Faculty of Agriculture, Food and Environmental Quality Sciences, HUJI

Faculty of Agriculture, Food and Environment Quality Sciences

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture

Home

Tel: 972-8-948-9251
Fax: 972-8-948-9899
E-mail: shimony@agri.huji.ac.il

<- Hanokh Czosnek Home

ABSTRACTS OF PUBLICATIONS (1997-2009)

Czosnek H and Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses. Archives of Virology 142:1391-1406.

The name tomato yellow leaf curl virus (TYLCV) has been given to several whitefly-transmitted geminiviruses affecting tomato cultures in many tropical and subtropical regions. Hybridization tests with two DNA probes derived from a cloned isolate of TYLCV from Israel (TYLCV-ISR) were used to assess the affinities of viruses in naturally infected tomato plants with yellow leaf curl or leaf curl symptoms from 25 countries. Probe A which included most of the intergenic region was expected to detect only isolates closely related to TYLCV-ISR, especially after high stringency washes. In contrast probe B, which included the full-length genome, was expected to detect a wide range of whitefly-transmitted geminiviruses. Tomato samples from six countries in the Middle East, from Cuba or the Dominican Republic proved to be closely related to TYLCV-ISR and probably were infected by strains of the same virus. Samples from Senegal and Cape Verde Islands were also related to the Middle Eastern virus. Samples from nine other countries in the western Mediterranean area, Africa, or South-East Asia were more distantly related and probably represent one or more additional geminivirus species. Samples from five countries in Africa, Central or South America gave hybridization signals with the full-length viral genome, only after low stringency wash, indicating that these samples were infected by remote viruses. These results were supported by DNA and protein sequence comparison, which indicate that tomato geminiviruses fall into three main clusters representing viruses from 1) the Mediterranean / Middle East / African region, 2) India, the Far East and Australia, and 3) the Americas. Within the first cluster, two sub-clusters of viruses from the western Mediterranean or from the Middle East / Caribbean Islands were distinguished. The incidence of tomato yellow leaf curl diseases has increased considerably between 1990 and 1996.

Rubinstein G and Czosnek H (1997) Long-term association of tomato yellow leaf curl virus (TYLCV) with its whitefly vector Bemisia tabaci: effect on the insect transmission capacity, longevity and fecundity. Journal of General Virology 78:2683-2689.

The association between tomato yellow leaf curl geminivirus (TYLCV, Israeli isolate) and its insect vector, the whitefly Bemisia tabaci, was investigated. Insects that emerged during a 24 h period were caged with TYLCV-infected plants for a 48 h acquisition access period, then with eggplants - a TYLCV non-host - for the rest of their lives. While TYLCV DNA was associated with the whiteflies during their entire adult life, the amount of capsid protein rapidly decreased and was no more detectable in the insect after approximately 12 days of age. The ability of the infected whiteflies to transmit TYLCV to tomato test plants steadily decreased with age but did not disappear completely. Transmission by viruliferous insects decreased from 100% to 10-20% during their adult life time, compared with 100% to 50% for non-viruliferous insects. The association of TYLCV with adult B. tabaci lead to a reduction of 17 to 23% in their life expectancy compared with insects that have not acquired the virus, and to a 40 to 50% decrease in the mean number of eggs laid. These results suggest that TYLCV has some features reminiscent of an insect pathogen.

Ghanim M, Morin S, Zeidan M and Czosnek H (1997) Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector the whitefly Bemisia tabaci. Virology 240:295-303.

The whitefly Bemisia tabaci is the only vector of the tomato yellow leaf curl geminivirus (TYLCV). The insect transmits the virus in a persistent-circulative manner. TYLCV DNA was detected by PCR and by Southern blot hybridization in progeny (eggs, first and second instars, adults) of single viruliferous whiteflies that developed on eggplant or on cotton (two TYLCV non-host plants). Furthermore TYLCV DNA was present in progeny of the insects that have acquired the virus through the egg. The adult progeny of the viruliferous insects and their own progeny were able to infect tomato test plants, producing typical disease symptoms. Ovaries and maturing eggs of viruliferous insects contained viral DNA as did eggs laid by viruliferous insects on artificial diet. Eggs laid by non-viruliferous whiteflies on cotton plants previously caged with viruliferous insects did not acquire viral DNA from the plant. Hence, TYLCV can be transmitted through the egg for at least two generations. In the absence of an available plant host, the whitefly may serve as a reservoir of virus between growing seasons.

Kunik T, Palanichelvam K, Czosnek H, Citovsky V and Gafni Y (1997) Nuclear Import of a geminivirus capsid protein in plant and Insect Cells: Implications for the Viral Nuclear Entry. The Plant Journal 13:121-129.

Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of the Tomato Yellow Leaf Curl Virus (TYLCV) capsid protein (CP). This protein is the only known protein that serves as a component of the viral coat. The complete sequence of the capsid protein gene was joined to the 3' end of the entire b-glucoronidase (GUS) gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into petunia protoplasts followed by in situ localization of b-glucoronidase activity. When complete sequence of the TYLCV capsid protein was fused to b-glucoronidase, normally a cytoplasmic protein, nuclear location of the fused protein was observed. This was also the case when the first 30 amino acids of the 260 amino acids of the capsid protein were fused to -glucoronidase. One functionally active, bipartite, nuclear localization signal (NLS) have been identified in the N-terminal side of the TYLCV capsid protein. Deletion mutagenesis demonstrated that this single NLS is sufficient for targeting the corresponding TYLCV CP/b-glucoronidase chimera into the nucleus. Removal of this sequence completely blocked the nuclear import of the resulting TYLCV CP/b-glucoronidase fusion protein, which instead became evenly distributed in the cytoplasm. When the capsid protein alone was fluorescently labeled and microinjected into Drosophila embryos, the protein localized to the cell nuclei. These observations characterize the TYLCV CP as a karyophilic protein in both plant and insect cells. We suggest a role of the CP in nuclear import of the virus both in plants and in insects.

Michelson I, Zeidan M, Zamski E, Zamir D and Czosnek H (1997) Localization of tomato yellow leaf curl virus (TYLCV) in susceptible and tolerant nearly isogenic tomato lines. Acta Horticulturae 447:407-414.

The Israeli isolate of tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted monopartite geminivirus. We have developed two nearly isogenic tomato breeding lines. Line 52 containing the TYLCV tolerance allele Ty-1, introgressed from the wild tomato species Lycopersicon chilense was tolerant to TYLCV; line 50 containing the ty-1 L. esculentum allele, was sensitive to the virus. The effect of Ty-1 on the establishment of the TYLCV disease was studied by comparing the structural changes and the location of the virus in tissue sections of inoculated leaves from plants of the two lines. A collapse of the spongy mesophyll cells of the susceptible line (but not of the tolerant line) was the first striking cytopathy observed two weeks after inoculation. TYLCV was located to these collapsed cells by in situ hybridization. TYLCV was also found in the phloem and mesophyll cells of both lines, indicating that this virus is not phloem limited in tomato.

Atzmon G, van Hoss H and Czosnek H (1998) PCR-amplification of tomato yellow leaf curl virus (TYLCV) from squashes of plants and insect vectors: application to the study of TYLCV acquisition and transmission. European Journal of Plant Pathology 104:189-194.

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm² were squashed onto a nylon membrane. A 1x2 mm strip containing the squash was introduced into a 25 ml PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.

Vidavsky F and Czosnek H (1998) Tomato breeding lines immune and tolerant to tomato yellow leaf curl virus (TYLCV) issued from Lycopersicon hirsutum. Phytopathology 88:910-914.

Two TYLCV-resistant plants from accessions LA 1777 and LA 386 of the wild tomato species Lycopersicon hirsutum have been crossed. The resulting resistant F1 plants were crossed with the domesticated tomato L. esculentum and a series of selfing was performed. At each generation, individuals were selected for resistance (no symptoms and undetectable viral DNA) and tolerance (no symptoms but with detectable viral DNA), following controlled massive and repeated inoculations with viruliferous whiteflies. A stable BC1F4 line (denominated 902) which does not segregate for resistance was obtained. This line does not support virus accumulation even upon extensive whitefly-mediated inoculation of young seedlings and does not need protection with nets and/or insecticides. Another stable BC1F4 line (denominated 908) was tolerant to the virus. Both lines have good horticultural characteristics and bear 80-120 g red fruits. Analysis of segregation of susceptibility, tolerance and resistance during the BC1F1 to BC1F4 crosses indicated that tolerance is controlled by a dominant major gene and resistance by 2 to 3 additive recessive genes. The resistant and tolerant lines do not need to be protected by insecticides and/or nets.

Morin S, Ghanim M, Zeidan M, Czosnek H, Verbeek M and van den Heuvel JFJM (1999) A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of Tomato yellow leaf curl virus. Virology 30:75-84.

Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A ~63kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that TYLVC particles displayed a specific affinity for the B. tabaci 63 kDa GroEL homologue. Feeding whiteflies with anti-Buchnera GroEL antiserum prior to acquisition of virions reduced TYLCV transmission to tomato test plants by more than 80%. In the haemolymph of these whiteflies TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph.

Rubinstein G, Morin S and Czosnek H (1999) Long-term effect of imidacloprid on mortality of the whitefly Bemisia tabaci caged with treated eggplant and tomato, and on transmission of tomato yellow leaf curl geminivirus (TYLCV) to tomato. Journal of Economical Entomology 92:658-662.

