1.12.3
VIRAL AND HOST FACTORS INFLUENCING CUCUMBER MOSAIC CUCUMOVIRUS-COWPEA INTERACTION

A KARASAWA1, Y CHIDA1, K AKASHI1, Y NASU1,2, S HASE1 and Y EHARA1

1Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai 981, Japan; 2Iwate Biotechnology Research Center, Narita 22-174-4, Kitakami 024, Japan

Background and objectives
Cucumber mosaic cucumovirus (CMV) is one of the most destructive plant viruses for crop production in the world. To control CMV-induced diseases, it is necessary to understand the resistance mechanism of plants against CMV. Cowpea (Vigna unguiculata) has long been used as the indicator plant for CMV identification. Upon infection with CMV, cowpea usually responds hypersensitively, and necrotic local lesions occur by 24 h post- inoculation. In this study we investigated the viral and plant factors influencing symptoms on cowpeas.

Materials and methods
CMV strains Y (CMV-Y) and L (CMV-L) were used in this study. CMV-Y elicits the hypersensitive response (HR) on cowpea cultivar Kurodane-Sanjaku (KS), whereas CMV-L does not, but infects systemically. To assign HR determinants on CMV genomic RNAs, we used full-length cDNAs against each RNA segment. Although KS has the Cry resistance locus, cultivar PI 189375 (PI) does not have it, allowing systemic infection [1]. To identify molecular markers linked to the Cry locus, we adopted RAPD analysis and R gene sequence-based PCR.

Results and conclusions
Viral determinants: based on the re-assortant tests, we found that inoculations using infectious Y2 (=CMV-Y RNA 2) transcripts resulted in the HR irrespectively of RNA 1 or 3 species, whereas inocula containing L2 (=CMV-L RNA 2) caused systemic infections [2]. The HR-inducing domain on Y2 was determined near the RdRp core sequence by constructing hybrid molecules between Y2 and L2 cDNAs. Sequence analysis and site-directed mutagenesis of Y2 and L2 cDNAs, resulting in mutated 2a protein expression, showed that Phe631 of the CMV-Y 2a protein was responsible for the HR elicitation. If RNA 2 showed avr(-) activity, RNA 3 could modify the symptoms on KS. Systemic mosaic symptoms were observed on KS when L2L3-containing inocula were used, whereas necrotic symptoms were induced by L2Y3-inocula. Necrosis-inducing activity was located on the 3' half of the coat protein (CP) gene. We could map this activity on the amino acid Ser129 of the CP by site-directed mutagenesis.
Cowpea determinants: to isolate the Cry gene by map-based cloning, several RAPD and RFLP markers were mapped near the Cry locus. RAPD marker WA3-850 was located 5.8 cM from the Cry locus. RFLP clone CRGA5, which was obtained by R gene sequence-based PCR for the nucleotide-binding site, was also mapped near the Cry locus. Several other markers will be mapped in the near future.

References
1. Nasu Y et al., 1996. Phytopathology 86, 946-951.
2. Karasawa A et al., 1997. Annals of the Phytopathological Society of Japan 63, 289-297.