Faculty of Agriculture, Food and Environmental Quality Sciences, HUJI

Faculty of Agriculture, Food and Environment Quality Sciences

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

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RECENT AND CURRENT RESEARCH PROGRAMS

REGIONAL (ISRAELI-ARAB) COOPERATION, plant breeding for disease resistance

1. Development of regional indexation and certification program for plant propagation materials in the Middle East.

With D. Maxwell D and M. Nakhla (University of Wisconsin at Madison USA, H. Mazyad and M. Shalaby (Agricultural Research Center, Giza, Egypt), N. Iraqi (Bethlehem University, Palestinian Authority), G. Anfoka (Al-Balqua' Applied University, Al-Salt Jordan), M. Sedegui (National Agronomic Research Institute, Rabat, Morocco), M. Marrakchi (Faculty of Science of Tunis, Tunisia).
Funded by the US AID-CDR Middle East Research and Cooperation Program (1998-2006).

Background:

Fruit trees and vegetables are among the most important horticultural commodities in the Middle East. The process of exchanging plant materials is laborious, expensive and time consuming. In most cases quarantine procedures significantly delay the release of imported materials. Traditional procedures for examining plant materials are quite slow and often insufficient to detect diseases, especially virus and viroid diseases.

Currently, there is an increasing demand in the Middle Eastern countries for locally produced fruits and vegetables, and for their propagation materials, both for export and local markets. The successful modern horticultural industry demands a continuing supply of new cultivars for introduction into the existing markets and to meet the needs of anticipated markets. The need for producing and releasing virus and viroid-free germplasm from imported, as well as local stocks require the development and implementation of an innovative research program that utilizes state-of-the-art biotechnology for the detection and control of viruses and viroids affecting these horticultural crops.

Vegetatively propagated plants, such as potato, stone fruits, and citrus are severely damaged when grown from infected plant materials. The most important virus problems affecting the movement of agricultural crops in the Middle East are: for tomato -- tomato yellow leaf curl virus (TYLCV), tomato spotted wilt virus (TSWV), tomato mosaic virus (ToMV), cucumber mosaic virus (CMV); for potato -- potato virus Y (PVY), potato leafroll virus (PLRV), potato spindle tuber viroid, potato virus A and potato virus X; for cucurbits -- cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus-2 (WMV-2), and cucumber vein yellow virus (CVYV); for citrus -- citrus tristeza virus (CTV), Psorosis, citrus xyloporosis virus, and citrus exocortis viroid (CEVd); and for stone fruits -- plum pox virus (PPV).

Since most measures for control of virus and viroid diseases are based on prevention rather than cure, it is essential to have reliable and sensitive methods for pathogen detection. Virologists and plant pathologists have found serological techniques to be extremely useful for the identification and the routine diagnosis of virus diseases. The main advantages of serological diagnosis of infections are specificity, rapidity, and reliability. In recent years, several serological methods have been developed for specific and rapid detection of plant viruses. The sensitivity and simplicity of ELISA (Enzyme-Linked Immunosorbent Assay), as well as the low cost and long shelf-life of the necessary reagents, make this technique well suited for mass indexing. With the introduction of molecular techniques it is now possible to obtain large amounts of viral coat protein using a bacterial expression system engineered to produce the coat protein of the virus. The coat protein is easily purified and can be used to produce a very specific antiserum. Once this is achieved, the antiserum will be incorporated into ELISA detection kits. This system has worked well for several RNA and DNA viruses. Because viroids lack the antigenic protein coat that encapsidates viral RNAs, they pose a problem in diagnosis.

A practical diagnostic test for the detection of viroids based on nucleic acid hybridization has been developed. Nucleic acid spot hybridization has been shown to be a highly sensitive, rapid, simple, and inexpensive technique for identifying RNA or DNA viruses and plant viroids. A drawback of molecular hybridization tests is that they can be applied only with difficulty to stone fruit crude extracts, either because of the low titer of target nucleic acids or because of the presence of interfering substances that generate a high background or false positive signals. Some modification of methods is therefore necessary. This may consist either of using purified total nucleic acid extracts as test samples, or amplifying target nucleic acid through reverse transcription-polymerase chain reaction (RT-PCR). This latter technique was successfully used for the detection of some stone fruit viruses and is expected to have an increasingly wider application in the immediate future. The polymerase chain reaction (PCR) has the advantage of being rapid, reliable, and more sensitive than many procedures presently in use for plant pathogen detection. PCR is especially useful for differentiating between strains of endemic and exotic viruses and viroids, for detecting viroid infections, and for detecting viruses present at low concentration in infected cells and for sorting out non-specific reactions in serological tests.

Objectives:

Develop and evaluate virus detection methods for potatoes, grapes, stone fruits, and tomatoes among the partners.
Foster confidence in the virus testing among the Middle East countries by standardizing the results from virus detection.
Protect agricultural crops from new diseases by testing imported plant materials with rapid and reliable detection procedures.
Expand collaborative research efforts between Arab countries and Israel.
Build adequate confidence in certification techniques through training and exchange visits and meetings between scientists from the different collaborating countries.
Establish a collection of virus antisera and viral detection probes for distribution to private sector and other public and government institutions.
Transfer virus detection methods to the appropriate administrative unit in each country for certification and quarantine.
See website: http://www.plantpath.wisc.edu/InVirLab/docs/ virusindexing.htm

2. Developmentment of tomatoes with resistance to Tomato yellow leaf curl virus (TYLCV) using both virus-derived resistance and molecular-assisted breeding.

