Complete Nucleotide Sequence and Construction of an Infectious Clone of a Tunisian Isolate of Tomato yellow leaf curl Sardinia virus

The full‐length genome of a Tunisian isolate of Tomato yellow leaf curl Sardinia virus (TYLCSV) was engineered and submitted to sequence analysis. The Tunisian isolate has 99% sequence identity with TYLCSV‐Sicilian (Sic), results thus providing further evidence for the inclusion of this isolate in the TYLCSV‐Sic group. A 1.7‐mer construct of the virus was obtained and efficiently agroinfiltrated into tomato and tobacco plants to induce symptoms indistinguishable from those of natural infection.     

Identification and Analysis of Resistance‐like Genes in the Tomato Genome

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world. However, the tomato production is severely affected by many diseases. The use of host resistance is believed to be the most effective approach to control the pathogens. In this study, a total of 1003 resistance‐like genes were identified from the tomato genome using individual full‐length search and conserved domain verification approach. Of the predicted resistance genes, serine/threonine protein kinase was the largest class with 384 genes followed by 212 genes encoding receptor‐like kinase, 107 genes encoding receptor‐like proteins, 68 genes encoding coiled‐coil–nucleotide‐binding site (NBS)–leucine‐rich repeat (LRR) and 19 genes encoding Toll interleukin‐1 receptor domain‐NBS‐LRR. Physical map positions established for all predicted genes using the tomato WGS chromosomes SL2.40 information indicated that most resistance‐like genes clustered on certain chromosomal regions. Comparisons of the sequences from the same resistance‐like genes in S. pimpinellifolium and S. lycopersicum showed that 93.5% genes contained single nucleotide polymorphisms and 19.7% genes contained insertion/deletion. The data obtained here will facilitate isolation and characterization of new resistance genes as well as marker‐assisted selection for disease resistance breeding in tomato.     

Distribution of tomato-infecting begomoviruses and Bemisia tabaci biotypes in Morocco

Tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) as well as their whitefly vector Bemisia tabaci were reported from the south‐west and central regions of Morocco. To establish a more comprehensive view of tomato begomoviruses and B. tabaci biotypes throughout Morocco, 32 tomato fields were surveyed for tomato yellow leaf curl disease (TYLCD) symptoms in southern and northern regions, and 54 samples of leaves from cultivated plants or weeds and 35 B. tabaci individuals were collected and analysed by PCR, randomly amplified polymorphic DNA and sequencing. Only TYLCV or TYLCSV were detected. TYLCV was detected in 15 plant samples whereas TYLCSV only in 4. Sequence analyses revealed the presence of the ‘Spanish’ strain of TYLCSV and distinguished two genetically distinct strains of TYLCV. The begomovirus infections were unevenly distributed throughout Morocco. In the north‐west and north‐central regions where tomato plants exhibiting TYLCD symptoms were rarely observed, only 1 sample out of 13 tested positive for the presence of a begomovirus. In the north‐east region, the ratio of begomovirus‐positive samples was higher, 6/13, and in the south‐west region, it was the highest, 13/14. Consistently the frequency of plants exhibiting TYLCD‐like symptoms in the northern regions was lower than that in the south‐west region. B. tabaci biotype Q is present throughout the country and in Algeria, whereas biotype B, identified for the first time in Morocco, was detected only in the north‐east region.     

Preference of the vector thrips Frankliniella occidentalis for plants infected with thrips‐non‐transmissible Tomato spotted wilt virus

The effect of a thrips‐non‐transmissible Tomato spotted wilt virus (TSWV) on insect–host interactions between thrips and Arabidopsis thaliana was analysed. A wild‐type TSWV virulent isolate and a TSWV isolate that induces mild symptoms on inoculated plants (TSWV‐Mo) were used in this study, and TSWV‐Mo isolate was obtained by single local lesion isolation using Petunia x hybrid after several passages on Nicotiana rustica plants. In transmission test, although wild‐type TSWV (TSWV‐wt) was transmitted by two thrips species (transmission ratio; Frankliniella occidentalis, 25%; Thrips tabaci, 10%; and T. palmi, 0%), none of the thrips transmitted TSWV‐Mo. Feeding damage by F. occidentalis in A. thaliana plants was more extensive on TSWV‐wt‐infected plants than on TSWV‐Mo‐infected plants, despite comparable preference. Among the markers of plant defences, salicylic acid‐regulated genes were upregulated threefold to sixfold by TSWV‐wt or TSWV‐Mo infection. In contrast, jasmonate‐regulated genes and jasmonate/ethylene‐regulated genes were not affected by the infections. Pull assays showed that adjacent TSWV‐Mo‐infected plants were preferred over uninfected plants. In conclusion, our results showed that the transmissibility by thrips of TSWV is not related to preference of vector thrips and suggested that TSWV‐Mo‐infected plants may be used as attractants for behaviour control of thrips.     

Truncated Rep gene originated from Tomato yellow leaf curl virus-Israel [Mild] confers strain-specific resistance in transgenic tomato

Transgenic tomato plants carrying a truncated replication associated protein (T‐Rep) gene of the mild strain of Tomato yellow leaf curl virus‐Israel (TYLCV‐Is [Mild]) were prepared. The transgene encoding the first 129 amino acids of Rep conferred resistance only against the virus strain from which it was derived, while these plants were susceptible to the severe strain of TYLCV‐Is. This strain‐specific effect may be the result of high sequence divergence within the N‐terminal domains of the Rep genes of the two virus isolates which share a mere 78% sequence identity at the nucleotide level and 77% at the amino acid level. Although the transgenic tomato plants were totally resistant to whitefly inoculation with the mild strain of TYLCV‐Is, agroinoculation with the same virus strain resulted in variable resistance responses in the tested plants: while 21% of plants were totally immune to the virus, 33% were susceptible and 46% expressed a wide range of intermediate resistance characteristics. The applicability of TYLCV‐Is derived resistance in tomato is discussed.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus‐encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro‐Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2‐green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2‐GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)‐C2 displayed chlorotic lesions and stunted growth. PVX‐C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host‐defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3–2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3–2 gene and pNbCMT3–2::GUS (β‐glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation.     

Development of plants resistant to tomato geminiviruses using artificial trans‐acting small interfering RNA

RNA interference (RNAi), a conserved RNA‐mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter‐strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down‐regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans‐acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single‐step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro‐infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro‐infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA‐generating constructs, and T₁ plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.     

DNA helicase activity is associated with the replication initiator protein rep of tomato yellow leaf curl geminivirus

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3' overhang and 3'-to-5' polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.     

Interaction between a geminivirus replication protein and the plant sumoylation system

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 (N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.