Yusuf Zafar

Diversity of DNA β, a satellite molecule associated with some monopartite begomoviruses

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.

Geminivirus disease complexes: an emerging threat

Small circular single-stranded DNA satellites have recently been isolated from plants infected with whitefly-transmitted monopartite begomoviruses. The satellites, named DNA beta, depend on the helper viruses for their proliferation and, in turn, are required for helper virus accumulation and symptom expression. They are highly diverse yet retain an overall conserved structure with respect to potential coding regions and regulatory elements. The begomovirus-satellite disease complexes are associated with economically important diseases, and have been isolated from vegetable and fibre crops, ornamental plants and weeds throughout Africa and Asia. Their widespread distribution and diversity, coupled to the global movement of plant material and the dissemination of the whitefly vector, suggests that these disease complexes pose a serious threat to tropical and sub-tropical agro-ecosystems worldwide.

Toward Sequencing Cotton (Gossypium) Genomes

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β

Summary For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the “common region”, which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus (Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA β. The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA β to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA β is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA β has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard “Coker” varieties and the diploid G. arboreum L. var. Ravi (old world cotton).

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.

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease

Summary. Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA β responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5′ half of the viral DNA replication gene, AC1, [ii] the 3′ half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T1 − T3 generations]: symptom-free and no replication of DNA A or DNA β even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

The Nuclear Shuttle Protein of Tomato Leaf Curl New Delhi Virus Is a Pathogenicity Determinant

ABSTRACT The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.

Mobilisation into cotton and spread of a recombinant cotton leaf curl disease satellite.

Summary.Analysis of a DNA β satellite associated with a recently identified cotton leaf curl disease (CLCuD) strain indicated it to be recombinant, with most of the molecule originating from CLCuD DNA β but with some sequence from a satellite isolated from tomato. Analysis of both archival (pre 2001) and recent cotton samples, shows the recombinant satellite is confined to a small area but was not present in cotton prior to 2001. This indicates that the recombinant DNA β was recently mobilized into cotton, likely from tomato, and that recombination plays a role in the evolution of these satellites.

Molecular phylogeny of Gossypium species by DNA fingerprinting

Abstract Total genomic DNA from 31 available Gossypium species, three subspecies and one interspecific hybrid, were analysed to evaluate genetic diversity by RAPD, using 45 random decamer primers. A total of 579 amplified bands were observed, with 12.9 bands per primer, of which 99.8% were polymorphic. OPJ-17 produced the maximum number of fragments while the minimum number of fragments was produced with primer OPA-08. Cluster analysis by the unweighted paired group method of arithmetic means (UPGMA) showed six main clusters. Cluster ’A’ consisted of two species and one subspecies of the A-genome, with a 0.78–0.92 Nei’s similarity range. Cluster B, composed of all available tetraploid species and one interspecific hybrid, showed the same sister cluster. Nei’s similarity ranged from 0.69 to 0.84. The B-genome formed the UPGMA sister cluster to the E-genome species. Cluster ’C’ consisted of five Gossypium species of which three belong to the B-genome, with Nei’s similarity values of 0.81 to 0.86. Although there was considerable disagreement at lower infra-generic ranks, particularly among the D- genome (diploid New World species) and C-genome (diploid Australian species) species. The sole F-genome species Gossypium longicalyx was resolved as a sister group to the D-genome species. Gossypium herbaceum and G. herbaceum Africanum showed the maximum Nei’s similarity (0.93). Minimum similarity (0.29) was observed between Gossypium trilobum and Gossypium nelsonii. The average similarity among all studied species was 50%. The analysis revealed that the interspecific genetic relationship of several species is related to their centre of origin. As expected, most of the species have a wide genetic base range. The results also revealed the genetic relationships of the species Gossypium hirsutum to standard cultivated Gossypium barbadense, G. herbaceum and Gossypium arboreum. These results correspond well with previous reported results. The level of variation detected in closely related genotypes by RAPD analysis indicates that it may be a more efficient marker than morphological marker, isozyme and RFLP technology for the construction of genetic linkage maps.