Interplay between the genetic clades of Micromonas and their viruses in the Western English Channel.

The genus Micromonas comprises distinct genetic clades that commonly dominate eukaryotic phytoplankton community from polar to tropical waters. This phytoplankter is also recurrently infected by abundant and genetically diverse prasinoviruses. Here we report on the interplay between prasinoviruses and Micromonas with regard to the genetic diversity of this host. For 1 year, we monitored the abundance of three clades of Micromonas and their viruses in the Western English Channel, both in the environment using clade-specific probes and flow cytometry, and in the laboratory using clonal strains of Micromonas clades to assay for their viruses by plaque-forming units. We showed that the seasonal fluctuations of Micromonas clades were closely mirrored by the abundance of their corresponding viruses, indicating that the members of Micromonas genus are susceptible to viral infection, regardless of their genetic affiliation. The characterization of 45 viral isolates revealed that Micromonas clades are attacked by specific virus populations, which exhibit distinctive clade specificity, life strategies and genetic diversity. However, some viruses can also cross-infect different host clades, suggesting a mechanism of horizontal gene transfer within the Micromonas genus. This study provides novel insights into the impact of viral infection for the ecology and evolution of the prominent phytoplankter Micromonas.

Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus.

Bacterial wilt caused by the soilborne bacterium Ralstonia solanacearum attacks hundreds of plant species, including many agriculturally important crops. Natural resistance to this disease has been found in some species and is usually inherited as a polygenic trait. In tomato, a model crop plant, genetic analysis previously revealed the involvement of several QTL (quantitative trait loci) controlling resistance and, in all of these studies with different strains of the pathogen, loci on chromosome 6 played the predominant role in controlling this trait. Using quantitative data collected from a greenhouse test F3 population, we identified a new locus on chromosome 12 that appears to be active specifically against a race 1 biovar 3 Pss4 bacterial strain endemic to Taiwan. Chromosome 6 still contributes significantly to the control of the resistance, and weaker associations of the trait to other regions of the genome are observed. These results are discussed in the context of current molecular knowledge about the strain specificity of disease resistance genes.

Temporal and multiple quantitative trait loci analyses of resistance to bacterial wilt in tomato permit the resolution of linked loci.

Ralstonia solanacearum is a soil-borne bacterium that causes the serious disease known as bacterial wilt in many plant species. In tomato, several QTL controlling resistance have been found, but in different studies, markers spanning a large region of chromosome 6 showed strong association with the resistance. By using two different approaches to analyze the data from a field test F3 population, we show that at least two separate loci approximately 30 cM apart on this chromosome are most likely involved in the resistance. First, a temporal analysis of the progression of symptoms reveals a distal locus early in the development of the disease. As the disease progresses, the maximum LOD peak observed shifts toward the proximal end of the chromosome, obscuring the distal locus. Second, although classical interval mapping could only detect the presence of one locus, a statistical "two-QTL model" test, specifically adapted for the resolution of linked QTL, strongly supported the hypothesis for the presence of two loci. These results are discussed in the context of current molecular knowledge about disease resistance genes on chromosome 6 and observations made by tomato breeders during the production of bacterial wilt-resistant varieties.

The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination.

Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene. The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences. In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants. The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.

virF, the host-range-determining virulence gene of Agrobacterium tumefaciens, affects T-DNA transfer to Zea mays.

The monocotyledonous plant Zea mays does not develop tumors after inoculation with Agrobacterium tumefaciens and is thus defined as nonhost. Agroinfection, Agrobacterium-mediated delivery of maize streak virus, demonstrates that transferred DNA (T-DNA) transfer to the plant does occur. Nopaline-type Agrobacterium strains such as C58 are efficient in the transfer process whereas the octopine-type strain A6 is unable to transfer T-DNA to maize. This phenotypic difference maps to the tumor-inducing (Ti) plasmid but not to the T-DNA. Steps preceding T-DNA transfer, such as attachment and induction of the virulence genes, were shown to take place in the octopine strain. The nopaline-plasmid-specific locus tzs and the octopine-plasmid-specific locus pinF (virH) are not involved in the strain specificity. However, mutations in the virF locus rendered the octopine strain agroinfectious on maize, whereas such virF-defective octopine strains, when complemented by virF on a plasmid, completely lost their agroinfectivity. We propose that VirF, known to increase the host range of the bacteria in other systems, acts as an inhibitor of T-DNA transfer to maize.

Genomic homologous recombination in planta.

A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process.

Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer.

A system for the analysis of independent T-DNA transfer events from Agrobacterium to plants is described. The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant, a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to the ends of T-DNA molecules. A rather conserved right border remnant of three nucleotides was found, whereas the sequences remaining at the left end were more variable. A point deletion in the left 25 bp sequence results in even less precise processing at the left end. In addition, many rescued T-DNA molecules carry small direct repeats between the joined T-DNA ends; linear T-DNA molecules are therefore transported to the plant.

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.

DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes.

"Agroinfection," an alternative route for viral infection of plants by using the Ti plasmid.

Most plant viruses are transmitted by insect vectors. We present an alternative method for the introduction of infectious viral DNA that uses the ability of Agrobacterium to transfer DNA from bacterial cells to plants. Cauliflower mosaic virus was chosen to develop this method because it is the best characterized plant DNA virus and can be introduced into plants via aphids, virus particles, viral DNA, or suitably treated cloned DNA. We show that systemic infection of turnips results from wounding and inoculation with strains of Agrobacterium tumefaciens in which more than one genome of cauliflower mosaic virus have been placed tandemly in the T-DNA of the tumor-inducing plasmid. Thus such constructions allow escape of the viral genome from the T-DNA once inside the plants. The combined use of the tumor-inducing plasmid and viral DNA opens the way to molecular biological approaches that are not possible with either system alone.

Recombination in a plant virus: template-switching in cauliflower mosaic virus.

A hybrid plasmid, containing tandemly arranged pieces of two different but well-defined cauliflower mosaic virus (CaMV) genomes, was used to study the mechanism by which infectious viral DNA can escape from transforming DNA. Systemic viral infection followed inoculation of Brassica plants with a strain of Agrobacterium tumefaciens containing the hybrid plasmid in its T-DNA. Restriction mapping of uncloned viral DNA from these plants, and sequencing of relevant portions of cloned viral DNA, showed that the majority of viral progeny were probably descendants of DNA produced by transcription/reverse transcription of the viral genome, thus providing further evidence for the hypothesis that this process is normally involved in viral replication. The reverse transcriptase enzyme, which is thought to undergo an intramolecular template switch during viral replication, is shown to move very close to the 5' end of the terminal repeat on the 35S RNA molecule before switching templates. The remaining minority of viral genomes can best be explained as arising from products of recombination between homologous regions of CaMV DNA.

Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants.

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3-4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.