12 Jul 2002 14:5 AR AR165-PY40-07.tex AR165-PY40-07.SGM LaTeX2e(2002/01/18)P1: IKH10.1146/annurev.phyto.40.030402.090559

Annu. Rev. Phytopathol. 2002. 40:169–89doi: 10.1146/annurev.phyto.40.030402.090559

Copyright c© 2002 by Annual Reviews. All rights reserved

COMPARATIVE GENOMIC ANALYSIS

OF PLANT-ASSOCIATED BACTERIA

M. A. Van Sluys1, C. B. Monteiro-Vitorello2,L. E. A. Camargo2, C. F. M. Menck3, A. C. R. da Silva4,J. A. Ferro5, M. C. Oliveira1, J. C. Setubal6, J. P. Kitajima6,7,and A. J. Simpson81Depto de Botanica, Instituto de Biociencias, Universidade de Sao Paulo;e-mail: mavsluys@usp.br and mcdolive@usp.br;2Escola Superior de AgriculturaLuiz de Queiroz, Universidade de Sao Paulo; e-mail: claudia@cena.usp.brand leacamar@carpa.ciagri.usp.br;3Depto. de Microbiologia, Instituto deCiencias Biomedicas, Universidade de Sao Paulo; e-mail: cfmmenck@usp.br;4Depto de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo;e-mail: acrasera@iq.usp.br;5Depto de Tecnologia, Fac. de Ciencias Agrarias eVeterinarias, Universidade Estadual Paulista; e-mail: jesus@fcav.unesp.br;6Institutode Computac¸ao, Universidade Estadual de Campinas; e-mail: setubal@ic.unicamp.br;7Centro de Biologia Molecular e Engenharia Genetica, Universidade Estadual deCampinas; e-mail: jpk@ic.unicamp.br;8Instituto Ludwig de Pesquisa sobre o CancerSao Paulo, Brazil; e-mail: asimpson@ludwig.org.br

Key Words bacteria, pathogen, symbiont, genome sequence, genome structure

■ Abstract This review deals with a comparative analysis of seven genome se-quences from plant-associated bacteria. These are the genomes ofAgrobacteriumtumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestrispv campestris, Xanthomonas axonopodispv citri , Xylella fastidiosa, andRalstoniasolanacearum. Genome structure and the metabolism pathways available highlight thecompromise between the genome size and lifestyle. Despite the recognized importanceof the type III secretion system in controlling host compatibility, its presence is not uni-versal in all necrogenic pathogens. Hemolysins, hemagglutinins, and some adhesins,previously reported only for mammalian pathogens, are present in most organisms dis-cussed. Different numbers and combinations of cell wall degrading enzymes and genesto overcome the oxidative burst generally induced by the plant host are characterizedin these genomes. A total of 19 genes not involved in housekeeping functions werefound common to all these bacteria.

0066-4286/02/0901-0169$14.00 169

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170 VAN SLUYS ET AL.

INTRODUCTION

Understanding the genetically defined mechanisms by which plant pathogens at-tack their hosts is central to the study of plant pathology. Such understandingshould also lead to the generation of important clues on how to control the eco-nomically important diseases caused by these organisms. A recent approach to thedefinition of the genetic basis of disease is through genome sequencing. As in-creasing numbers of whole genome sequences become available, their comparisonwill be a powerful approach to deducing the relevance and importance of specificgenes and gene families. This review deals with such comparative analysis of theplant-associated bacterial genomes described below.

The first agriculturally relevant bacterial genome sequence published was thatof the causal agent of citrus variegated cholorosis,Xylella fastidiosa(Xf-CVC), inJuly 2000 (71). Between December 2000 and January 2002 four more genomesof plant-associated bacteria were published: the nitrogen-fixing plant symbiontsMesorhizobium loti(Ml) (40, 41) andSinorhizobium meliloti(Sm) (25); the causalagent of crown gall,Agrobacterium tumefaciens(At) (27, 78); and the wilt-causingRalstonia solanacearum(Rs) (65). The complete, annotated genome sequences oftwo additional plant pathogens were also available for our analysis (17): the causalagent of citrus cankerXanthomonas axonopodispv.citri (Xac) and the causal agentof black rot,Xanthomonas campestrispv. campestris(Xcc). A second isolate ofX. fastidiosathat causes Pierce’s disease in grapevine is about to be finished.These two sequences had all been completed by the ONSA group (70). Table 1lists information about all completed and ongoing genome sequencing projects ofplant-associated bacteria, as of March 2002.

