1.12.19
PATHOGEN-DERIVED RESISTANCE TO RICE YELLOW MOTTLE VIRUS [RYMV]
YM PINTO, R KOK and DC BAULCOMBE

Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UH, UK

Background and objectives
RYMV causes a serious disease affecting African rice production. The virus is a member of the Sobemovirus group. The symptoms caused by RYMV include yellowing, mottling and stunting of infected plants leading to incomplete emergence of the panicle with sterile or unfilled grains [1]. Where infection occurs early, the plant normally dies. RYMV is endemic to Africa. It predominantly affects large-scale irrigated lowland rice of Asian exotic parentage. Estimated yield reductions in susceptible varieties are up to 97% [2]. Pathogen-derived resistance has been achieved by the expression of pathogen genes in plants. In potato virus X, protection is offered at the level of the RNA and does not require production of functional viral proteins [3]. However, in this type of resistance there is a requirement for a high degree of homology between the transgenic DNA and the inoculated virus. The objective of this work was to transform cultivated African rice varieties with highly conserved regions of the RYMV genome to provide protection against RYMV infection. Two sequenced RYMV isolates allowed highly conserved regions of the genome to be identified (93.4%). The region chosen for transgene construction contained putative motifs for the RNA-dependent RNA polymerase gene. To ensure that these transgenes would be effective against field isolates of RYMV, a molecular evaluation of strain variation was undertaken to determine the sequence heterogeneity in isolates of RYMV from hotspot locations in Africa.

Results and conclusions
Twenty-four transformed rice lines (ITA 212, Bouake 189, BG90-2) were produced with a transformation efficiency of 3% of bombarded explants. Most lines exhibited GUS activity and lines that were GUS negative were shown to contain the transgene by PCR. Southern blot analysis on the primary transformed lines showed that 17 were independently transformed and contained RYMV transgenes. However, estimates showed complex copy numbers in some lines with rearrangements of the DNA. 33% of primary transformants were sterile. Resistance testing of five Tl Bouake 189 lines with dilute (160 ng) and high-dose (100 g) virion inocula revealed variable levels of resistance against RYMV, ranging from complete susceptibility to extreme resistance. In two of the T2 populations tested there was resistance against RYMV in nearly all of the lines.

Five of 27 isolates of RYMV from different African locations have been characterized by RT-PCR, restriction digestion and sequencing. Preliminary results in resistance-testing these isolates on transgenic plants has shown that there was some resistance against all the isolates, which was manifest either as a delay or suppression of virus accumulation and symptom formation. Preliminary results also indicate that the resistance mechanism involves an RNA-based mechanism in which functional viral proteins will not be produced. These results are being confirmed and additional transgenic lines and RYMV isolates will be characterized and tested. It is anticipated that if lines are resistant to all the RYMV isolates tested they will be released for field testing in Africa.

References
1. Bakker W, 1970. Netherlands Journal of Plant Pathology 76, 53-63.
2. Fomba SN, 1988. Plant Disease 72, 641-642.
3. Mueller E, Gilbert J, Brigneti G et al., 1995. Plant Journal 7, 1001-1013.