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.

Mejía , 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 Methods. 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.

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 Aspergillusniger ß-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 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.

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 ìsignal transductionî defense response pathways.

Maxwell DP and Czosnek H (2007)
International networks to deal with tomato yellow leaf curl virus disease: 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 Communautés Européennes, 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 www.plantpath.wisc.edu/GeminivirusResistantTomatoes/ index.htm).

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 ñ 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 Trialeurodesvaporariorum (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 Methods 147:118-126.

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 3í 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 microarrayñbased 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: 323ñ328.

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 ñassociated 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 differentorigins 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 Eretmocerusmundus (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). I n: Encyclopedia of Virology. 5:138-145. Third Edition. Mahy BWJ and Van Regenmortel M, Editors. Oxford, Elsevier.

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 TrichovirusGrapevine 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 programóone 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 where LeHT1 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 100:225-229.

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.

Rosell RC, Blackmer JL, Czosnek H and Inbar M (2010).
Mutualistic and dependent Relationships with other Organisms. In: Bemisia, Bionomics and Management of a Global Pest, Stansly PA and Natanjo SE (Editors), Springer. Pp. 161-183.

Whiteflies in general and Bemisia tabaci in particular are involved in complex interactions with the host plant, various microorganisms and arthropods (herbivores and natural enemies). These relationships are not only important to the ecology and evolution of B. tabaci but are also essential to understanding and developing innovative control strategies. Thus, we have included in this chapter, a discussion of symbiotic relationships, the functional roles of microbial symbionts in whiteflies, the role of endosymbionts in begomovirus transmission and the intra- and interspecific relationships that occur between whiteflies, other herbivores and their host plants. Much new information has been generated since the previous Bemisia books (Gerling 1990, Gerling and Mayer 1996) and thus we have focused this work on new findings and the potential they hold for developing new control tactics.

Czosnek H and Brown J (2010).
The whitefly genome - white paper. Proposal to Sequence Multiple Genomes of Bemisia tabaci (Gennadius). In: Bemisia, Bionomics and Management of a Global Pest, Stansly PA and Natanjo SE (Editors), Springer. Pp.503-532.

As genomics, proteomics, and metabolomics research is expanded beyond model organisms, the methodologies developed through studies of model arthropods are becoming available for application to non-model systems, many of which are important agriculture pests. Directing these technologies to solving applied problems offers new opportunities for developing approaches to reduce the damage they cause as agricultural pests. Elucidating the functional genomics of the B. tabaci complex has become essential for devising novel, sustainable pest control strategies, and directed interference of whitefly-mediated virus transmission. Hence, availability of the complete genome sequence is now crucial for facilitating valuable genomics, proteomics, and functional genomics applications (Eisen et al. 1998).

Whitefly genomics research is expected to open important avenues into the discovery of novel strategies for whitefly management based in an improved understanding of molecular, cellular, and biological processes. The genome sequence will synergize projects underway to develop and sequence B. tabaci expressed sequence tags (EST) or cDNA libraries for functional genomics and proteomics analysis. The benefits are far reaching and include their application to better resolution of the B. tabaci complex systematics and contribute to the hallmark plasticity of this complex, to identify genes that combat abiotic and biotic stresses that often leads to invasiveness and insecticide resistance, and to understand the basis for whitefly-virus specificity (De Risi et al. 1997; Werling and Jungi 2003).

An annotated genome sequence, together with microarray capability and other functional genomics tools, will facilitate the analysis of whitefly genetics and of metabolic pathways. It will help elucidate the functional characterization of genes, their differential expression, the localization of their transcripts in situ, and the determination of the proteome. Altogether, availability of whole genome sequence will be instrumental in elucidating the biochemical processes that contribute to pest status. Below we summarize in greater detail the rationale for undertaking The Whitefly Genome Project. The case was made above for the importance of B. tabaci as a serious, new insect pest that undermines food and fiber production. We describe a number of unique biological and genetic attributes that will be elucidated at a deeper level than is presently possible.

Díaz-Pendón JA, Cañizares MC, Moriones E, Bejarano ER, Czosnek H and Navas-Castillo J (2010).
Tomato yellow leaf curl viruses: ménage à trois between the virus complex, the plant, and the whitefly vector. Molecular Plant Pathology 11:441-450.

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci.

