Diverse begomovirus-betasatellite complexes cause tomato leaf curl disease in the western India.

In the Indian sub-continent, tomato leaf curl disease (ToLCD) of tomato caused by begomoviruses has emerged as a major limiting factor for tomato cultivation. Despite the spread of this disease in the western India, a systematic study on the characterization of virus complexes with ToLCD is lacking. Here, we report the identification of a complex of begomoviruses including 19 DNA-A and 4 DNA-B as well as 15 betasatellites with ToLCD in the western part of the country. Additionally, a novel betasatellite and an alphasatellite were also identified. The recombination breakpoints were detected in the cloned begomoviruses and betasatellites. The cloned infectious DNA constructs cause disease on the tomato (a moderately virus-resistant cultivar) plants, thus fulfilling Koch's postulates for these virus complexes. Further, the role of non-cognate DNA B/betasatellite with ToLCD-associated begomoviruses on disease development was demonstrated. It also emphasizes the evolutionary potential of these virus complexes in breaking disease resistance and plausible expansion of its host range. This necessitates to investigate the mechanism of the interaction between resistance breaking virus complexes and the infected host.

Molecular insights into mechanisms underlying thermo-tolerance in tomato.

Plant productivity is being seriously compromised by climate-change-induced temperature extremities. Agriculture and food safety are threatened due to global warming, and in many cases the negative impacts have already begun. Heat stress leads to significant losses in yield due to changes in growth pattern, plant phonologies, sensitivity to pests, flowering, grain filling, maturity period shrinkage, and senescence. Tomato is the second most important vegetable crop. It is very sensitive to heat stress and thus, yield losses in tomato due to heat stress could affect food and nutritional security. Tomato plants respond to heat stress with a variety of cellular, physiological, and molecular responses, beginning with the early heat sensing, followed by signal transduction, antioxidant defense, osmolyte synthesis and regulated gene expression. Recent findings suggest that specific plant organs are extremely sensitive to heat compared to the entire plant, redirecting the research more towards generative tissues. This is because, during sexual reproduction, developing pollens are the most sensitive to heat. Often, just a few degrees of temperature elevation during pollen development can have a negative effect on crop production. Furthermore, recent research has discovered certain genetic and epigenetic mechanisms playing key role in thermo-tolerance and have defined new directions for tomato heat stress response (HSR). Present challenges are to increase the understanding of molecular mechanisms underlying HS, and to identify superior genotypes with more tolerance to extreme temperatures. Several metabolites, genes, heat shock factors (HSFs) and microRNAs work together to regulate the plant HSR. The present review provides an insight into molecular mechanisms of heat tolerance and current knowledge of genetic and epigenetic control of heat-tolerance in tomato for sustainable agriculture in the future. The information will significantly contribute to improve breeding programs for development of heat tolerant cultivars.

Bhendi Yellow Vein Mosaic Virus and Bhendi Yellow Vein Mosaic Betasatellite Cause Enation Leaf Curl Disease and Alter Host Phytochemical Contents in Okra.

Whitefly (Bemisia tabaci)-transmitted begomoviruses cause severe diseases in numerous economically important dicotyledonous plants. Okra enation leaf curl disease (OELCuD) has emerged as a serious threat to okra (Abelmoschus esculentus L. Moench) cultivation in the Indian subcontinent. This study reports the association of a monopartite begomovirus (bhendi yellow vein mosaic virus; BYVMV) and betasatellite (bhendi yellow vein mosaic betasatellite; BYVB) with OELCuD in the Mau region of Uttar Pradesh, India. The BYVMV alone inoculated Nicotiana benthamiana and A. esculentus cv. Pusa Sawani plants developed mild symptoms. Co-inoculation of BYVMV and BYVB resulted in a reduced incubation period, an increased symptom severity, and an enhanced BYVMV accumulation by Southern hybridization and quantitative real-time PCR. This is the first study that satisfies Koch's postulates for OELCuD in its natural host. Activities of various antioxidative enzymes were significantly increased in the virus-inoculated okra plants. Differential responses in various biochemical components (such as photosynthetic pigments, phenol, proline, and sugar) in diseased okra plants were observed. This change in phytochemical responses is significant in understanding its impact on virus pathogenesis and disease development.

Two distinct monopartite begomovirus-betasatellite complexes in western India cause tomato leaf curl disease.

