Noel T. Keen

Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria.

Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.

Plant-microbe interactions

Molecular cloning of plant disease resistance genes-- Gregory B. Martin Transgenic plants for disease control-- Luis Herrera-Hestrella, Laura Silva Rosales, and Rafael Rivera-Bustamante Systemic acquired resistance-- Urs Neuenschwander, Kay Lawton, and John Ryals Interactions of grasses with endophytic Epichloe species and hybrids-- Christopher L. Schardl Pathogenesis and sexual development of the smut fungi-- J.W. Kronstad Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites-- Linda S. Thomashow and David M. Weller Legume signals to rhizobial symbionts: a new approach for defining rhizosphere colonization-- Donald A. Philips and Wolfgang R. Streit Nodulation factors-- Jean Claude Prome and Nathalie Demont

Microbial phyllosphere populations are more complex than previously realized

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.

Bacteria expressing avirulence gene D produce a specific elicitor of the soybean hypersensitive reaction

Production of the avrD elicitor by P. s. pv. glycinea cells carrying the cloned avrD gene occurred independently of the hrp genes, considered important for pathogenicity and HR induction by certain P. syringae pathovars. The results indicated that expression of avirulence gene D in P. syringae pathovars and in E. coli causes them to produce a diffusible, elicitor-active molecule which initiates cultivar-specific induction of the HR

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-GalpA-([1→4]-α-D-GalpA)3-(1→4)-D-GalpA, is composed of five galacturonopyranose units (D-GalpA) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

Involvement of preformed antifungal compounds in the resistance of subtropical fruits to fungal decay

Fungal pathogens must perform precise functions and overcome several barriers before they are able to initiate disease in plants. First, the pathogen must locate and adhere to suscept tissue and then initiate infection (10,12,14). The first plant barriers encountered are generally the cuticle and cell wall, which may be breached by enzymatic (6) or physical (12) assault, or the pathogen may infect through wounds (27). Contact with underlying plant tissues presents the invading pathogen with a different set of barriers, most notably preformed antibiotic compounds and/ or morphologic barriers and phytoalexins induced by the plant (13,25,26). Pathogens that infect fruits are confronted by several problems not normally facing pathogens of vegetative plant tissues. Fruits are generally protected by differentiated integumentary structures, and their physiology changes markedly during development, particularly when ripening occurs. Pathogens frequently infect unripe fruits but cause relatively minor damage until ripening, when they may cause extensive decay. Such quiescent infections have been observed in tropical (l5), subtropical (8), and deciduous fruits (9). The resultant decays have great economic importance, since they reduce the shelf life of fruits during storage and transport (27). These disease problems have been further exacerbated by the development of pathogen resistance to fungicides and the withdrawal of pesticides on environmental grounds. Consequently, there is considerable interest in determining mechanisms accounting for the natural resistance of unripe fruits to fungal pathogens and extending its effectiveness to fruits after harvest. One of the possible mechanisms that may account for such differential

Development of the Particle Inflow Gun

A simple and inexpensive particle acceleration apparatus was designed for direct delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles directly in a helium steam. High levels of transient expression of theβ-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of maize and soybean, and leaf tissue of cowpea. Stable transformation of soybean and maize has also been obtained using this bombardment apparatus.

Colletotrichum gloeosporioides pelB Is an Important Virulence Factor in Avocado Fruit-Fungus Interaction

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Hydroxyphaseollin and related isoflavanoids in the hypersensitive resistance reaction of soybeans to pseudomonas glycinea

Abstract The hypersensitive reaction (HR) of soybean leaves to incompatible races of Pseudomonas glycinea was typified by rapid accumulation of the isoflavanoid compounds hydroxyphaseollin, coumestrol, daidzein and sojagol. The same compounds accumulated in response to inoculation with the non-pathogen P. lachrymans . In contrast, compatible races of P. glycinea led to delayed appearance of the isoflavanoids and the levels attained were 10% or less of those in resistant leaves. Accumulation of the isoflavanoids in resistant leaves was chronologically related to the restriction of bacterial multiplication. Cyclic-3′,5′-adenosine monophosphate at pH8 caused both the HR and isoflavanoid accumulation when supplied to leaves with normally compatible P. glycinea races. Hydroxyphaseollin possessed antibacterial properties in vitro at 25 to 100 parts/10 6 , concentrations that are less than 10% of those occurring in resistant soybean leaves. Coumestrol also inhibited colony development by P. glycinea in bioassays. Pronounced electrolyte loss did not occur in incompatible P. glycinea -soybean combinations, but was observed at the time of symptom appearance in leaves inoculated with compatible races. The data indicated that the HR of soybean leaves to P. glycinea was not related to permeability alteration but instead to the inducible accumulation of isoflavanoids.

Surface glycoproteins : evidence that they may function as the race specific phytoalexin elicitors of Phytophthora megasperma f.sp. glycinea

Abstract Glycoproteins were extracted from isolated cell walls of Phytophthora megasperma f.sp. glycinea (formerly P. megasperma var. sojae ) with 0·1 n NaOH at 0·C and elicited glyceollin in soybean hypocotyls with the same specificity as the fungus races from which they were obtained. Fractionation of the crude extracts on DEAE Bio-Gel and Bio-Gel A-5m columns showed that specific elicitor activity was associated with the presence of high molecular weight glycoproteins detected by SDS gel electrophoresis. The glycoproteins appeared to contain only glucose and mannose as neutral sugars. The elicitor activity of the glycoproteins was not diminished by boiling at 100°C or pronase treatment, but was destroyed by periodate, thus indicating that the carbohydrate portions are important for activity. The glycoproteins were the only concanavalin A reactive species detected in the crude cell wall extracts, and fluorescein labelled concanavalin A was hapten-specifically bound to living hyphae of the fungus and to native but not NaOH-extracted isolated cell walls. Therefore it was concluded that the glycoproteins are present at the surface of the fungus cell wall. Tunicamycin, which inhibits the glycosylation of eucaryote surface glycoproteins, was a potent inhibitor of mycelial growth of the fungus. The data supported the hypothesis that race specificity in the soybean— P. megasperma f.sp. glycinea system may be determined by specific plant recognition of fungus surface glycoproteins.