Gottlieb Y, Zchori-Fein E, Mozes-Daube N, Kontsedalov S, Skaljac M, Brumin N, Sobol I, Czosnek H,
Vavre F, Fleury F and Ghanim M (2010). The transmission efficiency of Tomato yellow leaf curl virus is correlated with the presence of a specific symbiotic bacterium species. Journal of Virology 84:9310-9317.

Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using cultures of B. tabaci populations collected in Israel. Virus-transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pull-down assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroELs, we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be essential for transmission of this virus.

Aboul-Ata A-AE, Anfoka G, Zeidan M and Czosnek H (2010).
Diagnosis of cereal viruses in the Middle East. International Journal of Virology 6:126-137.

Middle Eastern countries are major consumers of small grain cereals. For example, Egypt is the biggest bread wheat importer with 5.9 Million Tons (MT) although it itself produces 10.5 MT. Jordan and Israel import almost all the grains they consume. Viruses are the major factors that impair production in Middle East. They are transmitted in non persistent, semi persistent and persistent manners by insects (aphids, leafhoppers, and mites), and through soil and seeds. Hence there is a need to control insect-borne cereal viruses not only in the field but also through plant quarantine services for imported seed- and soil-borne viruses. Viruses need to be controlled in the frame of regional, collaborative activities involving the Middle Eastern countries. The means to be used to diagnose cereal viruses may include symptom observation, immunological technologies such as ELISA using polyclonal and monoclonal antibodies against the virus coat protein (raised against purified virions or against the virus capsid protein expressed in bacteria or in yeast), molecular techniques such as PCR (uniplex and and multiplex), RFLP, SSCP and microarrays. In this article, we explore the different diagnosis, typing and detection techniques of cereal viruses available to the Middle Eastern countries, and we review the ongoing collaborative research projects.

Czosnek H (2010).
Management of Tomato yellow leaf curl disease; a case study for emerging geminiviral diseases. In: Emerging Geminiviral Diseases and their Management. Eds: Sharma P, Gaur RK and Ikegami M. Nova Science Publishers Inc. pp. 37-57.

Although not the first virus geminivirus discovered, Tomato yellow leaf curl virus (TYLCV) has been one of the most studied. TYLCV impairs tomato cultures worldwide. Within less than 20 years TYLCV has spread from the Middle East to North America, Africa, Europe and Far East Asia. Molecular analyses have shown that the Tomato leaf curl disease is caused by several virus species and many strains related but different from the Middle Eastern TYLCV. This virus is an ideal object to study such topics in molecular virology as diverse as replication, expression, evolution, recombination and interaction with host factors. TYLCV is transmitted by the whitefly vector to plants in a circulative manner. The interactions between the virus, the vector and the target plant are the object of intense scrutiny. TYLCV causes a serious disease of tomato that needs to be avoided by all means. Hence ways to escape inoculation by whiteflies have been implemented, which include nets, chemical treatment to curb down whitefly populations, traps and natural enemies. Breeding for resistance has started already 30 years ago by introgressing resistance traits found in wild tomato species into the susceptible domesticated tomato. Breeding has been backed by genetic studies of resistance, discovery of QTLs and polymorphic DNA markers linked to resistance, and screening for resistance genes. Commercial lines with good tolerance to TYLCV are available; some are effective against several different TYLCV species, others are not. Altogether the study of TYLCV and the results achieved in molecular virology, genetics, plant-virus-insect interactions, IPM and breeding for resistance, can serve as model for similar studies involving other deleterious geminiviruses infecting important crops, both for basic and applied interests.

Czosnek H, Sade D, Gorovits R, Vidavski F, Beeri H, Sobol I and Eybishtz E (2011).
A RNAi-based genome-wide screen to discover genes involved in resistance to Tomato yellow leaf curl virus (TYLCV) in tomato. In: RNAi Technology. Eds: Gaur RK, Gafni Y, Sharma P, Gupta VK. Nova Science Publishers Inc. pp. 155-176.

The cultivated tomato Solanum lycopersicum is under the threat of diseases caused by the Tomato yellow leaf curl virus (TYLCV). Several wild tomato species are resistant to TYLCV. Five loci, coined Ty-1 to Ty-5, have been shown to be associated with resistance. Breeding for resistance consisted in introgressing resistance from the wild tomato species into S. lycopersicum. The genes conferring TYLCV resistance have not been identified so far.

