• Open Access

Genomics of plant-associated microbes

Authors

  • Peter Van Baarlen,

    1. Host-Microbe Interactomics, Wageningen University, 6709 PG Wageningen, The Netherlands.
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  • Roland J. Siezen

    Corresponding author
    1. Kluyver Centre for Genomics of Industrial Fermentation, TI Food and Nutrition, 6700AN Wageningen, The Netherlands.
    2. NIZO Food Research, 6710BA Ede, The Netherlands.
    3. CMBI, Radboud University Nijmegen, 6500HB Nijmegen, The Netherlands.
      *E-mail r.siezen@cmbi.ru.nl; Tel. (+31) 2436 19559; Fax (+31) 2436 19395.
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*E-mail r.siezen@cmbi.ru.nl; Tel. (+31) 2436 19559; Fax (+31) 2436 19395.

Plant-associated microbes and plant–microbe interactions can be largely divided in two types: detrimental (pathogenic) and beneficial (symbiotic) interactions. Neutral interactions also occur; this is the case for microbes that live in the rhizosphere (on roots) or phyllosphere (on leaves) without triggering any apparent plant response. Both pathogenic and symbiotic interactions are of relevance to industry because these may impact plant production in negative and positive ways respectively. Several plant-associated microbes produce cell wall-degrading enzymes which may be of industrial use in fermentation of plant products. Here we give a brief update of the current status of genome sequencing and genomics of microbes associated with plants.

Plant-pathogenic microbes

Most sequenced plant-associated microbes that produce cell wall-degrading enzymes exhibit pathogenic interactions with plants. Clear examples of these are the bacteria that belong to the genera Clavibacter, Xanthomonas and Xylella (Table 1). Apart from cell wall-degrading enzymes, bacteria such as Xanthomonas campestris may also produce more complex sugars. One of these is xanthan, the main ingredient of xanthan gum, a polysaccharide used as a food additive that is produced by X. campestris. With the availability and analysis of several Xanthomonas genomes, the biochemical pathways leading to xanthan have been shown to be based on a process involving fermentation of nucleotide sugars converted from intermediates of the pentose phosphate pathway (Vorhölter et al., 2008). The genome sequences of a related Xanthomonas species that causes the economically relevant rice blast disease, X. oryzae, also show presence of secreted plant cell wall-degrading enzymes (Lee et al., 2005). A recently sequenced highly pathogenic isolate belonging to this species displays diverse insertion sequences, genome rearrangements and presence of clustered regularly interspersed short palindromic repeats indicating many bacteriophage infections, presumably correlating with the strain-specific adaptations associated with high pathogenicity (Salzberg et al., 2008).

Table 1.  Selection of sequenced genomes of plant-associated microbes, not including lactic acid bacteria.
OrganismStrainClassImpactDiseaseHabitatENTREZ Genome Project IDDate
  1. Adapted from the GOLD Database (http://www.genomesonline.org; March 2009).

  2. A, agricultural; Ac, acidophile; AP, animal pathogen; AnP, antibiotic production; BC, biocontrol; BP, bioremediation of pollutants; BR, bioremediation; BT, biotechnological; D, denitrifying; E, environmental; FGI, Fungal Genome Initiative; FI, food industry; HP, human pathogen; M, medical; MP, mycotoxin producer; NF, nitrogen fixation; NFe, non-fermentative; NP, non-pathogen; P, pathogen; PD, pollutant degrader; PP, plant pathogen; RP, riboflavin producer; S, symbiotic; Sa, saprophyte; sPP, suppression of plant pathogens; vitC, vitamin C production.

