Many reports document the ability of certain Rhizobium strains to occupy nodules to the exclusion of other strains. The molecular basis of the superior nodulation competitiveness of any strain is not known, although important advances in recent years have been made (7-9, 12, 19, 20 (12,15,16,22,23,25,26).Hodgson et al. (12) have suggested that bacteriocin production by R. leguminosarum bv. trifolii CB782 is involved in its superior nodulation competitiveness. This was shown by co-inoculating CB782 with bacteriocin-sensitive and -resistant strains. However, these sensitive and resistant strains may differ in other respects that affect nodulation competitiveness aside from bacteriocin production. Transposon mutants of CB782 lacking bacteriocin production would demonstrate the role ofbacteriocin production in competitiveness, since Tn5 is a single point mutagen (3). We are studying the basis of the competitiveness expressed by R. leguminosarum bv. trifolii strain T24, which was isolated in Florida from the nodules of Trifolium dubium in 1937 (17, 25).In 1968, Schwinghamer and Belkengren (25) reported that T24 had three important properties: (a) it induces ineffective nodules on clover roots, (b) it produces a very potent antibiotic inhibitory toward strains of Rhizobium, and (c) it prevents nodulatiop by other strains of R. keguminosarum bv. trifolii. Schwinghamer and Belkengren (25) suggested that the basis for the nodulation competitiveness of T24 was its ability to produce an anti-rhizobial compound.The purpose of the experiments described here are to (a) determine using transposon mutagenesis the roles of antibiotic production and nodulation in the expression of nodulation competitiveness by T24, (b) determine the range of anti-rhizobial activity of the T24 antibiotic, and (c) determine whether T24 can induce effective nodules on any available clover species.

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Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the I8-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked TnS insertions and retained their resistance to the active toxin form, tabtoxinine-,-lactam (T,IlL). The wild-type DNA corresponding to the mutated region was cloned and found to restore the TnS mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10-3/CFU) in BR2. The Tox-deletion mutants also lost resistance to tabtoxinine-p-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox-derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease.

Pseudomonas syringae pv. coronafaciens, a pathogen of oats, was mutagenized with Tn5 to generate mutants defective in tabtoxin production. From a screen of 3,400 kanamycin-resistant transconjugants, seven independent mutants that do not produce tabtoxin (Tox-) were isolated. Although the Tn5 insertions within these seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic region of P. syringae. Instead, all of the insertions were within the P. syringae pv. coronafaciens lemA gene. The lemA gene is required by strains of P. syringae pv. syringae for pathogenicity on bean plants (Phaseolus vulgaris). In contrast to the phenotype of a P. syringae pv. syringae lemA mutant, the Tox- mutants of P. syringae pv. coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without the chlorosis associated with tabtoxin production. Northern (RNA) hybridization experiments indicated that a functional lemA gene was required for the detection of a transcript produced from the tblA locus located in the tabtoxin biosynthetic region. Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxin production. Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulation of tabtoxin production by lemA probably occurs at the transcriptional level.

We have identified a viable-yellow and a lethal-yellow chlorophyll-deficient mutant in soybean. Segregation patterns suggested single-gene recessive inheritance for each mutant. The viable- and lethal-yellow plants showed significant reduction of chlorophyll a and b. Photochemical energy conversion efficiency and photochemical reflectance index were reduced in the viable-yellow plants relative to the wildtype, whereas the lethal-yellow plants showed no electron transport activity. The viable-yellow plants displayed reduced thylakoid stacking, while the lethal-yellow plants exhibited failure of proplastid differentiation into normal chloroplasts with grana. Genetic analysis revealed recessive epistatic interaction between the viable- and the lethal-yellow genes. The viable-yellow gene was mapped to a 58kb region on chromosome 2 that contained seven predicted genes. A frame shift mutation, due to a single base deletion in Glyma.02g233700, resulted in an early stop codon. Glyma.02g233700 encodes a translocon in the inner membrane of chloroplast (GmTic110) that plays a critical role in plastid biogenesis. The lethal-yellow gene was mapped to an 83kb region on chromosome 3 that contained 13 predicted genes. Based on the annotated functions, we sequenced three potential candidate genes. A single base insertion in the second exon of Glyma.03G230300 resulted in a truncated protein. Glyma.03G230300 encodes for GmPsbP, an extrinsic protein of Photosystem II that is critical for oxygen evolution during photosynthesis. GmTic110 and GmPsbP displayed highly reduced expression in the viable- and lethal-yellow mutants, respectively. The yellow phenotypes in the viable- and lethal-yellow mutants were due to the loss of function of GmTic110 or GmPsbP resulting in photooxidative stress.