1.11.3S
THE MOLECULAR BASIS OF MAIZE STREAK VIRUS PATHOGENICITY

MI BOULTON, T HECKEL, H LIU, AP LUCY, AJ MAULE and JW DAVIES

John lnnes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Background and objectives
The geminivirus, maize streak virus (MSV), has a 2.7 kb monopartite genome of single-stranded (ss) DNA. Previous work [1] has shown that a sequence variant of the Nigerian strain of MSV (MSV-Nm) exhibits altered pathogenicity compared with MSV-NS. Only two nucleotide changes were responsible for changing the wide host range, severe symptom phenotype of MSV-Ns to a limited host range, narrow streak ('mild') symptom phenotype as seen in MSV-NM. One nucleotide (nt) substitution (nt 40) was located in the movement protein gene (MP), and although this was translationally silent it affected streak width. The other substitution (nt 2473) was present in the large intergenic region (LIR) and disrupted a potential promoter sequence (TATA) present in MSV-NS. The TATA was present 101 nt upstream of the translation initiation codon (AUG) of the replication-associated gene RepA (Cl), although two other TATA sequences were present closer to the AUG. Host range, severity of chlorosis and timing of symptom appearance were affected by nt 2473. The presentation will describe the studies undertaken to determine the basis of the differing pathogenicity characteristics, with particular reference to the functions of the virion-(V-) sense gene products required for virus movement, the coat protein (CP) and the MP.

Results and conclusions
Primer extension and RNase protection analyses were used to provide a high resolution map of transcripts produced from the V- and complementary (C-) sense of the virus genome. These showed that the major C-sense transcript, necessary for producing the replication-associated proteins Rep and RepA, was produced from TATA-101 (present in MSV-NS, but not MSV-Nm). Comparative analysis of RNA from MSV-NM and MSV-Ns-infected plants confirmed low levels of C-sense transcription for MSVNm. Furthermore, transient assays using P-glucuronidase (gus)-RepA gene replacement constructs showed that the enzyme activity produced from the -Ns LIR promoter was stronger than that from -Nm. These data strongly suggest that the limited host range, delayed time of symptom appearance and short streaks of MSV-NM were a result of limited amounts of the replication-associated products.

Comparison of the results obtained from primer extension and RNase protection analyses of the V-sense transcripts identified a novel RNA processing event, and RT-PCR confirmed the presence of an intron within the MP gene sequence [2]. When the V-sense transcripts present in MSV-NS- and MSVNm-infected tissue were compared using RT-PCR, it was observed that in -Nm-infected plants a significantly greater proportion of the transcripts was spliced, and therefore unable to produce MP. In situ hybridisation has shown that, in mature leaves, virus is located only in those areas of the leaf displaying chlorotic symptoms [3] and that a narrow streak phenotype reflects limited viral invasion of the leaf. We have shown that mutation of the MP gene results either in loss of systemic infection or in a narrow streak phenotype (and limited tissue invasion), suggesting a link between streak width and production of functional MP. Studies in our laboratory also indicate an interaction between MP and CP which may be important for regulating the intra- and inter-cellular movement of MSV. Transient expression studies suggest that the expression of CP is enhanced by the splicing event, and therefore the effect of the nt 40 substitution may be to increase CP expression in MSV-Nm-infected tissues. The regulation of MSV movement will be discussed and related to symptom phenotype.

References
1. Boulton MI, King DI, Donson J, Davies JW, 1991. Virology 183, 114-121.
2. Wright EA, Heckel T, Groenendijk J, Davies JW, Boulton MI, 1997. Plant Journal 12, 1285-1297.
3. Lucy AP, Boulton MI, Davies JW, Maule AJ, 1996. Molecular Plant-Microbe Interactions 9, 22-31.