Molecular genetic analysis of subterranean clover-microbe interactions

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Vol. 27, No. 415, pp. 485-490, 1995 Copyright 0 1995Elsevier8ciena Ltd Printed in Great Britain. Au ritthts rwrved 0038-0717/95-89.50 + 0.00

MOLECULAR GENETIC ANALYSIS OF SUBTERRANEAN CLOVER-MICROBE INTERACTIONS B. G. ROLFE,* M. A. DJORDJEVIC, J. J. WEINMAN, J. McIVER, E. GARTNER, H. CHEN, E. H. CREASER, K. BRITT, C. G. R. LAWSON, M. H. DE BOER, 1. A. MCKAY, M. V. SHOOBRIDGE, J. DE MAJNIK, C. PITTOCK, K. BRODERICK and T. DELBRIDGE Centre for Genetic Research, Plant-Microbe Interaction Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, ACT 2601, Australia Stnmuy-Trifolium subterrunewn (subterranean clover) is of considerable economic importance to the Australian rural industries as a pasture legume. In addition to its commercial value, it has a number of specific attributes--such as small seed size, diploidy, self-fertilization, the ability to be transformed and small genomc-which make it a prime target for the modem techniques of molecular genetics. We report genetic

and physiological factors that control the production and excretion of the lipooligosaceharide molecules formed by Iwlizobiumlegwnhosarum bv. rrifolii in the formation of the symbiosis with subterranean clover. These molecules, synthesized by the products of the nodulation (nod) genes, are a major determinant of nodule occupancy and the strain selection imposed by the host plant. In addition, we have investigated which plant genes and proteins are activated in subterranean clover when they are either physically wounded, infected with Rhizobium, or attacked by red-leggedearth mites. To analyse these interactions more precisely, we have cloned plant genes involved in the phenylpropanoid pathway and used their promoters to construct transgenic subterranean clover plants. Our studies provide an insight into the nature and consequences of the chemical exchange between plants and invading microbes.

INTRODUCTION

As we approach the 21st century there are major agricultural challenges to be faced. New plants will need to be developed to address specific agricultural needs of modern sustainable farming and the consequences of past practices. The challenges of the future include, the production of new plant varieties to check the proliferation of pests and pathogens in monoculture cropping, adverse environmental conditions in the soil (e.g. acidity, salinity, Al, Mn) and atmosphere (e.g. high CO1, high W) and the need to Iind alternatives to chemical pesticides, fertilizers and high-energy fuels. The contribution of biological Nz fixation is still essential, especially since added N fertilizer can have adverse environmental impacts. Molecular and genetic analysis is increasing our understanding of symbiotic N2 fixation. Rhizobium, Bradyrhizobium and Azorhizobium induce root nodules in which atmospheric Nz is fixed. The genetics of these rhizobia is now well characterized and they can be used as biological “probes” to investigate fundamental cellular processes in plants (Rolfe and Gresshoff, 1988; Long, 1989; Hirsch, 1992; Fisher and Long, 1992). Plant flavonoids (low molecular weight phenolics), which are often made in the plant’s response to pathogen infection, are involved in promoting Rhizobium infection of roots (Redmond et al., 1986).

*Author for correspondence.

Flavonoids induce rhizobia to produce a family of lipooligosaccharides (LOSS), which are necessary for infection and determine which host plant can be infected (Stacey et al., 1995). These signals, produced under the control of the nodulation (nod) genes, cause plant root hairs to grow abnormally (curled) and stimulate cell division in the root cortex. How LOSS cause these effects is not known (Hirsch, 1992). However, processes commonly induced by plant pathogens during infection, such as host cell death and induction of the hypersensitive reaction, are not characteristic of Rhizobium infection of legume roots. Some major unresolved issues which aifect the ability of the rural industries to take advantage of biological Nz fixation are: (i) the ability to deliver Rhizobium inoculants which compete with native rhizobia and form the root nodules; (ii) the effects of environmental factors on strain persistence and ability to nodulate; (iii) the effect of host genetics on nodulation and strain persistence; (iv) the utilization of cultivar attributes to increase resistance to pasture pests; (v) the use of genetic engineering to make new transgenic varieties of pasture plants by the introduction of new traits to combat disease or overcome plant nutritional limitations to growth; and (vi) the exploitation of microbes to produce biologically-active molecules of possible agricultural use, such as a new generation of biodegradable, ecologically sound, chemical “herbicides”. This review outlines some of the recent advances made in our laboratory which may be used to develop new

