (ORF 0, ORF 1, ORF 2, ORF 3, and ORF 5) in the positive-sense strand. The nucleotide sequence identity among the Japanese isolates and Spanish isolate VE-1 ranged from 98.0% to 99.8%. Comparison of the partial amino acid sequences of ten Japanese isolates and three Spanish isolates suggested that four amino acid residues, at positions of 83, 104, and 113 in ORF 2 and position 41 in ORF 5, might be unique to some Japanese isolates.

Ampeloviruses associated with incomplete flower syndrome and leaf-edge necrosis in Japanese apricot

Japanese apricot (Prunus mume Sieb. et Zucc.) trees produce popular fruits and are also used in Japan as ornamental flowering trees. Since the 1980s, graft-transmissible symptoms (e.g., incomplete flowers and leaf-edge necrosis) have been observed on ‘Nanko’ trees in Wakayama Prefecture. We here describe the detection of viruses associated with these symptoms in Japanese apricot trees using DECS analysis (i.e., double-stranded RNA [dsRNA] isolation, exhaustive amplification, cloning, and sequencing). We isolated dsRNA from symptomatic flowers, including dsRNA of plum bark necrosis stem pitting-associated virus (PBNSPaV), little cherry virus 2 (LChV-2), and a Luteovirus-like sequence. The reverse transcription polymerase chain reaction results suggested that PBNSPaV and LChV-2 may induce the development of incomplete flowers and leaf-edge necrosis. In contrast, the Luteovirus-like sequence is apparently unrelated to these symptoms. Additional investigations are necessary to clarify the relationship between the symptoms and viral infections.