Inheritance of Resistance to Crown Gall in Pisum sativum

We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10⁹ bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three `supersusceptible' genotypes, which developed very large tumors, also were identified.     

Sequence and analysis of the nodABC region of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean and other legumes.

We cloned and analyzed nodABC from Rhizobium fredii USDA257. These genes are thought to have common functions in initiation of nitrogen-fixing nodules by all rhizobia. In USDA257, they were located in a 9.2-kb EcoRI fragment that was not closely linked to either of two copies of the regulatory gene, nodD. nodABC was present in a 3,094-base pair (bp) sequenced region, which also included a consensus nod-box promoter. The three open reading frames contained 654, 642, and 1,239 bp, respectively, and encoded deduced proteins of 21.9, 23.4, and 44.7 kD. The sequence of the nodABC region of USDA257 was generally homologous with corresponding regions from other rhizobia, but it diverged significantly in the 5' non-translated region and in the 3'terminus of nodC. nodC was not translationally coupled to nodSU, as in another soybean symbiont, Bradyrhizobium japonicum, and the deduced NodC protein was the shortest of any such proteins yet described. Site-directed mutagenesis of the 9.2-kb EcoRI fragment confirmed that nodA, nodB, and nodC are essential for nodulation of soybean, but failed to identify other linked nod genes. Daidzein, a major isoflavone from soybean roots, was the most potent of nine tested flavonoids in activating a plasmid-borne nodC::lacZ fusion. The 9.2-kb fragment complemented nodA-, nodB-, and nodC- mutants of R. meliloti to the Nod+ phenotype on Medicago sativa, M. truncatula, and Trigonella foenum-graecum. Nodule numbers, percentage of nodulated plants, and shoot dry weights, however, were considerably less than in plants inoculated with mutants complemented with nodABC from R. meliloti.     

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.     

Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram

We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6.     

SLOUGHED PERIPHERAL ROOT CAP CELLS: YIELD FROM DIFFERENT SPECIES AND CALLUS FORMATION FROM SINGLE CELLS

Peripheral root cap cells can be isolated by gentle agitation of the root in water for less than 60 sec. Filtration of the detached cells through a 30-μm mesh screen results in a suspension of single cells. Yields of peripheral root cap cells from 28 species in 10 families ranged from fewer than 10 per root from tobacco to more than 5,000 per root from pine. The viability of freshly isolated cells from most species, including legumes and cereals, was 85% to 95%. Cultured cells from pea and soybean exhibited cytoplasmic streaming for more than a month. When incubated at an initial density of 10⁵ cells per ml of liquid B5(P) medium, 6% to 8% of isolated pea root cap cells divided and grew into callus.     

Variation in Binding and Virulence of Agrobacterium tumefaciens Chromosomal Virulence (chv) Mutant Bacteria on Different Plant Species

Chromosomal virulence (chv) mutants of Agrobacterium tumefaciens have been reported to be deficient in binding to cells of zinnia, tobacco, and bamboo. The mutants are nonpathogenic on stems of Kalanchoë, sunflower, tomato, Jerusalem artichoke, and tobacco, but they cause tumors on tubers of Solanum tuberosum. We used a root cap cell binding assay to test ability of cells from individual plants of 13 different plant species to bind parent or chv mutant bacteria. The same plants were then inoculated to test for disease response. Cells from nine of the plant species were grossly deficient in their abilities to bind mutant bacteria, and the plants inoculated with mutant bacteria failed to form tumors. In contrast, root cap cells as well as root hairs and root surfaces of S. tuberosum, S. okadae, and S. hougasii bound chv mutant bacteria as well as wild type. Nevertheless, S. tuberosum roots inoculated with mutant bacteria did not develop tumors. Although S. okadae plants inoculated with mutant bacteria formed a few tumors, and S. hougasii developed as many tumors in response to chv mutants as in response to the parent strain, the tumors induced by mutant bacteria were smaller.     

Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod⁺ on McCall] and USDA257 [Nod⁻ on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.     

nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.     

Immunogold localization of prolamines in developingHaynaldia villosa endosperm

Summary Haynaldia villosa is a wild grass belonging to the tribe Triticeae, which includes important crops such as wheat, barley, and rye. The alcohol-soluble proteins ofH. villosa have extensive immunological relatedness with wheat prolamines as visualized by Western blot analysis. Amorphous protein inclusions surrounded by a limiting membrane are commonly found in the vacuoles of endosperm and subaleurone layers ofH. villosa seeds. A layer of cells just beneath the aleurone layer is rich in ER. Unlike that in other cell types, the ER in these cells is highly dilated and contains materials at its swollen distal ends. These materials are structurally similar to substances found in the protein bodies. Protein A-gold immunocytochemical localization studies employing antibodies against wheat prolamine confirmed that the inclusions found in the lumen of the ER do not contain prolamines. This observation indicates that the ER does not act as the site of prolamine accumulation inH. villosa. Protein bodies found in the vacuoles and the vesicles associated with the Golgi complexes were specifically labeled. This suggests that Golgi complexes mediate the transport of prolamines into vacuoles ofH. villosa endosperm cells, in a fashion analogous to that of other vacuolar proteins of dicotyledonous plants.