Genetic engineering of rice for tungro resistance

O. Azzam, A. Klöti, F. Sta. Cruz, J. Fütterer, E.L. Coloquio, I. Potrykus, and R. Hull

Abstract

Genes encoding sense and antisense viral coat proteins, polymerases, and proteases have been successfully used to engineer resistance to several plant viruses. In this study, viral genes of rice tungro bacilliform virus and the coat protein 3 of rice tungro spherical virus were used to engineer resistance in rice against tungro infection. Rice varieties such as IR64, TN1, Taipei 309, and Kinuhikari were successfully transformed and fertile transgenic plants were evaluated at T1 and T2 generations for their ability to confer protection against tungro infection using insect inoculation assays. Unfortunately, none of the 71 transgenic lines tested provided protection against tungro infection. Possible factors for the lack of protection are discussed.

Introduction

Genetic engineering approaches expand the gene pool from which new and novel virus resistance genes can be selected. For complex diseases of rice, such as tungro, these approaches offer two advantages: (1) the ability to transfer single genes without any linkage to undesirable traits, and (2) the ability to introduce novel genes that have not been explored before in nature and that have potential to increase the durability of resistance. Rice tungro disease incidence is unpredictable, but when it occurs, it can cause catastrophic yield losses in farmers’ communities in the irrigated rice ecosystem. For the last 15 years, several institutions have invested substantial research efforts in studying the molecular biology of the two viruses that cause tungro, rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV), and to genetically engineer tungro resistance in rice. In this study, we report on the resistance tests done using insect inoculation assays to evaluate several of these antiviral strategies designed against both RTBV and RTSV. The transgenic rice plants were produced at the Institute of Plant Sciences, ETH, Zurich, Switzerland and John Innes Centre (JIC), Norwich. England, and the evaluation was done at the transgenic CL4 greenhouse facility at IRRI.Materials and methods

Tables 1 and 2 describe the first 19 transgenic lines from ETH and 20 lines from JIC, respectively. Some additional 32 lines from ETH carrying the antisense RNA constructs of RTBV ORF4 were also evaluated. Seeds from each transgenic line, positive controls (inoculated non-transgenic plants) and negative controls (uninoculated transgenic plants) were sown in sterile soil and seedlings were grown in the CL4 facility at IRRI. At 7–11 days after sowing (DAS), seedlings were inoculated with both tungro viruses by insect feeding using viruliferous green leafhoppers (3–5 insects/ seedling). Inoculated plants were then monitored for symptom expression and assayed for the presence or absence of virus particles at 20 and 40 days post-inoculation (DPI). For the evaluation of the first 19 transgenic lines from ETH, two sources of virus inocula were used. For later experiments, only the greenhouse virus inoculum was used.

Results

None of the initial 19 transgenic lines, which used either the greenhouse virus source or a locally collected virus source from Famy, 40 km northeast of Los Baños, showed resistance to either RTBV or RTSV (Tables 3 and 4). In addition, most of the inoculated plants showed severe symptoms such as stunted growth and leaf discoloration at 20 DPI, and their viral coat protein titers, as measured by the enzyme-linked immunosorbent assay (ELISA), were comparable with those titers from the non-transgenic control plants. Titers varied among individual plants from different lines. Some plants had high RTSV and RTBV titers while others, surprisingly, had low RTSV but high RTBV titers. Based on the ELISA results and visual scores, none of the test lines recovered at 40 DPI. The average symptom severity (SS) was about 7 per line, indicating that most individual plants within a line exhibited stunted growth and leaf discoloration. The 32 remaining lines from ETH were tested using only the greenhouse virus population and results were similar to those obtained earlier. None of the lines showed resistance to RTBV at 20 DPI and plants did not recover after 40 DPI (data not presented).

Furthermore, none of the lines from JIC showed any promising protection against either RTBV or RTSV. Based on ELISA and visual scores, the plants accumulated RTBV at a level similar to

that of the non-transgenic control plants. Generation T1 and T2 plants of the IRI and IRK lines did not show any protection against virus infection (Table 5).

Discussion

Four rice varieties, IR64, TNl, Taipei 309, and Kinuhikari, were successfully transformed with RTBV and RTSV gene constructs and fertile transgenic plants were generated. The coat protein, polymerase, protease, RNase H, and antisense RNA resistance strategies were used to confer protection against RTBV infection using the cauliflower 35S and RTBV promoters. The coat protein strategy was tried against RTSV infection using the 35S and ubiquitin promoters. Most of these strategies have been successful in other virus systems and they were expected to

be effective with DNA and RNA viruses. Unfortunately, the resistance tests showed that none of these strategies was effective against tungro infection. In the transformation experiments with the coat protein, polymerase, and protease strategies, most constructs were expected to express viral proteins when integrated in rice. Most transgenic plants, however, expressed the transgenes only at a very low level. In fact, transgene expression was either stopped or only expressed in a subset of cells. Such an irregular expression level could be responsible for the lack of protection. Novel transgene-expression strategies were thus designed and newly generated transgenic plants will be evaluated in the near future.

Another possible factor that could be responsible for the lack of protection is the quasi-species behavior of tungro viruses (Villegas et al 1996, Cabauatan et al 1999). The continuous supply of mutant and recombinant genomes during virus replication may permit great virus adaptability in overcoming selection pressures imposed on its replication or movement within a very short time. Our work on the genetic variation of RTBV and RTSV field populations (see Arboleda et a1 and Umadhay et al, this volume; Azzam et al 1999) suggests that effective protection mechanisms must be directed against highly conserved functions or sequences, which can be defined only by analyses of large numbers of field isolates, and that other sequence-specific protection mechanisms (like antisense RNA or silencing) are unlikely to be successful.

References

Azzam O, Yambao Ma. LM, Muhsin M, McNally K, Umadhay K. 2000. Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia. Archives of Virology 145 (6): 1183-1197.

Cabauatan PQ, Melcher U, Ishikawa K, Omura T, Hibino H. Koganezawa H, Azzam O. 1999. Sequence changes in six variants of rice tungro bacilliform virus and their phylogenetic relationships. Journal of General Virology 80(8):2229–2237.

Villegas LC, Druka A, Bajet NB, Hull R. 1996. Genetic variation of rice tungro bacilliform virus in the Philippines. Virus Genes 15: l–7.

Notes

Authors’ addresses: O. Azzam. F. Sta. Cruz, E.L. Coloquio. International Rice Research Institute, MCPO Box 3127, Makati City 127l, Philippines: A. Kliiti, J. Futterer, I. Potrykus, Institute of Plant Sciences, ETH, CH-8092 Zurich. Switzerland; and R. Hull, John Innes Centre, Colney, Norwich NR4 7UH, UK.

Citation: Azzam O, A. Klöti, F. Santa Cruz, J. Fütterer, E.L. Coloquio, I. Potrykus, and R. Hull. 1999. Genetic engineering of rice for tungro resistance. p. 38-44. In: Chancellor TCB, Azzam O, Heong KL (editors). Rice tungro disease management. Proceedings of the International Workshop on Tungro Disease Management, 9–11 November 1998, International Rice Research Institute, Los Baños, Philippines, 166 p.