With S. Vidavski (The Hebrew University of Jerusalem, Israel), M. Lapidot (The Volcani Center, Bet Dagan, Israel), D. Maxwell D and M. Nakhla (University of Wisconsin at Madison, USA), H. Mazyad and M. Shalaby (Agricultural Research Center, Giza, Egypt), N. Iraqi (Bethlehem University, Palestinian Authority), G. Anfoka (Al-Balqa Applied University, Al-Salt Jordan), M. Sedegui (National Agronomic Research Institute, Rabat, Morocco).
Funded by the US AID-CDR Middle East Research and Cooperation Program (2005-2009).

Background:

Tomato production in the Middle-East as well as around the entire Mediterranean basin is limited by the endemic presence of the Tomato yellow leaf curl virus (TYLCV). This virus is transmitted by an insect, the whitefly Bemisia tabaci. In Egypt, 100% of the fall-grown tomato plants are usually infected and production losses may reach 80%, rendering tomato production during the autumn unprofitable. In Jordan all field-grown tomato plants are severely infected and heavy losses occur. In Israel, much of the tomato production has been transferred to greenhouses covered with insect-proof nets. Multiple applications of insecticides, sometimes daily, in attempt to control the insect vector have failed to eradicate the virus. The most efficient way to increase tomato production is the introduction of resistant cultivars.

Resistance can be achieved during a breeding program by introgressing resistance genes found in wild tomato species into the domesticated tomato or by genetically engineering tomato cultivars. Classical breeding can be facilitated by using DNA markers closely linked to the resistance gene(s).

Because whiteflies do not know borders and because the virus is genetically identical in the countries of all participants in this project, a regional program to fight the disease makes the most sense. In a concerted effort we will pool the resistance genes available in various germplasm and will apply molecular markers to facilitate their introgression into one group of resistant cultivars. Similarly genetically engineered resistant tomato may be crossed with the resistant genotypes to produce a superior tomato suitable for the entire region. The partners will profit from exchange of technology and implementation of modern breeding techniques. The availability of tomato cultivars resistant to TYLCV will allow small farmers as well as big producers to increase their earnings and to conquer new markets. Production and marketing of superior seeds will be done either by a new regional company or will be subcontracted to an existing company with enhanced marketing possibilities.

Objectives:

Develop tomato cultivars resistant to TYLCV with characteristics suitable for the markets of collaborating countries by applying virus-derived and marker-assisted breeding strategies.
Introduce DNA marker-assisted breeding technology to Middle Eastern countries, using tomato and resistance to geminiviruses as a model.
Expand collaborative research efforts between Arab countries and Israel through training, collaborative research, exchange visits, joint meetings, and workshops.
Establish a collection of tomato germplasm resistant to TYLCV for distribution to private sector, or other public and government institutions.
Improve the socioeconomic status of farmers and growers in the participating nations.
see web site http://www.plantpath.wisc.edu/GeminivirusResistantTomatoes/MERC/ HomePage.html

3. Monitoring of cereal virus and virus-like diseases for prevention through regional detection and quarantine systems.

With Aboul-Ata NE (Agricultural Research Center, Giza, Egypt), G. Anfoka (Al-Balqa Applied University, Al-Salt Jordan).
Funded by the US AID-CDR Middle East Research and Cooperation Program (2008-20011).

Background:

Pathogens such as the leafhopper-borne Maize yellow stripe virus (MYSV) and the aphid-borne Barley yellow dwarf luteovirus (BYDV) cause frequent disease outbreaks in maize, sorghum, wheat and barley in Egypt (Aboul-Ata et al., 1996a), Israel (Solomon, 2003) and Jordan (Makkouk et al., 1983). In the proposed project we aim at assisting the cereal industry to produce virus and virus-like free crops for the good of the participating countries, facilitating the safe exchange of plant materials between Egypt, Jordan, and Israel, and the import/export from/to other countries.

Objectives:

Develop diagnostic tools of cereal virus and virus-like diseases, especially those which are the object of quarantine in the participating countries. In particular we will produce antibodies fine-tuned to the local pathogens using virus coat protein antigens expressed in bacteria. In addition we will develop a 70-mer oligonucleotide microarray for the high throughput diagnosis of cereal pathogens.
Use these tools to establish a regional system for virus early prediction in maize, wheat, barley, sorghum and other cereals, by monitoring the occurrence of viruses and their insect vectors in fields
Manage outbreaks through a quarantine network involving the participant countries.
Identify local and exotic resistant genotypes using a simple inoculation system, independent of the virus vector.
Establish a web-based database of viruses affecting maize, sorghum, wheat and barley in the Middle East, their characteristics, their recommended diagnostic means, a list of genotypes tested for resistance.
Integrate efforts, exchange scientists and students, allow the flow of information among partners to ensure fast implementation from the lab to the field; all will be instrumental in consolidating the Peace Process in the region

INTERNATIONAL COOPERATION, plant breeding for disease resistance

1. Molecular marker-assisted breeding for resistance to whitefly-transmitted geminiviruses infecting tomato in Guatemala.

