Combining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentum mill)

This study was undertaken to develop tomato plants with broad resistanceto tospoviruses which are a major limiting factor to tomato productionworldwide. A nontransgenic tomato line Stevens-Rodale (S-R), six transgenictomato lines expressing the nucleocapsid (N) protein gene of the lettuceisolate of tomato spotted wilt virus (TSWV-BL), and progeny of the crosses between S-Rand three of the transgenic lines homozygous for the N gene were evaluated fortheir resistance to tospovirus infection in greenhouse inoculation tests. S-Rhas the Sw-5 gene that confers resistance to several TSWVisolates. The six transgenic lines showed high levels of resistance wheninoculated with either TSWV-BL or a tomato isolate from Hawaii (TSWV-H).However, these same plants were highly susceptible to the Brazilian isolate ofgroundnut ringspot virus (GRSV-BR). Plants with the Sw-5gene were resistant to TSWV-BL and GRSV-BR, but were susceptible to TSWV-H.When inoculated with any of the three viruses, the F₁ progeny of thecrosses exhibited a susceptible, tolerant, or resistant phenotype with a higherproportion of the plants being either tolerant or resistant. When F₂progeny from F₁ resistant plants of each cross were inoculated withany of the three viruses, a higher proportion of tolerant and resistant plantswas observed compared to the F₁ progeny. Our results show thepotential to obtain broad resistance to tospoviruses by combining transgenicand natural resistance in a single plant.     

Density thresholds for Nesidiocoris tenuis (Heteroptera: Miridae) in tomato crops

Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) is common in vegetable crops of the Mediterranean area, with an increasing worldwide range of geographical distribution. This omnivore is a reputed predator of small arthropod pests, but also produces injuries on vegetative and reproductive plant parts. The aim was to estimate density thresholds based on N. tenuis and whitefly abundance for the management of N. tenuis in tomato crops. The assay was carried out in mesh-walled and plastic greenhouses in southern Spain during 2004 and 2007. The natural population dynamics of N. tenuis and whitefly were monitored, and impact on yield quantified. The economic injury level and intervention threshold were predicted based on the zoophytophagous response of N. tenuis and the yield compensation of tomato plants. The proportion of aborted flowers on the tomato plants was related directly to the abundance of N. tenuis and inversely to the interaction between the number of N. tenuis and the number of whitefly immatures. Over-compensation of fruit weight was predicted for flower abortion rates due to N. tenuis lower than 0.171. No yield reduction is expected for values <0.65 N. tenuis per leaf, independent of the whitefly abundance, nor for up to 5 N. tenuis and >26 whitefly immatures per leaf. For intermediate N. tenuis levels, the outcome depends on the prey density. The probability of N. tenuis producing yield loss in tomato crops increases at N. tenuis:whitefly ratios >0.168. Yield reduction is expected after N. tenuis population peaks, when whitefly numbers have been reduced.     

Effect of nitrogen supply rate on disease resistance in tomato depends on the pathogen

The aim of this study was to investigate the effect of tissue nitrogen concentration, as a consequence of nitrogen supply rate, on the susceptibility of tomato plants to three pathogens. We varied tissue N concentration by supplying N at different rates by adding nitrate in different, exponentially increasing amounts to the nutrient solution on which the tomato plants were grown. Separate experiments were carried out to test susceptibility of tomato plants to the bacterial speck-causing Pseudomonas syringae pv tomato, to the wilt agent Fusarium oxysporum f.sp. lycopersici and to tomato powdery mildew caused by Oidium lycopersicum. The effect of tissue N concentration appeared to be highly pathogen-dependent: there was no effect on susceptibility to F. oxysporum, but susceptibility to P. syringae and O. lycopersicum increased significantly with increasing N concentration. We have previously demonstrated the opposite for susceptibility to Botrytis cinerea: decreasing susceptibility with increasing N concentration. The apparent contradictory effects are discussed in relation to the effect of N supply on both the nutritional value of the plant tissue to the pathogen and on the concentration of resistance-related compounds. We conclude that the effect of changing both characteristics on disease susceptibility is highly pathogen-specific and is probably dependent on differences in resource requirements of the pathogen or the sensitivity of the pathogen to plant resistance reactions or on both these factors. This revised version was published online in August 2006 with corrections to the Cover Date.     

High resolution RFLP map around the root knot nematode resistance gene (Mi) in tomato

Summary In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F₂ plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers — GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome — a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.     

Response of tomato genotypes to bacterial speck (Pseudomonas syringae pv. tomato)

Two genotypes of tomato A 100 and Ontario 7710 which were inoculated separately with four strains of Pseudomonas syringae pv. tomato differed significantly in disease severity (susceptibility) to bacterial speck. At both concentrations of inoculum of each strain used (10⁷ and 10⁸ cfu/ml) A 100 appeared to be highly susceptible whereas Ontario 7710 showed very low or no susceptibility. The significant differences in virulence between strains and in response of tomato plants in three replicate experiments were found. Generally, concentration of inoculum 10⁷ cfu/ml was too low to induce consistent level of disease severity. The obtained results indicate the importance of consistent and favorable conditions for disease development in screening of tomato resistance to bacterial speck.     

