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We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F2 cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively.

The elicitation of the hypersensitive response (HR) is known to depend on the interaction between a resistance gene of a host plant and a corresponding avirulence gene of a pathogen. The cv. Kurodane-Sanjaku of cowpea (Vigna unguiculata) has the Cry locus that confers resistance against cucumber mosaic virus strain Y (CMV-Y). The resistance is overcome by infection with a legume strain of CMV (CMV-L). RNA 2, which codes for the 2a protein, a subunit of the viral replicase components, has been known to control virulent/avirulent phenotypes. We generated chimeric constructs of full-length cDNA clones of RNA 2 of both strains and inoculated infectious transcripts to delimit the domain controlling symptoms. A 243-base pair fragment containing a coding region for the GDD RNA-dependent RNA polymerase core sequence was shown to be responsible for the phenotypic differences. From sequence alignment analysis, two amino acids (Phe631 and Ala641) of the HR-type 2a protein encoded in this fragment were specifically exchanged to Tyr and Ser, respectively, in the 2a proteins of resistance-breaking strains. Point mutations introduced into RNA 2 backgrounds of both strains that were designed to change the amino acid at position 631 resulted in a change of symptoms, indicating that a single nucleotide change determines the reactions elicited by both strains. Analysis for one additional mutant RNA 2 showed that symptom determination may be correlated with the nature of the lateral chain of amino acid 631.

Transposon Tn4431 was used to introduce a luciferase (lux) operon into Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174. Two transconjugants which were highly bioluminescent and indistinguishable in pathogenicity with the parental strain were selected. The transconjugants produced more bioluminescence in the logarithmic phase in liquid cultures. Bioluminescence was also detected from rice leaves infected with the transconjugants. In the compatible rice cultivar IR24 bacteria multiplied and moved prior to the expression of disease symptoms. In contrast, in the incompatible rice cultivar IR-BB2, which has resistance genes Xa1 and Xa2, bioluminescence was restricted near the inoculation sites, but light produced by the transconjugants was detected even 3 weeks after inoculation, suggesting that bacteria could multiply, although restrictedly, and remain viable. No bacterium was reisolated from the leaf sections beyond the light-producing regions. The detection of bioluminescence makes it possible to examine the actual location or infection process of X. o. pv. oryzae continuously without destroying the infected leaves.

Ralstonia solanacearum OE1-1 was transformed with pNP126 carrying a luxCDABE operon of Vibrio fisheri and a promoter region derived from the genomic DNA of Burkholderia glumae. Intensity of bioluminescence from YN5 thus obtained positively correlated with growth of the bacteria from the lag phase to the stationary phase in vitro. In this study, we used the VIM camera equipped with the ARGUS 50 to successively observe bioluminescence and the development of bacterial wilt in singly and doubly grafted tomato plants in various combinations of the resistant LS-89 and the susceptible Oogata-Fukuju. The degree of bacterial growth in the roots and collars was one of the determinants for the induction of bacterial wilt in tomato plants. Resistance of LS-89 resulted from suppression of bacterial proliferation in the roots and stems below the first leaf. In grafted tomato plants using LS-89 as the rootstock, all rootstocks were latently infected with the bacteria. The bacteria were also recovered from susceptible scions even though the plants did not wilt. In some plants, the bacteria proliferated in the susceptible scions. These plants wilted heavily. Taken together, suppression of bacterial proliferation in the roots and the stems below the first leaf of resistant rootstocks affected the bacterial growth in the susceptible scions and quantative control of resistance against development of the disease.