With L. Mejia (University of San Carlos, Guatemala City, Guatemala) and D. Maxwell and M. Nakhka (University of Wisconsin at Madison).
Funded by the US AID CDR (2001-2004).

Background:

Whitefly-transmitted geminiviruses are currently the main biotic constraint to vegetable production in Guatemala and many other countries in the tropical and subtropical regions of the world. In an attempt to control this insect vector producers are using increasing amounts of pesticides, which pose a threat to health and environment, and there has been little benefit from their use and abuse. Resistance of the whitefly to the newer and most effective insecticides seems to be occurring, as evidenced by recent increases in the whitefly populations in some parts of Guatemala; thus recreating the crisis that was experienced about ten years ago, before the advent of these insecticides. Producers have responded by increasing their levels of applied technology, using more chemicals and buying virus-free seedlings. Production costs have therefore increased, yet crop yields have gone down. The use of genetic resistance in integrated pest management is an alternative, which has not been utilized in Guatemala or Central America in the development of strategies for the control of the whitefly-geminivirus complex. This project offers a unique opportunity to teach and apply technology, which was primarily developed in Israel, from diverse fields, virology, molecular biology, genetics and plant breeding, in the solution of a pressing agricultural problem in Guatemala and Central America and to provide additional tools towards the development of sustainable production systems in a developing country.

The use of molecular markers linked to genes for resistance is a tool, which can be used efficiently in plant breeding, and its principles and applications will be taught and utilized as part of this project. Tomato germplasm with resistance to the Eastern Hemisphere whitefly-transmitted geminiviruses TYLCV and ToLCV has been shown to also confer tolerance to three recently identified geminiviruses from Guatemala. Resistance to TYLCV and ToLCV has been associated with introgressions from the wild species L. hirsutum, L. chilense and L. pimpinellifolium in chromosomes 6 and 11 of the tomato genome. It is important to know if the resistance that is expressed by the tomato lines selected in Guatemala can be traced to the same chromosomal regions, or if not, yet un-described genetic loci could be involved. The use of molecular markers linked to different genes for resistance would allow the introduction of resistance from different sources into a single cultivar, thus making it stronger and more durable. This will be the first time that there is a molecular study of resistance genes in tomato to Central American geminiviruses and the first introduction of this technology into an educational program in Guatemala.

A Master of Science program in Plant Biotechnology was initiated by the College of Agronomy at San Carlos University in Guatemala City in August 1999. This program is currently being expanded to include Animal Sciences and Microbiology. One of the areas, which is being emphasized in the program, is the application of biotechnology in plant breeding. As part of the program, a laboratory for the students was taught in January 2000 and some basic technology in molecular biology was implemented. This project will be an extremely important addition to the Ms. Sc. program in Biotechnology.

The application of new technology in this project, which proposes innovative approaches to the sustainability of food production, the interaction with scientists and research centers in Israel and the U.S.A., will result in significant advancements in the application of biotechnology in agriculture in Guatemala and Central America. Additionally, a substantial contribution will be made towards the solution of an urgent and long pressing problem in vegetable production, which has been in existence for over a decade. This project offers a unique opportunity to transfer information and tomato germplasm from Israel to Guatemala and eventually Central America. Currently, there is no other mechanism in place to achieve these benefits for agriculture in Central America.

Objectives:

Introduce the principles of molecular marker-assisted breeding technology into a research program and into the M. Sc. Degree Program at Universidad de San Carlos, Guatemala City.
Determine whether the markers used to map genes for resistance to TYLCV and ToLCV (Eastern Hemisphere geminiviruses) identify identical loci in the tomato breeding lines selected for resistance to the Guatemalan geminiviruses.
Identify molecular markers linked to genes for resistance to whitefly-transmitted geminiviruses from Guatemala that are different than those in objective 2.
Determine if the field-selected resistant tomato germplasm is resistant to all three tomato-infecting whitefly-transmitted geminiviruses in Guatemala.
Combine the independent sources of resistance to whitefly-transmitted geminiviruses and evaluate this material in the field in Guatemala and Israel.
Use molecular markers to combine genes for resistance to whitefly-transmitted geminiviruses and other pathogens into improved resistant genotypes of tomatoes suited for the Guatemalan market.
The results are posted at the web site: http://www.plantpath.wisc.edu/ GeminivirusResistantTomatoes/CDR/GT.htm

2. Identification of the whitefly-transmitted geminivirus complexes affecting cassava production in the southern regions of Africa and development of cassava resistant lines.

With C. Rey and M. Gray, University of the Witwatersrand, Johannesburg, South Africa), N. Cossa (Agriculture Research Institute, Maputo, Mozambique) and J. Brown (The University of Arizona, Tucson, USA).
Funded by the US AID-CDR (2003-2009).

