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Specificity in Rhizobium-legume interactions Objective:

Specificity in Rhizobium-legume interactions Objective: To explore two levels of specificity that contribute to establishing the cross-inoculation groups for Rhizobium - legume interactions. Topics: A. Role of the NodD proteins of Rhizobia in recognition of flavonoids from legume hosts.

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Specificity in Rhizobium-legume interactions Objective:

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  1. Specificity in Rhizobium-legume interactions Objective: To explore two levels of specificity that contribute to establishing the cross-inoculation groups for Rhizobium - legume interactions. Topics: A. Role of the NodD proteins of Rhizobia in recognition of flavonoids from legume hosts. B. The production of legume-specific signals (Nod factors) by Rhizobia in response to plant flavonoids. C. What functions do nod genes encode? D. How is nod factor specificity achieved?

  2. A. Role of the nodD proteins of Rhizobia in recognition of flavonoids from legume hosts. Basic Message- Different Rhizobia species (representing different cross-inoculation groups) respond to different flavonoid inducers from legume roots. The differences in responses are determined by differences in the nodD gene products (NodD proteins) produced by the bacteria. The bacteria generally respond best to the flavonoids from their specific host plants (that they can nodulate). The nodD gene encodes the NodD protein that is constitutively produced in Rhizobia cells. The NodD protein is a sensor for flavonoid inducers; it binds flavonoids and becomes active to turn on the transcription of the other nod genes that encode the enzymes to make and modify nod factors.

  3. 1. Illustration of specificity differences for NodD Three nodD genes in Sinorhizobiummeliloti Sinorhizobium meliloti - three hosts - alfalfa (Medicago), sweet clover (melilotus) and Trigonella Genetic approach to explore the role of the three nodD genes: Make mutations in each of the nodD genes in Sinorhizobium meliloti and assay the mutants for nodulation ability on alfalfa and clover. Figure 3 - HONMA AND AUSUBEL 1987 Proc. Natl. Acad. Sci. USA. 84: 8558-8562. Three nodD genes in Sinorhizobiummeliloti Fig. 3 shows the data for the various mutant combinations in terms of nodulation on alfalfa and white clover.

  4. (sweet clover) (alfalfa) no D2 mutants no D1 and D2 no D1 no D1 and D3 no D3 no D1 and D3 no D2 and D3 no NodD no NodD

  5. The data show that NodD2 allows a delayed response on alfalfa but no response on sweet clover. The conclusion is that there is some specificity, thus raising the possibility that different NodD proteins respond to different compounds from hosts. Conclude: Different NodD proteins recognize different flavonoid inducers. Why mulitple nodD genes in Sinorhizobium meliloti? Perhaps this situation reflects the adaptation of the bacterium to different hosts. Sinorhizobium meliloti nodulates three genera of plants: alfalfa (Medicago), sweet clover (Melilotus) and Trigonella. Perhaps three NodD proteins are needed to optimize the interaction with specific flavonoids in the root exudates of the three different hosts.

  6. 2. Another demonstration that different nodD genes have different specificities Spaink et al. 1987. Nature. 328: 337-340. nodD genes from different Rhizobium species can control host range The nodD gene products of various fast growing Rhizobium species differ from each other in that they confer different responsiveness in a species-specific way to different sets of flavonoids and exudates. Fast growing species: Sinorhizobium meliloti R. leguminosarum bv. viciae R. leguminosarum bv. trifolii

  7. Table 2. Rhizobium trifolii as the recipient bacterium % nodulated plants Ave. # of nodules per nodulated plant Red CloverWhite CloverRed CloverWhite Clover R. trifolii (nodD- mutant) 0 0 - - R. trifolii nodD mutant + nodD (Rt) 90 100 2 4 R. trifolii nodD mutant + nodD (Rl) 3 100 1 4 R. trifolii nodD mutant + nodD1 (Sm) 0 70 - 2

  8. Table 2 - Spaink et al. Clear demonstration that host range (as reflected by nodulation ability) depends on nodD genes. nodD of R. leguminosarum bv. viciae or Sinorhizobium meliloti only able to complement the nodD mutation in the trifolii strain ANU851 on white clover (nodD of R. meliloti is not able to completely restore activity). See narrowing of host range of R. leguminosarum bv. trifolii when its own nodD gene is replaced with that of R. leguminosarum bv. viciae or Sinorhizobium meliloti. Other things to note: 1. Flavonoids can also stimulate the growth of the bacteria (not known how). 2. Combinations of flavonoids can act synergistically. 3. Some flavonoids also act as anti-inducers - inhibit activation.

