In this thesis the genetic analysis of a plant virus with a divided genome is described.
In chapter 1 a review is given of RNA plant viruses with multipartite genomes (Table 1.1). If in plant viruses with heterogeneous RNA complements specific marker properties can be assigned to specific RNAs a functional specialization of these RNAs is suggested. Such a specialization provides evidence that the virus has a divided genome.
Location of genetic markers in multicomponent plant viruses may be accomplished by constructing in vitro new genotypes through the exchange of corresponding genome parts between strains of the virus which differ with respect to the marker property. Comparing the properties of the reciprocal new genotypes with those of the parent strains may indicate through which genome part the marker is transferred to the new genotypes.
Many markers have been located in a number of multicomponent plant viruses (Tables 1.2 and 1.3). The data indicate that the RNAs of these viruses are functionally specialized and thus are parts of split genomes.
Similar investigations with mutants of cowpea mosaic virus (CPMV) are reported here. CPMV has small icosahedral particles and a bipartite RNA genome. The two RNAs, with molecular weights of 1.37 and 2.02 x 10 6
are encapsidated in different particles. The two particle types can be separated by density gradient centrifugation, after which they are designated middle(M)- and bottom(B)-component. As they contain only part of the genome the purified components are not infectious alone but after mixing they are again highly infectious.
CPMV is well suited for research on the molecular aspects of virus multiplication. As with many vertebrate and bacterial viruses the use of defective mutants may be fruitful in this research. Therefore the isolation of such mutants of CPMV was one of the objectives of the work reported here.
In chapter 2 methods are described for infectivity tests, purification of virus and of the components and for isolation of the viral RNA. In addition, mutagenic treatment and isolation of mutants are described.
In chapter 3 the mutagenic treatment is discussed. Upon treatment with nitrous acid the virus rapidly lost its infectivity (Fig. 3.1). Inactivation of purified B-component was faster than inactivation of purified M-component (Fig. 3.1). Compared with virus, the inactivation of isolated RNA was very slow (Fig. 3.21, suggesting that with virus an effect on the coat protein was involved in inactivation. Thus the loss of infectivity of the virus would only partly reflect changes in the RNA. This is in accordance with the relatively low frequency with which mutants were found among local lesion isolates surviving the treatment of virus as compared with the mutation frequency of isolated RNA (Table 3.1). Therefore for the induction of mutants the treatment of RNA was preferred.
B-RNA was inactivated by nitrous acid at a faster rate than M-RNA (Fig. 3.3). Apparantly Inactivating deaminations occurred less in M-RNA than in B-RNA. Yet the number of mutants having mutations in their M-component was not lower than the number of mutants with a mutated B-component (Table 9.1).
In chapter 4 the isolation of the mutants is described and biological properties of the mutants are compared with those of the wild type.
Mutants were isolated from necrotic local lesions on Pinto beans. For maintaining mutants local lesion transfers on Pinto were preferred to bulk transfers in the systemic host Blackeye cowpea because with the latter method wild type virus often accumulated in the mutant population. This appearance of wild type virus was considered to result from reversion of the mutant genotype and subsequent preferential multiplication of the resulting wild genotype. The symptoms on four differential hosts are given; the kidney bean varieties Pinto and Noordhollandse Bruine and the cowpea varieties Blackeye Early Ramshorn and Early Red.
On the hypersensitive host Early Red, two spontaneous mutants were differentiated: S1 and S8. These mutants infected Early Red systemically giving mosaic and necrosis, respectively. Eight other mutants, designated by N-numbers, were isolated after nitrous acid treatment of virus or RNA. From one of these induced mutants (N142) a spontaneous submutant S15 was isolated.
Almost all induced mutants caused altered symptoms on all the differential hosts. On Pinto the local lesions were slightly smaller (N3, N142, S15) or much smaller (N123, N163, N168, N172), white instead of brown (N163, N168), or less necrotic (N140, N142).
The local spots on the primary leaves of Noordhollandse Bruine were smaller (N123, N163) and less necrotic (N163). With some mutants (N3, N57, N123) systemic infection of this bean variety did not occur.
In Blackeye the local and systemic symptoms were either milder (SI, N57, N163, N168, S15) or absent (N123, N140, N142, N172). One mutant (N3) produced a relatively large amount of top(T)- component, which consists of particles devoid of RNA (Fig. 4.1). Another mutant (N168) appeared to be temperature sensitive for the induction of symptoms (Fig. 4.21 and for virus production (Table 4.2). One mutant (N172), inducing no symptoms at either low or high temperature and having low virus production at low temperature, failed to produce virus at high temperature.
In Early Red the necrotic local lesions were either white instead of brown (N140, N168, N172) or they did not occur at all (N123). Four mutants caused smaller lesions on this cowpea variety (N163, N168, N172, S15). One nitrous acid mutant (N142) caused local chlorotic spots and systemic mosaic in Early Red, like the spontaneous mutant S1.
