Prof. Rafael Perl-Treves

MOLECULAR GENETICS OF CUCURBITS

Overview of Research Projects

Our laboratory carries out molecular genetic research, mostly in plants of the Cucurbitaceae family. We have analyzed the melon and cucumber genomes using mapping approaches, and investigated the interaction of these plants with pathogens and insects. We also studied sex differentiation and fruit set in cucumbers at the molecular level. Our projects ask genetic and developmental-genetic questions on plant reproduction, and on disease resistance genes, and yield biotechnological assets in the form of diagnostic DNA markers to assist breeding, and cloned genes of biotechnological interest.

Genetic mapping and genome analysis in Cucumis

Broad scale analysis of eukaryotic genomes is one of the challenges of modern biology, starting from the construction of detailed genetic maps, and culminating in reverse-genetic approaches to isolate and study the function of economically important genes. Our lab contributed to the development and use of such discipline for crops of the cucurbit family, namely Cucumis sativus (cucumber) and Cucumis melo (melon). We have become recognized among the few laboratories that developed markers and performed genome analysis in Cucumis crops, and we collaborate with other groups and breeders, in Israel and elsewhere.

Genetic diversity studies

Seed collections of wild and primitive varieties of a given crop species represent a valuable resource for the future, requiring adequate protection and characterization. We have addressed the structure and distribution of genetic variation in the melon germplasm, that harbors impressive variation in fruit size, shape, color and taste, as well as aroma and ripening physiology. We used DNA fingerprinting to quantify such variation, related it to morphological and agronomic traits, and portrayed melon taxonomy at the infra-specific level with such tools (refs. 13, 14, 15). Variation in plant and fruit morphology and in DNA profiles were analyzed in a set of 60 melon varieties from all over the world (Fig. 1). We described and analyzed the variation in fruit sugars and in acid invertase, an enzyme that is developmentally modulated to determine sucrose levels (ref. 14; Fig. 2), and showed that potentially useful genetic variation in all three sugars is found in "exotic" botanical variants of melon that are currently under-utilized in breeding. These studies continued earlier studies with Prof. Galun on inter-specific classification of the Genus Cucumis (refs. 1, 2). A pre-breeding project that attempts to utilize variation in shape, size and taste of melons for generating novel fruit types is running in our lab. For more information on the Melon Gallery Project (Fig. 3) please contact us. 

Another project relates to the watermelon, Citrullus lanatus: using a small grant from the Israeli Gene Bank, we collected wild accessions of Citrullus in Israel, in order to characterize them in terms of genetic diversity and potential use for breeding (Fig. 4).  In addition, in the framework of a collaboration via Dr. Salih Kafkas of Chukrova University in Turkey, we applied molecular markers to characterize wild Pistacia species, that serve as rootstock for high value pistachio cultivars (refs. 20, 22-25).

Genetic mapping in melon

A melon genetic map was generated based on the F₂ generation of the cross P.I. 414723 x cultivar TopMark (Fig. 5; ref. 19, 29). We also prepared a Recombinant Inbred Lines (RIL) collection from the same cross. The map included ~180 loci obtained by different techniques. Several markers anchored our linkage groups to those of more saturated reference maps.

A great achievement of plant molecular biology involves cloning of disease resistance genes, responsible for pathogen recognition and signaling. We cloned and characterized resistance gene homologs (RGH) in melon. Together with the group of Drs. Pitrat and Dogimont at INRA and Prof. Gary Thompson in Little Rock we tested linkage between our clones and several disease and pest-resistance loci. These included Vat (aphid resistance), two Fusarium resistance loci, Fom-1 and Fom-2, and resistance to papaya ringspot virus (refs. 21, 26, 28, 33). Such markers are used as diagnostic tools to breed for insect and disease resistance (Fig. 6, Fig. 7).  We focused on the Fom-1 /Prv locus and on the Zym loci (resistance to the zucchini yellow mosaic virus) and generated high resolution maps of these loci (ref. 33). Linked markers were used as starting points for a chromosome walk that culminated in cloning of the resistance genes (Fig. 7B; ref. 48); this was done in collaboration with Drs. Pitrat, Dogimont and Bendahmane at INRA and Dr. Katzir at A.R.O.