Background:

Cassava is the most important crop in Africa in terms of dietary needs. Cassava mosaic disease (CMD), a disease caused by whitefly transmitted begomoviruses, is prevalent in all countries in Africa, and causes enormous reductions in yield. Furthermore, new evidence exists for a geographic distribution of a range of begomoviruses causing CMD. The need for resistant cultivars adapted to these local viruses is critical. Begomoviruses, and their vectors, have become important problems in the production of cassava cropping systems in southern Africa, including South Africa, Zimbabwe and Mozambique, due to the recent establishment of the B type whitefly vector in these regions.

Recent preliminary studies have revealed, for the first time, huge biodiversity in cassava-infecting begomoviruses, and furthermore, that several B. tabaci populations exist on cassava in southern Africa. This problem is further demonstrated by the recent epidemic of a new, devastating begomovirus-induced disease on tomato in eastern parts of South Africa, which is also probably due to the presence of a new biotype of Bemisia tabaci. The problem of whitefly-transmitted begomoviruses is exacerbated in that they exhibit high frequencies of recombination, and the hypothesis suggests that different whitefly biotypes may have corresponding variable virus transmission and disease inducing capabilities. This has a profound impact on disease control strategies and on production of crops in southern Africa. The objectives of this study are therefore to focus on the characterization of the viruses and vectors involved in cassava disease and to elucidate the relationship between begomoviruses and their whitefly vectors in order to select for the appropriate cultivars.

Training and capacity building are an integral part of this project through postgraduate student training and farmer participation. Focus will be on historically disadvantaged peoples as much as possible. Furthermore, exchange of methodology and expertise will benefit all countries enormously. Farmers will be trained to perform field tests in the various cassava growing regions, to evaluate crops, and to collect and transmit data.

Objectives:

To identify the biotype distribution of begomoviruses and B. tabaci vectors associated with cassava cropping systems in southern Africa using molecular markers, and to develop quick diagnostic methods.
To investigate the possibility of re-association and recombination among begomoviruses toward the emergence of new chimeric viruses having greater virulence and disease capability
To select cassava germplasm for resistance to existing begomoviruses and experimental chimeric constructs.
To transfer resistant cultivars and breeding lines to farmers the soonest.

3. Develop tomato breeding lines with resistance to Ralstonia solanacearum and begomoviruses for Guatemala and Central America

With L. Mejia (University of San Carlos, Guatemala City, Guatemala), Vidavski F (Tomatech Co, Israel), Lavi U and Tsror L (ARO, Israel), Scott JW (University of Florida, USA), Hanson P, Green S, Graham D and Wang JF (AVRDC, Taiwan), and Maxwell D (University of Wisconsin at Madison, USA).
Funded by the US AID CDR (2006-2010).

Background:

Begomoviruses infect vegetables in all tropical countries and they cause major losses on tomatoes in most of the countries on the CDR target country list. Insecticides are mainly used for management and as many as 25 applications may be made per crop cycle. Unfortunately, still 100% losses commonly occur. The importance of bacterial wilt (BW) varies but is also a major factor limiting tomato production in these CDR target countries. In Guatemala, BW causes major losses and was a problem in our most recent field trail for begomovirus resistance. These two pathogens cause the two most difficult diseases to manage in tomatoes. The resistant germplasm from this project will be made available to an appropriate seed company in Guatemala. By combining resistance to these two major diseases, tomatoes will again become a profitable option in these target countries. Our efforts will have a direct impact on the small vegetable growers as well as the larger growers. This will also have a direct benefit to all members of the family and local communities, as the level of toxic insecticides used can be reduced. The target countries for this project are Central America, but it is likely that this resistant germplasm will be of value in other developing countries in Africa and Asia.

Objectives:

Field test bacterial wilt-resistant tomato germplasm for resistance in Guatemala.
Characterize the Ralstonia solanacearum strains present in tomato.
Evaluate grafting desirable tomato cultivars onto bacterial wilt resistant root stock
Develop PCR-based markers for the BW resistance genes-associated with germplasm selected in Guatemala.
Develop PCR-based markers for the begomovirus resistance genes-associated with Guatemalan tomato breeding lines.
Combine resistance genes for both begomoviruses and RS into hybrids.

4. Classical and molecular-assisted breeding of tomatoes resistant to Tomato curly stunt virus (ToCSV) and related viruses for small-scale farmers in South Africa and Mozambique

With C. Rey (University of the Witwatersrand, Johannesburg, South Africa), Pietersen G (Agricultural Research center, Pretoria, South Africa), Mondjana A (Universidade Eduardo Mondlane, Maputo, Mozambique).
Funded by the US AID-CDR (2007-2010).

Background:

Tomatoes form an essential vegetable in a number of the developing countries of southern Africa (Mozambique, Swaziland, Zimbabwe and South Africa). Currently, Mozambique, Swaziland and South Africa are experiencing an epidemic of a newly described virus, Tomato curly stunt virus (ToCSV), a genetically close relative to the Tomato yellow leaf curl virus (TYLCV) complex. It was considered extremely important in Mozambique already in the mid 1990s (Anonymous, 1995), whereas in South Africa losses of up to 100% were experienced (Pietersen et al., 2000).