  9. Overview of NodD specificity The table shows a survey of the responses of different NodD proteins to flavonoids of different stucture. The assay is to measure the ability of different NodD proteins to activate the transcription of nod genes in response to each of the flavonoids.

  10. B. The production of legume-specific signals (Nod factors) by Rhizobia in response to plant flavonoids. 1. Early evidence for diffusible Nod factors. a. Dutch Group - Lugtenberg and Spaink In 1982, a research group in Leidenreported that R. leguminosarum, when cocultivated with vetch on agar medium, would cause a change in root growth - thick short roots (TSR). A subsequent report by van Brussels et al., (1986 J. Bact. 165: 517-522) showed that in the R. leguminosarum - Viciae sativa (common vetch) interaction, the pSym plasmid controlled the production of a diffusible factor that causes thick short roots on vetch. This TSR phenotype and a related Hac (hair curling) phenotype required the pSym plasmid and functional nodABC genes. This work identified the TSR factor as a chemically undefined product that was exported by the bacterium and that influenced root morphology.

  11. b. Australian group - Rolfe Additional early evidence that the nodABC genes play role in triggering root hair curling came from Rossen et al. 1984. Nucl. Acids Res. 12: 9497 This group was also working withR. leguminosarum. They found a diffusible factor whose production is blocked by mutations in the nodABC genes. It was also found that this compound or compounds from Sinorhizobium meliloti will also stimulate mitosis in plant cells - cell division was observed in protoplasts prepared from various plant species

  12. c. French Group - Truchet and Denarie At the same time, a French group was trying to find the same type of exported activity from S. meliloti. Faucher et al. J. Bact. 179: 5489-5499. The problem was that S. meliloti did not cause the thick short root phenotype so it was difficult to assay for exported factors. The big break came when it was found that nodH mutants of S. meliloti do cause the TSR phenotype. This provided evidence that S. meliloti was making an activity similar to that of R. leguminosarum. It was also found that S. meliloti culture supernatants would cause root hair deformation (HAD) (HAD factor that causes changes in root hair morphology) and this assay was more sensitive for factors in culture supernatants compared to the thick short root assay. French group won the race to publish the structure of the nod factor, the compound that is responsible for the HAC, HAD and TSR activities. Classic paper: Lerouge et al 1990 Nature 344: 781-784.

  13. French Group Lerouge et al 1990 Nature 344: 781-784. Structure of NodRm-1 Assay for nod factor = HAD activity of alfalfa seedlings Luteolin - induced cultures of S. meliloti. HAD activity purified by high performance liquid chromatography. Amounts too small for structural analysis. Trick: Put a plasmid into S. meliloti that carried all of the nod genes. The plasmid had a copy number of 5 - 10 per cell so each nod gene was present in 5-10 copies. This resulted in a strain of S. meliloti that produced 100 time more HAD activity on alfalfa. This provided enough of the Nod factor to allow structural analysis - Ten litres of induced bacterial culture yielded 4 mg of Nod factor.

  14. C. What functions do nod genes encode? Nod factors Lipochitooligosaccharides (LCOs) nodC gene encodes NodC protein - an enzyme with N-acetyl-glucosaminyltransferase activity. Synthesis of the N-acetyl-glucosamine backbone. nodB gene encodes the NodB protein - a deacetylase enzyme that removes an acetyl group from the Nod factor. nodA gene encodes the NodA protein - an acyltransferase that adds a fatty acid chain to the site deacetylated by NodB The LCO structure made by NodABC does have nodulation activity on certain legumes.

