Studies on interference between newly defined bean-infecting potyviruses.

Bean common mosaic virus (BCMV) and blackeye cowpea mosaic virus (BICMV) belonging to the genus Potyvirus of the plant virus family Potyviridae (Barnett, 1991, 1992) are of great economic importance. A large number of strains of BCMV and BlCMV are found to occur in nature, either in single or in mixed infections (Vetten & Allen, 1991). The latter may result in interactions among the strains and sometimes lead to antagonistic (Bercks, 1959; Quantz, 1961) or synergistic effects.Aim of the present research was to resolve the chaotic and contradictory taxonomic status of different isolates of BCMV (NL1, NY15, NL3) and BICMV (W) and to gain an insight into the mechanism of antagonism observed in Phaseolus vulgaris cv. Bataaf infected with both NY15 and NL3, by using these viruses as models.When this investigation was started, it was impossible to serologically distinguish the different strains of BCMV and BlCMV. As distinction of viruses in mixed infections was a prerequisite to study their behaviour in the plant, a novel serological technique was applied. To this end, antibodies directed towards N- and C-, or N-terminal peptide domains of the coat proteins of these viruses were prepared. The N-terminal targeted antibodies thus obtained, enabled a clear distinction of the strains in mixed infection. The specificity of these antibodies had some implications on the taxonomy of these viruses (Chapter 3). According to N-terminal serology NL1 of BCMV and W of BICMV should not be considered strains of the two different viruses but of the same virus viz. BCMV, whereas NL3 should be a strain of a distinct potyvirus. Although N-terminal serology enabled distinction between NY15, NU and W in mixed infections, it did not give a clue to their exact taxonomic positions. In some cases there may be unexpected serological relationships (Shukla et al. , 1989) whereas in others there is lack of expected serological relationships (Shukla et al. , 1992).The understanding of the coat protein structure in recent years has greatly contributed to potyvirus taxonomy. During the course of this investigation, some information on high performance liquid chromatography (HPLC) digests of the coat proteins of BCMV and BICMV became available. Therefore, an attempt was made to tentatively classify the strains of BCMV and BICMV into a proposed BCMV subgroup using biological, serological and HPLC characteristics as taxonomic parameters.Although subgroups have no official taxonomic status, for practical purposes it is easier to deal with closely related viruses at subgroup level (Chapter 4).Some of the results obtained with N-terminal sexology were contrary to those with HPLC. The former had shown that NY15 and W were different viruses (Chapter 3), whereas according to the latter these were strains of one virus (McKern et al. , 1992). We should realise that N-terminal serology and HPLC are both based on characteristics of the coat proteins. To resolve these conflicting findings, nucleotide sequences of the coat protein genes and 3'-nontranslated regions of the genomes of NL1, NL3, NY15 and W were determined (Chapter 5). The deduced amino acid sequences revealed that the coat proteins of NL1, NY15 and W were identical in size and exhibited a high percentage of sequence similarity (94-97%). Moreover, this high percentage of similarity was also reflected in their 3'- nontranslated regions (93-96% similarity) confirming that NL1, NY15 and W are not strains of two different virus species but of one single species only, viz. BCMV. It has been proposed to designate these strains BCMV-NL1, BCMV-NY15 and BCMV-BIC/W respectively. On the other hand, NL3 had a shorter coat protein and displayed a lower percentage of sequence similarity both in the coat protein (87-89% similarity) and 3'-nontranslated region (56-63% similarity) with the other investigated strains. This different molecular make-up of NU combined with earlier serological and HPLC findings justified the conclusion that NL3 should no longer be considered a strain of BCMV, but of a different virus, for which the name bean black root virus (BBRV) has been proposed.It is clear that the lack of serological relationship between NY15 and W, as reported earlier, can not be attributed to major differences in the nucleotide sequences of the N- terminal part of the coat protein genes and 3'-nontranslated regions. Regarding this, epitope mapping of mutated N-terminal domains expressed in heterologous systems, might give an insight into the complexity of the serological results.A special type of antagonism, henceforth referred to as interference, between strains NY15 and NU has been described in Chapter 6. It was found that, when a primary leaf of Phaseolus vulgaris cv. Bataaf was inoculated with the mosaic-inducing NY15 as inducer and one to eight days later with systemic necrosis inducing NU, as challenger, on the opposite leaf, the characteristic symptoms of NL3 i.e. wilting of first trifoliolate leaf followed by top necrosis, did not occur. The amount and distribution of both strains were analysed in the primary leaves of protected plants. It was remarkable to note that the amount of NL3 in the challenger-infected leaf of the protected plant was comparable to that of controls. At the histological level it was found, that NL3 arrived later in the xylem of petioles of challenger-infected leaves. In the light of these results, it was concluded that NY15 protected the plant without affecting the replication of NL3 in the inoculated leaf.Having analysed this unusual interference phenomenon in the primary leaves, the next logical step was to further investigate the exact localization and behaviour of NY15 and NL3 in the stem of protected plants (Chapter 7). Using dot-blot immunoassays, NL3 was detected seven days after its inoculation in protected (infected with NY15 and NL3 ) plants while in the controls NL3 infected only) its presence was established at four days. The later arrival of NL3 in the stems of protected plants was in fine with the earlier findings and could be explained by a hampered transport of NL3 from the inoculated leaf into the stem. Interestingly, the amount of NW in the stem of protected plants was less than that in the singly inoculated controls, in contrast to no difference in challenger inoculated leaf as reported in Chapter 6.In the light of these observations, it can be hypothesized that an early arrival of NL3 in the xylem might lead to an impaired water transport due to its (NW) deleterious effects on the water conducting-system resulting in wilting, withering and top necrosis. The fact that no necrosis occurred in protected plants might be due to the abundant presence of NY15 hampering the invasion of NL3 from the inoculated leaf into the stem, thus preventing the deleterious effects of NW on the water-conducting system. Furthermore, the presence of NY15 in the xylem vessels did not seem to have any adverse effects on the functioning of these vascular elements, possibly due to the different genetic make-up of the two viruses.The differences in nucleotide sequences of the 3'-nontranslated genomic regions between NW on the one hand, and BCMV strains on the other, may be reflected in their symptomatology. Such a correlation has been established in case of tobacco veinmottling virus possessing a determinant of disease symptoms in its 3'-nontranslated region (Rodriguez- Cerezo et al. , 1991).Besides the 3'-nontranslated regions, also the coat protein gene may be involved in the physiological interactions between the virus and its host, as has been shown for tobacco mosaic virus (TMV) mutants (Dawson et al., 1988). Moreover, a single point mutation in the coat protein gene of TMV, leading to a single amino acid substitution, has been shown to be responsible for the induction of hypersensitive reaction in Nicotiana sylvestris , possessing the N'N' genotype (Culver & Dawson, 1989). In contrast, induction of the N gene-mediated hypersensitive reaction caused by TMV, was mapped to the 126-KDa (polymerase) gene as reported by Padgett and Beachy (1993).In view of the possible involvement of more than only one single viral gene in symptomatology, site directed mutagenesis in different regions of the BCMV and BBRV genomes might give a clue to the different reactions of these viruses in bean plants