The impact on small scale farmers and communities of controlling such a virus disease in Southern Africa will be enormous. Several commercially available hybrids with tolerance to TYLCV and related begomoviruses, such as ToCSV, have been tested and are being incorporated directly into commercial farming practices when their agronomic properties are suitable. More will be produced by introducing natural resistance genes through breeding into varieties suitable for cultivation and use by small-scale farmers in Southern Africa. Studies in which breeding lines and cultivars carrying resistance genes introgressed from these wild tomato species have been inoculated with begomoviruses from the New and Old World, including ToCSV, indicate that these genes provide broad resistance to begomoviruses. Consequently, we anticipate that the resistance genes introgressed by crossing and marker assisted selection are highly likely to confer broad spectrum resistance against not only ToCSV, but also to close relatives or strains of TYLCV prevalent in Malawi and Tanzania and other southern African countries.

Objectives:

Screen TYLCV-resistant tomato germplasm in Soputh Africa and Mozambique and select ToCSV plants.
Cross resistant plants with local varieties.
Develop protein and DNA biomarkers for marker assisted selection.
Train Mozambique students in plant breeding and molecular virology.

Functional genomics of the whitefly Bemisia tabaci

1. Use of DNA microarrays to discover insect genes involved in the circulative transmission of geminiviruses in their insect vector, the whitefly Bemisia tabaci.

Funded by The Israel Science Foundation (ISF) (2002-2006).

Background:

The whitefly Bemisia tabaci is one of the most damaging insects to agriculture because of its feeding habits and because it transmits many plant viruses to many economically important crops worldwide. B. tabaci comprises a complex of biotypes with distinct genetic polymorphism that differ in plant-host range, fecundity, and ability to transmit begomoviruses. The B type (synonym B. argentifolii) is the resident biotype in Israel. Lately, the Q biotype has been identified in our country. The frequent use of insecticides has resulted in the establishment of resistant populations, exacerbating the damage caused by the insect.

Despite its extreme economical importance, the molecular genetics of B. tabaci is an almost virgin territory that needs to be explored. The project is aimed at using functional genomics to discover genes underlying biological processes, which make B. tabaci one of the major pests to agriculture: development from egg to adult, gender determination, plant-host selection, resistance to insecticides, and virus circulative transmission.

Objectives:

To determine:

Which genes are expressed during the development of B. tabaci?
Which genes are involved in the circulative transmission of begomoviruses?
Which genes are responsible for the detoxification of insecticides in resistant populations?
Which genes determine the host range of the various B. tabaci biotypes?

2. Functional genomics characterization of the whitefly Bemisia tabaci begomovirus interaction: an EST and array-based transcript profiling approach.

With R. Shatters and C. McKenzie (USDA, ARS, USHRL, Fort Pierce, Florida, USA), and J. Brown (The University of Arizona, Tucson, USA)

Funded by the United States-Israel Binational Agricultural Research and Development Fund (BARD) (2003-2007).

Background:

We propose the first US-Israel Binational genomics project on a homopteran insect pest to agriculture, the whitefly Bemisia tabaci. This insect transmits many viruses to a variety of economically important crops in the USA, Israel, and worldwide. The B type (synonym B. argentifolii) is resident to Israel, and has invaded the Southern USA, reaching epidemic proportions. Despite its extreme economic importance, there is little molecular genetics information on B. tabaci. This project proposes the development and public presentation of a B. tabaci expressed sequence tag (EST) library and associated cDNA high-density arrays, and to use the arrays to analyze how begomoviruses influence the whitefly transcript profile, emphasizing: 1) begomovirus circulative transmission, and 2) virus induced changes in whitefly immune defense and fecundity. The cDNA libraries will be constructed by pooling populations of B biotype from Israel, Florida, and Arizona selected before and after they acquire mono partite (Tomato yellow leaf curl virus, TYLCV-in Israel) and bipartite (Tomato mottle virus, ToMoV-in U.S.) begomoviruses. Genes whose transcript abundance changes in response to acquisition and circulative movement of begomoviruses through the whitefly will be identified using array-based hybridizations. Since experimental evidence suggests that the TYLCV actively produces viral transcripts and perhaps replicates, while ToMoV does not, differences in how these viruses influence whitefly transcript profiles will be studied. In silico analysis, Northern blot, real-time RT-PCR and in situ hybridization will be performed on clones of interest. This project combines expertise in B. tabaci biology, plant-virus-vector relationship, genomics and bioinformatics, and is presented with the knowledge that understanding the molecular mechanisms underlying the biology of B. tabaci is a prerequisite for the creation of new strategies to curb the disastrous effects of this insect on many crops worldwide.

Objectives:

Generate a normalized cDNA library from a pool of mixed population of adult male and female B. tabaci biotype B from Israel, AZ and FL, before and after they acquired for various periods of time monopartite and bipartite begomoviruses of tomato from Israel (TYLCV) and Florida (ToMoV), respectively.
Use an economical method to enable prescreening of randomly selected cDNA clones against an initial 500 whitefly clones for which the sequence has been obtained.
Employ high-density arrays in hybridization experiments to identify cDNA clones representing genes whose transcript abundance changes in response to virus acquisition.
Combine in silico data-mining of EST sequence, and array-based transcript profiling. Clones will be characterized using Northern blot analysis and real-time RT-PCR.
Develop a public website containing the whitefly EST sequence database, including sequences with chromatograms and BLAST search results.
Results posted at ad hoc NCBI website: http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=18077