  15. C. cont. What functions do host specific nod genes encode? 1. Sulfation (nodH, noeE). Sulfotransferases that modify Nod factors (e.g., in S. meliloti). 2. Fatty acid addition (nodEF). Different NodEF proteins from different Rhizobium species can put different fatty acids onto Nod factors and change specificity. 3. Arabinosylation (noeC). The NoeC protein adds arabinose to Nod factors - can influence number of nodules that are formed on host plants. 4. Fucosylation (e.g.,nodZ). Fucose addition changes specificity of nodulation for some symbionts such as Bradyrhizobium japonicum. 5. Acetylation (e.g., nodL). Acetylation can influence Nod factor activity for R. leguminosarum and B. japonicum NGR234 6. Methylation and Carbamoylation (e.g., nodS). Mutation changes host range for R. tropici, R. loti, R. fredii and B. japonicum.

  16. D. How is nod factor specificity achieved? 1. The nodHPQ story from the S. meliloti-alfalfa interaction. nodH encodes an enzyme involved in modification of nodRm-2 to give nodRm-1. The enzyme transfers activated sulfate to the nod factor. NodH- mutants lose the ability to produce nodRM-1, instead the mutants make nodRm-2 which is a more hydrophobic compound that is active on vetch, not on alfalfa. The NodP and NodQ enzymes are involved in activating sulfur for transfer to the nod factor.

  17. Model for difference in activity of nodRm-1 and nodRm-2

  18. 2. R. leguminosarum - the nodFE story - example of host specific modification based on changes in the fatty acid side chain. nodFE - involved in fatty acid biosynthesis, thought to mediate the transfer of a fatty acid group from the fatty acid biosynthetic pathway to nod factors. If one transfers nodE from one Rhizobium to another, one can change host range. nodE appears to be the only determinant of specificity between the two biovars R. leguminosarum bv. trifolii (C18:1 fatty acid) and R. leguminosarum bv. viciae (C18:4 fatty acid). Conclude: modification of nod factors in another way besides sulfate addition can influence host range. The type of fatty acid side chain on the nod factor influences specificity.

  19. How do plants detect Nod factor? Nod factor binding sites, NFBS1 and NFBS2, have been identified in Medicago plants. A similar binding activity has been found in tomato plants. These proteins also bind both sulfated and non-sulfated nod factors (inactive in Medicago species). It is possible that they are part of a binding complex. A lectin-nucleotide phosphohydrolase (LNP) has also been found to bind Nod factors. This protein is present at the surface of root hairs and it accumulates at root hair tips upon inoculation with Rhizobia. Unclear how it might function as a receptor. Ion fluxes are observed in root hair cells within a few minutes of Nod factor treatment. These include calcium influx and chloride and potassium efflux. These changes in membrane polarization provide a useful early indicator of the plant cellular response to Nod factor treatment. In particular, calcium “spiking” (oscillations in concentration) can be measured by fluorescence techniques. Genetic approaches using loss of function nodulation mutants identified candidate receptor genes NFR1 and NFR5.

  20. Oldroyd and Downie. 2008. Ann. Rev. Plant Biol. 59: 519-546 Legend on next slide

  21. Oldroyd and Downie. 2008. Ann. Rev. Plant Biol. 59: 519-546

  22. Radutoiu et al. 2007. EMBO J. 26: 3923- Lotus japonicusMedicago truncatula Medicago truncatula + LjNfr1 + LjNfr5 Mesorhizobium loti Nodules No Nodules Nodules Sinorhizobium meliloti No Nodules Nodules Nodules LjNfr1 + LjNfr5 = candidate Nod factor receptors from Lotus japonicus

  23. Summary Part of the molecular explanation for cross-inoculation groups: 1. NodD protein recognition of different compounds in root exudates (flavonoids) contributes to host specificity. 2. The nod genes encode functions that are involved in the regulation gene expression (e.g., NodD), producing the core Nod factor (NodABC) and decorating nod factors (hsn,e.g., NodH, NoDE). 3. Differences in Nod factor decoration (side chain modifications) contribute to specificity. 4. Specific plant receptors have been identified that recognize Nod factors.

  24. Specificity is a due to a combination of factors: 1. Types of flavonoids made by host plants 2. Nod D sensor proteins in the bacterial symbionts 3. Types of Nod factors produced by the bacteria 4. Amounts of Nod factors produced by the bacteria 5. Exopolysaccharide - for indeterminant nodules (role unclear) 6. Plant hydrolases (e.g. chitinases that may degrade Nod factors) 7. Plant lectins - carbohydrate binding proteins (possible role in attachment of bacteria to plant cells) 8. Plant receptors for Nod factors

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