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A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrBo and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrBI, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrBo and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, TnS transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB, from race 1 interacts with the soybean disease resistance locus, Rpgl.

Pectate lyases are secreted by pathogens and initiate soft-rot diseases in plants by cleaving polygalacturonate, a major component of the plant cell wall. The three-dimensional structure of pectate lyase C from Erwinia chrysanthemi has been solved and refined to a resolution of 2.2 angstroms. The enzyme folds into a unique motif of parallel beta strands coiled into a large helix. Within the core, the amino acids form linear stacks and include a novel asparagine ladder. The sequence similarities that pectate lyases share with pectin lyases, pollen and style proteins, and tubulins suggest that the parallel beta helix motif may occur in a broad spectrum of proteins.

The cloning of avirulence genes has greatly aided our understanding of plant-pathogen specificity. It has proven that the gene-for-gene relationship first noted by Flor is correct--single avirulence gene encoding single protein products indeed are the genetic elements that interact with plant disease resistance genes. Furthermore, firm genetic evidence has provided insight into how two cloned avirulence genes (the TMV coat gene and avrD) cause the HR. The differences in structure of pathogen elicitors also indicates that plants have evolved diverse recognitional mechanisms to detect pathogens. It is appealing to speculate, therefore, that elicitors represent the plant equivalent of antigens in vertebrates. Another consequence of these results has been the establishment of firm genetic and biochemical evidence supporting the elicitor-receptor model for recognition of incompatible pathogen races by plants. In both TMV and bacterial pathogens, we are also beginning to understand how avirulence genes are altered to confound plant recognition of the pathogen. The next few years should yield additional information on avirulence gene structure as well as the important questions of their function in the pathogen and the molecular mechanisms whereby plant recognition occurs. The marked successes in cloning avirulence genes underscore only more forcefully the pressing need to clone and characterize plant disease resistance genes. Certainly an understanding of these genes is required to further our basic knowledge of active defense in plants and to permit their manipulation for improved control of plant diseases in practical agriculture.

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97-100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.A ll plant species in natural habitats have associated epiphytic microflora comprising the so-called phyllosphere (1, 2). The composition and quantity of nutrients, including carbohydrates, organic acids, and amino acids that support the growth of epiphytic bacteria, are affected by the plant species, leaf age, leaf physiological status, and the presence of tissue damage (3). Similarly, host plants, leaf age, leaf position, physical environmental condition, and availability of immigrant inoculum have also been suggested to be involved in determining species of microbes in the phyllosphere (4-7).There has been much interest in life forms that inhabit extreme environments such as the phyllosphere. With the repeated, rapid alteration of environmental conditions occurring on leaf surfaces, the phyllosphere has been recognized as a hostile environment to bacteria (8). Leaf surfaces are often dry and temperatures can reach 40-55°C under intense sunlight. During the night, however, leaves are frequently wet with dew and at relatively cool temperatures (5-10°C). Strong UV radiation during the day and sparse nutritional (oligotrophic) conditions also contribute to stressful conditions in the phyllosphere (8). More than 85 different species of microorganisms in 37 genera have been reported in the phyllospheres of rye, olive, sugar beet, and wheat, all by culture-based methods (8-10). Most of these bacteria establish large populations with no apparent effect on the plant, but a few of them can infect the leaves and cause disease (1).Microbial ecologists have devoted much effort to investigating microbial diversity and studying biological interactions between species in the environment. Microor...

A genomic library of Pseudomonas syringae pv. glycinea race 6 DNA was constructed in the mobilizable cosmid vector pLAFRl and maintained in Escherichia coli HB101. Completeness of the library was estimated by assaying clones for the expression of ice-nucleating activity in E. coli. Ice-nucleation activity was represented approximately once in every 600 clones. Six hundred eighty random race 6 cosmid clones were mobilized from E. coli by plasmid pRK2013 in individual conjugations to a race 5 strain of P. s. glycinea. A single clone (pPg6L3) was detected that changed the race specificity of race 5 from virulent (compatible) to avirulent (incompatible) on the appropriate soybean cultivars. The clone was also mobilized from E. coUl into race 1 and race 4 strains of P. s. glycinea, and it conferred on these transconjugants the same host range incompatibility as the wild-type race 6 strain.

The pelB and pelE genes from Erwinia chrysanthemi EC16, which encode different pectate lyase enzymes, were sequenced and expressed at a high level in Escherichia coli. The genes possessed little similarity to each other in 5' signal regions, signal peptide sequences, coding sequences, or 3' noncoding regions. Both genes contained their own promoters as well as sequences 3' to the coding regions with considerable secondary structure which may function as rho-independent transcriptional termination signals. High-level expression plasmids were constructed with both genes, which led to 20% or more of E. coli cellular protein. The pectate lyases were secreted efficiently to the periplasm and, to ra lesser extent, the culture medium. The mature proteins in E. coli periplasmic fractions were obtained in milligram amounts and high purity with a single-column affinity purification method. E. coli cells which produced high amounts of the pelE protein macerated potato tuber tissue as efficiently as E. chrysanthemi EC16 cells but cells producing high amounts of the pelB protein were less effective. Thus, the pelE gene product is an important pathogenicity factor which solely enables E. coli to cause a soft-rot disease on potato tuber tissue under laboratory conditions. We cloned genes coding for two different pectate lyase (EC 4.2.2.2) enzymes from the phytopathogenic bacterium Erwinia chrysanthemi EC16 (13) and observed their expression in Escherichia coli. Pectate lyases have previously been shown to account largely or entirely for the maceration or soft rotting of plant tissue caused by Erwinia spp. (4). Confirming this, E. coli cells containing the cloned pectate lyase genes macerated plant tissue, albeit less efficiently than E. chrysanthemi (13). Several groups subsequently cloned similar genes from other strains of E. chrysanthemi (5,14,28,34) and the related bacterium Erwinia carotovora (18,29, 40). The genes that we cloned did not cross-hybridize (13), but coded for enzymes with similar physical properties (molecular weights of ca. 40,000 and isoelectric points of 8.8 and 9.8) which both catalyzed the random eliminative cleavage of sodium polypectate. These enzymes were efficiently secreted to the periplasm and, to a lesser extent, the culture medium of E. coli. For reasons discussed below, the cloned DNA fragments appear to contain pelB and pelE, described by others, and the mature proteins which they encode are PLb and PLe, respectively. Plasmids containing our pel genes were named pPL in the previous paper (13), but this designation was found to be already entered in the Plasmid Reference Center (17). Accordingly, our pel gene plasmid constructs have been renamed pPEL, a designation that we have registered in the Plasmid Reference Center (17).The cloned pelB and pelE genes were both regulated by catabolite repression in E. coli (13) but were not induced by sodium polypectate, as occurs in E. chrysanthemi (4). Due to their pathogenic importance in diseases caused by Erwinia spp. and to their regulation propertie...