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agricultural production techniques which can be integrated into classical strategies already in use. FINDINGS AND DISCUSSlON

Rhizobium production chemicals

of novel biologically-active

Rhizobium produces potent LOS signal molecules in

the presence of the host plant roots. These LOS molecules have specific structures, depending on the bacterial strain that produces them, and are biochemically synthesized by the products of the nodulation (nod) genes. At least 10 LOSS are synthesized by Rhizobium leguminosarum bv. trtfolii strains (McKay and Djordjevic, 1993). The nodA, nodB and nodC gene products are required to synthesize the basic LOS structures and the proteins from the nodF, nodE and nodL genes add various substitutions to the LOS molecule. The nodl and nod] genes are essential for the excretion of LOS molecules from the cells (McKay and Djordjevic, 1993) and explains why nodland nodlmutants have pronounced nodulation-defective phenotypes (Huang et al., 1988). Addition of these LOS signals to legume roots induces specific plant-gene expression (Spaink et al., 1991). Plant genes and their promoters that are LOS-responsive are being isolated and characterized. The promoters of these LOS-responsive genes can be used to generate specific transgenic plants with nodule-specific expression of the introduced gene. For example, the expression of rhizopine genes could be tied to the expression of these LOS-responsive promoters to enhance the survival of particular (superior) rhizobia present in applied inocula [see the rhizopine concept (Murphy et al., 1995)]. Further, some plants previously exposed (Ward et al., 1991) to a non-host parasitic or pathogenic microbe are more resistant to a real pathogen when added later. LOSS applied to plants exogenously may trigger or prime the plant’s defence system. Nod signal production is afSected by physiological factors affectingfield nodulation

The failure of compatible rhizobia and legumes to form nodules as a result of certain environmental conditions is an ongoing commercial problem. Production and particularly excretion of LOSS from rhizobia was strongly influenced by the growth environment. Both the number and amount of the metabolites produced were affected by the pH, the concentration of phosphate, the concentration and form of the N source and the growth temperature (McKay and Djordjevic, 1993). The recognition that LOS molecule accumulation is a complex system of production and excretion, with each component responding differently to changes in environmental conditions, has many consequences, both at the molecular level and in the field. Other environmental stresses, such as high concentrations of Al or salinity,

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may well affect nodulation by reducing the production and excretion of such molecules. The production and release of LOS molecules under stress conditions is likely to be a major determinant of nodule occupancy and should be considered when screening strains suitable for adverse environments. The advantages of experimental system

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From study of the interaction between R. 1. bv. clover, we now have an extensive knowledge of the molecular basis of plant defence systems in subterranean clover. This has been aided by a number of features of subterranean clover that make it useful as a model system. These are summarized in Table 1. These include the ease with which plants can be grown in the laboratory, self-fertilization, the availability of advanced tissue culture techniques, diploidy, transformation ability and the small genome size. We have developed several genomic libraries of cv. Karridale and have focused our studies on this and also cv. Woogenellup. Transformation and regeneration techniques were developed first in the laboratory of Dr T. J. V. Higgins (Heath et al., 1993; Khan et al., 1993) using Agrobacterium vectors. Transformed plants can be generated in 14 weeks--considerably faster than for other legumes-and this approaches the flexibility offered by tobacco systems. Further there is also extensive molecular genetic information on the fast growing, microsymbiont [Weinman et al. (1991) and Table 11. A wide variety of certified cultivars are available through the Australian National Sub Clover Improvement Program coordinated by the Western Australian Department of Agriculture. trtfolii and subterranean

Cloning of genes induced by microbial interaction: isolation of clover gene promoters that can be used to direct transgenes to specific tissues