Acidovorax avenae citrulliAAC00-1PA, PPBacterial fruit blotchHost157082007
Agrobacterium radiobacterK84PA, HP, M, PPNoneHost, root nodule134022009
Agrobacterium tumefaciensC58-UWash/ C58-CereonP, NFA, PPPlant tumoursHost, root nodule282/2832001
Agrobacterium vitisS4PA, PPCrown gallHost, root nodule133722009
Ashbya gossypiiATCC 10895P, RPA, BT, PP  138342004
Azoarcus sp.BH72NF, NPBT, ANoneObligate endophyte, soil, host132172006
Bacillus amyloliquefaciensFZB42 BT, AnP, sPP, ANoneRhizosphere-colonizing, Soil134032007
Burkholderia ambifariaMC40-6P, NF, PDHP, M, A, BC, BTCepacia syndromeHost, rhizosphere174112008
Burkholderia cepaciaAMMDNF, PDBC, ANoneHost, rhizosphere134902006
Burkholderia cepacia383 (R18194)P, NFM, E, BR, AP, HP, BC, A, BPNecrotizing pneumonia, chronic infectionHost, rhizosphere106952005
Candidatus Phytoplasma aster yellowsAY-WBPA, PPAster yellows, Witches' BroomHost134782006
Candidatus Phytoplasma australiense PA, PPAustralian grapevine yellowsHost294692008
Candidatus Phytoplasma maliATPPP, AApple proliferation diseaseHost253352008
Candidatus Phytoplasma onion yellowsOY-MPPP, AOnions yellowHost96152003
Clavibacter michiganensis michiganensisNCPPB 382PA, PPRing rot, tuber rot, wilting diseaseHost196432007
Clavibacter michiganensis sepedonicusATCC 33113PA, PPRing rot, tuber rotHost1842008
Cupriavidus taiwanensisLMG19424NF, SANoneHost, root nodule157332008
Enterobacter sp.638 ANoneEndophyte, host174612007
Frankia sp.CcI3NF, NPA, BTNonePlant symbiont, soil139632006
Frankia sp.Mbj2, EAN1pecNF, NPBNonePlant symbiont, soil139152007
Fusarium graminearumPH-1MP, PA, FGI, PPHead blight 138392007
Gluconobacter oxydans621HAcBT, VitCNonePlants, fruits, wine, beer133252005
Leifsonia xyli xyliCTCB07PPP, ARatoon stuntingHost2122004
Magnaporthe grisea70-15PFGI, PP, ARice blast 138402005
Methylobacterium populiBJ001 BT, ANoneHost, endophyte195592008
Pectobacterium atrosepticumSCRI1043PA, PPSoft rotHost3502004
Phytophthora ramorumPr102, UCD Pr4PA, PPSudden oak deathUndefined125712006
Phytophthora sojaeP6497PA, PPRoot rotUndefined179892006
Pseudomonas aeruginosaPAO1HPM, HP, PP, ANosocomial infectionSoil, fresh water, host, wastewater3312000
Pseudomonas fluorescensPf0-1NPE, BRNoneSoil122005
Pseudomonas fluorescensPf-5NPBR, BT, AnP, ENoneSoil, fresh water3272005
Pseudomonas putidaKT2440NP, SaE, BRNoneSoil2672002
Pseudomonas putidaW619NPA, BR,ENoneEndophyte, host, soil170532008
Pseudomonas stutzeriA1501D, NPE, A, BRNoneHost, rice roots, soil168172007
Pseudomonas syringae phaseolicola1448A/Race 6PA, PPPlant rot, halo blightPlants124162005
Pseudomonas syringae syringaeB728aPA, PPPlant rotPlants3232005
Pseudomonas syringae tomatoDC3000PA, PPPlant rot, speck diseaseEndophyte, host, soil, fresh water3592003
Ralstonia solanacearumGMI1000PA, PPPlant rot, wilting diseaseSoil132002
Rhizobium etliCFN42NFA , BTNoneHost, root nodule139322006
Rhizobium etliCIAT 652NFA, BTNoneHost, root nodule280212008
Rhizobium leguminosarum3841NF, NPBT, ANoneHost, root nodule3442006
Rhizobium leguminosarum bv trifoliiWSM2304NP, NFA, BTNoneRoot nodule, host201792008
Ustilago maydis521PA, FGI, PPCorn smut 14462007
Xanthomonas axonopodis pv. citriXV101, 306PA, PPCitrus cankerHost2972002
Xanthomonas campestris campestris8004PA, PPBlack rot, citrus cankerHost152005
Xanthomonas campestris campestrisATCC 33913/ B100PA, PPBlack rotHost296/
29801
2002/
2008
Xanthomonas campestris vesicatoria85-10PA, PPBacterial spotHost136492005
Xanthomonas oryzaeMAFF 311018PA, PPLeaf blight, rice blightHost162972006
Xanthomonas oryzae pv. oryzaeKACC10331PA, PPBlight diseaseHost129312005
Xanthomonas oryzae pv. oryzaePXO99APA, PPRice blightHost281272008
Xylella fastidiosaM12/ M23PA, PPCitrus variegated chlorosisHost17823/
18457
2008
Xylella fastidiosa CVC8.1.b clone 9.a.5.cPA, PPCitrus variegated chlorosis, Pierces diseaseHost2712000
Xylella fastidiosa-grapeTemecula1PA, PPBlack rot, citrus cankerHost2852003