We have proposed to use a TRV-VIGS RNAi-based genome screen to uncover the genes and gene networks underlying TYLCV resistance in tomato. We have postulated that genes preferentially expressed in resistant plants (compared to susceptible), and upregulated upon TYLCV infection, are part of resistance gene network(s). Moreover if a given gene has a critical role in the resistance network, silencing this gene will lead to the collapse of resistance. To decipher the networks we have used two inbred tomato lines issued from the same breeding program that used S. habrochaites as a source of resistance: one was resistant (R), the other was susceptible (S) to the virus. Sixty nine genes preferentially expressed in R tomatoes were identified by differential screening of cDNA libraries from infected and uninfected R and S tomato plants. From the twenty genes silenced so far, six answered to this criterion. Hence, not all the genes that have been found to be preferentially expressed in R plants have the same cardinal role in the establishment of resistance to TYLCV.

We are presenting the results obtained with three genes: a permease, a sucrose transporter and a lipocalin-like protein. If we define a pathogen resistance gene as a gene that if silenced the host becomes susceptible, then it is obvious that TYLCV resistance is controlled by more than one gene. At this time there is no apparent biochemical and physiological link between these genes and the way each one affects the expression of the others is not known. Future investigations will focus on the known resistance pathways in plants and in the hierarchy of the genes we have discovered. Since, in our case, resistance to TYLCV has been introgressed from the wild tomato species S. habrochaites, it will be of interest to find out whether the genes preferentially expressed in R tomato plants have been introgressed from this wild tomato species.

Czosnek H and Ghanim M (2011).
Bemisia tabaci - Tomato yellow leaf curl virus interaction causing world wide epidemics. In: The Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) interaction with geminivirus-infected host plants - Bemisia tabaci, host plants and geminiviruses. Thompson WMO Ed. Springer. Pp. 51-67.

Tomato yellow leaf curl virus (TYLCV)] is a begomovirus that threatens tomato production worldwide. TYLCV is transmitted in a circulative manner by the whitefly Bemisia tabaci. Once ingested, TYLCV was detected in the insect midgut after 1 h, in the haemolymph after 1.5 h, and in the salivary glands after 7 h. Whiteflies were able to infect tomato plants after 8 h. TYLCV survival in the haemolymph of B. tabaci is ensured by the interaction of virus particles with a GroEL homologue produced by the whitefly endosymbionts. The relation between TYLCV and its vector are intricate. Following a short acquisition period, the virus remains associated with B. tabaci for the four weeks-long adult life of the insect. During this period, infectivity decreased from 100 to 10-20%. The long-term presence of tylcv in B. tabaci was associated with a decrease in longevity and fertility. Similar results were reported with Tomato yellow leaf curl China virus (TYLCCNV). The question of whether TYLCV is expressed and replicates in its vector is not settled. Transcripts of TYLCV genes encoded by the genome strand as well as by the genome complementary strand have been detected in B. tabaci following virus acquisition. TYLCV was found to be transmitted transovarially up to the adult stage of the first progeny generation, as did Tomato yellow leaf curl Sardinia virus (TYLCSV); however contrary to TYLCV, the whiteflies were not able to transmit the disease to tomato plants. TYLCV and TYLCCN could be transmitted with various efficiency during mating. A cDNA microarray was constructed to discover the genes involved in TYLCV-B. tabaci interactions.

Czosnek H (2011)
Ethics in Agriculture. In: Research Ethics, Landau R and Shefler G Eds, The Hebrew University Magnes Press, Jerusalem. Pp.334-360.

Over the last 30 years worldwide agriculture has witnessed changes comparable to the green revolution that occurred in the 1960s. Advances in scientific research during this period have, among other things, brought about the development of new species of most known plants, including major food sources such as wheat and rice. While many view these developments in agricultural research as a blessing and consider them a great contribution to humanity, others warn of the destructive impacts of their applications. Their concern is directed primarily at the short- and long-term effects of this research on the ecological systems of numerous countries around the globe. The consequences of scientific research in agriculture may have economic and social implications that, among other things, will affect the nature of agricultural work as well as the standard of living for farmers and agriculturists worldwide. Moreover, scientific research applications in agriculture may also have a significant impact on the health of the general public, for whom agricultural produce is a major source of nourishment. This chapter discusses the ethical dilemmas raised by scientific research and its applications in the field of agriculture and vegetable growth and examines the concomitant opportunities and risks.

