V. G. Malathi

Molecular diversity of the DNA-β satellites associated with tomato leaf curl disease in India

DNA-β satellites, referred to here as betasatellites, were found associated with tomato leaf curl disease (ToLCD) in India. The size of eight betasatellites isolated from different geographical locations in India varied from 1353 to 1424 nt; these molecules had an ORF βC1, an adenine-rich region, and a satellite conserved region. Their nucleotide sequence identity varied from 45 to 93%. In phylogenetic analysis, these betasatellites grouped according to their geographic locations rather than the host species. Two new betasatellites, tomato leaf curl Bangalore betasatellite and tomato leaf curl Maharashtra betasatellite, were identified.

Tomato leaf curl Bangalore virus (ToLCBV): infectivity and enhanced pathogenicity with diverse betasatellites

Genomic components of a begomovirus isolated from tomato plants showing leaf curl and stunting symptoms in farmer’s fields at Hessarghatta village near Bangalore, India, were cloned by rolling-circle amplification. The virus was identified as a variant of strain C of the species Tomato leaf curl Bangalore virus and designated as Tomato leaf curl Bangalore virus-C[India:Hessarghatta:2008], ToLCBV-C[IN:Hess:08]. The betasatellite isolated from these samples belongs to the betasatellite species Tomato leaf curl Bangalore betasatellite. ToLCBV-C[IN:Hess:08] induced severe symptoms in Nicotiana benthamiana and Solanum lycopersicum plants when co-inoculated with the cognate betasatellite, Tomato leaf curl Bangalore betasatellite–[India:Hessarghatta:2008], ToLCBB-[IN:Hess:08] and with two other non-cognate betasatellites, Cotton leaf curl Multan betasatellite–[India:SriGanganagar:2002] and Luffa leaf distortion betasatellite–[India:Luffa:2004].

Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation

Yellow mosaic disease (YMD) of legumes endemic to South Asia are caused by begomoviruses transmitted by whiteflies. Based on molecular characterization, two distinct viruses – Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) – were found previously to be the etiological agents of YMD in legumes. Here, host range studies with a soybean isolate of MYMIV (MYMIV-[Sb]) were carried out by both whitefly transmission and agroinoculation. MYMIV-[Sb] was similar to a cowpea isolate of MYMIV (MYMIV-[Cp]) in its ability to infect cowpea, thus differing from blackgram (MYMIV) and mungbean (MYMIV-[Mg]) isolates, which do not infect cowpea. Genomic analysis of DNA A and DNA B components of these MYMIV isolates show characteristic differences in complete DNA B nucleotide sequence correlating with host range differences.

Cowpea golden mosaic disease in Gujarat is caused by a Mungbean yellow mosaic India virus isolate with a DNA B variant.

It has long been assumed that cowpea golden mosaic disease (CGMD) in southern Asia is caused by a begomovirus distinct from those causing disease in other legumes. The components of a begomovirus causing CGMD in western India were isolated, cloned and sequenced. Analysis of the sequences shows the virus to be an isolate of Mungbean yellow mosaic India virus, but with a distinct DNA B component with greater similarity to components of a second legume-infecting begomovirus occurring in the region, Mungbean yellow mosaic virus. The clones of the virus were readily infectious to cowpea, mungbean, blackgram and French bean by agroinoculation. However, the wild-type isolate was shown to be easily transmissible by whiteflies between cowpea plants but not to blackgram and mugbean, suggesting that the insect vector plays a major role in determining the natural host range of these viruses.

Adaptation to new hosts shown by the cloned components of Mungbean yellow mosaic India virus causing cowpea golden mosaic in northern India

The causal virus of golden mosaic of cowpea in northern India has been identified as a variant of Mungbean yellow mosaic India virus (MYMIV) and designated as MYMIV-[Cp]. MYMIV-[Cp] was transmissible by whitefly (Bemisia tabaci) only to cowpea (Vigna unguiculata), yard long bean (V. unguiculata subsp. sesquipedalis), and French bean (Phaseolus vulgaris) and not to any other leguminous hosts. However, agroinoculation of the cloned DNA A and DNA B components of MYMIV-[Cp] produced systemic symptoms in blackgram (V. mungo), mungbean (V. radiata), and mothbean (V. aconitifolia). The severity of disease symptoms and infectivity rate depended on the genotype tested. The progeny virus of agroinoculated plants was whitefly transmissible between blackgram, mungbean, cowpea, and French bean plants. Though the virus was transmissible to blackgram and produced severe symptoms, very low levels of viral DNA components were seen, indicating only limited adaptation to the host. The work communicated here describes, for the first time, the aetiology of golden mosaic of cowpea in northern India and demonstrates adaptation to new hosts of a cloned virus, through agroinoculation.