The ability of whiteflies (Bemisia tabaci Gennadius, B biotype) to transmit tomato yellow leaf curl geminivirus (TYLCV, Israeli isolate) to imidacloprid-treated and untreated tomato plants was investigated. Viruliferous whiteflies were caged with treated tomato plants held in a nethouse. Insect mortality and tomato infection was assessed during the summer and the winter seasons, following insecticide application. In summer, insects that were given access to tomato plants 3 and 11 d after insecticide treatment died within 80 min. This period increased to 150 min 18 d after treatment. The insecticide lost its potency 25 d after application. In winter the lethal effect of the insecticide lasted longer than in summer. In summer as well as in winter, 70% of the tomato plants caged with viruliferous whiteflies 3 d after insecticide treatment became infected. These figures increased to 80% 11 d after treatment and to nearly 100% 18 d after treatment. Insecticide-treated plants were as prone to infection as non-treated plants. Approximately 48 min of access to a treated tomato plant was sufficient for a single viruliferous insect to inoculate the virus with an efficiency rate of 20% or more. Transmission efficiency was similar to that achieved on non-treated plants. Therefore, viruliferous insects had enough time to inoculate TYLCV to imidacloprid-treated plants before they died.

Ghanim M and Czosnek H (2000) Tomato yellow leaf curl geminivirus (TYLCV-Is) is transmitted among whiteflies (Bemisia tabaci) in a sex-related manner. Journal of Virology 74: 4738-4745, 2000.

Tomato yellow leaf curl virus (TYLCV) is the name given to a complex of geminiviruses infecting tomato cultures worldwide. TYLCV is transmitted by a single insect species, the whitefly Bemisia tabaci. Herein we show that a TYLCV isolate from Israel (TYLCV-Is) can be transmitted among whiteflies in a sex-dependant manner, in the absence of any other source of virus. TYLCV was transmitted from viruliferous males to females and from viruliferous females to males, but not among insects of the same sex. Transmission took place when insects were caged in groups or in couples, in a feeding chamber or on cotton plants, a TYLCV-non-host. The recipient insects were able to efficiently inoculate tomato test plants. Insect to insect virus transmission was instrumental in increasing the number of whiteflies capable of infecting tomato test plants in a whitefly population. TYLCV was present in the haemolymph of whiteflies caged with viruliferous insects of the other sex; therefore the virus follows, at least in part, the circulative pathway associated with acquisition from infected plants. Taken as a whole, these results implicate that a plant virus can be sexually transmitted among its insect vector.

Morin S, Ghanim M, Sobol I, and Czosnek H (2000) The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of transmissible and non-transmissible begomoviruses in the yeast two-hybrid system. Virology 276:404-416.

We have previously suggested that a GroEL homologue produced by the whitefly Bemisia tabaci endosymbiotic bacteria interacts in the insect haemolymph with particles of Tomato yellow leaf curl virus from Israel (TYLCV-Is), ensuring the safe circulative transmission of the virus. We have now addressed the question of whether the non-transmissibility of Abutilon mosaic virus from Israel (AbMV-Is) is related to a lack of association between GroEL and the virus coat protein (CP). Translocation analysis has shown that while TYLCV-Is DNA is conspicuous in the digestive tract, haemolymph and salivary glands of B. tabaci 8 h after acquisition feeding started, AbMV-Is DNA was detected only in the insect digestive tract, even after 96 h. To determine whether AbMV-Is particles were rapidly degraded in the haemolymph as a result of their inability to interact with GroEL, we have isolated a GroEL gene from B. tabaci and used a yeast two-hybrid assay to compare binding of the CP of TYLCV-Is and AbMV-Is to the insect GroEL. The yeast assay showed that the CPs of the two viruses are able to bind efficiently to GroEL. We therefore suggest that, although GroEL-CP interaction in the haemolymph is a necessary condition for circulative transmission, the non-transmissibility of AbMV-Is is not due to lack of binding to GroEL in the B. tabaci haemolymph, but most likely to an inability to cross the gut/haemolymph barrier.

Ghanim M, Rosell RC, Campbell LR, Czosnek H, Brown JK and Ullman DE (2001) Digestive, Salivary and Reproductive Organs of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Biotype B. Journal of Morphology. 248:22-40.

A microscopic analysis of the morphology and ultrastructure of the digestive, salivary and reproductive systems of adult Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type was conducted using light, scanning and transmission electron microscopy. The internal anatomy of B. tabaci was found to be similar to that reported for Trialeurodes vaporariorum. In a microscopic analysis of the salivary glands, we have shown that each primary salivary gland is composed of at least 13 cells varying in morphology and staining differentially, while the accessory salivary glands are composed of four morphologically similar cells. We have analyzed the course of the alimentary canal in B. tabaci, demonstrated the internal morphology of the organs and clarified the location of the filter chamber relative to other organs in the whitefly. Our observations confirm that the pair of structures extending from the connecting chamber are caeca that may aid in fluid movement through the midgut and are not malphighian tubules as previously suggested. We confirm an earlier finding that the whitefly lacks Malphigian tubules, having instead, specialized Malphigian-like cells within the filter chamber at the juncture with the internal ileum. Finally, we provide the first scanning electron microscopic analysis showing the reproductive organs of B. tabaci. Our investigation provides clarified terminology for several components of the digestive and excretory system. We also provide drawings and micographs that will aid future researchers in localizing the internal organs of B. tabaci. We expect our analysis to provide a valuable tool for studying B. tabaci/plant virus interactions and physiological and biological aspects of this insect.

Czosnek H., Ghanim M., Rubinstein G, Morin S, Fridman V and Zeidan M (2001) Whiteflies: vectors or victims ? of geminiviruses. In Avances in Virus Research , Maramorosch K Ed., Academic Press. Vol 57, pp. 291-322.

Most of viruses infecting plants rely on insects to move from one host to another. Some remain associated with the mouth parts and can be inoculated within seconds or minutes (non-circulative transmission). Others are transmitted only several hours after acquisition (circulative transmission). During this latent period, virions translocate into the insect alimentary tract, cross the gut into the primitive blood stream and reach the salivary glands. Contrary to many circulative mammalian viruses, circulative plant viruses by en large do not replicate in their arthropod vector and apparently do not adversely affect their insect host. Nonetheless several plant viruses have evolved, or conserved, mechanisms allowing replication (circulative propagative) in both plant and animal hosts. Three of the five families of the phytoarbovirus taxonomic group, the rhabdoviruses, reoviruses and bunyaviruses, contain members which do replicate in both their host plants and in their aphid or leafhopper vector. In addition, the tomato spotted wilt tospovirus replicates in thrips. Most of these viruses depend on their arthropod host for survival and do not adversely affects them. However a number of phytoarboviruses are deleterious and impair the insect longevity and fecundity. Virus replication and deleterious effects on the insect host have been associated with transovarial transmission to insect progeny. In most cases it is not known whether the virus genes necessary for replication in plants and in insects are the same, and whether identical strategies are implemented. Yet in reoviruses the gene products required for replication in the insect and in the plant are different. It is not fully understood how viruses became pathogens of both plants and insects. Recombination between a virus infecting plants and another infecting animals may be the driving force enabling plant viruses to extend their host range to insects. Indeed evidence has been recently presented suggesting that a plant virus has acquired a vertebrate host by recombining with a vertebrate-infecting virus. Plant viruses such as luteoviruses and geminiviruses seem to use recombination as a way to broaden their host range and possibly to diversity their functions. In particular, geminiviruses may constitute a family of plant viruses who are in the process of acquiring, or loosing, abilities to interact actively with their insect vector, the whitefly Bemisia tabaci, to a point reminiscent of an host-pathogen relationship. This assumption is particularly pertinent when examining the relationship between B. tabaci and an isolate of the tomato yellow leaf curl geminivirus from Israel (TYLCV-Is)). Some of our last decade investigations will be presented and discussed in this review in the light of the geminivirus insect-pathogen hypothesis.

Czosnek H., Morin S, Rubinstein G, Fridman V, Zeidan M and Ghanim M. (2001) Tomato yellow leaf curl virus, a sexually transmitted disease of whiteflies. Pp. 1-27. In Virus-Vector-Plant Interactions , Harris KF, Smith OP, and Duffus JE Eds., Academic Press.

Tomato yellow leaf curl virus from Israel (TYLCV-Is) is a geminivirus (family: Geminiviridae, genus: Begomovirus) transmitted by the whitefly Bemisia tabaci, biotype B. This virus has many features reminiscent of an insect pathogen. TYLCV-Is is acquired and transmitted within 5-10 min. The latent period is 8-10 h. The virus that circulates in the insect is found in the midgut 1 h after the beginning of acquisition feeding, in the haemolymph after 1.5 h and in the salivary glands after 7h. Circulative transmission of TYLCV-Is is mediated by a GroEL homologue synthesized by the whitefly endosymbiotic bacteria and released into the haemolymph. B. tabaci GroEL and TYLCV-Is coat protein interact in the yeast two-hybrid system, indicating that a similar interaction may occur in the haemolymph of the whitefly. Disturbing this interaction in the haemolymph leads to the degradation of the virus and to a dramatic decrease in virus transmission. Following a 48 h acquisition access period, most of the virus remains associated with B. tabaci. While the viral DNA is detectable during the entire life span of the insect, the coat protein vanishes after 2 weeks. Although infectivity decreases with time, insect s are able to inoculate tomato plants for their entire life. The life-long association of TYLCV-Is with B. tabaci is associated with a marked decrease in longevity and in fertility. Besides these remarkable effects on the biology of the insect, TYLCV-Is can be transmitted among whiteflies in a sex-dependant manner, in the absence of any other source of virus. The virus can be transmitted from viruliferous males to females and from viruliferous females to males, but not among insects of the same sex. The recipient insects were able to efficiently inoculate tomato test plants. Insect to insect virus transmission was instrumental in increasing the number of whiteflies capable of infecting tomato test plants in a whitefly population. Based on the features of TYLCV-Is, we may consider this virus as sexually transmitted insect virus.