Scholthof K-BG, Adkins S, Czosnek H, Palukaitis P, Jacquot E, Hohn T, Hohn B, Saunders K, Candresse T, Ahlquist P, Hemenway C and Foster GD (2011).
Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology 12:938-954.

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a ëTop 10í based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology.The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Bromemosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Aboul-Ata AE, Mazyad H, El-Attar AK, Soliman AM, Anfoka G, Zeidan M, Gorovits R, Sobol I and Czosnek H (2011).
Diagnosis and control of cereal viruses in the Middle East. Advances in Virus Research 81:33-61.

Middle Eastern countries are major consumers of small grain cereals. Egypt is the biggest bread wheat producer with 7.4 million tons (MT) in 2007, but at the same time, it had to import 5.9 MT. Jordan and Israel import almost all the grains they consume. Viruses are the major pathogens that impairs grain production in the Middle East, infecting in some years more than 80% of the crop. They are transmitted in non persistent, semi persistent and persistent manners by insects (aphids, leafhoppers, and mites), and through soil and seeds. Hence cereal viruses have to be controlled, not only in the field but also through the collaborative efforts of the plant quarantine services inland and at the borders, involving all the Middle Eastern countries. Diagnosis of cereal viruses may include symptom observation, immunological technologies such as ELISA using polyclonal and monoclonal antibodies raised against virus coat protein expressed in bacteria, and molecular techniques such as PCR, microarrays and deep sequencing. In this chapter, we explore the different diagnosis, typing and detection techniques of cereal viruses available to the Middle Eastern countries. We highlight the plant quarantine service and the prevention methods. Finally we review the breeding efforts for virus resistance, based on conventional selection and on genetic engineered.

Kontsedalov S, Abu-Moch F, Lebedev G, Czosnek H, Horowitz AR and Ghanim M (2012).
Bemisia tabaci biotype dynamics and resistance to insecticides in Israel during the years 2008-2010. Journal of Integrative Agriculture 11:312-320.

The sweetpotato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is an extremely polyphagous insect pest that causes significant crop losses in Israel and worldwide. B. tabaci is a species complex of which the B and Q biotypes are the most widespread and damaging worldwide. The change in biotype composition and resistance to insecticide in Israel was monitored during the years 2008-2010 to identify patterns in population dynamics that can be correlated with resistance outbreaks. The results show that B biotype populations dominate crops grown in open fields, while Q biotype populations gradually dominate crops grown in protected conditions such as greenhouses and nethouses, where resistance outbreaks usually develop after several insecticide applications. While in previous years, Q biotype populations were widely detected in many regions in Israel, significant domination of the B biotype across populations collected was observed during the year 2010, indicating the instability of the B. tabaci population from one year to another. Reasons for the changing dynamics and the shift in the relative abundance of B. tabaci biotype, and their resistance status, are discussed.

Czosnek H and Ghanim M (2012)
Back to basics: are begomoviruses whitefly pathogens? Journal of Integrative Agriculture 11:225-234.

Begomoviruses and whiteflies have interacted for geological times. An assumed long-lasting virus-vector intimate relationship of this magnitude implies that the partners have developed co-evolutionary mechanisms that insure on one hand the survival and the efficient transmission of the virus, and on the other hand the safeguard of the insect host from possible deleterious effects of the virus. Several studies have indicated that viruses belonging to the Tomato yellow leaf curl virus family from China, Israel and Italy (TYLCVs) are reminiscent of insect pathogens. TYLCVs like all begomoviruses are transmitted in a circulative manner by the whitefly Bemisia tabaci. The survival of the virus in the haemolymph of B. tabaci is ensured by a GroEL homologue produced by a whitefly secondary endosymbiont. Following acquisition and transfer to non-host plants, the virus may remain associated with the insect for its entire 4-5 weeks-long adult life. During this period, the ability of the insects to inoculate plants steadily decreased, but did not disappear. The long-term presence of tylcvs in B. tabaci was associated with a decrease in the insect longevity and fertility. Viral DNA was transmitted to progeny, but seldom infectivity. TYLCV transcripts were found associated with the insects, raising the possibility of replication and expression in the vector. TYLCVs may spread amidst whiteflies during copulation. Functional genomics tools such as microarrays, deep sequencing, quantitative PCR and gene silencing allow revisiting the proposition that TYLCVs have retained, or acquired, some characteristics of an insect pathogen.

