Optimized RNA-Silencing Strategies for Rice Ragged Stunt Virus Resistance in Rice

Rice ragged stunt virus (RRSV) is one of the most damaging viruses of the rice culture area in south and far-eastern Asia. To date, no genetic resistance has been identified and only expensive and non-environmentally friendly chemical treatments are deployed to fight this important disease. Non-chemical approaches based on RNA-silencing have been developed as resistance strategies against viruses. Here, we optimized classical miRNA and siRNA-based strategies to obtain efficient and durable resistance to RRSV. miRNA-based strategies are involved in producing artificial miRNA (amiR) targeting viral genomes in plants. Classically, only one amiR is produced from a single construct. We demonstrated for the first time that two amiRs targeting conserved regions of RRSV genomes could be transgenically produced in Nicotiana benthamiana and in rice for a single precursor. Transgenic rice plants producing either one or two amiR were produced. Despite efficient amiR accumulations, miRNA-based strategies with single or double amiRs failed to achieve efficient RRSV resistance in transformed rice plants. This suggests that this strategy may not be adapted to RRSV, which could rapidly evolve to counteract them. Another RNA-silencing-based method for viral resistance concerns producing several viral siRNAs targeting a viral fragment. These viral siRNAs are produced from an inverted repeat construct carrying the targeted viral fragment. Here, we optimized the inverted repeat construct using a chimeric fragment carrying conserved sequences of three different RRSV genes instead of one. Of the three selected homozygous transgenic plants, one failed to accumulate the expected siRNA. The two other ones accumulated siRNAs from either one or three fragments. A strong reduction of RRSV symptoms was observed only in transgenic plants expressing siRNAs. We consequently demonstrated, for the first time, an efficient and environmentally friendly resistance to RRSV in rice based on the siRNA-mediated strategy.

Resistance of Rice Varieties and Accessions to Dwarf Viruses

Identifikasi ketahanan plasma nutfah padi terhadap virus kerdil bertujuan untuk mendapatkan informasi mengenai varietas dan aksesi yang tahan terhadap virus kerdil padi, yaitu Rice ragged stunt virus (RRSV) dan Rice grassy stunt virus (RGSV). Penelitian dilakukan di Rumah Kaca Balai Besar Penelitian Tanaman Padi pada MT1/MT2 tahun 2018. Materi genetik yang diuji yaitu 19 varietas padi yang sudah dilepas dan 50 aksesi plasma nutfah padi koleksi Balai Besar Penelitian Tanaman Padi Badan Litbang Pertanian-Kementerian Pertanian. Pengamatan mengikuti metode skoring SES IRRI 2014. Respons tanaman uji terhadap RRSV dapat dikelompokkan menjadi rentan (1 varietas dan 22 aksesi), agak tahan (18 varietas dan 22 aksesi), dan tahan {2 aksesi, yaitu MDK Karawang (800 butir/Malai) (10597), dan Pulo Hitam (10615}. Respons tanaman uji terhadap RGSV dapat dikelompokkan menjadi rentan (16 varietas dan 34 aksesi), agak tahan (3 varietas dan 11 aksesi), dan tahan (1 aksesi, yaitu Ketik 1-1062). Lebih lanjut, aksesi padi yang tahan terhadap RRSV dan/atau RGSV dapat digunakan sebagai tetua dalam perakitan varietas yang tahan terhadap virus kerdil.

Transmission Effectivity of Rice Yellow Stunt Disease by Imidacloprid-Resistant and Susceptible Brown Plant Hopper

The brown plant hopper (BPH) is a major pest of rice and as a vector of Rice ragged stunt virus (RRSV) and Rice grassy stunt virus (RGSV). Curently, numerous rice yellow stunt disease symptoms are found in the field that caused by the single and simultaneous infection of these two viruses. Brown plant hopper population correlate with the incidence and severity of the disease. Misuse of insecticides, would cause of BPH resistances to imidacloprid. This study aimed to investigate the ability of BPH imidacloprid-resistant and susceptible to transmit of rice yellow stunt disease on rice plants. The variables tested were the acquisition period, inoculation period, number of infesting BPH, and lifespans of the viruliferous BPH that used in this research. Experiments were set as separated Completely Randomized Design with 10 replications for each treatment within an experiment. The results showed that both resistant and susceptible BPH to imidacloprid was able to transmit the virus to healthy plants. The acquisition and inoculation period test showed the BPH could transmit the virus with the shortest acquisition time for 30 minutes followed 24 hours of inoculation, as well as the acquisition time of 10 days with the shortest inoculation time for 30 minutes. Based on the incubation time, symptoms variation, and disease severity, susceptible BPH were more effective in transmitting rice yellow stunt disease than imidacloprid-resistant BPH. Single imidacloprid-resistant or susceptible BPH was proven able to transmit rice yellow stunt disease to healthy plants during its lifespan. Lifespans BPH viruliferous of imidacloprid-resistant were shorter than susceptible, which was 16 days for resistant BPH and 21 days for susceptible BPH.