Ghanim M, Morin S and Czosnek H (2001) Rate of Tomato Yellow Leaf Curl Virus (TYLCV) Translocation in the Circulative Transmission Pathway of its Vector, the Whitefly Bemisia tabaci Phytopathology 91:188-196.

Whiteflies (Bemisia tabaci, biotype B) were able to transmit the Tomato yellow leaf curl virus (TYLCV) 8 h after they were caged with infected tomato plants. The spread of TYLCV during this latent period was followed in those organs thought to be involved in the translocation of the virus in B. tabaci. After increasing acquisition access periods (AAPs) on infected tomato plants, the stylets, the head, the midgut, an haemolymph sample and the salivary glands dissected from individual insects were subjected to PCR without any treatment; the presence of TYLCV was assessed using virus-specific primers. TYLCV DNA was first detected in the head of B. tabaci after a 10 min AAP. The virus was found in the midgut after 40 min and was first detected in the haemolymph after 90 min. TYLCV was found in the salivary glands 5.5 h after it was first detected in the haemolymph. Subjecting the insect organs to immunocapture-PCR showed that the virus capsid protein was found in the insect organs at the same time as the virus genome, suggesting that at least some TYLCV translocates as virions. Although females are more efficient as vectors than males, TYLCV was found in the salivary glands of males and of females after approximately the same AAP.

Goldman V and Czosnek H (2002). Whiteflies (Bemisia tabaci) issued from eggs bombarded with infectious DNA clones of Tomato yellow leaf curl virus from Israel (TYLCV) are able to infect tomato plants. Archives of Virology 147:787-801.

We have reported previously that Tomato yellow leaf curl virus from Israel (TYLCV) penetrates the reproductive system of its vector, the whitefly Bemisia tabaci biotype B, and may be transmitted to progeny. In order to mimic this phenomenon and to understand how TYLCV accompanies the development of the insect, we have bombarded B. tabaci eggs with an infectious DNA clone of TYLCV. After a linear full-length genomic copy of TYLCV DNA was delivered to eggs, the DpnI-sensitive DNA became circular and DpnI resistant. When a dimeric copy of TYLCV DNA was delivered to eggs, the viral DNA was detected in all the whitefly developmental stages. Adult insects that developed from the treated eggs were able to infect tomato test plants with variable frequency. Viral DNA was detected in the progeny of whiteflies that developed from eggs bombarded with TYLCV. Similarly, when insect eggs were bombarded with a dimeric copy of an infectious clone of the genome of Tomato yellow leaf curl virus from Sardinia, Italy (TYLCSV), adults that eclosed from the treated eggs were able to infect tomato test plants.

Brown JK and Czosnek H (2002). Whitefly transmission of plant viruses. Advances in Botanical Research. Plumb RT Ed. Vol. 36. pp. 65-100. Academic Press.

Presently, three whitefly species, Bemisia tabaci (Genn.), Trialeurodes vaporariorum, and T. abutilonea, are recognized as vectors of plant viruses; B. tabaci is the most important, having been associated with more than 100 plant viral diseases, damaging food, fiber, and ornamental plants. The relationship between the virus and whitefly vector is often specific for vector and viral species. Plant viral and insect associated factors, which are crucial to successful virus-vector interactions and transmission, have been maintained with great constancy, owing to continual co-evolution within virus/vector populations. Whitefly-mediated transmission of circulative plant viruses involves highly specific, co-evolved intramolecular interactions between viral-encoded determinants and receptor-like molecules of insect origin that interact to confer virus-vector specificity. Virus nucleocapsid structure and specific amino acids or particular functional motifs, are probably involved in the binding of key proteins of whitefly origin. In the vector tissue-specific tropisms, putative membrane-bound, receptor-like molecules of vector origin and endocytosis-exocytosis mechanisms, and protection of virions while in transit to the salivary glands by soluble, chaperonins or helper molecules found in the haemolymph, are vital for successful transmission. Mechanisms involved in semipersistent viral transmission by whiteflies are not well understood. It is likely that specificity is conferred by binding of virus to specialized sites in the whitefly foregut, and there is evidence that the viral coat protein together with the minor coat protein of criniviruses are essential for whitefly-mediated transmission. For non-circulative, non-persistent systems, viral epitopes have been identified that interact with a specific motif on a viral-encoded helper protein, which subsequently interacts with specific tissues in aphid mouthparts to facilitate potyvirus transmission by aphids, and similar mechanisms are thought likely for potyviruses transmitted by whiteflies.

Czosnek H, Ghanim M and Ghanim M (2002) Circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci - insights from studies with Tomato yellow leaf curl virus. Annals of Applied Biology. 140:215-231.

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 labeling was also associated with food in the lumen. The microvilli and electron-dense material in the epithelial cells of the gut wall were also labeled 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.

Czosnek H (2002) Tomato yellow leaf curl virus. CABI Crop Protection Compendium. 2002 Edition.

Disease first reported in Israel in 1939-40 associated with outbreaks of Bemisia tabaci. The causal agent was described in 1964 and named tomato yellow leaf curl virus (TYLCV) (Cohen and Harpaz, 1964). The virus was isolated in 1988 (Czosnek et al., 1988) and its genome sequenced in 1991 (Navot et al., 1991). The virus isolate from Israel is denoted as TYLCV-Is.

A virus exclusively transmitted by the whitefly Bemisia tabaci (synonym B. argentifolii) in natural conditions (Cohen and Nitzany, 1966) (Fig. 1). Geminate particle approximately 20x30 nm in size (Fig. 1) encapsidating a single molecule of covalently closed circular ssDNA of 2787 nucleotides (Navot et al., 1991). The virus is not sap transmissible and is not transmissible through seeds. It infects a wide range of wild (weeds, wild tomato species) and domesticated plants (bean, lisianthus, petunia, tomato). TYLCV-Is is present in the Middle East, Spain, the Caribbean Islands and Florida. Other TYLCV strains are found in Southwest Europe, Northern and tropical Africa and Southeast Asia. If cultures are not protected in the affected regions by using insecticides or nets, losses may reach 100% of the yield.

Czosnek H (2002) Tomato yellow leaf curl virus Israel. Association of Applied Biologists (AAB) Description of Plant viruses DPV 368. www.dpvweb.net/dpv/ showdpv.php?dpvno=368.

Maruthi MN, Czosnek H, Vidavski F, Tarba S-Y, Milo J, Leviatov S, Venkatesh HM, Padmaja AS, Kulkarni RS and Muniyappa V. (2003) Comparison of resistance to Tomato leaf curl virus (India) and Tomato yellow leaf curl virus (Israel) among Lycopersicon wild species, breeding lines and hybrids. European Journal of Plant Pathology 109:1-11.

The objective of this study was to screen wild and domesticated tomatoes for their resistance to Tomato yellow leaf curl virus, Israel (TYLCV) and to an isolate of Tomato leaf curl virus from Bangalore, India (Tomato leaf curl Bangalore virus, isolate 4, ToLCBV-[Ban4]) to find sources of resistance to both viruses. Resistance was assessed by different criteria like disease incidence, symptom severity and squash-blot hybridization. A total of 34 tomato genotypes resistant/tolerant to TYLCV were screened for resistance to ToLCBV-[Ban4] both under glasshouse and field conditions at the University of Agricultural Sciences, Bangalore, India. All the tomato genotypes inoculated with ToLCBV-[Ban4] by the whitefly vector Bemisia tabaci (Gennadius) in the glasshouse were infected and developed disease symptoms. In some plants of the lines 902 and 910, originated from the wild tomato species Lycopersicon hirsutum, virus was not detected by hybridization. The tomato genotypes susceptible to ToLCBV-[Ban4] by whitefly-mediated inoculation were also found susceptible to the virus under field conditions, within six weeks after transplanting. However, there were substantial differences between different genotypes in disease incidence, spread, symptom severity and crop yield. Despite early disease incidence, many Israeli genotypes produced substantially higher yields than the locally cultivated hybrid, Avinash-2. Sixteen tomato genotypes from India resistant/tolerant to ToLCBV-[Ban4] were also tested for TYLCV resistance at the Hebrew University of Jerusalem, Rehovot, Israel. Accessions of wild species, L. hirsutum LA 1777 and PI 390659 were found to be the best sources of resistance to both viruses. Lines 902 and 910 resistant to TYLCV were only tolerant to ToLCBV-[Ban4] and accession Lycopersicon peruvianum CMV Sel. INRA resistant to ToLCBV-[Ban4] was only tolerant to TYLCV. Implications of using the resistant lines in breeding program are discussed.

Levy A and Czosnek H (2003). The DNA-B of the non-phloem limited Bean dwarf mosaic virus (BDMV) is able to move the phloem-limited Abutilon mosaic virus (AbMV) out of the phloem, but DNA-B of AbMV is unable to confine BDMV to the phloem. Plant Molecular Biology 53:789-803.