The bacteria analyzed here fall into three separate phylogenetic groups ac-cording to the subdivisions of proteobacteria.Sm, Ml, andAt belong to theαsubdivision (within the Rhizobiaceae family);Rsbelongs to theβ subdivision;andXanthomonasandXylella belong to an independent group at the basis of theγ subdivision. Figure 1 shows a 16S rRNA-based phylogeny of these organisms,together with a few other reference bacteria.

All of the plant-associated bacteria for which the genome has been sequencedare Gram-negative. In terms of disease symptoms, bacterial plant pathogens can beclassified as either tumorigenic or necrogenic (3). Tumorigenic pathogens induceabnormal tissue growth; the classical example is the crown gall tumor causedby At. Although not considered pathogens because their interaction with theirhosts results in overall benefit, one may also classifySmandMl as tumorigenicbecause they cause tumorlike nodules.Xanthomonas, Ralstonia, andXylella arenecrogenic pathogens because they cause plant cell death instead of abnormalgrowth.

The comparative analyses presented in this review are based primarily on the re-sults of published genome papers and the publicly available databases. The genomefeatures for the genomes that are being considered in this review are summarized inTable 2. The vast majority of such results come from computational analyses, notexperimental analyses, and are thus in essence hypotheses, not verified biological

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GENOME COMPARISON OF PHYTOBACTERIA 171

TABLE 1 Genome sequencing of plant-associated bacteria

Organism Host Disease Status Website

Agrobacterium Tobacco Crown gall Completed www.agrobacterium.orgtumefasciens (27,78)C58

Mesorhizobium Lotus (Nitrogen-fixing Completed http://www.kazusa.or.jp/rhizobase/loti symbiont) (40)

Ralstonia Tomato Vascular wilt Completed sequence.toulouse.inra.fr/R.solanacearumsolanacearum (65)

Sinorhizobium Alfafa (Nitrogen-fixing Completed sequence.toulouse.inra.fr/S.meliloti.htmlmeliloti 1021 symbiont) (25)

Xanthomon as Citrus Asiatic canker Completed cancer.lbi.ic.unicamp.br/xanthomonas/axonopodis (17)pv citri

Xanthomonas Crucifers Black rot Completed cancer.lbi.ic.unicamp.br/xanthomonas/campestrispv (17)campestris

Xylella fastidiosa Citrus Citrus variegated Completed aeg.lbi.ic.unicamp.br/xf/9a5C chlorosis (71)

Xylella fastidiosa Grapevine Pierce’s disease Completed aeg.lbi.ic.unicamp.br/xf-grape/Temecula1 (75)

Leifsonia xyli Sugarcane Ratoon stunting In progress aeg.lbi.ic.unicamp.brsubsp. Xyli disease

Pseudomonas Tomato Bacterial speck In progress www.tigr.orgsyringae diseasepv. tomatoDC3000

Spiroplasma Maize Corn stunt In progress www.genome.ou.edu/spiro.htmlkunkelii

Xanthomonas Citrus Cancrosis B In progress www.lbm.fcav.unesp.br/aurantifolii B

Xanthomonas Citrus Cancrosis C In progress www.lbm.fcav.unesp.br/aurantifolii C

facts. Computational analysis of a genome sequence basically involves gene iden-tification and putative functional assignment (genome annotation). Such assign-ments rely extensively on the concept of sequence similarity. The most widelyused similarity-detection tool is the BLAST program (4), generally used in con-junction with publicly available databases such as GenBank. BLAST results (hitsor matches to database sequences) have an associated measure of statistical signif-icance called e-value. When a new gene matches database sequences that have noassigned function it is defined as a conserved hypothetical. When no significantmatches are found, the new gene is referred to as hypothetical. All prokaryoticgenomes sequenced to date have a fairly high fraction (between 20 and 40%) ofgenes for which no function has been assigned.

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172 VAN SLUYS ET AL.

Figure 1 Consensus neighbor-joining phylogenetic tree constructed using SSU rDNA (16S)sequences. Black numbers at the branches represent bootstrap values for parsimony (top, 1000replicates), maximum likelihood (middle, 100 replicates) and neighbor-joining (bottom, 1000replicates). Numbers in italics represent bootstrap values (1000 replicates) for the commonbranches from parsimony analysis from amino acid sequences for recA (top) and rpoA(bottom). Only bootstrap values above 70 are shown in the tree. Sequence’s accession numbersare in parentheses.