Moshe A, Pfannstiel J, Brotman Y, Kolot M, Sobol I, Czosnek H and Gorovits R (2012)
Stress responses to Tomato yellow leaf curl virus (TYLCV) infection of resistant and susceptible tomato plants are different. Metabolomics S1:006. doi:10.4172/2153-0769.S1-006 .

Two genetically close inbred tomato lines, one resistant to Tomato yellow leaf curl virus (TYLCV) infection (R), the other susceptible (S), showed completely different stress response upon TYLCV infection. S plants were stunted and do not yield, while R plants remained symptomless and yielded. Comparison of protein profiles and metabolites patterns in TYLCV infected R and S tomatoes revealed a completely different host stress response. S plants were characterized by higher levels of reactive oxygen species (ROS) and ROS compounds, the anti-oxidative, pathogenesis-related (PR) and wound-induced proteins were predominant. In contrast, infection of R tomatoes did not drastically activate the same host defense mechanisms as in S plants, while R homeostasis was much more effectively maintained by protein and chemical chaperones. Sources of carbon and nitrogen were less affected by TYLCV in R than in S plants, which could make R plants more balanced and more fit to sustain infection. Even though both tomato types contained comparable amounts of TYLCV at the specified stage of infection, the cellular immune responses were different. Presented results are preliminary and indicate not so much the concrete data but the global tender in understanding of the cellular response to virus stress at the background of resistance and susceptibility to TYLCV.

Sade D, Eybishtz A, Gorovits R, Sobol I and Czosnek H (2012)
A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance. Plant Molecular Biology. 80:273-287.

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene, termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip) was expressed in leaves during a 15-day window starting about 40 days after sowing. SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and a necrotic response along the stem and petioles. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLipwas inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.

Götz M, Popovski S, Kollenberg M, Gorovitz R, Brown JK, Cicero J, Czosnek H, Winter S and Ghanim M (2012)
Implication of Bemisia tabaci heat shock protein 70 in begomovirus - whitefly interactions. Journal of Virology 86:13241-13252.

The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome, and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative non-propagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these co-evolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR and western blot analyses indicated that a B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture Polymerase Chain Reaction, protein coimmunoprecipitation and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in-situ hybridization and immunolocalization showed co-localization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 protein within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly

Esterhuizen LL, Mabasa KG, van Heerden SW, Czosnek H, Brown JK, van Heerden H and Rey MEC (2013)
Genetic identification of members of the Bemisia tabaci cryptic species complex from South Africa reveals native and introduced haplotypes . Journal of Applied Entomology 137:122-136.

The whitefly Bemisia tabaci cryptic species group is an important agricultural pest and virus vector. Members of the complex have become serious pests in South Africa (SA) because of their feeding habit and their ability to transmit Begomovirus species. Despite their economic importance, studies on the biology and distribution of B. tabaci in SA are limited. To this end, an analysis was made of the current diversity and distribution of B. tabaci cryptic species in SA using the mitochondrial cytochrome oxidase I (mtCOI) sequences. Phylogenetic analysis revealed the presence of members from two endemic sub-Saharan Africa (SSAF) subclades co-existing with two introduced putative species. The SSAF-1 subclade includes cassava host-adapted B. tabaci populations, whereas the whiteflies collected from cassava and non-cassava hosts formed a distinct subclade, referred to as SSAF-5, and represent a new subclade among previously recognized southern Africa clades. Two introduced cryptic species, belonging to the Mediterranean and Middle East-Asia minor 1 clades were identified and include the B and Q types. The B type showed the widest distribution, being present in five of the eight provinces explored in SA, infesting several host plants and predominating over the indigenous haplotypes. This is the first report of the occurrence of the exotic Q type in SA alongside the more widely distributed B type. Furthermore, mtCOI PCR-RFLP was developed for the SA context to allow rapid discrimination between the B, Q and SSAF putative species. The capacity to manage pests and disease effectively relies on knowledge of the identity of the agents causing the damage. Therefore, understanding the South African B. tabaci species diversity and host plant distribution contribute to the development of knowledge-based disease management practices.

Gorovits R, Moshe A, Kolot M, Sobol I and Czosnek H (2013)
Progressive aggregation of Tomato yellow leaf curl virus coat protein in systemically infected tomato plants, susceptible and resistant to the virus. Virus Research. 171:33-43.

