C. A. Wijffelman

Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria.

A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

Amino acid synthesis is necessary for tomato root colonization by Pseudomonas fluorescens strain WCS365

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600-607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Small leguminosae as test plants for nodulation of Rhizobium leguminosarum and other rhizobia and agrobacteria harbouring a leguminosarum sym-plasmid

Abstract Small plants from the pea cross-inoculation group were selected for rapid nodulation on agar in test tubes. Plants from the genus Vicia were best; strains of the species hirsuta, tetrasperma, sativa and lathyroides nodulated respectively 4, 5, 4 and 7 days after inoculation with Rhizobium leguminosarum RBL 1. Rapid nodulation, visibility of the root system and in situ acetylene reduction makes the culture tube system ideal for genetic studies of Rh. leguminosarum. A number of bacteria including Agrobacterium tumefaciens containing a Sym-plasmid from Rh. leguminosarum formed root nodules. A Vicia sativa strain formed thick and short roots (Tsr) when inoculated with a bacterium harbouring a Sym-plasmid from Rh. leguminosarum. Crown gall formation could also be investigated by wounding of axenic Vicia hirsuta in test tubes.

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non‐polar transbilayer alpha‐helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid‐inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5′ part of the R.leguminosarum nodE gene and a 3′ part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non‐identical amino acids, determines the difference in the host‐specific characteristics of the two NodE proteins.

Repression of Small bacteriocin excretion in Rhizobium leguminosarum and Rhizobium trifolii by transmissible plasmids

SummaryAll of the fast growing Rhizobium leguminosarum and R. trifolii strains studied, except four, excrete a small bacteriocin (small) into the culture medium. Only the four non-excreting strains harbour highly transmissible plasmids among which are the Sym plasmids pRL1JI (pea cross-inoculation group) and pRtr5a (clover cross-inoculation group). Small production genes were demonstrated in three of these strains and a plasmid function preventing excretion of small was present in all four strains (Rps). Two of the plasmids rendered the cells sensitive to small (Sbs). The plasmid functions Rps, Sbs, and medium bacteriocin production (Mep) on pRL1JI were expressed in R. leguminosarum, R. trifolii, R. phaseoli and A. tumefaciens. The formation of thick and short roots (Tsr) on Vicia sativa and nodulation (Nod) were also expressed in these hosts. No expression of above mentioned functions was found in R. meliloti.

Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum

The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular basis of the development of bacterium to bacteroid, we wanted to know whether these outer membrane protein differences are conserved in various plant-Rhizobium combinations, For this purpose we developed a faster assay in which cell lysates instead of isolated cell envelopes were used to analyze these outer membrane changes, With this method the previously described low levels of antigen groups II and III in isolated bacteroid cell envelopes were confirmed, Moreover the described decrease in antigen groups II and III was also found in bacteroids of Rhizobium leguminosarum by. viciae with a mutated nifA or nifK gene as well as in the non-fixing pea mutant FN1 inoculated with the wild-type strain 248, This indicates that the decrease in the antigen levels is not restricted to effective nodules, The results also showed that the decrease in antigen group II not only occurs in bacteroids from pea, but also in bacteroids from vetch, broadbean, white clover, and common bean, Antigen group III, however, remained present in bacteroids from common bean, It is concluded that the changes in antigen group II are not restricted to a specific cross-inoculation group but represent a general phenomenon in the rhizobial bacteroid differentiation process, Of the tested plants, the decrease in antigen group III was not found in bacteroids from common bean and appeared to be restricted to bacteroids from indeterminate nodules. Therefore one should expect that at least two molecular mechanisms are responsible for these outer membrane protein changes and that elucidation of these mechanisms will contribute to our understanding of bacteroid development.

Isolation of ropB, a gene encoding a 22-kDa Rhizobium leguminosarum outer membrane protein.