Up to 8% of fixed C can be channelled through the phenylpropanoid pathway (PPp/w) in plants. The Table I. Valuable characteristics of subterranean clover Attribute Seed size Ploidy Genome size Breeding characteristics Cultivar availability Life cycle Agrobacrerium transformation Tissue culture propagation Regeneration Pathogens available: Bacterial Fungal Viral Rapidity of nodulation Minimum genetic requirement for nod&ion Genomic library available Spectrum of symbiotic flavonoid signals Bioactivity of fiizobium EPS

Subterranean clover Small Diploid Small Self-fertilizing Many certified cultivars Annual Yes Yes Yes Many Many Numerous characterized isolates 9-12 days nodDAECI.IL

Yes Complex Demonstrated

Genetics of subterranean clover-Rhizobirun interactions

Physical Wounding

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Fig. 1. Specific induction of chalcone synthase genes in subterranean clover roots. An increase in total CHS message was detected following Northern blot analysis after inoculation with R. 1.bv. triloliiANU843 and physical wounding of root tissue (5 mm longitudinal cut into root). This was measured using the highly conserved second exon fragment of CHS from subterranean clover and quantified using a phosphorImager and are recorded on the right-hand side of the panel (dotted histograms). The peak of CHS induction was at 2 h after wounding and 6 h after Rhizobiuminoculation. The mRNA from these treatments was then copied into cDNA using reverse transcriptase with a T(17) modified primer incorporating the T3 primer site and a restriction enzyme site. The cDNA was then amplified for 30 cycles using a conserved CHS upstream primer and the T3 incorporated into the reverse transcription primer. The products were cloned into plasmid vector pBluescriptSK( +), sequenced using dye-labelled primers and compared to genomic clones. The results showed that CHSS and CHS6 were highly represented in the pool of sequenced clones 2 h after wounding (top box). CHSS and CHSl were present in the pool of sequencing clones (lower box) 6 h after Rhizobiuminoculation. All other known CHS genes (CHS2 to CHS4 and CHS7 to CHSlO shown by arrows) were not detected. Collectively these results indicate that CHSS and CHS6 and CHSS and CHSl were induced upon wounding and Rhizobiuminoculation treatments respectively.

genes coding for certain enzymes of the PPp/w are modulated in a complex and coordinated manner during plant development, plant defence and in response to environmental stimuli. Using a bioassay system we have shown that an accumulation of PPp/w intermediates occurs following Rhizobium and pathogeninfection(Rolfeetal., 1988; Weinman etal., 1991). The end points and intermediates of the PPp/w have roles in microbial defence, protection from UV radiation, stimulation of Rhizobium infection, internal gene regulation and phyto-oestrogenic effects on ewes (Lloyd-Davies, 1985). Gene sequences from our genomic library have been used to monitor responses induced in legumes by microbes. We have investigated the metabolic channelling and the regulation of the synthesis of flavonoids-isoflavonoids in Trifolium when subjected to pathogen and Rhizobium attack and have isolated various PPp/w genes from our genomic library. These include the genes for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI). The enzyme PAL was found to be encoded by a small gene family of at least 4 copies in T. subterraneum. The enzymes coded for by the CHS genes catalyse the first (committed) reaction in the branch pathway of plant phenylpropanoid metabolism, leading to the biosynthesis of the flavonoids and isoflavonoids. We have identified 10

copies of the CHS genes and at least 3 copies of the CHZ genes. Molecular studies of these cloned genes have yielded sequence data from 10 of the CHS genes, 1 of the PAL genes and internal sequence data from the CHZgenes (Arioli et al., 1994; Howles et al., 1994).