Tomato yellow leaf curl virus (TYLCV) coat protein (CP) accumulated in tomato leaves during infection. The CP was immuno-detected in the phloem associated cells. At the early stages of infection, punctate signals were detected in the cytoplasm, while in the later stages aggregates of increasing size were localized in cytoplasm and nuclei. Sedimentation of protein extracts through sucrose gradients confirmed that progress of infection was accompanied by the formation of CP aggregates of increasing size. Genomic ssDNA was found in the cytoplasm and in the nucleus, while the dsDNA replicative form was exclusively associated with the nucleus. CP-DNA complexes were detected by immuno-capture PCR in nuclear and cytoplasmic large aggregates. Nuclear aggregates contained infectious particles transmissible to test plants by whiteflies. In contrast to susceptible tomatoes, the formation of large CP aggregates in resistant plants was delayed. By experimentally changing the level of resistance/susceptibility of plants, we showed that maintenance of midsized CP aggregates was associated with resistance, while large aggregates where characteristic of susceptibility. We propose that sequestering of virus CP into midsized aggregates and retarding the formation of large insoluble aggregates containing infectious particles is part of the response of resistant plants to TYLCV.

Czosnek H, Eybishtz A, Sade D, Gorovits R, Sobol I, Bejarano E, Rosas-D?az T and Lozano-Durán R (2013)
Discovering host genes involved in the infection by the Tomato yellow leaf curl virus complex and in the establishment of resistance to the virus using Tobacco rattle virus-based post transcriptional gene silencing. Viruses 5:998-1022.

The development of high-throughput technologies allows evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infectionof susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, that over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the twenty five genes studied so far, silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicate that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular mechanisms underlying plant infection and resistance to infection by begomoviruses.

Sade D, Brotman Y, Eybishtz A, Cuadros-Inostroza A, Fernie AR, Willmitzer L and Czosnek H (2013)
Involvement of the hexose transporter gene LeHT1 and of sugars in resistance of tomato to Tomato yellow leaf curl virus . Molecular Plant 5:1707-1710.

Tomato yellow leaf curl virus(TYLCV) is a whitefly-transmitted geminivirus infecting tomato crops (Czosnek, 2007). TYLCV-resistant (R) and susceptible (S) lines with the same genetic background have been bred using Solanum habrochaites as the resistance source. Previously, we demonstrated that the hexose transporter gene LeHT1 is up-regulated upon infection in R plants and its silencing in R plants (RH) leads to the collapse of resistance (Eybishtz et al., 2010). To uncover the role of LeHT1 in resistance we (I) analyzed the transcriptome re-programming in leaves of S, R and RH plantsusing a home-designed microarray, before and 7 days after TYLCV inoculation (0, 7dpi), and (II) measured the concentration of sugars and their derivatives in S, R and RH leaves at 1 and 7dpi because LeHT1 is transporting both glucose and fructose. ). Upon infection, the genes differentially expressed in S vs. R plants, are also those differentially expressed in Si vs Ri plants. In Ro plants - the highly expressed genes were related to biotic stress, jasmonic acid and ethylene biosynthesis, signal transduction, and RNA regulation and processing. LeHT1 silencing has only a minor effect on R plant gene expression (RHo is similar to Ro). Most of the genes upregulated in R plants upon LeHT1 TRV-mediated silencing are involved in response to pathogens; nonetheless, induction of these genes did not prevent collapse of resistance of the RH plants upon TYLCV infection. TYLCV infection significantly modified gene expression in S, R and RH tomatoes (Si vs. So, Ri vs. Ro, RHi vs. RHo). At 7dpi, the transcriptional pattern of RHi plants resembled more that of Ri than that of Si. These results might indicate that these Ri-specific genes are not involved in general stress response, but rather contribute to TYLCV resistance. At 1dpi, when LeHT1 is strongly induced in Ri plants (Figure 1D), the sugar content of Si and Ri plants was similar, but different from that of RHi plants. Hence when TYLCV attacks a tomato plant, it initiates rapid changes resulting in the decline in photosynthesis accompanied by an increase in invertase expression, release of hexose that activates defense response or, if failing, promoting pathogen replication and disease expression. In R plants where LeHT1 has been silenced, the hexose transporter is not expressed, and hence hexoses cannot be internalized into the cell in order to act as defense signaling molecules, likely decreasing the PTM effect, and inducing the collapse of resistance. In this respect, sugar metabolism in RH and S plants are similar, but remarkably differ from R plants.