Some plant pathogenic bacteria are relevant to industry and science for multiple reasons. Agrobacterium tumefaciens (Rhizobiaceae) infects woody rosaceous plants including Rosa, but also stone fruit and nut trees and causes cancerous deformations (crown galls) and growth defects. Agrobacterium tumefaciens achieves this by transferring a small part of its bacterial genome to the plant and co-opting the plant to produce factors that the bacterium uses for its own subsistence and propagation. The genome sequence of A. tumefaciens, published in 2001 (Goodner et al., 2001; Wood et al., 2001), resulted in a framework to understand how bacteria may evolve to integrate bacterial DNA into the nuclei of eukaryote hosts (Fig. 1). Interestingly, A. tumefaciens is able to transfer its DNA into eukaryote hosts through a type IV secretion system in such a way that the host is not alerted to the activity of the bacteria, nor to the fact that it is essentially becoming transformed (McCullen and Binns, 2006). Expression microarray analysis during infection of plants by A. tumefaciens has started to shed light on how the bacteria achieve their manipulation of plant hosts (Ditt et al., 2006).

Figure 1.

Schematic example of plant–microbe interactions. Agrobacterium tumefaciens responds to plant-derived signals and transfers DNA and proteins into the host plant cell. Reproduced from McCullen and Binns (2006).

Use of A. tumefaciens to transform plants or as a heterologous transient in planta expression system is not only widespread in plant sciences but is also used to transform industrially important fungi such as Aspergillus niger (that produces complex sugar-degrading enzymes and enzymes involved in diverse fermentation processes) and the commonly sold edible mushroom Agaricus bisporus (de Groot et al., 1998). Agrobacterium tumefaciens is therefore commonly used in biotechnological companies and academic research groups for plant and fungal transformation. Plasmids and an exceptional genome organization and maintenance have enabled A. tumefaciens to evolve its sophisticated lifestyle, but the evolutionary prerequisites leading to this have been elusive. This year, genome sequences of the two related species Agrobacterium radiobacter and Agrobacterium vitis, which are also of agricultural and industrial importance, have become available (Slater et al., 2009). Agrobacterium radiobacter is commercially being sold as a biological agent for the control of soil-borne plant-pathogenic bacteria, whereas A. vitis is an important pathogen of grapes. The published genomes of these two bacteria may help to understand how A. tumefaciens may have evolved as a pathogen with complex intragenomic rearrangements and the ability to transfer some of its coding sequences to organisms from other kingdoms of life.

Neutral associations

One group of plant-associated non-pathogenic bacteria belongs to the lactic acid bacteria (LAB; Schroeter and Klaenhammer, 2009). Sequenced genomes of plant-associated LAB include Lactobacillus plantarum (Kleerebezem et al., 2003), Lactococcus lactis (Siezen et al., 2008), Pediococcus pentosaceus, Leuconostoc mesenteroides and Oenococcus oeni (Makarova et al., 2006). The plant-associated Lactococcus lactis strain KF147 was shown to encode numerous enzymes that can degrade complex plant polymers (Siezen et al., 2008). Many LAB are used in starter cultures for fruit and vegetable fermentations (Cogan et al., 2007). A recent study also evaluated the efficacy of LAB isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi X. campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa and Botrytis cinerea (Trias et al., 2008).

