Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or ii) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and ii alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

Gene-for-gene disease resistance typically includes a programmed cell death response known as the hypersensitive response (HR). The Arabidopsis thaliana dnd1 mutant was previously isolated as a line that failed to produce the HR in response to avirulent Pseudomonas syringae pathogens; plants homozygous for the recessive dnd1-1 mutation still carry out effective gene-for-gene resistance. The dnd1-1 mutation also causes constitutive systemic resistance and elevated levels of salicylic acid. In the present study, a positional cloning approach was used to isolate DND1. DND1 encodes the same protein as AtCNGC2, a cyclic nucleotide-gated ion channel of previously unknown organismal function that can allow passage of Ca(2+), K(+) and other cations [Leng, Q., Mercier, R. W., Yao, W. & Berkowitz, G. A. (1999) Plant Physiol. 121, 753-761]. By using a nahG transgene, we found that salicylic acid is required for the elevated resistance caused by the dnd1 mutation but that removal of salicylic acid did not completely eliminate the dwarf and loss-of-HR phenotypes of mutant dnd1 plants. A stop codon that would severely truncate the DND1 gene product was identified in the dnd1-1 allele. This demonstrates that broad-spectrum disease resistance and inhibition of the HR can be activated in plants by disruption of a cyclic nucleotide-gated ion channel.

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

A complex regulatory network controls virulence genes of Pseudomonas solanacearum. Analysis of the transposon-generated mutant AW1-83 suggests that a new locus, designated phcB, may play a role in this network. AW1-83 (phcB83) produced at least 30-fold less than the wild type of extracellular polysaccharide (EPS I, encoded in part by eps) and at least seven extracellular proteins, but these traits were fully restored in response to one or more extracellular factors (EF) released by wild-type P. solanacearum. Presence of EF increased transcription of a genomic eps::lacZ fusion in a phcB83 background more than 50-fold, restoring wild-type expression. The EF made by P. solanacearum was present in both the aqueous and the vapor phases. Millimolar levels of methanol (but not larger alcohols) and micromolar levels of C14- to C18-fatty acid methyl esters (but not larger or smaller methyl esters) also restored nearly wild-type expression of eps::lacZ in a phcB83 background. The methoxy group was essential for this increase, since neither free fatty acids nor the ethyl or propyl esters were active. Growth with the C16-methyl ester restored normal production of EPS I and extracellular proteins by AW1-83. The wild-type phcB locus was subcloned on a 4-kb fragment and delimited to less than 2 kb by transposon inactivation and complementation studies. Genomic phcB::Tn3HoHo1 mutations appeared to eliminate EF production but did not uniformly reduce production of EPS I and extracellular proteins. Site-specific recombination of the phcB83 allele into the genome of five other P. solanacearum strains revealed that they have a structurally and functionally conserved phcB locus. Although all 80 wild-type strains of P. solanacearum tested made some EF, out of seven genera of bacteria tested, only Agrobacterium produced an EF-like activity that stimulated visible EPS production by AW1-83. Our results suggest that the EF may be an extracellular signal molecule in P. solanacearum that is different from the acyl-homoserine lactone signal compounds produced by Vibrio fischeri and other Gram-negative bacteria.

A complex regulatory network controls production of some extracellular macromolecules that Pseudomonas solanacearum strain AWl needs for full virulence on tomato. Expression of genes for extracellular polysaccharide (EPS) and other virulence factors is coordinately controlled by phcA, since inactivation of this gene results in a pleiotropic change called phenotype conversion (PC). PhcA is likely to be a trans-acting DNA-binding protein because it has strong similarity at the amino acid level to the LysR family of transcriptional activators. At least three different insertions were detected within phcA after spontaneous PC, suggesting that random inactivation of this gene is responsible for this phenomenon. Another locus, phcB, is necessary for production by wild-type P. solanacearum strains of a volatile inducer compound that appears to be required for full activity of PhcA. Cell density, and thus levels ofthe endogenous inducer, appear to mediate expression of a Phc.4-regulated eps::lacZ fusion. Expression of eps genes also depends on three additional loci (xpsR, vsrA. and vsrB), two of which encode membraneassociated proteins and thus may serve as environmental sensors. These findings suggest that current concepts regarding the physiology, ecology, and possible strategies for controlling P. solanacearum should be re-evaluated. IN the last ten years there has been substantial progress in elucidating the genetic and biochemical bases of pathogenesis of P. solanacearum. For example, the genes encoding several of the putative virulence factors identified in the 1950s were cloned and subsequently inactivated to test their contribution to the typical wilt symptoms. It is now clear that, in planta, the extracellular polysaccharide (EPS) is the primary virulence factor (Denny and Baek 1991; Kao and Sequeira 1992). whereas the extracellular endoglucanase (EG) and en do-polygalacturonase (endo-PG) enzymes. which may act on plant cell walls, are relatively minor virulence factors (Denny et aL 1990). In contrast to these factors that enhance virulence but are dispensable, the hrp genes are essential for pathogenesis on compatible hosts and the hypersensitive response on incompatible hosts (Boucher et al. 1992). Unlike virulence mutants, strains with one or more hrp genes inactivated grow very poorly in planta (Macol 1989; ·Departments of Plant Pathology and Microbiology. 2105 Plant Science Building, University of Georgia, Athens GA 30602,USA. Trigalet and Demery 1986) and cause no disease symptoms. Despite these and other significant advances, our understanding of the pathogenic processes of P. solanacearum is still incomplete. Not all of the putative virulence factors have been examined (Denny and Schell 1992) and even those mentioned above have not been tested for their role in other aspects of pathogenesis or saprophytic survival. Even less is known about regulation of genes encoding pathogenesisspecific molecules. This paper will discuss some of our recent research on phenotype conversion in P. solanacearum and the associated regulatory net work that controls virulence. Phenotype Conversion Nearly four decades ago Kelman (1954) reported that when P. solanacearum spontaneously changes from a mucoid to a nonmucoid colony morphology there is a concomitant loss of its capacity to wilt plants. These non-mucoid strains are not hrp mutants, because they stilI grow in planta (Denny and Baek 1991) and cause

Pseudomonas solanacearum produces an acidic, nitrogen-rich, extracellular polysaccharide (EPS) that is required for wilting and killing of infected plants Biosynthesis of EPS is partially encoded by the 18-kb eps locus; additional loci near eps are also required for EPS production, but only under specific growth conditions. All these loci are transcriptionally controlled by an interacting regulatory network involving the products of at least five distinct regulatory loci: phcA, phcB, vsrA,vsrB, and xpsR. This network, which also regulates other virulence genes, may control transcription in response to various environmental signals, such as nutritional status and cell density.