Molecular Cloning and Characterization of FATTY ACID ELONGATION1 (BjFAE1) Gene of Brassica juncea

The Brassica juncea homologue of Arabidopsis thaliana FAE1 gene, which is responsible for elongation of fatty acid chain length from C18 to C20 and C22 was amplified via PCR (Polymerase Chain Reaction) using heterologous primers. The PCR product was cloned into pGEM-T vector, subcloned and sequenced. The BjFAE1 has 1536-nucleotides and shares 93.6% homology with the A. thaliana counterpart. Southern analysis, using the PCR product as probe, indicated that FAE1 gene is of the same size in all the cultivated Brassica species, i.e. B. juncea, B. nigra, B. campestris, B. oleracea, B. napus and B. carinata. It expresses strongly only in the developing seed and podwall.

Tomato leaf curl Joydebpur virus: a monopartite begomovirus causing severe leaf curl in tomato in West Bengal

A begomovirus was isolated from tomato plants showing leaf curl and stunting symptoms in farmers’ fields near the district of Kalyani, West Bengal, India. Viral genomic components amplified by rolling-circle amplification were cloned and sequenced. The genome organization of this virus was found to be similar to those of Old World monopartite begomovirus, with DNA A and a betasatellite component. Neither alphasatellite nor DNA B component was detected. The begomovirus showed highest sequence identity of 93.6% to tomato leaf curl Joydebpur virus (ToLCJoV-[IN:Kal:Chi:06]) and was thus identified to be an isolate of ToLCJoV. The betasatellite isolated from these samples was identified as tomato leaf curl Joydebpur betasatellite. ToLCJoV-[IN:Kal:Tom:08] alone induced severe symptoms in Solanum lycopersicum, N. benthamiana and N. glutinosa plants, and its severity was enhanced when co-inoculated with the cognate betasatellite. ToLCJoV-[IN:Kal:Tom:08] trans-replicated four more non-cognate betasatellites and induced severe symptoms in N. benthamiana and tomato. Since DNA A replicated efficiently and caused systemic symptom expression, it is hypothesized that ToLCJoV is essentially a monopartite virus, which could have acquired a betasatellite from an unknown source.

Infectivity analysis of a blackgram isolate of Mungbean yellow mosaic virus and genetic assortment with MYMIV in selective hosts

Yellow mosaic disease in grain legumes in Indian subcontinent is caused by two important virus species viz. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), belonging to the genus Begomovirus of the family Geminiviridae. The genomic components of a begomovirus causing yellow mosaic disease in blackgram in southern India were cloned and sequenced. Nucleotide sequence comparison of DNA A component shows the virus isolate to be a variant of Mungbean yellow mosaic virus:–(MYMV-[IN:Vam:05]). However, DNA B component of the present virus isolate has greater similarity (92%) to Mungbean yellow mosaic India virus. Agroinoculations of the viral clones produced typical yellow mosaic symptoms in blackgram and mungbean, severe leaf curl and stunting in French bean, similar to blackgram isolate of MYMIV. Blackgram isolates of both the virus species were only mildly infectious on cowpea, produced atypical leaf curl symptoms and not yellow or golden mosaic. In agroinoculations done by exchanging genomic components, symptom expression was seen only in French bean. In cowpea, blackgram and mungbean there was no visible symptoms though viral DNA could be detected by PCR.

Absence of interaction of genomic components and complementation between Mungbean yellow mosaic India virus isolates in cowpea

Summary.Agroinoculations were performed with DNA A and DNA B components of Mungbean yellow mosaic India virus (MYMIV) isolates differing in their infectivity on cowpea. Exchange of genomic components of the MYMIV isolates occurred in all the leguminous species but not in cowpea. Extremely low viral DNA accumulation and atypical leaf curl symptoms produced by reassortants in cowpea suggest barriers both for replication and systemic movement despite genetic similarity.

Mutagenesis in ORF AV2 affects viral replication in Mungbean yellow mosaic India virus

Mungbean yellow mosaic India virus (MYMIV) is a whitefly-transmitted begomovirus with a bipartite genome. We investigate the functions of the MYMIV-AV2 protein, the open reading frame present upstream of the coat protein gene in DNA A component. The ability of MYMIV-AV2 mutants to replicate, spread and cause symptoms in legume hosts, blackgram, cowpea and French bean was analysed. Plants agroinoculated with mutants K73R, C86S and the double mutant C84S,C86S showed increase in severity of symptoms compared with the wild type. However, mutants W2S and H14Q,G15E caused marked attenuation of symptoms. While the double mutants C84S,C86S caused a 50-fold increase in double-stranded supercoiled and single-stranded DNA accumulation, the mutations W2S and H14Q,G15E showed a decrease in double-stranded supercoiled and single-stranded viral DNA accumulation. Because AV2 mutants affect the ratio between open circular and supercoiled DNA forms, we hypothesize that these mutations may modulate the functions of the replication initiation protein.