Microbial growth-promoting symbionts

A different group of bacteria related to Agrobacterium within the Rhizobiaceae family are of relevance to industry, not as pathogens but as beneficial symbionts. Bacteria from the genera Rhizobium or Sinorhizobium contribute to plant production by fixating nitrogen, one of the major elements that are essential for plant growth and agro-production. It has been suggested several times that it would be very attractive for biotech companies to identify the major genes controlling the trait of nitrogen fixation in selected rhizobia and transfer these to other plant-associated microbes (Zahran, 2001). Since the publication of the first genome of Rhizobium leguminosarum (Young et al., 2006), 12 more rhizobial genomes are either completed or underway together with four genomes of the closely related Bradyrhizobium and two genomes of Sinorhizobium. The fact that these symbiotic rhizobia are related to pathogenic Agrobacterium sp. makes them ideal objects for comparative expression profiling. Indeed, transcription profiling of soybean nodulation by Bradyrhizobium japonicum showed that this bacterium is able to reduce plant defence responses during nodule development (Brechenmacher et al., 2008).

Use of the very well-characterized model plant Arabidopsis has shown that it is possible to further identify innate defence-associated transcripts that are not directly relating to pathogenic infection. In root colonization experiments with the general plant growth-promoting bacterium Pseudomonas thivervalensis, the interactions of roots and bacteria led to an increase of defence-related transcripts in the shoots of plants with colonized roots. Interestingly, plants colonized by P. thivervalensis were more resistant to subsequent infections by virulent Pseudomonas syringae pv. tomato (Cartieaux et al., 2003). Comparison of the genome sequences of pseudomonads, rhizobia and agrobacteria together with host expression profiling will doubtlessly lead to discovery of a wealth of novel bacterial effectors that play roles in symbiotic or neutral plant colonization, pathogenic infection and modulation of host responses. Some of these effectors may also play important roles in infection of humans.

One different class of microbes is the fungi that form symbiotic interactions with trees and shrubs. The basidiomycete Laccaria bicolor is an ectomycorrhizal and saprophytic fungus that is commercially used in forest nurseries to promote growth of tree seedlings. Its genome sequence was published recently (Martin et al., 2008) and transcriptomes of different tissues and developmental stages have been obtained (NCBI GEO Datasets record GSE9784). Moreover, inoculation studies of scotch pine trees with Laccaria bicolor and pathogenic fungi resulted in the identification of genes specifically differentially expressed in the pathogenic, saprotrophic and symbiotic interactions (NCBI GEO Datasets record GSE5410). Such data help to further an understanding of the critical events that are necessary for successful interactions of trees with beneficial symbiotic microbes. A better understanding may contribute to tree management, e.g. by faster screening for optimally symbiotic partners, using biomarkers derived from expression information of both the host and microbe during their interaction. Optimal colonization of tree root systems by Laccaria symbionts has beneficial impact on growth and sometimes also protection against stresses including drought-induced salt tolerance and pathogen infection. Interestingly, Laccaria encodes enzymes that can hydrolyse sugars and proteins of microbial, decaying organic matter and small arthropod origin, but not of plant cell-wall origin like pathogenic microbes can (Martin and Selosse, 2008).

Plant–microbe interactions relevant to human clinical studies

Plant models can contribute to the study of human health and disease (Jones et al., 2008). Plants, like animals, are in possession of an innate immune system that uses pattern recognition receptors in order to detect and eliminate potentially harmful microbes. Some features of the plant and animal innate immune systems show important similarities at the molecular level. Indeed, some pathogens infect plants as well as animals, including humans, by remarkably similar molecular pathways (van Baarlen et al., 2007a). These similarities make it possible to use plants as an alternative model host to investigate pathogenicity of human pathogens such as Staphylococcus aureus, an important human pathogen of which 30 genomes will soon be available (NCBI Genome Project lists 14 projects as completed, 8 in progress and 8 as draft assembly). Staphylococcus aureus genomes are characterized by large between-isolate genetic variation with clear pathological relevance (Melles et al., 2004) but the contribution of genetic variation to specific pathological traits are not well understood, partly because testing experimental animals is costly and ethically unfavourable. To accommodate this, Arabidopsis plant models can be used to study differential pathogenicity of S. aureus isolates (van Baarlen et al., 2007b). Upon infection of Arabidopsis by S. aureus isolates that differ in clinical pathology, the bacteria induce rotting symptoms that differ in severity and morphology (Fig. 2), correlating to a certain extent with disease severity in humans. This forms the basis for experiments where the genetic basis of plant innate resistance against S. aureus isolates is investigated and potentially correlated with genomic differences of S. aureus isolates. Such a plant model-driven approach may accelerate the identification of microbial drug targets. Functional genomic tools as expression profiling make it possible to compare plant and human transcriptomes during infection by microbes such as S. aureus. In silico tools for such comparative analyses are available for in animal and plant sciences (van Baarlen et al., 2008) and the results of such tools can be successfully integrated with other omics and molecular biology tools (van Esse et al., 2008).