As judged from immunochemical detection, the levels of outer membrane antigen groups II and III of Rhizobium leguminosarum bv. viciae strain 248 decrease during bacteroid differentiation (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). Using a newly developed colony blot assay, a cosmid clone expressing the Mab8 epitope of the outer membrane antigen group II of R. l. bv. viciae strain 248 was selected in Rhizobium meliloti LPR2120. From this cosmid the gene encoding this epitope was cloned and characterized. An open reading frame of 636 nucleotides was found and predicted to encode a protein with a calculated molecular mass of 22.5 kDa. After subtraction of the predicted 23 amino acid signal peptide, a M(r) of 20.3 kDa was calculated for the mature protein. This gene, designated ropB, was not active in Escherichia coli under the control of its own promoter. The C-terminal amino acid of the protein is a phenylalanine residue which is required for efficient translocation of outer membrane proteins. Membrane spanning amphipathic beta-sheets are predicted to represent a major part of the secondary structure of the protein. A model of the topology of the protein is presented. We determined the start of transcription in order to analyze the promoter region. No homology was found with other known promoter sequences. The ropB gene appeared to be well-conserved in R. leguminosarum and Agrobacterium tumefaciens strains. An attempt was made to mimic the immunochemical decrease of RopB ex planta. Neither the various growth conditions tested nor the addition of nodule or plant extracts resulted in a reduction of the Mab8 epitope to bacteroid levels.

Perspectives in Plant Cell Recognition: The Rhizobium trap: root hair curling in root–nodule symbiosis

Introduction The root-nodule bacteria Rhizobium, Bradyrhizobium and Azorhizobium (collectively rhizobia) invade and nodulate the roots of their host plants via either wounds or root hairs. The choice is made by the host plant, e.g. the same rhizobial strain infects Vigna roots via root hairs and Arachis roots via wounds (Sen & Weaver, 1984), whereas another strain infects Parasponia via root epidermal cracks and Macroptilium via root hairs (Marvel et al ., 1985). Shortly before or during root invasion, rhizobia induce cell divisions in the root cortex, resulting in formation of a nodule primordium. Through infection threads (tip-growing tubular structures containing invading rhizobia) and/or between cortical cells the rhizobia migrate towards the growing primordium, are endocytosed by young nodule cells, and differentiate into dinitrogenfixing bacteroids (see also Brewin et al ., this volume). Rhizobial invasion of most agronomically important legumes such as pea ( Pisum sativum ), soybean ( Glycine max ) and bean ( Phaseolus vulgaris ) occurs through root hairs. Infection of a living plant cell is an unusual phenomenon in plant–bacteria interactions. Plants are open organisms. At many sites, the intercellular space of a plant is in direct contact with the environment, e.g. in stomata, hydathodes or wounds resulting from emergence of lateral roots. A plant is used to regular visits of (plant-associated) bacteria to its interior. Therefore, wound-infection by rhizobia is a normal phenomenon whereas root hair infection is special.

Recent Ideas on the Physiology of Infection and Nodulation

The establishment of an endosymbiosis is the result of a complicated series of interactions for which D.C. Smith (1981) postulated a scheme of four stages: a contact, including events leading to contact, b incorporation into host matrix, c integration into regulatory mechanisms and d development of nutrient flows between (endo) symbiont and host. All stages may contain specific interactions which are only possible between homologous partners. However, if such specific interactions, which contain an element of recognition, are part of an essential step, their failure will prohibit all further stages. This failure will be most dramatic if it occurs early in the total sequence of interactions. These interactions form a very subtle, intricate pattern, for a main part operating at a subcellular level. This makes their analysis extremely difficult. Much information may be derived from comparisons with the deviations in the normal development, when less compatible combinations are studied. The progress in the microbial genetics of Rhizobium has markedly improved possibilities for such comparisons and enabled a further analysis of the interactions during infection and nodulation.

The Expression of Sym-Plasmids and Ti Plasmids in Rhizobia and Agrobacteria

Agrobacteria and rhizobia belong to the bacterial family of Rhizobiaceae.The classification of bacteria belonging to this family into species is based on phytopathogenic and symbiotic properties. In the genus Agrobacterium three species can be distinguished viz A. tumefaciens, the causative agent of the crown gall disease, A.rhizogenes,which induces hairy root in plants, and A.radiobacter, which is non-pathogenic. The genus Rhizobium contains species such as R.trifolii, which forms root nodules on clovers, R.phaseoli, which nodulates beans, and R.leguminosarum, which induces nodulation on peas, lathyrus and vetches.