Abutilon mosaic virus (AbMV) and Bean dwarf mosaic virus (BDMV) are two phylogenetically related bipartite begomoviruses. While AbMV is restricted to phloem, BDMV spreads to non-phloem tissues. Cell-to-cell and long-distance movement of AbMV and BDMV were investigated after replacing the coat protein (CP) gene with the reporter gene encoding the green fluorescence protein (GFP). The DNA-A and DNA-B genomic components of AbMV and BDMV, and their pseudorecombinants (PR), were delivered to bean (Phaseolus vulgaris) seedlings and detached leaves using DNA-coated microprojectiles. Virus-associated fluorescence was observed using the confocal microscope. Delivery of AbMV and BDMV GFP reporters showed that the epidermal tissue was the main recipient of the viral DNA; the DNA-A of the two viruses was unable to move out of the recipient cells. AbMV DNA-A co-inoculated with AbMV DNA-B did not move cell-to-cell in the epidermis and did not reach the phloem. However, co-inoculation of AbMV DNA-A with BDMV DNA-B resulted in PR cell-to-cell movement out of the epidermis and long-distance movement in the phloem. In contrast, BDMV DNA-A moved cell-to-cell and long distance when co-inoculated with either its own DNA-B or with the DNA-B of AbMV. Therefore, the DNA-B of the non-phloem-limited BDMV overcame the phloem limitation of AbMV. In the reciprocal case, the DNA-B of the phloem-limited AbMV did not confine the non-phloem limited BDMV to the phloem. Hence, we assume that the DNA-A component of BDMV includes determinants involved in the movement pattern of the virus in addition to the DNA-B encoded BC1 and BV1, previously shown to be involved in virus movement. The results also confirm that the CP is not necessary for virus movement; however, replacing the CP of AbMV and BDMV with GFP resulted in a decrease in symptom severity. DNA-B was involved in symptom severity; the B component of BDMV produced symptoms more severe than those induced by that of AbMV, whether in wild-type PRs, or in PRs with CP-GFP replacement. It is interesting to note that when the GFP gene under the control of the CaMV 35S promoter (35S-GFP) was delivered to the bean tissue, with or without the DNA-B component of BDMV, GFP was expressed but did not move cell-to-cell. However, when the 35S-GFP was delivered together with BDMV DNA-A and DNA-B, GFP moved cell-to-cell in the epidermis, but was restricted to these cells. Hence, infection of cells with a functional bipartite begomovirus may facilitate cell-to-cell movement of macromolecules.

Akad F, Dotan N. and Czosnek H (2004). Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly Bemisia tabaci. Archives of Virology. 149:1481-1497.

To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) - a whitefly-transmitted geminivirus - was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll virus (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.

Hadidi A, Czosnek H and Barba M. (2004). DNA microarrays and their potential applications for the detection of plant viruses, viroids, and phytoplasmas. Journal of Plant Pathology. 86:97-104.

DNA microarrays were first described in 1995 for simultaneous analysis of a large-scale gene expression patterns. Since then, they have moved to center stage in many areas of biological research and now assuming an increasingly important role in diagnostics, genomics, pharmacology, cancer and other biomedical research, among others. In this article, we discuss the scientific background and principle of microarrays; describe their types, several technical steps needed for obtaining microarray data, and their current applications. The potential applications of DNA microarrays in detection and identification of plant pathogens, especially viruses, viroids, and phytoplasmas are presented.

Brown JK, Lambert GM, Ghanim M, Czosnek H and Galbraith DW (2005). Nuclear DNA Content of the Whitefly Bemisia tabaci (Genn.) (Aleyrodidae: Homoptera/ Hemiptera) Estimated by Flow Cytometry. Bulletin of Entomological Research. 95:309-312.

The nuclear DNA content of the whitefly Bemisia tabaci Gennnadius [Aleyrodidae; Homoptera/Hemiptera] was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA = 980 Mbp) indicate that the haploid genome of B. tabaci is made up of 1,020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen [Drosophilidae; Diptera]. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.

Akad, A., Teverovsky, E., Gidoni, D., Elad, Y., Kirshner, B., Rav-David, D., Czosnek, H., and Loebenstein, G. (2005). Resistance To Tobacco Mosaic Virus And Botrytis Cinerea In Tobacco Transformed With cDNA Encoding An Inhibitor Of Viral Replication (IVR)-Like Protein. Annals of Applied biology 147:89-100.

We have previously cloned a 1016 bp-long cDNA clone (named NC330) encoding an inhibitor of virus replication (IVR)-like protein from induced-resistant leaves of Nicotiana tabacum cv. Samsun NN. In the present work NC330 cDNA was cloned in an Agrobacterium binary vector and introduced into the genome of N. tabacum cv. Samsun nn that is susceptible to TMV. Eight R₀ were highly resistant, with TMV titers less than 2% of the controls. The progeny of these primary transformants resulted in four TMV-resistant and four susceptible lines. The progeny of the plant with highest resistance for TMV was selfed during four generations. In the R₂ generation out of 112 plants tested, three plants showed excellent resistance and five a lesser degree of resistance. NC330 transcripts were detected in these R₂ plants as well as in their R₁ and R₀ parents. Two R₂ plants were self-pollinated to obtain the R₃ generation. All the 25 R₃ progeny plants of one line contained PCR-amplifiable NC330, compared with only 6 of the 23 progeny plants from the other line. Following TMV inoculation, virus was undetectable in 7 of the 8 tested R₃ progeny of the first line and 5 of the 6 R₃ progeny plants of the second line. NC330 transcripts were detected in the TMV-resistant transgenic plants but also in some susceptible plants. TMV resistance was suppressed when the plants were kept at higher temperatures (32-34 C) returning the plants to lower temperatures (24-26 C) restored resistance. Transgenic plants resistant to TMV were also resistant to B. cinerea following inoculation of seedlings or of whole plants.

The presence of the NC330 DNA was correlated with changes in the physiology of the plant. Seed germination was inhibited at high temperatures and in the dark. Rootlets of germinating seeds of the transgenic grew significantly faster than those of control seeds. These observations were correlated with a significantly higher concentration of abscisic acid (ABA) in the seeds and seedlings of the transgenic plants.

In silico analysis suggests that the NC330 protein translated from the NC330 cDNA is a tetratricopeptide repeats (TPR) protein and that it may be involved in protein-protein interaction as part of the pathogen resistance mechanism.

Mej a, L., R.E. Teni, F. Vidavski, H. Czosnek, M. Lapidot, M. K. Nakhla and D. P. Maxwell (2005). Evaluation of tomato germplasm and selection of breeding lines for resistance to begomoviruses in Guatemala. Acta Horticulturae. 251-255.

Tomato genotypes with resistance to begomoviruses derived from different wild species were evaluated in Guatemala. Selection of individual plants for several generations resulted in breeding lines with high levels of resistance. Resistance derived from L. hirsutum was dominant, while resistance from the other sources was more recessive in nature. Crosses among resistant lines resulted in higher levels of resistance for F1 populations than crosses between resistant and susceptible lines. Resistant lines were crossed to susceptible lines with other traits of interest, namely resistance to other pathogens and desirable fruit characters. Improved breeding lines with begomovirus resistance have been selected from these hybrids. These breeding lines are currently being used in the production of begomovirus-resistant hybrids with acceptable market quality and yields.

Habib, S., Galiakparov, N., Goszczynski, D.E., Batoman, O., Czosnek, H. and Mawassi, M. (2006). Engineering the genome of Grapevine Virus A into a Vector for Expression of Proteins in Herbaceous Plants. Journal of Virological Mehtods. 132:227-231.

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a ~ 7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. The broad host range of GVA makes it probably the virus of choice for engineering a vitivirus-based expression vector in grapevine and other plants. We have engineered the genome of GVA as a vector containing duplication of heterologous sequences that contain the promoter of the movement protein-sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool to transiently express foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild type of GVA. It was successfully used to express the coat protein gene of Citrus tristeza virus and the reporter gene beta-glucuronidase (GUS) in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines. It may allow vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.

Levy A and Czosnek H (2006) Replacing the AC2/AC3 genes of Abutilon mosaic virus (AbMV) with those of Bean dwarf mosaic virus (BDMV) greatly enhances AbMV accumulation, movement and symptom severity in bean. Journal of Plant Pathology 88:37-50.

