M. Ali Rezaian

Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

A Single Complementary-Sense Transcript of a Geminiviral DNA β Satellite Is Determinant of Pathogenicity

Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

A NAC Domain Protein Interacts with Tomato leaf curl virus Replication Accessory Protein and Enhances Viral Replication

Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.

Host Responses to Transient Expression of Individual Genes Encoded by Tomato leaf curl virus

The six open reading frames of Tomato leaf curl virus (TLCV) were expressed in host Nicotiana species using a Tobacco mosaic virus vector. Each of the genes, except that encoding the viral coat protein, produced a phenotypic effect when expressed in planta, but the corresponding untranslatable mutant genes were asymptomatic. The C1 (Rep) gene invoked a hypersensitive response in Nicotiana clevelandii that restricted the viral construct to sites of infection. Expression of the C2 gene in N. benthamiana produced necrotic lesions on inoculated leaves as well as severe veinal necrosis on systemically infected leaves. This gene was also able to suppress post-transcriptional gene silencing in N. tabacum. C4 induced viruslike symptoms in host plants tested, providing further evidence for the involvement of this gene in symptom expression. Expression of the V1 and C3 genes caused severe stunting of N. benthamiana plants, indicating they may also have a role in symptom development. These results reveal that a complex set of interactions between the TLCV gene products and host factors occurs in planta, and these are discussed in relation to our current understanding of TLCV gene function.

Analysis of Silencing Escape of Tomato leaf curl virus: An Evaluation of the Role of DNA Methylation

RNA silencing is a sequence-specific mechanism regulating gene expression and has been used successfully for antiviral defense against RNA viruses. Similar strategies to develop resistance against DNA containing Tomato leaf curl virus (TLCV) and some other geminiviruses have been unsuccessful. To analyze this silencing escape, we transformed tomato plants with a hairpin construct from the TLCV C2 open reading frame (ORF). The transgenic plants showed a strong RNA silencing response, and following TLCV inoculation, their infection was delayed. However, the viral infection was not prevented and TLCV DNA accumulated to the levels found in nontransgenic plants. To determine the fate of a transgene carrying homology to the virus, we used transgenic plants carrying the TLCV C4 gene, which induces a distinct phenotype. Upon TLCV infection, the phenotype was abolished and C4 transcript disappeared. Concurrently, TLCV-specific small interfering RNAs were produced. In situ hybridization showed abundant levels of TLCV DNA in phloem cells of TLCV-infected C4 transgenic plants. However, the C4 transcripts were no longer detectable in nonvascular cells. Analysis of the transgene by methylation sequencing revealed a high level of de novo methylation of asymmetric cytosines in both the C4 ORF and its 35S promoter. A high level of methylation also was found at both symmetric and asymmetric cytosines of the complementary-sense strand of TLCV double-stranded DNA. Given the previous finding that methylated geminiviral DNA is not competent for replication, we provide a model whereby TLCV evades host defense through a population of de novo synthesized unmethylated DNA.

Interaction with a Host Ubiquitin-Conjugating Enzyme Is Required for the Pathogenicity of a Geminiviral DNA β Satellite

DNA beta is a single-stranded satellite DNA which encodes a single gene, betaC1. To better understand the role of betaC1 in the pathogenicity of DNA beta, a yeast two-hybrid screen of a tomato cDNA library was carried out using betaC1 from Cotton leaf curl Multan virus (CLCuMV) DNA beta as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between betaC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the betaC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-beta-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing betaC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of betaC1 with SlUBC3 is required for DNA-beta-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

Grapevine viroid 1B, a new member of the apple scar skin viroid group contains the left terminal region of tomato planta macho viroid

GV1B is one of five viroids that have recently been purified from grapevines. GV1B has now been sequenced and its 363 nucleotide residues can potentially form the typical rod-like structure of viroids with 67% of nucleotides base-paired. GV1B has highest sequence similarity with grapevine yellow speckle viroid (GYSV; 73%) and has a central sequence which is conserved in GYSV and apple scar skin viroid (ASSV) which have been reported to constitute the ASSV group. Therefore, we have placed GV1B into the ASSV group. GV1B contains a direct repeat sequence at the terminal portions of its T1 and T2 regions. GV1B also contains a sequence of 69 nucleotides in the terminal portion of its T1 region which is almost identical to the corresponding region in tomato planta macho viroid (TPMV). This provides further evidence of the importance of RNA recombination in viroid evolution.

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types

We have previously shown that the soil‐borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication‐competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low‐level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using β‐glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

Identification and Characterization of a Host Reversibly Glycosylated Peptide that Interacts with the Tomato leaf curl virus V1 Protein

Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in␣vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.