In India, begomovirus infection causing tomato leaf curl disease (ToLCD) is a major constraint for tomato productivity. Here, we have identified two distinct monopartite begomovirus and betasatellite complexes causing ToLCD in the western part of India. A new monopartite begomovirus (Tomato leaf curl Mumbai virus, ToLCMumV) and betasatellite (Tomato leaf curl Mumbai betasatellite, ToLCMumB) were isolated from the Mumbai sample. A distinct Tomato leaf curl Gandhinagar virus (ToLCGanV) and Tomato leaf curl Gandhinagar betasatellite (ToLCGanB) were identified from the Gandhinagar sample. Both of the cloned begomoviruses were recombinants. The demonstration of systemic infection caused by begomovirus (ToLCGanV or ToLCMumV) alone in N. benthamiana and tomato (a virus resistant variety) emphasizes that they were monopartite begomoviruses. Co-inoculation of cognate begomovirus and betasatellite reduces the incubation period and increases symptom severity. Thus, Koch's postulates were satisfied for these virus complexes. Further, an enhanced accumulation of ToLCGanV was detected in the presence of cognate ToLCGanB, however ToLCMumB did not influence the level of ToLCMumV in the agro-inoculated tomato plants. Our results indicate that the cloned viruses form potential virus resistance breaking disease complexes in India. This necessitates to investigate the spread of these disease complexes to major tomato growing regions in the country.

A novel monopartite begomovirus and satellites associated with yellow mosaic disease of Sida spp. in India.

Begomoviruses (family Geminiviridae) cause severe diseases in many economically important crops and non-cultivated plants in the warmer regions of the world. Non-cultivated weeds have been reported to act as natural virus reservoirs. In January 2016, Sida plants with yellow mosaic symptoms were found at the edge of an agricultural field in Gujarat, India. Sequence analysis of the viral genomic components cloned from a diseased Sida plant indicated the presence of a distinct monopartite begomovirus (proposed as sida yellow mosaic Gujarat virus) along with a betasatellite (ludwigia leaf distortion betasatellite) and an alphasatellite (malvastrum yellow mosaic alphasatellite). Our results emphasize that this weed may harbor a begomovirus-alphasatellite-betasatellite complex. This host serves as a potential source of virus inoculum, which can be transmitted by whiteflies to other cultivated crops.

Silencing of tomato CTR1 provides enhanced tolerance against Tomato leaf curl virus infection.

Tomato leaf curl virus (ToLCV) belonging to Begomovirus family of Geminivirus is known to cause one of the most destructive diseases in tomato. Amongst various ToLCVs, a monopartite Tomato leaf curl Joydebpur virus (ToLCJoV) is most prevalent in eastern part of India. In the present study, we observed induced expression of one of the negative regulators of ethylene signaling pathway gene (LeCTR1) in ToLCJoV infected plants. The Tobacco rattle virus (TRV) induced silencing of the LeCTR1 gene provided enhanced tolerance to ToLCJoV infections. The leaf curling as well as ROS accumulation was significantly reduced in the viral infected LeCTR1 silenced plants. Induction of several defense marker genes (NPR1, PR1, PR5, AOS2, EIN2, EIN3 and ERF5) reinforced enhanced tolerance against ToLCJoV infection in the LeCTR1 silenced tomato. Overall, the present study provides evidence that silencing of LeCTR1 can be deployed to protect tomato from ToLCJoV infections.

CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops.

Horticultural crops are an important part of agriculture for food as well as nutritional security. However, several pests and diseases along with adverse abiotic environmental factors pose a severe threat to these crops by affecting their quality and productivity. This warrants the effective and accelerated breeding programs by utilizing innovative biotechnological tools that can tackle aforementioned issues. The recent technique of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) has greatly advanced the breeding for crop improvement due to its simplicity and high efficiency over other nucleases such as Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases. CRISPR/Cas9 tool contains a non-specific Cas9 nuclease and a single guide RNA that directs Cas9 to the specific genomic location creating double-strand breaks and subsequent repair process creates insertion or deletion mutations. This is currently the widely adopted tool for reverse genetics, and crop improvement in large number of agricultural crops. The use of CRISPR/Cas9 in horticultural crops is limited to few crops due to lack of availability of regeneration protocols and sufficient sequence information in many horticultural crops. In this review, the present status of applicability of CRISPR/Cas9 in horticultural crops was discussed along with the challenges and future potential for possible improvement of these crops for their yield, quality, and resistance to biotic and abiotic stress.