STRUCTURAL COMPARISONS BETWEEN THE GENOMES

Whole genome alignment can be attempted for organisms that are sufficientlyclose phylogenetically. Such analysis can provide important clues about evolution-ary relationships, revealing large-scale gene order conservation. For the genomesunder study this analysis yields basically two kinds of results. The first is noconservation if one compares the main chromosome ofXacwith the main chro-mosome ofRs. Although these organisms have much in common in terms ofgene content, there is no significant conservation of gene order. The second resultis large-scale gene order conservation, with varying degrees of rearrangements.The chromosomes ofXacandXcc(17), and the circular chromosomes ofAt, Sm,andMl are related in this way (78). Thus, these results indicate that the organisms

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174 VAN SLUYS ET AL.

under study form the following separate clusters: (a) Rhizobiaceae; (b) the twoXanthomonas; (c) the Xylella; and (d) Ralstonia. These considerations closelymirror the phylogenetic relationships shown in Figure 1.

MECHANISMS OF DNA EXCHANGE

Horizontal gene transfer (HGT) occurs via transposons, integrons, plasmids, andprophages of temperate viruses. Such elements can carry genes that code for aspecific ecological adaptation or for new features associated with pathogenicity orbacterium-host interaction (36, 53, 74). Thus, rapid alterations of phenotype canoccur via HGT. Regions of distinct GC content and codon usage in a genomeare usually associated with mobile genetic elements and are plausibly the conse-quence of HGT (22, 42, 55). Such regions are called genomic islands; when theisland contains pathogenicity genes it is called a pathogenicity island (30, 58).The size of genomic islands may vary from a few Kb to as many as 500 Kb.At the upper extreme is the 500-Kb genomic island found inMl, which carriesthe genes responsible for symbiosis (74). Five ACURs (alternative codon usageregions) described forRshave the typical features of pathogenicity islands andinclude candidate genes for type III secretion system–dependent effectors. ThevirB and T-DNA regions ofAt both show relatively low GC content as do threeother regions containing hypothetical ORFs surrounded by insertion sequencesand phage integrases. BothXanthomonasalso contain several regions with an al-tered GC content that can be as low as 41%, in contrast to the genome average of64%. These regions include the type IV secretion system present in both strains aswell as strain-specific genes encoding hemolysin, hemagglutinin, hemin storageproteins, syringomycin, avrPphE, and cvgSY homologous proteins. These pro-teins are relevant to pathogenicity and may account for many of the phenotypicdifferences betweenXacandXcc.

GENERAL METABOLISM

Table 3 shows a comparative distribution of the genes per functional category. Be-low, we present some specific aspects of several of these categories, including thepresence or absence of specific pathways and genes. Although the basic metabolicpathways responsible for energy production and the synthesis of both micro- andmacromolecules are very highly conserved among bacteria, not all pathways arepresent in all organisms. The presence or absence of particular pathways directlyrelates to the adaptation of the organism to its environment(s), together with thecontinual selective pressure to maintain the genome at its smallest viable size toreduce the metabolic load of replication. In this view,Xylella’s smallest genomecan be accounted for by its essentially invariant environment within the xylemand the limited metabolic options that this implies. On the basis of the genespresent, energy can be deduced to be derived strictly from simple sugars; as a

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GENOME COMPARISON OF PHYTOBACTERIA 175

TABLE 3 Comparative analysis of gene category distribution of the genomes ofplant-associated bacteria

Categorya,c Xcc Xac Xf-CVC Ml At Sm Rs

I. Intermediary 16.7%b 16.5% 10.3% 19.2% 23.2% 22.5% 17.4%metabolismI.D. Regulatory 6.7% 6.7% 3.3% 7.8% 9.4% 9% 7.5%functions

