A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40?days after sowing (20?days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.     

Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus

A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program—one susceptible (S), one resistant (R—that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance.     

Hexose transporters of tomato: molecular cloning,expression analysis and functional characterization

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H⁺/hexose symporter. The K _m of the symporter for glucose is 45 M.     

Lower incidence and severity of tomato virus in elevated CO2 is accompanied by modulated plant induced defence in tomato

Elevation in atmospheric CO₂ concentration broadly affects plant phenology and physiology, and these effects may alter the performance of plant viruses. The effects of elevated CO₂ on the susceptibility of tomato plants to Tomato yellow leaf curl virus (TYLCV) were examined for two successive years in open top chambers (OTC) in the field. We experimentally tested the hypothesis that elevated CO₂ would reduce the incidence and severity of TYLCV on tomato by altering plant defence strategies. Our results showed that elevated CO₂ decreased TYLCV disease incidence (by 14.6% in 2009 and 11.8% in 2010) and decreased disease severity (by 20.0% in 2009 and 10.4% in 2010). Elevated CO₂ also decreased the level of TYLCV coat protein in tomato leaves. Regardless of virus infection, elevated CO₂ increased plant height and aboveground biomass. Additionally, elevated CO₂ increased the leaf C:N ratio of tomato, but decreased soluble protein content in leaves. Notably, elevated CO₂ increased the salicylic acid (SA) level in uninfected and infected plants. In contrast, elevated CO₂ reduced jasmonic acid (JA) in uninfected plants while it increased JA and abscisic acid (ABA) in virus‐infected plants. Furthermore, combined exogenous SA and JA application enhanced resistance to TYLCV more than application of either SA or JA alone. Our results suggest that the modulated antagonistic relationship between SA and JA under elevated CO₂ makes a great contribution to increased tomato resistance to TYLCV, and the predicted increases in tomato productivity may be enhanced by reduced plant virus susceptibility under projected rising CO₂ conditions.     

Differential responses between a vector species Bemisia tabaci and a nonvector species Trialeurodes vaporariorum following ingestion of tomato yellow leaf curl virus

In transmitting plant viruses, insect vectors undergo physiological and behavioral alterations. The whitefly Bemisia tabaci is a vector of tomato yellow leaf curl virus (TYLCV), causing severe damages to various horticultural crop plants. To determine whether whitefly alteration is specific to vector species, the responses to TYLCV ingestion were compared between B. tabaci and Trialeurodes vaporariorum, a nonvector for TYLCV. The two species were reared on TYLCV‐infected and noninfected tomato, a host of TYLCV, and their longevity and fecundity were determined while rearing in either tomato or eggplant, a nonhost of TYLCV. TYLCV‐ingested B. tabaci increased their developmental rates but reduced fecundity when they were reared in either tomato or eggplant compared with those of TYLCV‐free ones. In contrast, TYLCV‐ingested T. vaporariorum did not show any of the aforementioned changes when reared on both plant species. In addition, TYLCV‐ingested B. tabaci increased their levels of three heat shock protein genes ( hsp20, hsp70, and hsp90) against thermal stress, whereas TYLCV‐ingested T. vaporariorum did not. The presence of TYLCV virions was identified in two colonies of both species via polymerase chain reaction analysis. TYLCV was detected in the whole body, saliva, and eggs of B. tabaci, while TYLCV was detected only in the whole body but not in the saliva and eggs of T. vaporariorum. The present results strongly indicated that TYLCV specifically manipulate physiological processes of the vector species, B. tabaci.     

Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O₂ supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y

ADG2 is a DNA sequence mapped to a resistance (R) gene-rich region at the distal end of chromosome XI in potato (Solanum tuberosum subsp. andigena). The gene, in which ADG2 represents the predicted nucleotide-binding domain (NBS), was cloned and characterized. The coding region of the gene (designated as Y-1) is 6,187 bp long and structurally similar to gene N that confers hypersensitive resistance to Tobacco mosaic virus in Nicotiana spp. Both belong to the TIR-NBS-LRR class of genes and show 57% identity at the amino acid sequence level. The introns of Y-1 were spliced as predicted from the sequence. Y-1 cosegregated with Ry(adg), a gene for extreme resistance to Potato virus Y (PVY) on chromosome XI, as tested in a potato-mapping population and with independent potato cultivars. Leaves of the transgenic potato plants expressing Y-1 under the control of Cauliflower mosaic virus 35S promoter developed necrotic lesions upon infection with PVY, but no significant resistance was observed, and plants were systemically infected with PVY.     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.     

Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance

Tobacco mosaic virus-resistant tobacco was selected in vitro using callus tissues induced from axillary buds of systemically infected tobacco plants. Callus lines in which the virus was continuously multiplying were first isolated and redifferentiated into shoots. By the procedure, non-diseased, healthy shoots were successfully isolated from diseased shoots, which showed typical mosaic symptoms of the virus, and regenerated into intact plants.These regenerated plants showed resistance to virus inoculation, and selfed progeny of virus-resistant regenerants segregated the resistance and susceptibility according to the Mendelian system.     

Occurrence of whitefly-transmitted geminiviruses in crops in Burkina Faso, and their serological detection and differentiation

Whitefly-transmitted geminiviruses were found to be associated with four diseases of crop plants in Burkina Faso: cassava mosaic, okra leaf curl, tobacco leaf curl and tomato yellow leaf curl. Tomato yellow leaf curl is an economically serious disease, reaching a high incidence in March, following a peak population of the vector whitefly, Bemisia tabaci, in December. Okra leaf curl is also a problem in the small area of okra grown in the dry season but is not important in the main period of okra production in the rainy season. The geminiviruses causing these four diseases, African cassava mosaic (ACMV), okra leaf curl (OLCV), tobacco leaf curl (TobLCV) and tomato yellow leaf curl (TYLCV) viruses, were each detected in field-collected samples by triple antibody sand-wich-ELISA with cross-reacting monoclonal antibodies (MAbs) to ACMV. Epitope profiles obtained by testing each virus isolate with panels of MAbs to ACMV, OLCV and Indian cassava mosaic virus enabled four viruses to be distinguished. ACMV and OLCV had similar but distinguishable profiles. The epitope profile of TobLCV was the same as that of one form of TYLCV (which may be the same virus) and was close to the profile of TYLCV from Sardinia. The other form of TYLCV reacted with several additional MAbs and had an epitope profile close to that of TYLCV from Senegal. Only minor variations within each of these four types of epitope profile were found among geminivirus isolates from Burkina Faso. Sida acuta is a wild host of OLCV.     

Screening additional wild tomatoes for resistance to the whitefly-borne tomato yellow leaf curl virus

Plants of 25 wild Lycopersicon accessions were screened in the greenhouse for resistance to the whitefly-borne tomato yellow leaf curl virus (TYLCV). High levels of resistance were detected in 7 of 9 accessions of L. peruvianum and in all 5 accessions of L. chilense tested. In contrast, plants of 7 accessions of L. hirsutum and 3 of 4 accessions of L. pimpinellifolium were highly susceptible. Plants of accession CIAS 27 (L. pimpinellifolium) showed moderate resistance to TYLCV.     

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci— insights from studies with Tomato yellow leaf curl virus

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent unpublished data. We describe the cells and organs encountered by begomoviruses in B. tabaci. We show immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the descending midgut, and TYLCV‐specific labelling was also associated with food in the lumen. The microvilli and electron‐dense material in the epithelial cells of the gut wall were also labelled by the anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a putative site of long‐term virus storage. We describe the path of begomoviruses in their vector B. tabaci and in the non‐vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating, probably by exchange of haemolymph. We show that following a short acquisition access to infected tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is undetectable after a few hours in the non‐vector T. vaporariorum. The implications of the long‐term association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors are discussed.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.