Biotechnological Advancements and Begomovirus Management in Okra (Abelmoschus esculentus L.): Status and Perspectives.

Despite the importance of okra, as one of the important vegetable crop, very little attention has been paid to its genetic improvement using advanced biotechnological tools. The exploitation of marker assisted breeding in okra is often limited due to the availability of a few molecular markers, the absence of molecular genetic-map(s), and other molecular tools. Chromosome linkage-groups were not yet constructed for this crop and reports on marker development are very scanty and mostly hovering around cultivar characterization. Besides, very little progress has been observed for transgenic development. However, high throughput biotechnological tools like chromosome engineering, RNA interference (RNAi), marker-assisted recurrent selection (MARS), genome-wide selection (GWS), targeted gene replacement, next generation sequencing (NGS), and nanobiotechnology can provide a rapid way for okra improvement. Further, the etiology of many deadly viral diseases like the yellow vein mosaic virus (YVMV) and okra enation leaf curl virus (OELCV) in okra is broadly indistinct and has been shown to be caused by various begomovirus species. These diseases cause systemic infections and have a very effective mode of transmission; thus, preventing their spread has been very complicated. Biotechnological interventions have the potential to enhance okra production even under different viral-stress conditions. In this background, this review deals with the biotechnological advancements in okra per se along with the begomoviruses infecting okra, and special emphasis has been laid on the exploitation of advanced genomic tools for the development of resistant varieties.

Molecular diversity, recombination and population structure of alphasatellites associated with begomovirus disease complexes.

The genus, begomovirus (family Geminiviridae) includes a large number of viruses infecting a wide range of plant species worldwide. The majority of monopartite begomoviruses are associated with satellites (betasatellites) and/or satellite-like molecules (alphasatellites). In spite of the Indo-China region being regarded as the centre of origin of begomoviruses and satellites, a detailed study on the emergence and evolution of alphasatellites in India has not yet conducted. Our present analysis indicated the association of 22 alphasatellites with monopartite and bipartite begomovirus-betasatellite complexes in India. Based on sequence pairwise identity, these alphasatellites were categorized into five distinct groups: Cotton leaf curl alphasatellite, Gossypium darwinii symptomless alphasatellite, Gossypium mustelinum symptomless alphasatellite, Okra leaf curl alphasatellite and an unreported Chilli leaf curl alphasatellite (ChiLCA). Furthermore, infectivity analysis of the cloned ChiLCA along with the viral components of either cognate or non-cognate chilli-infecting begomoviruses on Nicotiana benthamiana suggested that ChiLCA is dispensable for leaf curl disease development. It is noteworthy that in the presence of ChiLCA, a marginal decrease in betasatellite DNA level was noticed. Additionally, high genetic variability and diverse recombination patterns were detected among these alphasatellites, and the nucleotide substitution rate for the Rep gene of ChiLCA was determined to be 2.25×10-3nucleotides/site/year. This study highlights the genetic distribution, and likely contribution of recombination and nucleotide diversity in facilitating the emergence of alphasatellites.

Identification and molecular characterization of a new recombinant begomovirus and associated betasatellite DNA infecting Capsicum annuum in India.

Capsicum annuum (Chilli) is a perennial herbaceous plant that is cultivated as an annual crop throughout the world, including India. Chilli leaf curl disease (ChiLCD) is a major biotic constraint, causing major losses in chilli production. During 2014, leaf samples of chilli plants displaying leaf curl disease were collected from the Ahmedabad district of Gujarat, India. These samples were used to isolate, clone and sequence viral genomic DNA and an associated betasatellite DNA molecule. Sequence analysis showed 90.4 % nucleotide sequence identity to the previously reported chilli leaf curl virus-[India:Guntur:2009] (ChiLCV-[IN:Gun:09]. As per ICTV nomenclature rules, ChiLCV-Ahm represents a new species of begomovirus, and we therefore propose the name chilli leaf curl Ahmedabad virus-[India:Ahmedabad:2014] (ChiLCAV-[IN:Ahm:14]). The associated betasatellite DNA showed a maximum of 93.5 % nucleotide sequence identity to a previously reported tomato leaf curl Bangladesh betasatellite and may be named tomato leaf curl Bangladesh betasatellite-[India:Ahmedabad:Chilli:2014].