Quantitative resistance to Fusarium oxysporum race 1.2 in melon

Race 1.2 of melon Fusarium is controlled by multiple genes and is more difficult to manipulate in breeding. We have characterized a breeding line, BIZ (property of Zeraim Gedera Ltd.) that harbors strong and reliable resistance towards Israeli strains of race 1.2. We showed that two main recessive genes confer full resistance (Ref. 40) and began mapping them using QTL analysis.  We have used a GFP strain of the fungus to visualize the colonization of melon seedlings in vivo (Fig. 8), and observed quicker and higher expression of defense genes in the resistant seedlings, compared to susceptible ones (Ref. 44).

More plant-pathogen and plant-pest interactions

Arabidopsis-rhizoctonia interaction: During a Sabatical at CSIRO, Perth, West Australia (2002), I collaborated with Prof. Karam Singh and Dr. Rhonda Foley on interactions between Arabidopsis and the fungus Rhizoctonia, using the luciferase reporter system. We made real-time observations of the physical contact between the fungus and the living plant, which responds to the challenge by emitting bioluminescence (Fig. 9; ref. 30, 38).

Plant response to broad mites: This project addresses plant-herbivore interactions, in collaboration with A.R.O. entomologists Dr. Victoria Sorocker. We studied the interaction of cucurbit crops with a serious pest, the broad mite. We described its damage at the microscopical level (Fig. 10), and documented the plant responses, including induction of defense genes (ref. 34). We used tomato mutants defective in defense signaling to demonstrate the importance of a defense hormone, jasmonic acid, in defense against broad mites. We have shown for the first time the broad mites are able to choose a favorable host and set up elegant bioassays to monitor the mite-plant interaction on detached leaves (Ref. 43). Presently, the three-party interaction between tomato, broad mite and the whitefly that serves as a vector in mite dispersal (Fig. 11), is being investigated in collaboration with Prof. L. Walling from Riverside, California.

Molecular studies on sex expression in cucumber

This project focuses on flower differentiation in plants that have a specific mechanism to separate their male and female organs. We have utilized two interesting features of cucumbers to approach such a complex developmental process. One is the availability of well characterized genotypes with striking differences in sex expression (Fig. 12). The second involves the possibility to physiologically induce dramatic sex changes by gibberellin and ethylene application (ref. B2, B3, B5). Using two contrasting genotypes, male and female cDNA libraries were prepared from very young floral buds, where "developmental decisions" are probably being made (ref. 36).

Cucumber genes that govern floral architecture and determine sex-hormone synthesis and perception

A family of three cDNAs that are homologous to the transcription factor AGAMOUS (that controls pistil and stamen formation in flowers) has been cloned and characterized (ref. 11). They probably are part of the flower's differentiation program after its fate has been determined by more "upstream" genes. A family of three ACC oxidase cDNAs, encoding the final step of ethylene synthesis, has been cloned (ref. 16). The role of ethylene in cucumber sex-modification is well established, and we studied the expression of these genes in relation to sex patterns of different cucumber genotypes. Distinct patterns of ACC oxidase transcripts in floral organs suggested localized, stage-specific roles for ethylene in floral organ differentiation (Fig. 12). In addition, we have cloned ethylene receptor-homologues from cucumber and analyzed their expression. In collaboration with other researchers in Israel (Dr. Zelcer, A.R.O.) and India (Dr. Ganapathy, Dr. Rajagopalan, Tiruchirappalli), we set-up cucumber transformation in our lab (Fig. 13; refs. 32, 35). A construct for over-expression of ERS, an ethylene receptor gene, was indeed shown to enhance femaleness of transgenic cucumbers, establishing the importance of ethylene sensitivity for sex expression (Fig. 14; ref. B5).

We were also involved in map-based cloning of the cucumber sex determining genes. We prepared several genetic populations, each segregating for a different sex determining locus. In collaboration with INRA researcher Dr. Bendahmane, the Monoecious gene has been cloned using this approach. It was shown to encode an ACC synthase isoform that is expressed in the carpels of pistillate flowers, and inhibits the developing stamen (ref. 44). Together with theFemale gene cloned years ago by other groups, this finding greatly advanced our view on sex determination mechanism in cucumber. As a tool for functional characterization of cloned genes, we have prepared and tested a TILLING population for cucumber (Proceedings ref. 7; Fig. 15).

Control of cucumber fruit set

In the past two years we have begun to investigate the control of fruit set at the whole-plant level. Following fertilization, what factors determine whether an ovary will set fruit, or remain inhibited and senesce? Preliminary data relating to the "First-Fruit Inhibition phenomenon, being developmentally distinct from "regular senescence" has been obtained (Proceedings ref. 5, 8).