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.

Development of reproductive tissue and control of cell division are common challenges to all sexually reproducing eukaryotes. The Arabidopsis thaliana TSO1 gene is involved in both these processes. Mild tso1 mutant alleles influence only ovule development, whereas strong alleles have an effect on all floral tissues and cause cell division defects. The tso1 mutants described so far carry point mutations in a conserved cysteine-rich domain, the CRC domain, but the reason for the range of phenotypes observed is poorly understood. In the present study, the tesmin/TSO1-like CXC (TCX) proteins are characterized at the biochemical, genomic, transcriptomic, and functional level to address this question. It is shown that the CRC domain binds zinc, offering an explanation for the severity of tso1 alleles where cysteine residues are affected. In addition, the phylogenetic and expression analysis of the TCX genes suggested an overlap in function between AtTSO1 and the related gene AtTCX2. Their expression ratios indicated that pollen, in addition to ovules, would be sensitive to loss of TSO1 function. This was confirmed by analysis of novel tso1 T-DNA insertion alleles where the development of both pollen and ovules was affected.

BackgroundIron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron.ResultsHere we reveal the distribution of iron in seeds of three Phaseolus species including thirteen genotypes of P. vulgaris, P. coccineus, and P. lunatus. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of P. vulgaris and P. coccineus seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 μg g-1 of iron, depending on the genotype. In contrast to P. vulgaris and P. coccineus, we did not observe iron accumulation in the cells surrounding the provascular tissues of P. lunatus cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype.ConclusionsThe presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.

Summary Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40‐ to >100‐fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60‐ to >100‐fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA‐binding proteins and RNase H. The products of two other mating‐induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating‐induced gene was also expressed in a self‐fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self‐fertile heterokaryon. Besides the mating‐induced genes, two extrachromosomal RNA elements were identified.

Sarpo Mira, a potato variety with high resistance against the late blight pathogen Phytophthora infestans, is being used in breeding programs to increase late blight resistance in commercial varieties. Discovering genes that are important for P. infestans resistance will assist in the development of molecular markers for the selection of new resistant cultivars and the use of resistant varieties will reduce the environmental, health and financial costs associated with the use of pesticides. Using complementary DNA amplified fragment length polymorphism analyses, differentially expressed genes involved in the potato-P. infestans interaction were identified in the susceptible Bintje and in the resistant Sarpo Mira potato cultivars. Forty-eight differentially expressed transcript derived fragments (TDFs) were cloned and sequenced. The expression profiles of some of these genes were analyzed in detail using quantitative RT-PCR at seven time points: 1, 4, 17, 24, 30, 41 and 65 hours after inoculation (hai). We found that five transcripts with homologies to pathogenesis/defense-related genes and two TDFs with homology to transcription factors were significantly induced to higher levels in the resistant cultivar at very early stages of the infection (1 hai). Interestingly, most of these genes showed different expression profiles throughout the whole infection process between both cultivars. Particularly during its biotrophic growth phase, P. infestans triggered the down-regulation of infection responsive genes in the susceptible but not in the resistance cultivar. Our results suggest that these newly identified early-induced transcripts may be good candidates for conferring Sarpo Mira's resistance to late blight and they could be useful molecular markers for the selection of new resistant cultivars.