Comparative analysis of microsatellites in chloroplast genomes of lower and higher plants.

Microsatellites, or simple sequence repeats (SSRs), contain repetitive DNA sequence where tandem repeats of one to six base pairs are present number of times. Chloroplast genome sequences have been  shown to possess extensive variations in the length, number and distribution of SSRs. However, a comparative analysis of chloroplast microsatellites is not available. Considering their potential importance in generating genomic diversity, we have systematically analysed the abundance and distribution of simple and compound microsatellites in 164 sequenced chloroplast genomes from wide range of plants. The key findings of these studies are (1) a large number of mononucleotide repeats as compared to SSR(2-6)(di-, tri-, tetra-, penta-, hexanucleotide repeats) are present in all chloroplast genomes investigated, (2) lower plants such as algae show wide variation in relative abundance, density and distribution of microsatellite repeats as compared to flowering plants, (3) longer SSRs are excluded from coding regions of most chloroplast genomes, (4) GC content has a weak influence on number, relative abundance and relative density of mononucleotide as well as SSR(2-6). However, GC content strongly showed negative correlation with relative density (R (2) = 0.5, P < 0.05) and relative abundance (R (2) = 0.6, P < 0.05) of cSSRs. In summary, our comparative studies of chloroplast genomes illustrate the variable distribution of microsatellites and revealed that chloroplast genome of smaller plants possesses relatively more genomic diversity compared to higher plants.

Recent evolution of a novel begomovirus causing tomato leaf curl disease in the Al-Batinah region of Oman.

For last two decades, begomoviruses (family Geminiviridae) have been a major constraint for tomato production in Oman, particularly in the Al-Batinah region, the major agricultural area of Oman. Farms in the Al-Batinah region were surveyed during January-March and November-December in 2012 and January-February in 2013. Leaf samples of tomato plants showing typical leaf curl disease symptoms were collected and analyzed for begomoviruses. Out of fifteen begomovirus clones sequenced, seven were shown to be tomato yellow leaf curl virus strain Oman (TYLCV-OM); three, chili leaf curl virus strain Oman (ChLCV-OM); and one, tomato leaf curl Oman virus (ToLCOMV) - viruses that have previously been shown to occur in Oman. Four sequences were shown to have relatively low percent identity values to known begomoviruses, with the highest (86 %) to isolates of pepper leaf curl Lahore virus, indicating that these should be included in a new species, for which the name "Tomato leaf curl Al Batinah virus" (ToLCABV) is proposed. Although the betasatellite tomato leaf curl betasatellite (ToLCB; 7 full-length sequences isolated) was identified with some isolates of ChLCV-OM, TYLCV-OM and ToLCOMV, it was not identified in association with any of the ToLCABV isolates. Analysis of the sequences of the TYLCV-OM and ToLCOMV isolates characterized here did not show them to differ significantly from previously characterized isolates of these viruses. The three isolates of ChLCV-OM characterized were shown to have a recombination pattern distinct from earlier characterized isolates. ToLCABV was shown to have resulted from recombination between ChLCV-OM and ToLCOMV. A clone of ToLCABV was infectious by Agrobacterium-mediated inoculation to Nicotiana benthamiana and tomato, inducing symptoms typical of those seen in tomato in the field. Additionally, ToLCABV was shown to be able to interact in planta with ToLCB, resulting in a change in symptom phenotype, although the betasatellite did not appear to affect viral DNA levels.

Genetic diversity and distribution of a distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman.

