Transgenic strategies to confer resistance against viruses in rice plants

Abstract

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.

Keywords: RNA interference; Reoviridae; Tenuivirus; forage rice cultivar; transgenic rice.

FIGURE 1

Genome structure of Rice dwarf virus (RDV). Map of segments 1 through 12 of the RDV genome. Lines represent the viral genomic double-stranded RNA segments; boxes denote genes encoded by RDV genes. Arrows indicate 500-bp RNA interference (RNAi) trigger sequences that were amplified from the 5′-proximal regions of each RDV gene and cloned into the RNAi trigger plasmids used to transform the plants. RdRp, RNA-dependent RNA polymerase; Cap, capping enzyme; MP, movement protein; VSR, silencing suppressor.

FIGURE 2

Transgenic rice plants enhanced resistance against three rice-infecting reoviruses. (A) Phenotype of transgenic rice plants (cv. Nipponbare) that harbor the RNAi trigger sequence targeting the Rice dwarf virus (RDV) gene for Pns12 (from Shimizu et al., 2009). (B) Phenotype of transgenic rice plants that harbor the RNAi trigger sequence targeting the Rice gall dwarf virus (RGDV) gene for Pns9 (from Shimizu et al., 2012). (C) Phenotype of transgenic rice plants that harbor the RNAi trigger sequence targeting the Rice black streaked dwarf virus (RBSDV) gene for P9-1 (from Shimizu et al., 2011a). Ten-day-old transgenic rice seedlings were exposed to approximately 10–15 viruliferous vector insects per plant for 1 day and evaluate plant response to virus infection at 4 months after virus inoculation. Rice plants in pots from left to right: mock-inoculated non-transgenic rice plants (Nt) exposed to virus-free insect vectors, showing normal growth; virus-inoculated transgenic rice plants (T), showing healthy growth and fertility after inoculation; virus-inoculated non-transgenic rice plant (Nt), showing typical symptoms caused by RDV, RGDV or RBSDV infection. Bar, 30 cm.

FIGURE 3

Genome structure of Rice stripe virus (RSV). Map of RNAs 1 through 4 of the RSV genome. Upper and lower lines represent the virion-sense RNA segments and virion complementary-sense RNA segments, respectively, and boxes denote genes encoded by RSV genes. Arrows indicate 500-bp RNA interference (RNAi) trigger sequences that were amplified from the 5′-proximal regions of each RSV gene and cloned into the RNAi trigger plasmid for plant transformation. MP, movement protein; NCP, major non-capsid protein; NP, nucleocapsid protein; RdRp, RNA-dependent RNA polymerase; VSR, silencing suppressor.

FIGURE 4

Transgenic rice plants with enhanced resistance against two tenuiviruses. (A) Phenotype of transgenic rice plants (cv. Nipponbare) that harbored the RNAi trigger sequence targeting the Rice stripe virus (RSV) gene for pC3 (from Shimizu et al., 2011b). (B) Phenotype of transgenic rice plants that harbored the RNAi trigger sequence targeting the Rice grassy stunt virus (RGSV) gene for pC5 at 4 months after RGSV inoculation (from Shimizu et al., 2013). Ten-day-old transgenic rice seedlings were exposed to approximately 10–15 viruliferous vector insects per plant for 1 day and evaluate plant response to virus infection. Rice plants in pots from left to right: mock-inoculated non-transgenic rice plants (Nt) exposed to virus-free insect vectors, showing normal growth; virus-inoculated transgenic rice plants (T), showing healthy growth and fertility after inoculation; virus-inoculated non-transgenic rice plant (Nt), showing typical symptoms caused by RSV or RGSV infection. Bar, 30 cm.

FIGURE 5

Transgenic forage rice cultivars inoculated with Rice stripe virus (RSV) and Rice dwarf virus (RDV) (from Sasaya et al., 2013). Phenotypes of transgenic forage rice cultivars Tachiaoba (A) and Tachisugata (B) that harbored the RNAi trigger sequence targeting the RSV gene for pC3 and the RDV gene for Pns12 at 5 months after RDV and RSV inoculation. Ten-day-old transgenic forage rice seedlings were exposed to approximately 15 RSV-carrying viruliferous small brown hoppers and 10 RDV-carrying leaf hoppers per plant for each 1 day, and evaluate plant response to infection with RSV and RDV at 4 months after virus inoculation. The forage rice cultivars in pots from left to right are: mock-inoculated non-transgenic forage rice cultivars (Nt) exposed to virus-free insect vectors, showing normal growth; RDV-inoculated non-transgenic forage rice cultivars (Nt), showing typical symptoms caused by RDV infection; RSV-inoculated non-transgenic forage rice cultivars (Nt), showing typical symptoms caused by RSV infection; RDV+RSV-inoculated transgenic forage rice cultivars, showing healthy growth and fertility. Bar, 30 cm.