II. Biosynthesis 8.4% 7.9% 9.9% 7.5% 8% 7.5% 7.9%small molecules

III. Macromolecule 12% 11.2% 13.9% 6.4% 7.1% 6.7% 7.5%metabolism

IV. Cell structure 4.7% 4.6% 5.7% 2.6% 2.8% 2.8% 3.6%

V. Cellular processes 9% 8.8% 5.3% 12.6% 17.6% 15.5% 9.3%V.A. Transport 6.4% 6.3% 4.2% 11.6% 15.9 % 14.1% 7.5%

VI. Mobile genetic 4.1% 4.3% 6 % 3.1% 1.7% 3.1% 4.6%elements

VII. Pathogenicity, 6.8% 6.9% 5.9% 3.1% 3.8% 3.3% 5.3%virulence andadaptation

VIII.A. Conserved 34.5% 33.4% 38.5% 32.3% 25.2% 30.7% 31.1%hypothetical

VIII.B. Hypothetical 3.4% 6.3% 4.4% 13% 10.5% 7.8% 13.2%(orphan genes)

aCategory names and numbers from Simpson et al. 2000 (http://aeg.lbi.ic.unicamp.br/xf/).bPercentage of genes per category.cIn order to organize the information related to the genomes analyzed in this article, a relational database was createdcontaining: [1] general data of each genome; [2] data of each replicon of each genome; [3] data of each putative Open ReadingFrame (ORF); and [4] information about BLASTP hits of each ORF against NCBI NR protein database. An automaticcategorization, based on BLAST, was performed on genomes not managed by the ONSA Network Brazilian group (theseexternal genomes areMesorhizobium loti, Sinorhizobium meliloti, andRalstonia solanacearum).

supplementary glucose source, plant cell walls can be degraded, liberating glu-cose for import. There are no pathways for fatty acid degradation. The genomecontains a complete pathway for glycolysis and TCA cycle as well as a com-plete set of genes for ATP synthesis by oxidative phosphorylation. WhenXylellais placed in a more nutrient-rich environment, such as laboratory growth media,it is unable to switch to more rapid growth, because it apparently is condemnedto the fastidious behavior responsible for its name. This is further evidence thatXylella’s relatively small genome, lacking metabolic capacity to take advantageof additional nutrients, is adapted for survival in a constant but nutritionally diluteenvironment.

Xanthomonas,Rs, andAt, with much larger genomes thanXylella, contain genesfor an extensive arsenal of metabolic pathways, and this reflects their adaptation

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176 VAN SLUYS ET AL.

to a variety of different environmental conditions.Xanthomonasis capable ofliving as an epiphyte, whereasRsandAt live freely in the soil. When infection isestablished,Xac, Xcc, andRsinvade the xylem and/or colonize the mesophyll, arich environment with high sugar concentration and amino acids. These bacteriaexhibit accelerated growth when exposed to nutrient-rich environments with variedpotential of metabolic substrates; this is made possible by the presence of genes forthe conversion of amino acids, fatty acids, and some alcohols into glucose, amongothers.SmandMl can survive in the soil, as canRsandAt, but they also existin a form where they are completely dependent on plant cells for their nutrientand oxygen requirements. Indeed, as a result of the interaction with the plant thebacteria exchange their outer membrane, possibly facilitating nutrient uptake forthe energy-consuming process of nitrogen fixation in low tension of oxygen (9). Asa consequence of this dichotomy of lifestyles, bothSmandMl have large genomesthat encode a wide variety of metabolic pathways (39).

DNA METABOLISM

At first sight we might expect DNA metabolism to be the most conserved ofall categories of bacterial metabolism and relatively independent of environmentor lifestyle. In the genomes under study, most of the genes necessary for DNAreplication, repair and recombination, as well as RNA and protein synthesis, arevery similar to those reported for other Gram-negative bacteria. Nevertheless,differences in the genes for some aspects of these processes are revealing. Oneexample is the SOS-induced DNA polymerases, whose expression is induced underconditions of general stress, thus increasing the mutation rate of the organism inorder to allow rapid adaptation to altered environmental conditions (52, 59). Onesuch polymerase, DNA polymerase IV (DinP), is absent inXylella but is presentin the other bacteria;At andSmhave two andMl three paralogs of this gene. Thissuggests thatAt, Sm, andMl would have an enhanced rate of evolution in the faceof stress and starvation; it would be of interest to ascertain experimentally whetherany of theDinP duplications are related to survival within the host.Xylella, onthe other hand,would be a rather unadaptative organism, possibly reflecting itsuniform environment.

