Edi Cecchini

Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies

Gene VI of cauliflower mosaic virus (CaMV) is an important determinant of symptom expression during infection. We have constructed a series of transgenic Arabidopsis lines that express gene VI protein (P6) from two CaMV isolates (Bari-1 and Cabb B-JI) that cause mild and severe symptoms, respectively, in Arabidopsis, and from a recombinant virus (Baji-31) with a hybrid gene VI that causes very severe symptoms. From 41 transgenic lines analyzed, 17 showed symptom-like phenotypes that ranged from mild vein chlorosis to severe chlorosis and stunting. P6 levels in transgenic lines varied from undetectable in the lowest expressors to levels greater than those in CaMV-infected plants. There was a strong correlation between phenotype severity and the level of P6, and with the gene VI origin in the order, Baji-31 > B-JI > Bari-1. This was similar to symptom severity in Arabidopsis infected with the respective CaMV variant. We also found that transgenic P6 accumulated in inclusion bodies that were similar to those found in infected plants but lacking virions. We conclude that expression of P6, in the absence of virus replication, elicits a subset of the host symptom responses normally observed during infection and that the level, sequence, and possibly the form of P6 are important in potentiating the process.

Altered Patterns of Gene Expression in Arabidopsis Elicited by Cauliflower Mosaic Virus (CaMV) Infection and by a CaMV Gene VI Transgene

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.

Arabidopsis mutants that suppress the phenotype induced by transgene-mediated expression of cauliflower mosaic virus (CaMV) gene VI are less susceptible to CaMV-infection and show reduced ethylene sensitivity.

Protein P6 is the main symptom determinant of cauliflower mosaic virus (CaMV), and transgene-mediated expression in Arabidopsis induces a symptom-like phenotype in the absence of infection. Seeds of a P6-transgenic line, A7, were mutagenized by γ-irradiation and M2 seedlings were screened for mutants that suppressed the phenotype of chlorosis and stunting. We identified four mutants that were larger and less chlorotic than the A7 parent but which contained an intact and transcriptionally active transgene. The two mutants with the strongest suppression phenotype, were recessive and allelic. The transgene was eliminated by back-crossing with wild-type Arabidopsis. In progeny lines that were homozygous for the putative suppressor mutation the proportion of plants becoming infected following inoculation with CaMV was 40% that of wild-type, although in plants that did become infected, levels of virus DNA in mutants and wild-type did not differ significantly. Symptoms in the mutants were milder and delayed although this was somewhat dependent on the virus isolate. This phenotype was inherited stably. Both mutant alleles showed a partially ethylene-insensitive phenotype in an ethylene triple response assay. P6-transgenic plants were also almost completely insensitive to ethylene in the triple response assay. We suggest that the chlorosis and stunting in P6-transgenic and CaMV-infected plants are dependent on interactions between P6 and components involved in ethylene signalling, and that the suppressor gene product may function to augment these interactions.

A kinetic model for subtractive hybridization

Nucleic acid sequences that differ in abundance between two populations (target sequences) can be cloned by multiple rounds of subtractive hybridization and amplification by PCR. These sequences can be cDNAs representing up-regulated mRNAs, or genomic DNAs from deletion mutants. We have derived an equation that describes the recovery of such sequences, and have used this to simulate the outcome of up to 10 rounds of subtractive hybridization and PCR amplification. When the model was tested by comparing its predictions with the published results from genomic and cDNA subtractions, the predictions of the model were generally in good agreement with the published data. We have modelled the outcomes of genomic subtractions, for a variety of genomes, and have used it to compare various strategies for enriching targets. The model predicts that for genomes of less than 5 x 10(8) bp, deletions of as small as 1 kbp should represent > 99% of the DNA after three to six rounds of hybridization (depending on the enrichment procedure). As genomes increase in size, the kinetics of hybridization become an important limiting factor. However, even for genomes as large as 3 x 10(9) bp, it should be possible to isolate deletions of 5 kbp using the appropriate conditions. These simulations suggest that such methods offer a realistic alternative to chromosome walking for identifying genomic deletions for which there are known phenotypes, thereby considerably reducing time and effort. For cDNA subtractive hybridization, the model predicts that after six rounds of hybridization, sequences that do not differ in abundance between the tester and driver populations (the background) will represent < 1% of the subtracted population, and even quite modestly upregulated cDNAs should be successfully enriched. Where several up-regulated cDNAs are present, the predicted final representation is dependent on both the initial abundance and the degree of up-regulation.

Examination of the pipetting variation between individuals performing random amplified polymorphic DNA (RAPD) analysis

Abstract The similarity between random amplified polymorphic DNA (RAPD) profiles generated by nine experimenters is presented. Despite simultaneously using the same pipettes, the same solutions and the same thermocycler, no two RAPD profiles generated identical banding patterns. This result suggests that before using a particular primer, it is essential that all experimenters should obtain a reference profile for their own work, rather than comparing it to one generated by another researcher.