3. Identification of genes involved in circular transmission of begomoviruses in whiteflies

With H, Ghanim M (Israel), and Winter S (Germany)
Funded by The German Israeli Foundation for Research and Development (GIF) (2008- 2010)

Background:

The whitefly Bemisia tabaci complex is a major pest of agriculture. It transmits begomoviruses (a family of small geminate particles encapsidating a circular ssDNA genome) in a persistent-circulative manner. Our working hypothesis is that virions interact with a number of whitefly proteins during circulative transmission. They need to evade host immune responses, to breach multiple epithelial/barriers and to exploit receptors and chaperonins to translocate in their insect vector to ensure plant inoculation. Using vector biology, biochemistry, molecular and evolutionary biology, proteomics, genomics, and RNAi, we wish to identify and validate the role of those whitefly key proteins involved in the successful transmission of two begomoviruses, Tomato yellow leaf curl (TYLCV) and Watermelon chlorotic stunt (WmCSV), which infect tomato and cucurbits, respectively, often causing total yield loss. Understanding these processes may help devise new countermeasures specifically designed to disturb key gene products, thereby avoiding the need to spray chemicals with wide-range effects and protecting the environment.

Objectives:

Use the B. tabaci microarray platform to discover the genes regulated during TYLCV and WmCSV transmission. A comparative study will be performed with the Myzus persicae-transmitted Potato leafroll virus (PLRV) to find out whether similar genes are involved in the same processes in two different insect/circulative virus systems, underlining their central role and their conservation.
Isolate sequence and identify the whiteflies proteins involved in begomovirus transmission, especially those interacting with the capsid from wild type and non-transmissible begomovirus mutants, and which may fit the definition of putative receptors mediating begomovirus translocations in the insect body. We will find out whether these proteins are represented in the whitefly and/or aphid microarray and verify their pattern of expression during virus acquisition, retention and transmission.

4. Dissection of whitefly-geminivirus interactions at the transcriptomic, proteomic and cellular levels

With Ghanim M (Israel), Cicero J, Brown J (USA)
Funded by The United States Israel Binational Agricultural Research and Development Fund (BARD) (2007- 2010)

Background:

The whitefly Bemisia tabaci (Gennadius)species complex and the plant viruses they transmit pose major constraints to vegetable and fiber production; including the arid SW deserts of the USA and Israel. Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV) (Begomovirus; Geminiviridae) are emerging viruses in tomato and cucurbits, respectively, and at times economic losses are 100%. TYLCV originated in the Middle East but now occurs in the USA, while SLCV originated in the SWUS, and has been introduced to Israel. Both are transmitted in a persistent-circulative manner by the B. tabaci complex. Begomoviruses have intimate and complex associations with their arthropod vector, and cytopathic effects, altered longevity and fecundity, and antimicrobial and/or stress molecules are demonstrated cellular responses to virus acquisition. The contributions of key molecular and cellular processes remain virtually unknown. This knowledge is pivotal for developing innovative approaches to modulate transmission and reduce virus spread in crops. Our working hypothesis is that virions interact directly and indirectly with a number of whitefly proteins during the phases of circulative transmission. And, as they are foreign to whitefly cells, they evade and/or combat host innate immune responses to breach multiple epithelial/barriers and exploit receptors.

In this project, we will advance the understanding of this complex process by identifying and validating key protein roles using vector biology, biochemistry, molecular and evolutionary biology, proteomics, genomics, and microinjection toolboxes. The long-term goal is to identify and characterize all proteins/genes involved in whitefly-mediated virus transmission. Studies will utilize B. tabaci biotypes and viruses that differ evolutionarily, genetically, and in transmission competency. We will employ established circulative transmission parameters to study begomoviruses in co-evolved and divergent B. tabaci biotypes (A, B, Q). Exploitation of this study system will produce information specific to the B. tabaci-virus complex, but also will have relevance to other virus-vector complexes of plants owing to the putative conservation of orthologous genes involved in these processes. Consequently, we expect to identify proteins involved in innate immunity, evasion/mimicry, membrane disruption, and receptor exploitation.

Objectives:

Identify genes expressed post-virus (SLCV, TYLCV) acquisition and retention in adult whitefly by microarray analysis.
Construct a Whitefly Proteome and cross-identify proteins withinsect ESTs.
Validate gene expression by RT-PCR, and subcellularly (in situ).
Develop and use newly established RNAi protocol to enable gene knockouts.