Gorovits R and Czosnek H (2013)
Insect symbiotic bacterial GroEL (Chaperonin 60) and plant virus transmission . B. Henderson (ed.), Moonlighting Cell Stress Proteins in Microbial Infections, Heat Shock Proteins 7, DOI 10.1007/978-94-007-6787-4_11. pp. 173-187.

GroEL is a multifunctional protein belonging to the conspicuous family of chaperones active in prokaryotic and eukaryotic cells. GroEL of Escherichia coli is a heat shock-like protein (Hsp60). It is involved in the correct folding of newly synthesized proteins, and participates in protein aggregation and in repair of damaged proteins. GroEL is essential for the morphogenesis and the capsid assembly of a number of E. coli bacteriophages. In eukaryotic cells, HSPs were shown to promote virus replication and survival. GroEL homologues are produced not only by free living bacteria but also by bacteria living in total symbiosis with insects and located in specialized eukaryotic cells called bacteriocytes. Symbiosis, which occurred some 200 million years ago, has lead to a reduction of the bacterial genome by two third, accompanied by the adaptation of the endosymbiotic bacteria to novel functions such as providing the host with essential amino acids and other nutrients. It seems that circulative plant viruses have taken advantage of the high production of GroEL by the endosymbionts to device a protective mechanism allowing viral particles to safely cross the haemolymph-filled body cavity of the insect, while translocating from the digestive tract to the salivary system. It seems that the relationship between chaperones and plant viruses, and the insects that vector them, have lasted for geological times. Here we analyze the relationship of endosymbiotic GroEL with viruses in a number of insect-circulative virus systems. Moreover, we show how we can exploit this relationship to devise diagnosis tests for a number of viruses and generate virus-resistant plants by expressing insect endosymbiotic GroEL proteins.

Luan J-B, Ghanim M, Liu S-S and Czosnek H (2013)
Silencing the ecdysone (synthesis and signaling) pathway genes disrupts nymphal development in the whitefly. Insect Biochemistry and Molecular Biology. 43:740-746.

Sap-sucking insects are important pests in agriculture and good models to study insect biology. The role of ecdysone pathway genes in the life history of this group of insects is largely unknown likely due to a lack of efficient gene silencing methods allowing functional genetic analyses. Here, we developed a new and high throughput method to silence whitefly genes using a leaf-mediated dsRNA feeding method. We have applied this method to explore the roles of genes within the molting hormone-ecdysone synthesis and signaling pathway for the survival, reproduction and development of whiteflies. Silencing of genes in the ecdysone pathway had a limited effect on the survival and fecundity of adult whiteflies. However, gene silencing reduced survival and delayed development of the whitefly during nymphal stages. These data suggest that the silencing method developed here provides a useful tool for functional gene discovery studies of sap-sucking insects, and further indicate the potential of regulating the ecdysone pathway in whitefly control.

Gorovits R, Moshe A, Ghanim M and Czosnek H (2013)
Recruitment of the host plant heat shock protein 70 by Tomato yellow leaf curl virus coat protein is required for virus infection. PLoS ONE. 8(7): e70280.

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.

Barba M, Czosnek H and Hadidi A (2014)
Historical perspective, development and applications of next-generation sequencing in plant virology. Viruses. 6:106-136.

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Gorovits R, Moshe A, Ghanim M and Czosnek H (2014).
Degradation mechanisms of the Tomato yellow leaf curl virus coat protein following inoculation of tomato plants by the whitefly Bemisia tabaci . Pest Management Science. doi : 10.1002/ps.3737 /