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2² resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2² resistance gene and the susceptible allele, tm-2. The Tm-2² gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2² gene survive. The Tm-2² locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2² sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2² allele. Between the tm-2 and the Tm-2² polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2² gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2² resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2²-encoded resistance is aimed at the Achilles' heel of the virus.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

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     

Engineering tomato for resistance to tomato leaf curl disease using viral <Emphasis Type="Italic">rep</Emphasis> gene sequences

Transgenic tomato resistant to tomato leaf curl disease (ToLCD) using replicase (rep) gene sequences of Tomato leaf curl virus in antisense orientation were developed via Agrobacterium-mediated transformation. A binary vector carrying the antisense rep gene (untranslatable full length sequence, 1086 bp) along with the npt II gene was used for transformation. High level of resistance and inheritability of the transgene was observed up to T₂ stage following challenge inoculation with the virus. The mechanism of resistance appears RNA-mediated, since the plants carried the untranslatable antisense rep gene. Progeny analysis of these plants showed classical Mendelian pattern of inheritance in two of the six transgenic lines having single transgene insertion.     

The tomato gene Sw5 is a member of the coiled coil,nucleotide binding,leucine-rich repeat class of plant resistance genes and confers resistance to TSWV in tobacco

Tomato spotted wilt virus is an important threat to tomato production worldwide. A single dominant resistance gene locus, Sw5, originating from Lycopersicon peruvianum, has been identified and introgressed in cultivated tomato plants. Here we present the genomic organization of a 35 250 bp fragment of a BAC clone overlapping the Sw5 locus. Two highly homologous (95%) resistance gene candidates were identified within 40 kb of the CT220 marker. The genes, tentatively named Sw5-a and Sw5-b, encode proteins of 1245 and 1246 amino acids, respectively, and are members of the coiled-coil, nucleotide-binding-ARC, leucine-rich repeat group of resistance gene candidates. Promoter and terminator regions of the genes are also highly homologous. Both genes significantly resemble the tomato nematode and aphid resistance gene Mi and, to a lesser extent, Pseudomonas syringae resistance gene Prf. Transformation of Nicotiana tabacum cv. SR1 plants revealed that the Sw5-b gene, but not the Sw5-a gene, is necessary and sufficient for conferring resistance against tomato spotted wilt virus.     

Characterization of induced resistance in tomato plants

We have characterized, using several types of bioassays, the resistance induced in young tomato plants by feeding of the corn earworm, Helicoverpa zea. Beet armyworm larvae, Spodoptera exigua, and leafminers, Liriomyza trifolii, were used to assay the induced resistance. In whole-plant experiments, damage localized to a single leaflet of fourleaf tomato plants induced a systemic increase in resistance such that beet armyworm larvae confined to previously damaged (induced) plants grew at a rate about half that of larvae raised on control plants and consumed less leaf tissue from induced plants than from control plants. In experiments using excised leaves, beet armyworm larvae suffered increased mortality when reared on leaves from induced plants. The strength of this induced resistance varied spatially relative to the damaged position; moreover, the spatial distribution of induced resistance changed over a three-week period following damage. Other experiments demonstrated that the mechanisms of induced resistance in tomato foliage involves both a decrease in larval preference for and a decrease in the nutritional value of induced foliage. Induction also retarded the oviposition and/or early development of leafminers. Thus, induced resistance has relatively severe effects on the biology of subsequent herbivores. These data should allow us to begin to elucidate cause-effect relationships between induced resistance and induced chemistry in tomato plants.     

番茄中Rpi-blb2同源基因的挖掘与鉴定

番茄晚疫病是番茄生产中的主要病害之一,经常会造成较大的经济损失。晚疫病生理小种的变异和进化常会导致番茄品种原有的遗传抗性丧失,因此不断挖掘新的抗性基因,改良番茄晚疫病抗性是番茄抗病育种的长期任务。该研究采用BLAST同源比对的方法,以马铃薯野生近缘种的晚疫病抗性蛋白序列Rpi-blb2为种子序列,在NCBI蛋白质序列数据库中检索得到11条番茄蛋白质序列,这些序列与种子序列相似性为78%~83%,属于番茄疾病抗性蛋白家族,并对该家族成员进行了基因结构、基因定位、序列保守结构域和进化关系等分析。结果表明:该家族中10条序列分布在第Ⅵ条染色体上,1条分布在第Ⅴ染色体上;6号染色体上的10序列呈现2个抗病基因簇分布,在染色体上分别占据2个和3个基因位点;10条同源蛋白是Rpi-blb2的共同垂直同源蛋白,但不具有平行同源关系,大多数成员定位于细胞质。按照蛋白质保守结构域和基因定位的不同可分为三类,第一类共4条系列,包含有DUF3542和NB-ARC两个保守结构域特征序列;第二类共6条序列,与马铃薯Rpi-blb2蛋白一样,仅包含NB-ARC保守结构域特征序列,在这2类蛋白序列的NB-ARC结构域均位于序列中部;第三类(仅包含XP_004239406.1)虽然也具有与第一类蛋白相似的DUF3542和NB-ARC结构域,但在结构域两端的非保守区序列较短,且位于5号染色体上,因此将其单独归为1类。前两类蛋白成员相应的基因具有1~2个内含子,第3类蛋白不含内含子。该研究结果为利用生物技术选育番茄抗性品种提供了理论基础。     