Abutilon mosaic virus (AbMV) and Bean dwarf mosaic virus (BDMV) are two phylogenetically related bipartite begomoviruses. While AbMV is limited to the plant host phloem, BDMV is not. We have previously provided evidence that the genomic DNA-A component of BDMV contains determinants involved in movement (Levy and Czosnek, 2003). We report here that the DNA-A-encoded genes AC2 and AC3 are involved in virus accumulation and spread. To follow AbMV and BDMV movement in inoculated bean plants, we have replaced their coat protein gene (CP) with the green or the red fluorescent protein (GFP, RFP) to create BDMV-CP:GFP, BDMV-CP:RFP, and AbMV-CP:GFP. Frame-shift mutations in BDMV AC2 and AC3 to produce BDMV-CP:GFP-mC23 resulted in inhibition of BDMV movement when co-inoculated with BDMV DNA-B. The mutation reversed to wild-type and movement was recovered when BDMV-CP:GFP-mC23 was co-inoculated with BDMV-CP:RFP and BDMV DNA-B, strongly suggesting that the AC2/AC3 region is important for BDMV movement. Consequently we replaced the AC2/AC3 region of AbMV-CP:GFP with that of BDMV to create AbMV-CP:GFP-C23:BDMV. AbMV-CP:GFP-C23:BDMV inoculated together with AbMV DNA-B moved cell-to-cell in the epidermis towards the phloem, and long-distance in the entire plant, a feature that AbMV-CP:GFP was unable to perform. Inoculation of bean with AbMV-CP:GFP-C23:BDMV and BDMV DNA-B resulted in the accumulation of very large amounts of viral DNA, in remarkably fast virus systemic movement, and in the early appearance of severe symptoms, which in most cases, resulted in inhibition of germination. These results suggest that the interaction between BDMV AC2 (transcriptional activator protein) located on the DNA-A component and AbMV BV1 (nuclear shuttle protein) on the DNA-B component influences the ability of AbMV to move in non-phloem cells. Furthermore, they suggest that the interaction between BDMV AC3 (replication enhancer protein) and AbMV AC1 (replication associated protein) results in enhanced AbMV replication, producing an overflow of virus overcoming the phloem barrier of AbMV, and leading to systemic spread and severe symptoms.

Leshkowitz D, Gazit S, Reuveni E, Ghanim M, Czosnek H, McKenzie C, Shatters RG Jr., and Brown JK (2006). Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries. BMC Genomics 7:79.

The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae). Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown.

To address this general shortage of genomic sequence information, we have constructed three cDNA libraries for non-viruliferous whiteflies (eggs, immature instars, and adults) and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato mottle virus (ToMoV). In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production of eggs.

This is the first functional genomics project involving a hemipteran (Homopteran) insect from the subtropics/tropics. The B. tabaci sequence database now provides an important tool to initiate identification of whitefly genes involved in development, behaviour, and B. tabaci-mediated begomovirus transmission.

Bar-Or C, Bar-Eyal M, Gal T, Kapulnik Y, Czosnek H and Koltai H (2006) Derivation of species-specific hybridization-like knowledge out of cross-species hybridization results. BMC Genomics, 7:110.

DNA microarrays constitute a powerful tool for quantitative elucidation of gene expression of numerous genes in a genome. One of the approaches to conducting genomics research in organisms that do not yet have a proper microarray template is to profile their expression patterns by using cross-species hybridization.

We used a tomato spotted cDNA array to examine the ability of cross-species hybridization to reflect a similar biological process in potato (and in tomato). Two RNA sources and two microarray platforms were used to generate three datasets from heterologous and homologous hybridizations. The results revealed difficulties in the use of transcriptomics data obtained from cross-species hybridization to reproduce the results obtained from the species-specific hybridization. Nevertheless, once the data had been filtered for those corresponding to matching probe sets, the cross-species data showed higher consistency with the species-specific data and facilitated the identification of significantly regulated genes, which were common to both the species-specific and cross-species hybridizations.

We have combined the comparison between species-specific and cross-species microarray hybridization of a certain biological process with data analysis that included filtering for matched probe sets. This study enabled us to outline some considerations relating to the performance of heterologous hybridization, which would lead to further refinement of the use of cross-species hybridization to reflect biological processes.

Bar-Or C, Czosnek H and Koltai H (2007). Cross-species microarray hybridizations: a developing tool for studying diversity. Trends in Genetics 23:200-207.

The use of cross-species hybridization (CSH) to DNA microarrays, in which the target RNA and microarray probe are from different species, has increased in the past few years. CSH is used in comparative, evolutionary and ecological studies of closely related species, and for gene-expression profiling of many species that lack a representative microarray platform. However, unlike species-specific hybridization, CSH is still considered a non-standard use of microarrays. Here, we present the recent developments in the field of CSH for cDNA and oligomer microarray platforms. We discuss issues that influence the quality of CSH results, including platform choice, experiment design and data analysis, and suggest strategies that can lead to improvement of CSH studies to investigate species diversity.

Bar-Or C, Novikov E, Reiner A, Czosnek H and Koltai H. (2007). Utilizing Microarray Spot Characteristics to Improve Cross-Species Hybridization Results. Genomics. 90:636-645.

Cross-species hybridization (CSH), i.e., the hybridization of a (target) species RNA to a DNA microarray that represents another (reference) species, is often used to study species diversity. However, filtration of CSH data has to be applied to extract valid information. We present a novel approach to filtering the CSH data, which utilizes spot characteristics (SCs) of image-quantification data from scanned spotted cDNA microarrays. Five SCs that were affected by sequence similarity between probe and target sequences were identified (designated as BS-SCs). Filtration by all five BS-SC thresholds demonstrated improved clustering for two of the three examined experiments, suggesting that BS-SCs may serve for filtration of data obtained by CSH, to improve the validity of the results. This CSH data-filtration approach could become a promising tool for studying a variety of species, especially when no genomic information is available for the target species.

Czosnek H. (2007). Ethics in agriculture. In: Research Ethics, Landau RL and Shefter G Eds, Magnes, The Hebrew University of Jerusalem (in Hebrew). Pp 279-289.

Akad F, Eybishtz A, Edelbaum D, Gorovits R, Dar-Issa O, Iraki N and Czosnek H (2007) Making a friend from a foe: Expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to Tomato yellow leaf curl virus. Archives of Virology. 152:1323-1339.

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus Tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In, vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato.

Ghanim M, Kontsedalov S and Czosnek H (2007) Tissue-specific gene silencing by RNA interference in the whitefly Bemisia tabaci (Gennadius). Insect Biochemistry and Molecular Biology. 37:732-738.

The Hemipteran whitefly Bemisia tabaci (Gennadius)species complex and the plant viruses they transmit pose major constraints to vegetable and fiber production, worldwide. The insect tissue- and developmental-specific gene expression has not been exhaustively studied despite its economic importance. In 2002, a functional genomic project was initiated, which generated several thousands expressed sequence tags (ESTs) and their sequence. This project provides the basic information to design experiments aimed at understanding and manipulating whitefly gene expression. In this communication, for the first time we provide evidence that the RNA interference mechanism discovered in many organisms, including in Hemiptera, is active in B. tabaci. By injecting into the body cavity long dsRNA molecules, specifically directed against genes uniquely expressed in the midgut and salivary glands, we were able to significantly inhibit the expression of the targeted mRNA in the different organs. Gene expression levels in treated insects were reduced up to 70% compared to whiteflies injected with buffer or with a Green Florescent Protein (GFP)-specific dsRNA. Phenotypic effects were observed in B. tabaci ovaries following dsRNA targeting the whitefly Drosophila chickadee homologue. Disruption of whitefly gene expression opens the door to new strategies aimed at curbing down the deleterious effects of this insect pest to agriculture.

El Mehrach K, Sedegui M, Hatimi H, Tahrouch S, Arifi A, Czosnek H, Nakhla MK and Maxwell DP (2007) Molecular characterization of a Moroccan isolate of Tomato yellow leaf curl Sardinia virus and differentiation of the Tomato yellow leaf curl virus complex by the polymerase chain reaction. Phytopathologia Mediterranea. 46:185-194.

The polymerase chain reactions (PCR) was used to identify an isolate of Tomato yellow leaf curl Sardinia virus (TYLCSV) from southwestern Morocco and to detect the members of the Tomato yellow leaf curl virus (TYLCV) complex. Thirty-five tomato samples with typical TYLCV symptoms were collected from infected tomato fields in the Souss-Massa region. PCR was performed with a general primer pair based on the coat protein (Cp) gene of the TYLCV complex, as well as with specific primer pairs for TYLCV and TYLCSV. Of the 35 samples tested, 29 generated a viral DNA product with the general primer pair, 29 samples gave a viral DNA product with the TYLCV-specific primers, and of these, 9 also gave a product with the TYLCSV primer pair; 6 samples did not give any PCR product with either of one primer pair or the other. The full-length genome of TYLCSV was amplified with overlapping primers at the unique NcoI site in the TYLCSV genome (GenBank accession number X61153). The full-length genome of the TYLCSV isolate from Morocco is 2,777 nucleotides long (accession number AY702650) and is almost identical (97% nucleotide identity) to a TYLCSV isolate from Murcia, Spain (accession number Z25751). A PCR-based diagnostic method was developed to distinguish between TYLCV and TYLCSV in Morocco. To diagnose the TYLCV/TYLCSV complex a general primer pair was designed that anneals to a conserved region of the Cp gene. To diagnose TYLCSV exclusively, two primer pairs were designed to anneal specifically to the replication-associated protein gene (Rep) of TYLCSV from Murcia. To detect TYLCV exclusively, a primer pair previously described to amplify the intergenic region (IR) of TYLCV was used. The PCR primers were tested for their effectiveness using DNA clones of the TYLCSV from Morocco and of the TYLCV from the Dominican Republic. PCR using these primers offers a rapid means to detect the TYLCV complex and to distinguish between the two TYLCV species present in Morocco.

Wei S, Semel Y, Bravdo B-A, Czosnek H and Shoseyov (2007) Expression and subcellular compartmentation of Aspergillus niger -glucosides in transgenic tobacco increase insecticidal activity on whiteflies (Bemisia tabaci), and modulate plant growth, density of leaf secretory glandular trichomes and metabolic profiles. Plant Science 172:1175-1181.