Chapter 5 describes the in vitro recombination (IVR) test. In this test heterologous combinations of components were composed by exchanging in vitro the purified components of mutants and wild type virus. Properties of the heterologous combinations were compared with those of the parent strains on the differential hosts. The test could be performed either by inoculating the mixtures of components directly on the differential hosts (Table 5.1; Fig. 5.2) or by inoculating the mixtures on the local lesion host Pinto and testing local lesion isolates taken from this variety on the differential hosts.
When each of the heterologous combinations had the phenotype of one of the parent strains it was clear which of the components was responsible for the differentiating propertie(s). In almost all IVR tests this proved to be the case.
Some mutants attained only low titres in Blackeye cowpeas and could not be purified in workable quantities (N123, N140, N142, N172). Thus purification of the components of these mutants was not possible and IVR tests could not be performed. Such mutants could be analyzed by the supplementation test which is described in chapter 6. In this test wild type components were separately added to homogenates of typical mutant local lesions from Pinto. When addition of wild type M-component restored the wild phenotype and addition of B-component did not (Fig. 6.2 en 6.3; Tables 6.1 - 6.41 it was concluded that the M-component carried one or more mutations. In the reciprocal case the B-component was considered to carry the mutation(s).
The supplementation test was less laborious than the IVR test but the interpretation of the results was also more equivocal. In supplementation tests the inoculum always contained three components: two of the mutant and one of the wild type. Whether the observed phenotype was the result of an interaction between all these components or only between two of them could not be established directly.
In chapter 7 experiments are described in which crude preparations of pairs of mutants were inoculated onto the differential hosts separately and after mixing. When the mutants carried their mutations in different components the unmutated components would together contain complete wild type genomes. In that case wild type infections would be initiated through reassortment of components (ROC) in the inoculum.
Subculturing local lesion isolates of virus from primary Blackeye leaves, inoculated with a mixture of N123 and N140 and showing local symptoms (Fig. 7.1), revealed that the virus resulting from this combination was indeed mainly wild type virus. This agreed with the results of supplementation tests with these mutants which showed that they carried their mutations for the absence of symptoms in Blackeye in the M- and B-component, respectively.
Normal wild type symptoms were also induced on Blackeye by a mixture of mutants (N123 and N142) having mutations in their M-components for absence of symptoms and for milder local and systemic symptoms, respectively. To explain this, the occurrence of two independent functional genetic units within the M-RNA was postulated. It was hypothesized that the mutants had their mutations in different functional units and could genetically complement each other in cells in which both M-components were present. Indeed local lesion isolates of virus from Blackeye plants doubly infected with these mutants mainly contained the M-component of either mutant.
Mutants may have mutations affecting the same phenotypic expression in both components. Occurrence of mutations in both components may be assumed if in IVR tests both heterologous combinations of components have phenotypes other than the parent strains or when in supplementation tests neither of the wild type components can completely restore the wild phenotype. In those cases the role of the mutations in the separate components may be evaluated by studying the properties of the two reciprocal hybrids of the mutant and the wild type. This is described in chapter 8 for the temperature sensitive mutant N168. Mutations in both components appeared to affect symptoms in three differential hosts (Table 8.1; Fig. 8.1). Temperature sensitivity was due to a mutation in the M-component. Recombining the components of the two hybrids mutually restored the genotypes of the wild type and the mutant N168.
In chapter 9 a general discussion and conclusions are given. Using four methods, i.e. in vitro recombination, supplementation, reassortment of components and isolation of hybrids, mutations in 11 mutants were located. Mutations affecting specific properties were found in both components, indicating that the components of CPMV are functionally specialized. This confirms that CPMV has a divided genome.
Phenotypic alterations assigned to the M-component were:
- smaller size and changed colour of local lesions on Pinto;
- smaller size of local spots and absence of systemic infection in Noordhollandse Bruine;
- smaller size and reduced chlorosis and absence of local spots and absence of systemic infection in Blackeye cowpeas;
- smaller size and even absence of local lesions in Early Red;
- production of a relatively large amount of T-component;
- temperature sensitivity.
Phenotypic alterations assigned to the B-component were:
- reduced necrosis of local lesions on Pinto;
- absence of systemic infection in Noordhollandse Bruine;
- reduced chlorosis of local spots and absence of local and systemic symptoms in Blackeye;
- changed colour of local lesions on Early Red and occurrence of systemic infection of this host;
- temperature sensitivity.
Apparently both components direct the formation of symptoms in all four hosts. Some changes were brought about by mutations in either component.
Research on the molecular basis of changes in phenotype may help to elucidate the process of virus multiplication and the distribution of functions between the virus components.