Interaction of fertilizer regime with host-plant resistance in tomato

The effect of fertilizer regime on trichome- and lamellar-based resistance in the wild tomato species, Lycopersicon hirsutum f. glabratum C.H. Mull accession PI 134417, to three insect pests of tomato, the tobacco hornworm, Manduca sexta (L.), the colorado potato beetle, Leptinotarsa decemlineata (Say), and the tomato fruitworm, Heliocoverpa zea (Boddie), was examined. Increasing the rate at which NPK fertilizer was applied, from 1.8 to 19.6 g/plant/week, reduced the trichome-based resistance of PI 134417 to M. sexta and L. decemlineata by lowering both the density of type VI (sensu Luckwill, 1943) glandular trichomes and the amount of 2-tridecanone contained in the tips of these trichomes. 2-Tridecanone is a toxic methyl-ketone responsible for glandular trichome-mediated resistance in PI 134417 to M. sexta and L. decemlineata. A similar increase in the application rate of NPK fertilizer reduced the lamellarbased resistance of PI 134417 to L. decemlineata and H. zea. The meachanisms for this reduction of resistance are unknown, but may be related to improved nutritional quality of hosts at higher fertilizer regimes.     

Genetic and physical mapping of the lateral suppressor (ls) locus in tomato

Tomato plants homozygous for the recessive lateral suppressor (ls) mutation show a number of phenotypic abnormalities among which the lack of lateral meristem initiation during vegetative growth and the absence of petals on the flower are the most prominent. As a first step towards the isolation of the Ls gene by means of map-based cloning, we have determined its position on the restriction fragment length polymorphism (RFLP) map of tomato. RFLP analysis of 527 F2 plants segregating for the ls allele allowed us to define an interval of 0.8 cM in which the Ls gene is located. Analysis of the physical distance between the two flanking RFLP markers by pulsed field gel electrophoresis revealed that they lie no further than 375 kb apart. Knowledge of the physical distance together with the availability of a tomato yeast artificial chromosome (YAC) library, makes it feasible to isolate the Ls gene by a map-based cloning approach.     

Use of the maize transposonsActivator andDissociation to show that phosphinothricin and spectinomycin resistance genes act non-cell-autonomously in tobacco and tomato seedlings

Cell-autonomous genes have been used to monitor the excision of both endogenous transposons in maize andAntirrhinum, and transposons introduced into transgenic plants. In tobacco andArabidopsis, the streptomycin phosphotransferase (SPT) gene reveals somatic excision of the maize transposonActivator (Ac) as green sectors on a white background in cotyledons of seedlings germinated in the presence of streptomycin. Cotyledons of tomato seedlings germinated on streptomycin-containing medium do not bleach, suggesting that a different assay for transposon excision in tomato is desirable. We have tested the use of the spectinomycin resistance (SPEC) gene (aadA) and a Basta resistance (BAR) gene (phosphinothricin acetyltransferase, or PAT) for monitoring somatic excision ofAc in tobacco and tomato. Both genetic and molecular studies demonstrate that genotypically variegated individuals that carry clones of cells from whichAc orDs have excised from either SPEC or BAR genes, can be phenotypically completely resistant to the corresponding antibiotic. This demonstrates that these genes act non-cell-autonomously, in contrast to the SPT gene in tobacco. Possible reasons for this difference are discussed.     

The effects of mullein plants (Verbascum thapsus) on the population dynamics of Dicyphus hesperus (Heteroptera: Miridae) in tomato greenhouses

The response of Dicyphus hesperus Knight (Heteroptera: Miridae) to whitefly populations in tomato greenhouses was measured in the presence and absence of mullein (Verbascum thapsus L.) as an alternative host plant. The dynamics of the D. hesperus population on tomato (Lycopersicon esculentum Mill.) and on mullein plants were followed through an entire growing season. In houses with mullein plants, more predators occurred on mullein when whitefly density was low on tomato. A mark-release-recapture experiment where rabbit IgG was used as an external marker showed that D. hesperus adults moved from mullein plants to tomato plants. D. hesperus was always more abundant in houses with mullein than in the houses with tomato plants alone. Movements between tomato and mullein plants are discussed as a strategy to optimize predator foraging. The use of mullein as an alternative host plant may contribute to the establishment of D. hesperus and help to preserve the predator population when prey on tomato crops is scarce.     

Position of the reduced mycorrhizal colonisation (<Emphasis Type="Italic">Rmc</Emphasis>) locus on the tomato genome map

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.