Transgenic tobacco plants expressing Aspergillus niger -glucosidase gene (BGL1) in different subcellular compartments [cell wall (Tcw), endoplasmic reticulum (ER) (Ter), and vacuole (Tvc)] were analyzed. Metabolic profiling indicated that 34 out of 56 compounds identified were significantly altered in transgenic plants. The majority of these compounds decreased significantly in Tcw plants compared to wild-type and other transgenic plants. Hierarchical cluster analysis (HCA) showed that wild-type plants were closer to Tvc and Ter compared with Tcw plants. Compared with wild-type control, Ter and Tvc transgenic plants did not exhibit any significant differences in seed germination, plant growth rate, plant height as well as flowering time. However, Tcw plants showed a significant delay of seed germination, beginning of flowering and decreased leaf area and plant fresh weight. Transgenic plants had a marked insecticidal effect on whiteflies (Bemisia tabaci) as determined by caging insects with mature plants and by confining insects with detached leaves in closed vials. Significant increase in density of secretory glandular trichomes was found in transgenic leaves compared with the wild-type. Our data indicates that expression of BGL1 in different subcellular compartments may significantly alter metabolic pathways, growth, morphology and plant insect interaction in transgenic tobacco.

Czosnek H. Editor (2007) Tomato yellow leaf curl virus Disease, Management, Molecular Biology, Breeding for Resistance. 420 pp. Springer, The Netherlands.

This book is dedicated to the farmers who suffer from the disease caused by the Tomato yellow leaf curl virus (TYLCV), especially those from developing countries where a diseased tomato field often causes economic and human tragedies. It is also dedicated to the scientists and the breeders who work together to understand all aspects of virus epidemiology and provide solutions to the growers.

The disease caused by the TYLCV family has become a major constraint to tomato production worldwide with losses often reaching 100 % of the yield. TYLCV is transmitted exclusively by the whitefly Bemisia tabaci. TYLCV outbreaks that were sporadic in the 1960s have become a serious economic problem, starting in the early 1970s. By the beginning of the 1980s, all regions of tomato culture in the Middle East were affected by the virus. In the 1990s, the viral disease has been described in Italy, then in Spain and Portugal, in North and West Africa, and in the Arabian Peninsula. At about the same time it was identified in the Caribbean Islands and almost immediately thereafter in the south western states of the USA. Recently it was detected in Arizona and in Mexico. TYLCV was also found in Thailand in the 1980s and in China and Japan in the late 1990s. Virions have been isolated and characterized in the late 1980s. The first sequence of the genome of a TYLCV isolate has been published in 1991. Until now close to sixty virus isolates have been sequenced. Sequence comparisons have shown that TYLCV includes a complex of closely as well as distantly related viruses affecting tomato. Well before identification of the causal agent of the tomato yellow leaf curl disease, breeding programs for resistance have been put in place in the late 1960s and have developing since. The first commercial cultivars tolerant to TYLCV were released in 1990. The first locus associated with resistance to TYLCV has been discovered in 1994. Today several virus-resistant cultivars are available commercially and more efforts are underway. In addition genetic engineering has been to work to produce resistant tomato plants. The relationship between TYLCV and its whitely vector have shown an intricate relationship which is far of been fully understood. Similarly the relationship between the virus and the plant, whether susceptible or tolerant, is still a black box. The host proteins involved in the systemic spread of the virus in the tomato plant are not known. Likewise, the molecular events triggering the appearance of symptoms and arrest of growth have not been identified. The genes underlying resistance and their mode of expression are still very mysterious.

The goal of this book is to evaluate the state of the art of the many aspects of TYLCV research. It is presented in six parts. The first part discusses aspects of the worldwide expansion of TYLCV related to the spread of the virus whitefly vector and describes the events accompanying the invasion of the virus in an insular environment. The second part presents a molecular analysis of the TYLCV genome, of the molecular biodiversity of the virus, and discusses the dangers of recombination between TYLCV strains or species. The third part of the book discusses the complicated interactions between the virus, the whitefly vector and the tomato plant. The mode of replication of the virus and its interaction with plants proteins is discussed. Localization studies of the virus in the plant and in the whitefly help us to understand the cells and organs involved in the systemic spread of the virus in the plant and the circulative transmission by the whitefly. Also an attempt is made to understand the physiologic state of the infected tomato plant, whether susceptible or tolerant to the virus. The fourth part of the book deals with integrated pest managements and protection of tomato cultures. It includes a review of the virus detection methods, presents new means to protect crops with special plastic covers, and demonstrates that a sound reinforced management program can provide excellent results. This part also deals with the dangers presented by the accelerated acquisition of resistance to the major pesticides by whiteflies. The fifth part of the book presents the efforts aimed at breeding TYLCV-resistant tomato. The status of classical breeding is reviewed; it includes methods to screen for resistance, the various wild tomato species serving as source of resistance genes and their exploitation, and the efforts to map the resistance genes on the tomato chromosomes. Then it deals with the novel approaches of genetic engineering, including gene silencing. The last part of the book presents the international efforts that have been put in place to deal with the TYLCV disease and reviews the programs and the agencies involved in these efforts.

I believe that this book will present precise answers to specialists, each in its area, and will also open wide perspectives to understand the numerous facets of one of the most deleterious viruses. It is addressed to breeders, epidemiologists, molecular biologists, virologists and entomologists. Each can found an updated view of their field of interest.

I understand that this book is not complete and does not cover all the many aspects of the TYLCV disease. Much is still not understood and many efforts are needed to progress. However, I hope that this book will constitute a reference for the years to come for researchers and for students who may find new ideas from reading the chapters of the book and from those that are missing.

I warmly thank all the contributors for their time and efforts for exquisite chapters. I also wish to than Springer who has understood the importance of the TYLCV problem, and has agreed to publish this book. And of course I wish to thank Zuzana Bernhart and Ineke Ravesloot for their consistent help. And finally I wish to thank Iris Sobol for precious help in putting the manuscripts in the right format.

Czosnek H. (2007) Interactions of Tomato yellow leaf curl virus with its insect vector. In: The Tomato Yellow Leaf Curl Virus Disease: Management, Molecular Biology And Breeding For Resistance. H. Czosnek Ed. Pp 157-170. Springer, The Netherlands

Whiteflies cause damages to many economically important agricultural crops because of their feeding habits and their begomovirus transmissions. The whitefly Bemisia tabaci is a genetically diverse group, which includes a large number of different biotypes. It is extremely prolific; a single female may lay approximately 400 eggs during her lifetime. Unfertilized eggs give rise to haploid males, whereas fertilized eggs develop into diploid females (arrhenotoky). The male/female ratio naturally changes throughout the course of the year, in fields and in insectaries (Horowitz & Gerling, 1992). B. tabaci develops into a flying adult from an egg, through four instars. Although B. tabaci nymphs are able to ingest and transmit begomoviruses, flying adults are those who spread the disease in the field (Gerling & Mayers, 1996). In this chapter we discuss the characteristics of acquisition, transmission, and retention of Tomato yellow leaf curl virus (TYLCV) and related begomoviruses by the whitefly vector B. tabaci.

Gorovits R and Czosnek H (2007) Biotic and abiotic stress responses in breeding tomato lines resistant and susceptible to Tomato yellow leaf curl virus. In: The Tomato Yellow Leaf Curl Virus Disease: Management, Molecular Biology And Breeding For Resistance. Czosnek H. (Ed). Pp 223-237. Springer, The Netherlands.

In the eyes of a tomato grower, resistance to TYLCV, as opposed to susceptibility, is defined by the absence of, or mild, disease symptoms, and acceptable yield. In resistant cultivars and breeding lines, the amount of virus that can be detected with molecular tools is usually smaller than that in the susceptible plants, especially during the first 4 weeks after inoculation. Genetic studies have indicated that several genes, expressed as quantative trait loci (QTL), are involved in providing the resistance phenotype described above. Several QTLs have been localized to tomato chromosomes using polymorphic DNA markers. However, the molecular basis of resistance to TYLCV remains totally unknown. Moreover, the physiological state of susceptible vs. resistant plants, before and after inoculation, has never been compared. To provide some clues on what makes a plant resistant to TYLCV and another susceptible, we have considered the virus as a particular case of stress, among many that a tomato plant may face, and resistance as a particular case of successful response to stress. The response of plants to biotic and abiotic stresses has been studied intensively. A stress response is initiated when plants recognize stress at the cellular level, activating signal transduction pathways that transmit information within the individual cell and throughout the plant, leading to the changes in the expressing of many gene networks. Hence plants respond to biotic and abiotic stresses by activation of R-gene mediated and signal transduction defense response pathways.

Maxwell DP and Czosnek H (2007) International networks to deal with tomato yellow leaf curl virus diseaseL the middle east regional cooperation program. In: The Tomato Yellow Leaf Curl Virus Disease: Management, Molecular Biology And Breeding For Resistance. H. Czosnek Ed. Pp 409-415. Springer, The Netherlands.

The Middle East is a major producer of both processing and fresh market tomatoes (Solanum lycopersicum); and tomatoes are a main component of the local cuisines. Since Tomato yellow leaf curl disease was first reported in Israel in the early 1950s, it has become one of the major, if not the most important, constraint to production. This disease has been reported in all countries of the Middle East, and the importance of this disease has been associated with the expanding range of vector Bemisia tabaci biotype B and of the pathogen, members of the Tomato yellow leaf curl virus complex. Management of this disease has mainly involved methods for reducing the vector population; and in many cases, this was primarily by the application of insecticides. Tomatoes with resistance to Tomato yellow leaf curl virus (TYLCV) would effectively reduce losses and reduce the quantity of insecticides needed to obtain satisfactory yields. Several breeding programs were initiated in the 1970s and in general, progress was slow. In all cases, resistance to TYLCV was based on introgressions of resistance loci from wild tomato species (e.g., S. chilense, S. habrochaites, and S. peruvianum). It was not until the 1990s that commercial hybrids with moderate levels of resistance were available.