Comparative genomics of virus circulative transmission by two Homopteran insect pests to agriculture, the whitefly Bemisia tabaci and the green peach aphid Myzus persicae

With Ghanim M
Funded by the Israel Science Foundation (ISF) (2008-2010)

Background:

The whitefly Bemisia tabaci (Gennadius) species complex, the green peach aphid Myzus persicae and the plant viruses they transmit pose major constraints to vegetable, flowers and fiber production; worldwide, including Israel. The two geminiviruses, Tomato yellow leaf curl (TYLCV), Squash leaf curl (SLCV) (Begomovirus; Geminiviridae) and the luteovirus Potato leaf roll virus (PLRV) (Polerovirus; Luteoviridae) are emerging viruses in tomato, cucurbits and potato, respectively, and at times economic losses are severe. TYLCV originated in the Middle East but now occurs in Europe and the US, while SLCV originated in the US, and has been recently introduced to Israel. PLRV in present in Israel and cause losses of two types: reduction in yields and poor quality tubers due to net necrosis. TYLCV and SLCV are transmitted in a persistent-circulative manner by the whitefly B. tabaci complex, while PLRV is exclusively transmitted by M. persicae. Begomoviruses and luteoviruses have intimate and complex associations with their arthropod vectors. Both virus classes cause cellular responses to virus acquisition, and in the case of begomoviruses, these are demonstrated by cytopathic effects, altered longevity and fecundity, and antimicrobial and/or stress molecules. The contributions of key molecular and cellular processes in virus transmission remain virtually unknown. This knowledge is pivotal for developing innovative approaches to modulate transmission and reduce virus spread in crops. Our working hypothesis is that virions interact directly and indirectly with a number of whitefly/aphid proteins during the phases of circulative transmission. And, as they are foreign to whitefly/aphid cells, they evade and/or combat host innate immune responses to breach multiple epithelial/barriers and exploit receptors.

Exploitation of this study will not only produce information specific to the whitefly/aphid-virus complexes, but also will have relevance to other plant virus-vector complexes owing to the expected conservation of orthologous genes involved in these processes. Consequently, we expect to identify proteins involved in innate immunity, evasion/mimicry, membrane disruption, and receptor utilization.

Objectives:

In this project, we will advance the understanding of this complex process by identifying and validating roles of key proteins using vector biology, molecular and evolutionary biology, genomics, and microinjection tools.
The long-term goal is to identify and characterize the genes involved in whitefly/aphid-mediated virus transmission. Studies will utilize B and Q B. tabaci biotypes (both present in Israel) and M. persicae, and viruses that differ evolutionarily, genetically, and in their transmission competency.
We will employ our rich experience in insect-virus interactions to study begomovirus/PLRV in co-evolved and divergent B. tabaci biotypes (B, Q) and in M. persicae.
Our objectives are to: (i) identify genes expressed post-virus (SLCV, TYLCV, PLRV) acquisition and retention in adult B. tabaci B, Q biotypes or in aphid adults by microarray analysis, (ii) validate gene expression by RT-PCR and qRT-PCR, and in situ, and (iii) develop and use newly established microinjection RNAi protocol to enable gene knockouts in whiteflies.
This multifaceted approach will enable us identifying genes in the aphid and the whitefly involved in virus circulative transmission.

NATO network agricultural bioterror countermeasures

1. Microarray-based detection of plant viral and viral-like pathogens

With Barba M (Plant Pathology Research Institute, Rome, Italy), Hadidi A (USDA, Hedgegrove MD, USA), Caglayan Kadriye (Mustafa Kemal University, Antakya, Turkey), Mazyad H (Agricultural Research Center, Giza, Egypt), Anfoka G (Al-Balqa' Applied University, Al-Salt, Jordan).

Funded by NATO Science for Peace Program (2006-2008).

Background:

The production of vegetables, fruits, and cereals are constantly threatened by diseases caused by hundreds of micro-organisms. National Plant Protection Organizations monitor the import of agricultural goods to avoid the spread of diseases which may cause extremely important economic losses. The diagnostic of plant pathogens is an increasingly demanding task because of growing international trade, the emergence of new pathogens and the concerns of agricultural bio-terrorist attacks. Hence there are needs for new and more efficient diagnostic tools. DNA microarrays (or DNA chip) is a new technology which allows the simultaneous detection and identification of a large number of pathogens. Microarrays are tiny probes placed on a piece of glass; each probe is sensitive to a specific pathogen. The arrays are flooded with a mixture of DNA or RNA from plant samples and individual probes react if a particular pathogen is present. This technology has been successfully used to detect human viruses and bacteria and it is considered as the most advanced diagnostic tool the in bio-terrorism countermeasures arsenal.

DNA microarrays for the detection of plant pathogen are not to be found yet. Our project is aimed at fulfilling this need. Scientists from three NATO countries (Italy, Turkey, USA) and three non-NATO countries (Egypt, Israel, Jordan) are cooperating to design a DNA microarray fit to detect the most important viruses, viroids and phytoplasmas infecting major agricultural crops. Though the exchange of scientists and via hands-on workshops, this project will allow transferring the DNA microarray technology to all partners, and to enhance cooperation between NATO and non-NATO partners and between Israel and neighbouring Arab nations. Once the technology is validated using known pathogens, the DNA chip will be offered to the national Plant Protection Services and to the scientific community to be used as a routine detection device in quarantine and certification programs, in bioterror countermeasures, and as a research tool.

Objectives:

Develop a DNA microarray chip for the detection and genotyping of plant viruses, viroids, and phytoplasmas.
Design a DNA microarray with the potential to simultaneously detect many viruses, viroids, and phytoplasmas, including pathogens which may threaten the stability and security of food supply in the countries involved in the project.
Validate the pathogen microarray for the detection of pathogens for plant protection services.

LOCAL PROJECTS

1. Breeding tomato resistant to Tomato yellow leaf curl virus.

With S. Vidavski (TomTech Co)
Funded by TomTech Co. (2004-2006).

