Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome ( approximately 90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%–4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

In this study, we examined the relationship between the virulence of Shigellaflexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Pcrmutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcrmutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2al40. In many of the mutants, concomitant loss of pigmentation, virulence, and pSf2al40 was accompanied by the appearance of a new plasmid, smaller than pSf2al40. We believe these new plasmids to be deletion derivatives of pSf2al40 and that loss of pigmentation and loss of virulence are associated with deletions in pSf2al40. We transduced Pcrmutants to Pcr+ and isolated transductants which suppressed the Pcr-phenotype. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr-phenotype were identified as mutations in cell wall biosynthesis. These results support our belief that although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence can also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells. However, loss of ability to bind Congo red is accompanied by loss of virulence.

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37°C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dvsenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30°C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33°C were only partially invasive in the Henle assay, whereas strains grown at 35°C were as invasive as strains grown at 37°C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37°C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37°C. Restoration of invasiveness after growth at 30°C required protein synthesis. When shigellae were grown at 30°C and shifted to 37°C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37°C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature. Bacteria of the genus Slhigella are capable of penetrating and multiplying within the epithelial cells of the colon (15). The infection produces ulcerative lesions in the colonic mucosa and results in the bloody mucous diarrhea characteristic of bacillary dysentery (7). Reliable model systems that measure the pathogenic potential of Shigella spp. have been developed, and these include infection of guinea pig conjunctiva (Sereny test [25]) and in vitro infection of epithelial cells in culture (9). In both assay systems, virulent strains of Shigella spp. which are capable of producing dysentery in humans can invade the epithelial cells and multiply within them, whereas avirulent strains do not invade these cells. These assay systems have been useful in defining genetic determinants required for virulence. For example. Sansonetti et al. have demonstrated that virulence is associated with the presence of a 120-megadalton plasmid in Shigella sonnei and a 140-megadalton plasmid (pSf2al40) in Shigellaflexneri 2a (23, 24). We are interested in examining the genetics of pathogenesis of S. flexneri 2a and in determining how expression of

summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5' end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.