Genetic Variability and Heritability of Green Super Rice (GSR) Lines Resistance to Rice Ragged Stunt Virus (RRSV) and Rice Grassy Stunt Virus (RGSV)

<p>Brown plant hopper (BPH) is one of the main pest of rice in Indonesia and it occurred every years with fluctuate acreage. BPH is also vector of Rice Ragged Stunt Virus (RRSV) and Rice Grassy Stunt Virus (RGSV). Green Super Rice (GSR) was designed to have resistance to major pests and diseases so that it need less pesticides and thus save ro environment. GSR was developed in IRRI and China and was tested in Indonesia since 2009 until now (2019). This research was aimed to study the genetic variability and heritability of 26 selected GSR lines to RRSV and RGSV along with four check varieties. The experiment was conducted in ICRR Sukamandi and Pusakanagara Experimental Station. The experiment was arranged following randomized complete block design with three replications. Transplanting was done into 21 days old seedling into 25 cm x 25 cm planting space of 1 m x 1 m plot. BPH and virus investation was occured narturally due to BPH outbreak along the season (DS 2010). Percentage of plant showing RRSV and RGSV symtom was measured as consideration the resistance of plant to the viruses. The results showed that the tested genotypes had high genetic variability and heritability classified as medium in the resistance to RGSV. The genotypes showed low genetic variability and heritability in the resistance to RRSV. It implies that breeder effort is feasible to develop resistant lines to RGSV. HUANGHUAZHAN and HHZ 12-Y4-Y3-Y1 are consistently resistant to RGSV and can be used as donors in further plant breeding activities. The selection of resistant individuals in populations of plant breeding material recommended to be done on earlier generations for RGSV and further generation for RSSV.</p>

DETECTION AND TRANSMISSION OF RICE STUNT VIRUS ON CIHERANG AND SITU BAGENDIT VARIETIES

Detection and Transmission of rice stunt virus on Ciherang and Situ Bagendit Varieties. The explosion of brown planthoppers recently has caused reduction of rice production in Indonesia. Brown planthoppers do not only act as pest, but also transmit Rice grassy stunt virus (RGSV) and Rice ragged stunt virus (RRSV). Detection of the existence of the two viruses in rice plants and vector insects is important to be done to ensure that the virus is infected with the vector. The aim of this research is to detect the existence of virus in varieties of Ciherang and Situ Bagendit as a result of transmission in the laboratory and to find out the ability of brown planthoppers to transmit stunt virus to both of the varieties. This research was compiled using Completely Randomized Design (CRD) with 4 treatments, namely healthy rice plants of Ciherang and Situ Bagendit varieties, Ciherang and Situ Bagendit varieties which were infested by brown planthoppers each with 5 repetitions. The parameters observed were incubation period, symptoms, plant height, number of leaves and incidence of disease. The data on plant height, number of leaves and incidence of disease were analyzed using ANOVA and continued with the Least Significant Difference (LSD) test at the level of 5%. The results showed that Ciherang and Situ Bagendit varieties were only positively infected by Rice ragged stunt virus. The results of the rice transmission showed that Ciherang variety had a faster incubation period of 10 DAI while Situ Bagendit was 14 DAI, but the two varieties showed an inhibition of growth in plant height and number of leaves compared to healthy plants with each incidence of 51.3% and 46.3%.

Impact of Two Reoviruses and Their Coinfection on the Rice RNAi System and vsiRNA Production

Both Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV) belong to the family Reoviridae, and synergistic infection of these two viruses commonly occurs in the field. This study revealed that both SRBSDV and RRSV affect the RNA interference (RNAi) pathway and form different virus-derived interfering RNA (vsiRNA) profiles in rice. Co-infection of rice by SRBSDV and RRSV up-regulated the expression of rice DICER-like (DCL) proteins but down-regulated the expression of rice RNA-dependent RNA polymerases (RDRs), and the accumulation of vsiRNAs of either RBSDV or RRSV was decreased compared with that in singly infected plants. The majority of SRBSDV vsiRNAs were 21 nt or 22 nt in length, whether plants were singly infected with SRBSDV or co-infected with RRSV. On the other hand, the majority of RRSV vsiRNAs were 20 nt, 21 nt, or 22 nt in length, among which those 20 nt in length accounted for the largest proportion; co-infection with SRBSDV further increased the proportion of 20 nt vsiRNAs and decreased the proportion of 21 nt vsiRNAs. Co-infection had no effects on the strand favoritism and hot spots of the vsiRNAs, but changed the bias of the 5′ terminal nucleotide significantly. This study provides a reference for further study on the pathogenesis and synergistic mechanism of SRBSDV and RRSV.