Because of the seriousness of this disease and the difficulty of managing it, international networks of scientists have been organized to provide solutions. Henri Laterrot from INRA, France, was the first to organize an international project, and it was funded by Commission des Communaut s Europ ennes, in the late 1980s (Laterrot, 1995). One goal was to test germplasm in different countries (Israel, Egypt, Jordan, Mali, and S n gal) and then to combine the resistant plants into a population that could be used in breeding programs (e.g., Pimpertylc, Chiltylc). Subsequently, several international projects have been organized to focus on the management of whiteflies and begomoviruses. This chapter will not attempt to describe them all, but will discuss mainly two international projects that have as their main goal the development of breeding lines resistant to begomoviruses in the Mediterranean Basin and Central America (see http://www.plantpath.wisc.edu/GeminivirusResistantTomatoes/ ).

Gorovits R, Akad F, Beery H, Vidavsky F, Mahadav A and Czosnek H (2007) Expression of stress-response proteins upon whitefly-mediated inoculation of Tomato yellow leaf curl virus (TYLCV) in susceptible and resistant tomato plants. Molecular Plant Microbe Interactions 20:1376-1383.

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as TYLCV resistance source (Vidavski and Czosnek 1998), one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response to stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related proteins (PRs). The two biotic stresses non-viruliferous whitefly feeding and virus infection with viruliferous insects - led to a slow decline in abundance of MAPKs, HSPs and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PRs, -1, 3-glucanase and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied followed by a decline; the virus-susceptible and resistant tomato lines behaved similarly in response to the fungus.

Ghanim M, Sobol I, Ghanim M and Czosnek H (2007). Horizontal transmission of begomoviruses between Bemisia tabaci biotypes. Arthropod-Plant Interactions. 1:195-204.

We have previously shown that Tomato yellow leaf curl virus (TYLCV), a begomovirus (family Geminiviridae, genus Begomovirus) infecting tomato plants can be transmitted in a sex-dependant manner among its insect vector the whitefly Bemisia tabaci (Gennaduis) (Aleyrodidae: Hemiptera) type B during mating. Viruliferous females were able to transmit the virus to non-viruliferous males and vice versa, in the absence of any other virus source. In this communication, we present evidence that two bipartite begomoviruses infecting cucurbits, Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV) can be transmitted in a sex-dependant manner among whiteflies. In addition we show that TYLCV can be transmitted during mating among individuals from the same biotype (from B-males to B-females and vice versa; and from Q-males to Q-females and vice versa). However, viruliferous males of the B biotype are unable to transmit the virus to females of the Q biotype (and vice versa); similarly, viruliferous males of the Q biotype are unable to transmit the virus to females of the B biotype (and vice versa). These findings support the hypothesis that a pre-zygotic mating barrier between the Q and B biotypes is the cause for the absence of gene flow between the two biotypes, and that virus transmission can be used as a marker for inter-biotype mating. To be transmitted during mating, the virus needs to be present in the haemolymph of the donor insect. Abutilon mosaic virus (AbMV), a bipartite begomovirus that can be ingested but not transmitted by B. tabaci, is absent in the whitefly haemolymph, and cannot be transmitted during mating. Mating was a precondition for horizontal virus transfer from male to female, or female to male. Virus was not transmitted when viruliferous B. tabaci were caged with the non-vector non-viruliferous whitefly Trialeurodes vaporariorum (Westwood) (Aleyrodidae: Hemiptera)and vice versa.

Peretz Y., Mozes-Koch R., Akad F., Tanne E., Czosnek H and Sela I. (2007). A universal expression/silencing vector in plants. Plant Physiology. 145:1251-1263.

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat, pepper, grapevine, citrus and olive. Expression or silencing develops within a few days in tomato, wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus (TYLCV), which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the TYLCV's natural insect vector. In addition, artificial satellites were constructed which require a helper virus for replication, movement and expression. With IL-60 as the disarmed helper "virus", transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral silencing suppressor of Grapevine virus A (GVA), resulting in GVA-resistant/tolerant plants.

Pasquini G., Barba M., Hadidi A., Faggioli F., Negri R., Sobol I., Tiberini A., Caglyan K., Mazyad H., Anfoka G. Ghanim M., Zeidan M and Czosnek H. (2008). Microarray-based detection and genotyping of Plum pox virus. Journal of Virological Methods147:118-126.

Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. Therefore the detection and identification of its strains is of critical importance to plant quarantine and certification programs world-wide. Currently, existing techniques to screen simultaneously strains of PPV suffer from several limitations. We have developed a genomic strategy for PPV screening to facilitate the detection and genotyping of the virus from infected plant tissue or biological samples. The cornerstone of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping of PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal) derived from the genome highly conserved 3 non-translated region detected all individual strains of PPV. This universal PPV probe combined with probes specific for each known strain could be used for new PPV strain discovery. Finally, by indirect fluorescent labelling of cDNA with cyanine in a separate step after cDNA synthesis, we were able to enhance the sensitivity of the virus detection without the use of PCR amplification step. Using the PPV microarray, we were able to efficiently detect and identify the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. The microarray based PPV detection method is versatile and has the potential to make the simultaneous detection of plant pathogens using microarray technology feasible and easier.

Moskovitz Y, Goszczynski DE, Bir L, Fingstein A, Czosnek H and Mawassi M (2008) Sequencing and assembly of a full-length infectious clone of grapevine virus B and its infectivity on herbaceous plants. Archives of Virology 153: 323 328.

Grapevine virus B (GVB) has been found associated with corky bark-diseased vines. Although the sequence of a 7.6-kb cDNA clone from a GVB isolate from Italy has been described, striking differences in sequences between GVB isolates prompted us to construct an additional full-length GVB clone from the isolate 94=971 and to determine its complete sequence. The cDNA of GVB 94=971 shared a nucleotide sequence identity of only 77% with the GVB isolate from Italy. The cDNA of GVB 94=971 was infectious on Nicotiana plants as demonstrated by symptoms and by means of Northern blot, Western blot and electron microscopic analyses.

Gorovits H. and Czosnek H. (2008) Expression of stress-response proteins upon abiotic stress in tomato lines susceptible and resistant to Tomato yellow leaf curl virus. Plant Physiology and Biochemistry. 46:482-492.

The defense response to several abiotic stresses has been compared in two tomato inbred lines issued from the same breeding program, one susceptible and the other resistant to Tomato yellow leaf curl virus (TYLCV) infection. The level of oxidative burst and the amounts of key regulatory stress proteins: pathogenesis-related proteins (PRs), heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs) was appraised following treatments with NaCl, H₂O₂, and ethanol. Significant differences in the response of the two tomato genotypes to these stresses have been found for HSPs and MAPKs patterns at the level of down-regulation but not activation. The higher abundance of HSPs and MAPKs in tomatoes resistant to TYLCV could result in enhanced defense capacity against abiotic stresses.

Anfoka G, Abhary M, Haj Ahmad F, Hussein AF, Rezk A, Akad F, Abou-Jawdah Y, Lapidot M, Vidavski F, Nakhla MK, Sobh H, Atamian H, Cohen L, Sobol, I, Mazyad H, Maxwell DP and Czosnek H (2008) Survey of tomato yellow leaf curl disease associated viruses in the eastern mediterranean basin. Journal of Plant Pathology. 90:311-320.

Tomato production in the Middle East and elsewhere is under the constant threat of the whitefly-transmitted geminivirus Tomato yellow leaf curl virus (TYLCV). Sequencing has indicated that the generic name TYLCV includes a large number of viruses and strains. Here we have investigated the distribution of the TYLCV complex in Egypt, Israel, Jordan and Lebanon. A simple and reliable multiplex polymerase chain reaction (PCR) has been developed that allowed detecting four different viruses and strains: Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl virus-Mild (TYLCV-Mld), Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl Sardinia virus from Malaga Spain (TYLCSV-[ES2]). We have sequenced the PCR products to confirm their identity. Subsequently we have sequenced the full-length genomes of TYLCV from Egypt, Jordan and Lebanon, of TYLCV-Mld from Jordan and Lebanon, and of TYLCSV (Sicily strain) from Israel. This is the first time that TYLCSV has been detected in Israel, and the first report of TYLCV-Mld in Egypt and Lebanon.

Vidavski F, Czosnek H, Gazit S, Levy D and Lapidot M (2008) Pyramiding of genes conferring resistance to Tomato yellow leaf curl virus from different wild tomato species. Plant Breeding. 127:625-631.

Tomato (S. lycopersicum) production in tropical and subtropical regions of the world is limited by the endemic presence of Tomato yellow leaf curl virus (TYLCV). Breeding programs aimed at producing TYLCV-resistant tomato cultivars have utilized resistance sources derived from wild tomato plants. So far all reported breeding programs have concentrated on a single source of resistance. Here we tested the hypothesis that pyramiding the chromosomal regions associated with resistance in lines from different origins might improve the degree of resistance of tomato to TYLCV. We have crossed TYLCV-resistant lines which were originated from different wild-type solanum progenitors, S. chilense, S. peruvianum, S. Pimpinellifolium, and S. habrochaites. The various parental resistant lines and the F1 hybrids were inoculated in the greenhouse using whiteflies. Control, non-inoculated plants of the same lines and hybrids were exposed to non-viruliferous whiteflies. Following inoculation the plants were scored for disease symptom severity, and transplanted to the field. Resistance was assayed by comparing yield components of inoculated plants to those of the control non-inoculated plants of the same variety.

