Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

To overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans-potato and -tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coverage of the P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pathogenicity, such as class III pectate lyases, despite the considerable evolutionary distance between oomycetes and fungi. Thirty-seven genes encoding components of flagella also were identified. Several genes not anticipated to occur in oomycetes were detected, including chitin synthases, phosphagen kinases, and a bacterial-type FtsZ cell-division protein. The sequence data described are available in a searchable public database.

Summary Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and elevation of transcript levels during P. infestans infection was confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to beta-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillus nidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail.

SUMMARY Suppression Subtractive Hybridization (SSH) was used to search for genes of Phytophthora infestans that are induced during the infection of potato. To avoid having to distinguish the genes of the pathogen from the plant genes involved in defence responses and to isolate the genes involved in the early stages of interaction, mycelium of P. infestans was induced by contact with the host plant and then separated from the plant tissue. A differential cDNA library was generated by SSH that compared such induced mycelium with mycelium incubated in water. The expression of about 100 cDNA fragments from this differential cDNA library was analysed by hybridization of the arrayed PCR products with mRNA from control and induced mycelium. Twenty per cent of them showed increased transcript levels in mycelium within the first 24 h after exposure to a potato leaf. For six of these cDNA clones the elevated expression in response to the potato leaf could be proven by RNA gel blot analysis. Five of these cDNA clones have predicted amino acid sequence homologies to entries in the databases, including an amino acid transporter, a sucrose transporter, a spliceosome-associated factor, an ABC transporter, and a cell division control protein. We showed that the genes corresponding to these six cDNA clones are differentially regulated during their life. Reliable gene expression analysis of Phytophthora in infected leaf tissue is not possible until c. 48 h post-infection, but for two of the genes we identified, induction during in planta growth was detectable by RNA gel blot analysis. Therefore the SSH library that we have created provides a basis for the identification of P. infestans genes that are up-regulated during the interaction with the plant, which could be important for pathogenicity.