Tomato and pepper are widely grown in Oman for local consumption. A countrywide survey was conducted during 2010-2011 to collect samples and assess the diversity of begomoviruses associated with leaf curl disease of tomato and pepper. A virus previously only identified on the Indian subcontinent, chili leaf curl virus (ChLCV), was found associated with tomato and pepper diseases in all vegetable grown areas of Oman. Some of the infected plant samples were also found to contain a betasatellite. A total of 19 potentially full-length begomovirus and eight betasatellite clones were sequenced. The begomovirus clones showed >96% nucleotide sequence identity, showing them to represent a single species. Comparisons to sequences available in the databases showed the highest levels of nucleotide sequence identity (88.0-91.1%) to isolates of the "Pakistan" strain of ChLCV (ChLCV-PK), indicating the virus from Oman to be a distinct strain, for which the name Oman strain (ChLCV-OM) is proposed. An analysis for recombination showed ChLCV-OM likely to have originated by recombination between ChLCV-PK (the major parent), pepper leaf curl Lahore virus and a third strain of ChLCV. The betasatellite sequences obtained were shown to have high levels of identity to isolates of tomato leaf curl betasatellite (ToLCB) previous shown to be present in Oman. For the disease in tomato Koch's postulates were satisfied by Agrobacterium-mediated inoculation of virus and betasatellites clones. This showed the symptoms induced by the virus in the presence of the betasatellite to be enhanced, although viral DNA levels were not affected. ChLCV-OM is the fourth begomovirus identified in tomato in Oman and the first in Capsicum. The significance of these findings is discussed.

A novel recombinant tomato-infecting begomovirus capable of transcomplementing heterologous DNA-B components.

The genome of a tomato-infecting begomovirus from Ranchi, India, was cloned, sequenced and analysed. The viral genome shared 88.3% sequence identity with an isolate belonging to the species Tobacco curly shoot virus (TbCSV), and this virus should therefore be considered a member of a new species, tentatively named Tomato leaf curl Ranchi virus (ToLCRnV). The DNA-ß molecule, which had 74.5% sequence identity with tomato leaf curl Bangladesh betasatellite (ToLCBDB), is named tomato leaf curl Ranchi betasatellite (ToLCRnB). Phylogenetic analysis revealed that ToLCRnV is related to tomato leaf curl Bangladesh virus (ToLCBDV), tobacco curly shoot virus (TbCSV) and tomato leaf curl Gujarat virus (ToLCGV). An infectivity study with ToLCRnV established the monopartite nature of the viral genome, whereas inoculation with ToLCRnB resulted in increased symptom severity. ToLCRnV could transreplicate DNA-B of tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl New Delhi virus (ToLCNDV), both in N. benthamiana and tomato, although DNA-B accumulation of was less than with the wild-type combinations. ToLCRnB could be efficiently replicated by DNA-A of both ToLCNDV and ToLCGV. A leaf disk assay suggests that DNA-A could transreplicate the homologous DNA-B and DNA-ß more efficiently than the heterologous one.

Molecular characterization of a new species of Begomovirus and betasatellite causing leaf curl disease of tomato in India.

A new tomato-infecting begomovirus and cognate betasatellite were characterized from the gangetic plain of northern India. Genome organization of this virus was found to be similar to those of other old world begomoviruses. The DNA-A molecule (2752nt) shared maximum (85.8%) identity with Tomato leaf curl Laos virus-[Laos] (ToLCLV-[LA]; AF195782) from Laos and betasatellite molecule (1349nt) shared maximum (75.8%) identity with Tomato leaf curl Joydebpur betasatellite (ToLCJoB-[BD:Gaz:05]; AJ966244) from Bangladesh. Interestingly, both these molecules showed less identity with known tomato-infecting begomoviruses and their satellites from India. The recombination detection program (RDP) revealed that these molecules are not an outcome of direct exchange of sequences between existing begomovirus species. According to International Committee on Taxonomy of viruses (ICTV) species/strains demarcations norms for viruses belonging to the family Geminiviridae, this is a new Begomovirus species and we named this virus as Tomato leaf curl Patna virus (ToLCPaV) and new beta species as Tomato leaf curl Patna betasatellite (ToLCPaB). Partial tandem repeats of ToLCPaV and ToLCPaB could induce typical leaf curl symptom on tomato (Solanum lycopersicum) and Nicotiana benthamiana. Although, DNA-A could alone infect tomato typical to a monopartite Begomovirus, co-inoculation of DNA-A and DNA-beta resulted more stunting and severe symptoms. Interestingly, association of ToLCPaB did not assist in increased ToLCPaV accumulation in systemic leaves. ToLCPaV neither transreplicate DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) nor of Tomato leaf curl Gujarat virus (ToLCGV), presumably due to difference in rep-binding sequences. However, ToLCPaB formed viable pseudorecombinant with mono-bipartite ToLCGV DNA-A infecting both N. benthamiana and tomato but could not cause systemic infection on natural host tomato when co-inoculated with ToLCNDV DNA-A, which is a bipartite Begomovirus.