Tomato yellow leaf curl virus(TYLCV) is a begomovirus infecting tomato cultures worldwide. TYLCV is transmitted to plants by the whitefly Bemisia tabaci. Once in the plant the virus is subjected to attack by the host plant defenses, which may include sequestration in aggregates, proteolysis, ubiquitination, 26S proteasome degradation, and autophagy. Elucidating how the virus avoids destruction will enable to understand infection and possibly devise counter-measures. The accumulation of viral coat protein (CP) and of viral DNA in plants are markers of a successful virus transmission by B. tabaci. In response to infection, tomato tissues display multiple ways to degrade TYLCV proteins and DNA. In this study, we show that CP (in soluble and insoluble states) is the target of protease digestion, 26S proteasome degradation and autophagy. The highest degradation capacity was detected among soluble proteins and proteins in large aggregates/ inclusion bodies; cytoplasmic extracts displayed higher activity than nuclear fractions. The very same fractions possessed the highest capacity to degrade viral genomic DNA. Separately, 26S proteasome degradation was associated with large aggregates (more pronounced in the nuclearthan in the cytoplasmic fractions), which are indicators of a successful abduction of plants by viruses. Autophagy/ lysosome/vacuole degradation was a characteristic of intermediate aggregates, sequestering the CP in the cytoplasm and retarding the development of large aggregates. Chloroplast proteases were active in soluble as well as in insoluble protein extracts. To the best of our knowledge, this study is the first attempt to identify elements of the virus-targeted degradation machinery, which is a part of the plant response to virus invasion.

Kliot A, Cilia M, Czosnek H and Ghanim M (2014)
Implication of the bacterial endosymbiont Rickettsia spp. in the whitefly Bemisia tabaci interactions with Tomato yellow leaf curl virus . Journal of Virology 88:5652-5660.

Numerous animal and plant viruses are transmitted by arthropod vectors in a persistent, circulative manner. Tomato yellow leaf curl virus (TYLCV) is transmitted by the sweet potato whitefly Bemisia tabaci. Here we report that infection with Rickettsia spp., a facultative endosymbiont of whiteflies, altered TYLCV- B. tabaci interactions. A B. tabaci strain infected with Rickettsia acquired more TYLCV from infected plants, retained the virus longer and exhibited nearly double the transmission efficiency than a non-infected strain, with the same genetic background. Temporal and spatial antagonistic relationships were discovered between Rickettsia and TYLCV within the whitefly. Along different time course experiments, the levels of virus and Rickettsia within the insect were inversely correlated. Fluorescence in situ hybridization analysis on Rickettsia infected midguts showed evidence for niche exclusion between Rickettsia and TYLCV. In particular, high levels of the bacterium in the midgut resulted in higher virus concentration in the filter chamber, a favored site for virus translocation along the transmission pathway, while low levels of Rickettsia in the midgut resulted in an even distribution of the virus. Taken together, these results indicate that Rickettsia, by infecting the midgut, increases TYLCV transmission efficacy, adding further insights into the complex association between persistent plant viruses, their insect vectors and microorganisms tenants that reside within these insects.

Sade D, Shriki O, Cuadros-Inostroza A, Tohge T, Semel Y, Haviv Y, Willmitzer L, Fernie AR, Czosnek H and Brotman Y (2014)
Comparative metabolomics and transcriptomics of plant response to Tomato yellow leaf curl virus infection in resistant and susceptible tomato cultivars. Metabolomics. Accepted.

In order to understand resistance to TYLCV we have performed a combined analysis of the metabolome and transcriptome of resistant (R) and susceptible (S) tomato plants both prior to and following TYLCV infection. Metabolites detected by GC-MS and LC-MS analysis, in leaves of R and S plants, at 1, 3, 7 and 14 days post infection and control plants, were used for the reconstruction of four independent metabolic networks. Measuring the network parameters revealed distinctive systemic metabolic responses to TYLCV infection between the R and S plants. Notably, the GC-MS metabolic network indicated that, following infection, the R plant exhibited tight coordination of the metabolome than the S plant. Clear differences in the level of specialized metabolites between the S and R plants were revealed; among them, substantial alteration in the abundance of amino acids and polyamines, phenolic and indolic metabolites, all leading to the synthesis of defense compounds. Integrating metabolome and transcriptome data highlighted differently regulated pathways in the R and S plants in response to TYLCV, including the phenylpropanoid, tryptophan/nicotinate and urea/polyamine pathways. Salicylic acid biosynthesis was additionally distinctively activated in R plants upon infection. Comparing the expression of genes of the urea and phenylpropanoid pathways in S, R and Solanum habrochaites, the resistance genitor wild species tomato, indicated a time-shift in the expression patterns, before and following infection, which on one hand reflected the genetic similarity between these plants, and on the other hand demonstrated that the resistant phenotype is intermediate between that of S and S. habrochaites.