Background:

Tomato yellow leaf curl virus (TYLCV) from the Middle East is a monopartite geminivirus, transmitted by the whitefly Bemisia tabaci. TYLCV and its relatives infect tomatoes in open field and greenhouse, causing up to 100% losses in crop production in many countries in the Middle-East, Southwest Europe, Tropical Africa and Southeast Asia. Lately the Middle Eastern strain of TYLCV has been identified in the Caribbean Islands, in Spain, in several US states:Florida Georgia Mississipi (late 1990s), Arizona and California (2006), and Mexico (2006).

Breeding programs aimed at producing tomato cultivars resistant to TYLCV and to other geminiviruses infected tomato have started in the late 1960s and have expanded since. These programs are based on the introduction of resistance or tolerance found in some accessions of wild tomatoes species into the domesticated tomato Solanum lycopersicum. Depending on the plant source, resistance was reported to be controlled by one to five genes, either recessive or partly dominant. The first commercial cultivar tolerant to TYLCV, TY20, incorporated resistance genes from S. peruvianum. It showed delayed symptoms and delayed accumulation of viral DNA. A TYLCV - tolerance gene originated from S. chilense LA 1969, Ty-1, has been mapped to tomato chromosome 6 and has been introgressed into a domesticated tomato line using RFLP markers. Another gene conferring tolerance to TYLCV originated from S. pimpinellifolium has also been mapped to tomato chromosome 6 (to a locus different from Ty-1) using RAPD markers. Recently a gene from S. habrochaites conferring tolerance to Tomato leaf curl virus from Taiwan has been mapped to tomato chromosome 11.

After more then 25 years of efforts, the best cultivars and breeding line available show tolerance to the virus rather than resistance. Upon infection, yields are far higher than those of susceptible cultivars. Diseases symptoms are absent or very mild, but the plant contain various amount of virus. These tolerant cultivar need to be protected from viruliferous insects during the first month after planting with insecticides and/or nets. Hence we have initiated a breeding program with the ultimate goal to obtain cultivars totally resistant to whiteflies-mediated inoculation that do not support virus replication, thereby rendering superfluous the use of insecticide or nets. We started from a cross between two S. habrochaites accessions completely immune to TYLCV. This F1 was crossed with S. lyxcopersicum and a series of selfing were performed. At each generation, asymptomatic individuals with no or very low amount of virus were selected following repeated, massive, controlled inoculation with viruliferous whiteflies. We have reached a stable line that does not segregate for resistance and has good horticulture characteristics (80-120 g, red fruit, fertility, self-compatibility and vigor). The F1 (Favi-9, Favi-13, Favi-17) between our highly resistant line and susceptible lines were found to be resistant to TYLCV and to other members of the geminivirus family. They were so far successfully tested in Jordan, Egypt, Sicily, India, Taiwan, Jamaica and Guatemala.

Objectives

Cross our resistant line with high-quality commercial variety in order to obtain segregating population with resistant to TYLCV.
Selecting highly performed individuals plants with resistant to TYLCV and to Verticillium, Fusarium race 1 &2 and TMV.
Advancing and stabilizing the most promising individual plants.
Preparing 100 experimental hybrids for field trial.
Evaluating the experimental hybrids under field condition against related geminiviruses in several locations in the world, and under controlled inoculation.
Establish near isogenic lines for mapping the resistance from S. habrochaites using DNA markers.

2. Macro- and microarray-based platforms for the detection and identification of plant pathogens

Funded by the Hebrew University of Jerusalem and Johnson &Johnson Fund for Innovative Science (2009-2010).

Background:

Our food supply is under the constant threat of pathogens - viruses, viroids, phytoplasma, fungi, bacteria - that may destroy crops on huge scales. World-scale movement of agriculture products enhances the dangers of epidemics. In addition agricultural bio-terrorism has become a major concern. All major agriculture goods imported or exported need compulsory pathogen free certification. Today, diagnosis is based mainly on low sensitivity serological methods that allow detecting the pathogen looked for; unexpected exotic pathogens usually escape diagnosis. Therefore the development of a platform displaying multiple pathogen probes is a must for early, rapid, efficient and specific pathogen diagnosis to prevent, circumvent and eradicate the spread of epidemics affecting our food supply. Hence we plan to develop macro- and microarray platforms that have the potential to diagnosis any of the major plant pathogens. As such, the layout of an array typically involves gene-specific 70-mer oligonucleotides representing the pathogens bound to a solid support in a spatially separated fashion. The RNA/DNA sample to be examined is usually fluorescently labeled in an enzymatic reaction and this fluorescent nucleic acid is hybridized to the array. The fluorescence allows the hybridization events on the solid support to be identified, and the identity of the gene is deduced from the position on the array

Objectives

Design and synthesize 70-mer oligonucleotide probes of at least 150 viruses, viroids, and phytoplasmas that threaten the major crops worldwide.
Develop DNA microarray chip printed on glass and macroarray printed on nitrocellulose membranes for simultaneous detection and genotyping of the above plant viruses, viroids, and phytoplasmas.
Validate the use of the arrays with plants infected with known pathogens and with cloned pathogen DNAs.