Several PPp/w promoters which are expressed in response to pathogen attack, Rhizobium infection or wounding have been extensively characterized (C. G. R. Lawson et al., unpubl.). These promoters are expressed highly and transiently and are being used in transgenic subterranean clover programs (unpubl.). These molecular studies of the PPp/w genes have enabled us to take a reverse transcriptase-polymerase chain reaction (RT-PCR) approach to isolate specific Rhizobium-induced PPp/w transcripts (C. G. R. Lawson, lot. cit.). These methods have shown that upon physical wounding, 2 of the 10 CHS genes (CHSS and CHS6) are rapidly induced within 2 h (Fig. 1). Incontrast,only2ofthe lOCHSgenes(CHS5 and CHSl) are extensively induced within 6 h when wild-type R. 1. bv. trifolii strain ANU843 infects clover roots. Other information suggests that Rhizobium infection results in a channelling of PPp/w intermediates to increase the pool size of flavonoids at the expense of later intermediates in the biochemical pathway (J. J. Weinman er al., unpubl.). Using RT-PCR technology we have found that 1 of 2

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transcripts for the dihydroxyflavonol-4-reductase gene is down-regulated after the addition of R. 1. bv. trtjofii to the clover roots. This enzyme follows the CHS step and is a committed step in anthocyannin synthesis (Martin and Gerats, 1993). Defence proteins of clover

Given that subterranean clover is an important agricultural pasture plant in Australia, it is surprising that little is known about pathogen and pest resistance. Infestations of red-legged earth mite (RLEM) and infection caused by pathogens (Phytophthora clandestina, Kabatiella caulivora) are major problems. An investigation of the proteins produced in response to RLEM feeding and pathogen infection has established that clover produces several of the recognized pathogenesis-related (PR) proteins. These include glucanases, chitinases and peroxidases, which are thought to provide protection against pathogens (Bowles, 1991). Furthermore, lignin deposition, another branch of the PPp/w, may form a defensive barrier in response to pathogen attack. Lignin, which is normally made as the major strengthening component of plant cell walls, is an insoluble polymer, resistant to most enzymic attacks. Lignin synthesis starts as a branch of the PPp/w at the cinnamic acid step and ends when the phenoxy monomers, made by cinnamyl alcohol dehydrogenase (CAD) and oxidized by H202 and peroxidase, spontaneously polymerize to lignin. In other plant species CAD can be used as a marker enzyme to study formation of lignin (Walter, 1992). We have shown that subterranean clover contains multiple species of CAD, some of which are organ specific, and have different specificities for the cofactors NAD and NADP. Previous surveys in other plants have not shown this multiplicity and we are very interested to find out what their role is in the biology of clovers. In subterranean clover the major enzyme of this class, called CADl, is present in roots, stems and leaves and has a molecular weight of approximately 80 kDa and uses NADP as a cofactor. The CAD1 enzyme increases in activity upon wounding suggesting that it could be an inducible defence protein. Construction of transgenie subterranean clovers

Although many plants can be successfully transformed with foreign DNA there are very few systems where transformation can be readily and easily achieved at high levels and with all cultivars and varieties. Until this is achieved, transformation strategies will rely on introducing the gene traits of interest via traditional breeding methods. Transformation systems in legumes are particularly underdeveloped with only Lotus offering a reasonably amenable system. Recently, subterranean

clover has been transformed using Agrobacteriurn mediated transformation via direct organogenesis from wounded hypocotyl tissue (Heath et al.. 1993). Some cultivars are readily transformed. These transformation procedures are being used to assay the activity of several native gene promoters from subterranean clovers using the /I-glucuronidase (GUS) enzyme as the reporter system [Jefferson (1989) and Fig. 21. We aim to identify promoters (such as the PPp/w genes CHS, CHI and CAD) which either express at high levels or in a tissue-specific manner. These promoters will be used to establish expression patterns in transgenic subterranean clovers, thereby enabling the timing and cellular location of molecular events that take place during pest and microbial infection and plant development. Genes for resistance to pests or pathogens could also be introduced from other organisms, and expressed in an appropriate manner using the promoters discussed above. Possible candidates for foreign genes that may increase resistance are the genes coding for the various insect-specific toxins from Bacillus thuringiensis or genes for PR proteins from other plants. Single gene traits in plants can be used to bias the nodtdation preference of the plant host towards the preferred inoculum strain