Mahadav A, Gerling D, Gottlieb Y, Czosnek H and Ghanim M (2008) Gene expression in the whitefly Bemisia tabaci pupae in response to parasitization by the wasp Eretmocerus mundus. BMC Genomics. 9:342.

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and the viruses it transmits, are a major constraint to growing vegetable crops worldwide. Although the whitefly is often controlled using chemical pesticides, biological control agents constitute an important component in integrated pest management programs, especially in protected agriculture. One of these agents is the wasp Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae). E. mundus lays its egg on the leaf underneath the second third instar nymph of B. tabaci. First instars of the wasp hatch and penetrate the whitefly nymphs. Initiation of parasitization induces the host to form a capsule composed of epidermal cells around the parasitoid. The physiological and molecular processes underlying B. tabaci-E. mundus interactions have never been investigated.

Czosnek H. (2008). Tomato yellow leaf curl virus (geminiviridae). In: Encyclopedia of Virology. Third Edition. Mahy BWJ and Van Regenmortel M, Editors. Oxford, Elsevier. Vol. 5:138-145.

In the late 1950s the tomato cultures in the Jordan valley of Israel were unexpectedly affected by a disease of unknown etiology. The disease was accompanied by large populations of whiteflies. The suspicion that the whiteflies were the vector of a viral disease was confirmed following controlled transmission experiments in the laboratory. The virus was named tomato yellow leaf curl virus (TYLCV). The virus was isolated and its genome sequenced in the late 1980s.

TYLCV is a member of the genus Begomovirus of the family Geminiviridae, which includes viruses transmitted by the whitefly Bemisia tabaci. Begomoviruses have a genome either split between two circular single-stranded DNA molecules of approximately 2700 nt each named DNA A and DNA B (bipartite) or with a single genomic DNA A-like molecule (monopartite). TYLCV is monopartite. The relationships between the virus, the vector, and the host tomato plant have been the object of many studies.

From the early 1960s tomato cultures have been under the constant threat of TYLCV-like begomoviruses worldwide. TYLCV has quickly spread to the Middle East, Central Asia, North and West Africa, Southeast Europe, the Caribbean islands, Southeast USA, and Mexico. TYLCV-related begomoviruses have been identified in Italy, the Maghreb and Western Africa, and the Arabian Peninsula. Breeding programs for resistance have started in the mid-1970s and several commercial varieties with adequate resistance have been released. Several loci tightly linked to TYLCV resistance have been assigned to the small arm of tomato chromosome 6. A variety of strategies have been devised based on the pathogen derived resistance concept, which involves the expression of functional as well as dysfunctional viral genes. RNA mediated virus resistance based on antisense RNA and post-translational gene silencing was efficient but was highly sequence dependent.

Czosnek H. (2008). Acquisition, circulation and transmission of begomoviruses by their whitefly vectors. In: Viruses in the Environment, Editors: Palombo EA and Kirkwood CD. Research Signpost, Trivandrum, Kerala, India. Pages 29-44.

Some geminiviruses are transmitted by the whitefly Bemisia tabaci in a circulative manner. These geminiviruses are assigned to the genus Begomovirus within the family Geminiviridae. The route followed by begomoviruses in their insect vector and the velocity of translocation of the various viruses seem to be intrinsic to the whitefly, not to the virus. Subsequent to ingestion during feeding on an infected plant, viruses are first associated with the stylet food canal and then to the proximal part of the descending midgut. Transmittable viruses cross the gut barrier into to haemolymph from where they reach the accessory salivary glands. A GroEL homologue produced by the whitefly endosymbiotic bacteria facilitates this journey. The virus is then egested with the saliva into the phloem of a host plant. The capsid seems to be the only viral determinant involved in particle translocation. Unidentified receptors interacting with the viral particles permit their passage across membranes of the insect digestive system and salivary glands. Begomoviruses remain associated with their vector for various periods of time, sometimes during their entire adult life. The long-term presence of some begomoviruses has deleterious effects on the longevity and the fertility of the insect host. Some begomoviruses have been shown to be transovarially transmitted to adult progeny. Monopartite as well as bipartite begomoviruses can also be transmitted during mating. A functional genomic project of the whitefly Bemisia tabaci initiated about three years ago may provide answers to questions related to cellular determinants involved in virus translocation in its vector, effect of virus on the insect host and others related to whitefly development, resistance to insecticide, and host plant preference.

Edelbaum D, Gorovits R, Sasaki S, Ikegami M and Czosnek H (2009) Expressing a whitefly GroEL protein in Nicotiana benthamiana plants confers tolerance to Tomato yellow leaf curl virus (TYLCV) and Cucumber mosaic virus (CMV), but not to Grapevine virus A (GVA) and Tobacco mosaic virus (TMV). Archives of Virology 154:399-407.

Transgenesis offers many ways to obtain plants resistant to viruses. However, in most cases resistance is against a single virus or viral strain. We have taken a novel approach that allows predicting plant response to viruses. It is based on the ability of a whitefly endosymbiotic GroEL to bind viruses belonging to several genera, in vivo and in vitro. We have expressed the GroEL gene in Nicotiana benthamiana plants, postulating that upon virus inoculation, GroEL will bind to virions, thereby interfering with pathogenicity and expression of symptoms. The transgenic plants were inoculated with the Begomovirus Tomato yellow leaf curl virus (TYLCV) and the Cucumovirus Cucumber mosaic virus (CMV), which both interacted with GroEL in vitro, and with the Trichovirus Grapevine virus A (GVA) and the Tobamovirus Tobacco mosaic virus (TMV), which did not. While the transgenic plants inoculated with TYLCV and CMV presented a high level of tolerance, those inoculated with GVA and TMV were susceptible. The amounts of virus in tolerant transgenic plants was lower by three orders of magnitude than those in non-transgenic plants; in comparison, the amounts of virus in susceptible transgenic plants were similar to those in non-transgenic plants. The sap of the tolerant plants contained GroEL-virus complexes. Hence, tolerance was correlated with trapping of viruses in planta. This study demonstrated that multiple resistances to viruses belonging to several taxonomic families could be achieved. Moreover, it could be predicted that plants expressing GroEL will be tolerant to those viruses that bind to GroEL in vitro.

Eybishtz A, Peretz Y, Sade D, Akad F and Czosnek H (2009) Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus. Plant Molecular Biology 71:157-171.

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 (TRV VIGS) 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.

Mahadav A, Kontsedalov S, Czosnek H and Ghanim M (2009) Thermotolerance and gene expression following heat stress in the whitefly Bemisia tabaci B and Q biotypes. Insect Biochemistry and Molecular Biology 39:668-676.

The whitefly Bemisia tabaci (Gennadius) causes tremendous losses to agriculture by direct feeding on plants and by vectoring several families of plant viruses. The B. tabaci species complex comprises over 10 genetic groups (biotypes) that are well defined by DNA markers and biological characteristics. B and Q are amongst the most dominant and damaging biotypes, differing considerably in fecundity, host range, insecticide resistance, virus vectoriality, and the symbiotic bacteria they harbor. We used a spotted B. tabaci cDNA microarray to compare the expression patterns of 6000 ESTs of B and Q biotypes under standard 25 C regime and heat stress at 40 C. Overall, the number of genes affected by increasing temperature in the two biotypes was similar. Gene expression under 25 C normal rearing temperature showed clear differences between the two biotypes: B exhibited higher expression of mitochondrial genes, and lower cytoskeleton, heat-shock and stress-related genes, compared to Q. Exposing B biotype whiteflies to heat stress was accompanied by rapid alteration of gene expression. For the first time, the results here present differences in gene expression between very closely related and sympatric B. tabaci biotypes, and suggest that these clear-cut differences are due to better adaptation of one biotype over another and might eventually lead to changes in the local and global distribution of both biotypes.

Eybishtz A, Peretz Y, Sade D, Gorovits R and Czosnek H (2010) Tomato yellow leaf curl virus (TYLCV) infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread and necrosis. Planta 231:537- 548.

To identify genes involved in resistance of tomato to Tomato yellow leaf curl virus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants whereLeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth, and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

Loebenstein G, Rav David D, Leibman D, Gal-On A, Vunsh R, Czosnek H and Elad Y (2010) Tomato plants transformed with the inhibitor-of-virus-replication (IVR) gene are partially resistant to Botrytis cinerea. Phytopathology, in press.

Tomato plants transformed with a cDNA clone encoding the inhibitor-of-virus-replication (IVR) gene were partially resistant to Botrytis cinerea. This resistance was observed as a significant reduction in the size of lesions induced by the fungus in transgenic plants, as compared with the lesions on the nontransgenic control plants. This resistance was weakened when plants were kept at an elevated temperature, 32 C, before inoculation with B. cinerea, as compared with plants kept at 17 to 22 C prior to inoculation. Resistance correlated with the presence of IVR transcripts, as detected by RT-PCR. This is one of the few cases in which a gene associated with resistance to a virus also seems to be involved in resistance to a fungal disease.