The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.

No abstract

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Rapid progress in gene technology has allowed, on the one hand, insight to be gained into the complex molecular mechanisms of plant/pathogen recognition and the natural defence strategies of host plants. On the other hand, this technology can also be used for the controlled and efficient generation of genetic variability and for the identification of desirable genotypes, far beyond the possibilities of classical breeding. The first successful attempts have been made to improve resistance against pathogenic viruses, bacteria, fungi and insects by engineering transgenic plants. The majority of these strategies were based on constitutively expressing single proteins that are either toxic to the pathogen/pest, or interfere with its reproductive cycle. More refined strategies, which are at the stage of testing, try to mimic and modify naturally-evolved defence reactions of plants and, thereby, will potentially confer a more durable resistance to a broad range of pathogens

MATERIALS AND METHODSZygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas relnhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quanftites and a method was developed for the extraction from zygotes of RNA that could be translated in vitro.The sexual cycle of Chlamydomonas reinhardtii involves the fusion of two gametes of opposite mating type resulting in the formation of a diploid zygote. The mature zygote can subsequently be induced to undergo germination. After meiosis, four spores are released (9). The individual steps within this life cycle (all at a single cell level) represent defined stages in cell differentiation. The morphological and physiological characteristics of the individual cell types have been investigated in some detail (3,12). Although analyses of the mating reaction and zygote formation have provided insights into the mechanisms of cell-cell recognition (1, 18) and signal transduction ( 14), the cellular stages of zygote maturation and germination have so far not been analyzed at a molecular or biochemical level in C. reinhardtii. The reasons were the lack of procedures to obtain mature zygotes (i.e. zygotes able to germinate) in sufficient quantities and the lack of methods for the efficient disruption of these thick walled cells. Two major problems have previously prohibited the production of large numbers of zygotes. First, zygotes appear to require contact to a firm surface (e.g. an agar plate) for maturation. Zygotes left in liquid medium are different from plate matured zygotes (3,5). More important, zygotes from liquid medium germinate very poorly (3). Second, when newly formed zygotes are plated on agar plates for maturation, the mature zygotes become embedded within the agar and are difficult to recover. We describe here a novel method for the production of large numbers of mature zygotes that overcomes both of these problems, and we also present a reliable procedure for extraction of functional zygote RNA.I Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB206). Strains and Culture ConditionsChlamydomonas reinhardtii wild-type strains 137c+ and 1 37c-(obtained from R. Matagne) were used for all experiments. One L cultures were inoculated from plates and grown in TAP2-medium (4) in 1 L round flasks with air bubbling and continuous illumination (30 uE m-2 s-') at 23C. Gametogenesis was induced by transferring vegetative cultures in midlog phase (cell density 1 -2 x 106 cells/mL) to nitrogen-free TAP-medium (16). Gametogenesis was performed under the conditions described above for the growth of vegetative cells. Production of Zygotes in Large QuantitiesEighteen to 20 h after induction of gametogenesis by nitrogen removal (cell density 4-8 x 106 cells/mL), equal numbers of mt+ and mt-gametes were mixed, usually 2 L of both. Portions (50-75 mL) of the mating mixture were then transferred to 100 mL Erlenmeyer flasks and incubated without shaking...