Figure 2.

Two different symptoms (leaf yellowing, upper arrow and wet rot (lower two arrows) induced in Arabidopsis thaliana by the human pathogen Staphylococcus aureus. Source: B. Thomma and P. van Baarlen, Laboratory of Phytopathology, Wageningen University, the Netherlands.

A similar approach has been used to identify the basis of plant susceptibility to the human pathogen Pseudomonas aeruginosa. This bacterium is among the three most often occurring causes of opportunistic infections of human. Because of its clinical importance, a complete genome sequence of highly pathogenic P. aeruginosa PAO1 has been available since 2000 (Stover et al., 2000). Part of its importance as a pathogen is determined by its resistance to antibiotics. Its relatively large genome sequence of over 6 Mb shows several classes of genes (transcription regulators, protein secretion systems, multi-drug efflux pumps) that are likely to be directly correlating with pathogenicity and antibiotics resistance. Using resistant and susceptible tobacco plants in infection experiments with P. aeruginosa PAO1, plant resistance, as in animal hosts, was found to correlate with salicylic acid (the active ingredient of aspirin) accumulation and the availability of micronutrients. Intriguingly, bacteria harvested from the intracellular fluid of plants that were either resistant or susceptible to infection by P. aeruginosa showed a differential modulation of bacterial global gene expression (Weir et al., 2008). A comparison of genes that were differentially expressed under these two conditions showed that in the resistant plant, especially P. aeruginosa genes involved in mobility and attachment, protein secretion and export, secreted factors and small molecule transport were downregulated (Weir et al., 2008). These classes of bacterial genes are likely to be involved in resistance against antibiotics and other compounds that are harmful to the bacterial fitness. A similar approach, but now using poplar cuttings in an in vitro system, showed that P. aeruginosa virulence factors are differentially transcribed in bacteria in presence of the tree host and are necessary for full virulence on poplar (Attila et al., 2008). The poplar cuttings responded to bacterial infection by differential transcription of nearly 1800 genes, modulating signal transduction, primary and secondary metabolism and molecular transport. Plant compounds may interfere with pathogenicity, e.g. by inactivating bacterial effectors. Interestingly, similar compounds may also be produced by related bacteria. For instance, at least two completely sequenced strains of the species P. fluorescens produce metabolites that suppress rhizosphere plant pathogens (Nelson et al., 2002; Paulsen et al., 2005). Associative comparisons of expression patterns for other bacteria during antibiosis might yield a better understanding of the function of such bacterial genes that often encode hardly characterized effectors. Furthermore, such genes may turn out to encode essential pathogenicity factors. Several genomes of related pseudomonads have been published in the last 2 years (Table 1). We expect that comparative analyses of the recently published genomes of the plant-pathogenic P. syringae, the non-pathogenic but related Pseudomonas putida and the non-pathogenic denitrifying Pseudomonas stutzeri, together with bacterial expression profiling under relevant in vivo (e.g. correlating with pathogenicity) and in vitro conditions may contribute to the identification of bacterial factors involved in diverse processes including virulence and pathogenicity, symbiosis, or merely neutral interactions. Some of these factors may turn out to be antibiotics, help cleaning up polluted soils, break down complex contaminating polymers, and may be amenable to large-scale production via genetic engineering.

Acknowledgement

This work was carried out within the research programme of the Kluyver Centre for Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.

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