Induction of plant cell division by beet curly top virus gene C4

Beet curly top virus (BCTV) is a small DNA virus that causes tumorigenic growths (enations) in infected plants by inducing division of phloem parenchyma cells (hyperplasia). It has previously been shown that BCTV C4 plays an important role in symptom development in sugarbeet and Nicotiana benthamiana , and it has been suggested that this gene is responsible for the induction of hyperplasia. Using in situ hybridization, we show that BCTV infection is closely associated with the vascular system in these hosts, although hyperplastic cells associated with wild-type virus infection frequently do not contain detectable levels of viral DNA. Extensive hyperplasia was not observed in plants infected with a C4 mutant, demonstrating a role for C4 in virus-induced cell proliferation. Ectopic expression of C4 in transgenic N. benthamiana resulted in abnormal plant development and the production of tumorigenic growths, confirming that this gene alone is sufficient to initiate cell division in permissive cells when removed from the context of the viral genome.     

Identification of a promoter motif involved in Curtovirus sense-gene expression in transgenic Arabidopsis

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bi-directional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.     

Resistance to African cassava mosaic virus conferred by a mutant of the putative NTP-binding domain of the Rep gene (AC1) in Nicotiana benthamiana

We constructed a mutation in DNA A of African cassava mosaic virus (ACMV) to alter the putative NTP-binding site in the replication- associated protein gene (AC1). When transgenic Nicotiana benthamiana plants expressing the mutated AC1 gene were infected with ACMV, the plants exhibited tolerance to infection consisting in a delay in symptom appearance and/or the presence of mild symptoms. In addition, the resistant plants accumulated less viral DNA than non-transgenic plants. As judged by northern blot analysis and symptom development of segregating progeny from different lines, a high level of expression of the mutated AC1 gene is essential for the development of resistance. Issues related to the use of different versions of AC1 for the control of ACMV are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetically engineered transgenic plants with the domain 1 sequence of tobacco mosaic virus 126 kDa protein gene are completely resistant to viral infection

In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.     

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.     

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.     

Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX-resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX-resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain-specific resistance to PVX; and the ability of the transgene to trans -inactivate homologous transgenes. This gene-silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX-susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage-dependent manner. The same association of strain-specific resistance and low-level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild-type versions of the RdRp gene. Strain-specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology-dependent gene silencing and transgenic resistance to PVX may be due to the same RNA-based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans -inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.     

Tête à tête of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei

Since 1997 two distinct geminivirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have caused a similar yellow leaf curl disease in tomato, coexisted in the fields of southern Spain, and very frequently doubly infected single plants. Tomatoes as well as experimental test plants (e.g., Nicotiana benthamiana) showed enhanced symptoms upon mixed infections under greenhouse conditions. Viral DNA accumulated to a similar extent in singly and doubly infected plants. In situ tissue hybridization showed TYLCSV and TYLCV DNAs to be confined to the phloem in both hosts, irrespective of whether they were inoculated individually or in combination. The number of infected nuclei in singly or doubly infected plants was determined by in situ hybridization of purified nuclei. The percentage of nuclei containing viral DNA (i.e., 1.4% in tomato or 6% in N. benthamiana) was the same in plants infected with either TYLCSV, TYLCV, or both. In situ hybridization of doubly infected plants, with probes that discriminate between both DNAs, revealed that at least one-fifth of infected nuclei harbored DNAs from both virus species. Such a high number of coinfected nuclei may explain why recombination between different geminivirus DNAs occurs frequently. The impact of these findings for epidemiology and for resistance breeding concerning tomato yellow leaf curl diseases is discussed.     

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).