Single gene traits in plants can control nodulation by the normal symbiont (e.g. Rj4) or infection by a particular pathogen. The inadvertent introduction of the Rj4 gene into soybean lines for the southern U.S.A. has prevented nodulation of these plants by Bradyrhizobium elkanii, which show suboptimal N2 fixation on a variety of soybeans. Improved N2 fixation is then provided by nodulation with B. japonicum strains, which are normally out-competed by B. efkanii (G. Stacey, pers. commun.). A similar phenomenon also occurs with subterranean clovers. The specific interaction of T. subterraneum cv. Woogenellup and R. 1. bv. trtfolii strain TAl gives a unique, temperature-sensitive, nodulation process that is dependent upon a gene-for-gene type mechanism commonly found in plant pathogen interactions (Djordjevic et al., 1987, 1992). Furthermore, this unique gene-for-gene system provides a powerful experimental tool to investigate the concept of incompatibility in plant-microbe interactions and plant defence responses to Rhizobium infection. cv. Woogenellup possesses a single recessive gene (rwtl) that “interacts” with at least 2 dominant bacterial genes, nodM and the cultivarspecific nodulation gene, csn 1, present in TAl resulting in nodulation failure at 22°C or less [Fig. 3 and Lewis-Henderson and Djordjevic (1991)]. Mutation of either nodM or the csnl gene of strain TAl enables nodulation of cv. Woogenellup without the loss of nodulation ability on other hosts. Strain TAl initiates infection threads containing

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Fig. 2. Transgenic subterranean clover containing chalcone synthase promoter. The promoter of the subterranean clover CHSl gene was fused to the GUS (/?-glucuronidase) gene and introduced into wounded hypocotyl tissue using Agrobacrerium tumefaciens. Shoots of cv. Larisa were developed from the transformed tissue under specific hormonal regimes. GUS activity was demonstrated in the leaf of regenerating shoots (tissues stained dark blue, right-hand side). Control plants containing no GUS expression appear on the left-hand side (show no blue stain).

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Fig. 3. Nodulation rate at 22°C on cv. Woogenellup plants inoculated with R. 1. bv. trifolii strain ANU794, a streptomycin-resistant derivative of strain TAl (0). the nodM mutant of strain TAl (m) and strain ANU843 (i), shown as the average number of nodules per plant (9 plants per treatment). An analysis of variance was calculated for 13- and 21.day-old plants and showed a significant difference (LSD = 1.27 and 2.08, respectively; P = 0.005) for the number of nodules induced by strain ANU794 compared to the nodh4 mutant and strain ANU843.

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bacteria and cortical cell division, but steps subsequent to that are not sustained and nodulation development terminates. Semi-thin sectioning of spot-inoculated Woogenellup roots showed that there is no evidence of a hypersensitive response (HR) leading to host cell death in the area of strain TAI infections. A HR reaction is commonly found in plant pathogen interactions. Moreover, we have found that another defence response, enhanced peroxidase activity, increased significantly in strain TAl-infected roots. Recent evidence suggests a possible mechanism for this resistance-to-nodulation phenotype. The LOS molecules produced by strain TAI are structurally distinguishable from those produced by R. 1. bv. trifolii strain ANU843 which can nodulate cv. Woogenellup and this may be responsible for the inability of strain TAl to nodulate cv. Woogenellup. We are investigating the effects of no&4 or csnl mutations on the pattern of LOS molecules formed by these mutants. Thisdiscoveryoflociin the R. I. bv. trifoliiwhichconfer cultivar-specific nodulation is consistent with a gene-for-gene interaction between the plant and the symbiont and illustrates the fine line between “incompatible” infections and symbiosis. This highlights the possibility of breeding such plant genes into clover lines to control nodulation preference in the field by excluding inferior native Rhizobium strains which give suboptimal or no Nz fixation. Such a practice could lead to improved cultivar-Rhizobium combinations. Acknowledgements-The authors (B.G.R., E.H.C., J.J.W.. M.A.D..C.G.R.L.andK.B.)arerecipientsofanMRCcore research grant; B.G.R.. M.A.D. and J.J.W. have been recipients of research grants from the AWC R&D Fund. REFERENCES

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