Several integrases and recombinases, required for DNA recombination, arefound in the genomes being discussed. Only theXanthomonasandXylellagenomes,however, possess exonuclease V, a complex of the RecB, RecC, and RecD pro-teins and known to participate in the repair of double-strand breaks producedduring DNA replication. Surprisingly, this complex has been recently associatedwith the virulence ofSalmonella enterica, possibly because it is required to me-diate recombinational repair within infected phagocytes (12). Indeed, this enzymeis also found in many pathogenic bacteria and may also be important for muta-genicity in bacteria exposed to stress conditions (10). These genes are certainlycandidates for further investigation of the infection processes ofXanthomonasandXylella.

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GENOME COMPARISON OF PHYTOBACTERIA 177

As discussed further below, one of the most important plant responses to in-fection and abiotic stress is the generation of oxidants, especially highly reactiveoxygen radicals such as superoxide and hydrogen peroxide, which are highlydamaging to DNA. The main forms of DNA damage caused by these agents arerepaired either by the nucleotide excision repair present in all species under dis-cussion or by specific DNA glycosylases and AP-endonucleases, partaking in theprocess known as base excision repair (35). Most of the enzymes related to thispathway in bacteria are found in the eight proteomes. In some cases, such as theAP-endonucleases known as Exo III (encoded byxthAandexoA), they are presentin several copies. The main enzyme for the removal of 8-oxoguanine, a crucialform of damage induced by oxygen radicals, is formamidopyrimidine DNA glyco-sylase, encoded by the genefpgor mutM. This gene is found in the eight genomesanalyzed, with two identical copies present in theXylella genome. A noteworthyand intriguing absence in the proteome ofMl is the geneudg, which encodes foruracil-DNA glycosylase. This enzyme, critical for DNA base-excision repair, rec-ognizes and removes uracil from DNA (57). Uracil is normally misincorporatedduring replication, and also may result from cytosine deamination. The way inwhich Ml deals with genomic uracil is a stimulating question deserving furtherinvestigation.

Another enzyme that merits specific mention is photolyase. This enzyme de-pends on light to remove pyrimidine dimers induced by the ultraviolet componentof solar radiation. Proteins of the photolyase family, either photolyases or cryp-tochromes, are very conserved and found in the three domains of life [Bacteria,Eukarya, and Archaea, (18)]. At least one gene from the photolyase family hasbeen found inXanthomonas, At, andMl, but none was found inXylella, Rs, andSm. The presence or absence of this gene may reflect the habitat preferences ofthese bacteria and their responses to light. Therefore, its absence inXylella maysimply be due to the fact that this bacterium lives exclusively inside plants or insectvectors. However, forRsandSmthe absence of photolyases is more difficult tointerpret, as these bacteria are also found in the soil and might be exposed to lightunder certain conditions. On the other hand, the existence of a photolyase-like genein Xanthomonas, At, andMl may indicate that illuminated habitats play importantroles in their life cycles.

PATHOGENICITY AND HOST-BACTERIA INTERACTIONS

The main focus of this review lies with pathogenicity and host-bacteria interac-tions. Pathogenicity, virulence, and adaptation is a category in Table 3.Xylella,Xanthomonas, andRs(which are necrogenic) have about 6% of genes in this cat-egory, whereas the Rhizobiaceae (which are tumorigenic) have about 3.5%. Thisdifference is significant considering the uniform method we used to categorizegenes in all seven genomes (see legend of Table 3), and suggests the existence ofspecific gene complements that determine the nature of plant-bacteria interactions.Different aspects of pathogenicity mechanisms are discussed below.

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Secretion Systems

Secretion systems are of fundamental importance for the correct translocation ofproteins and other molecules, so that synthesized proteins are properly targeted tothe location where they will function or be stored. For the purposes of this review,the most important secretion systems are those that mediate the translocation ofbacterial proteins to host cells. There are five different forms of secretion systems(SS) enumerated as types I-V, which are distinguished on the basis of the proteinsthat form them (24, 32, 45). The type I-SS is involved in the secretion of toxins suchas hemolysins, cyclolysin, and rhizobiocin. It is characterized by the presence ofABC (ATP-binding cassette) proteins involved in the export and import of a varietyof compounds using energy provided by ATP hydrolysis (7, 50). This system ispresent in all the genomes reported here.

The type II-SS is a common mechanism present in Gram-negative bacteria forthe extracellular secretion of a variety of proteins, including extracellular enzymes,proteases, toxins, and virulence factors. This system is a two-step mechanismwhere the exportation of the unfolded exoprotein to the periplasm occurs via thewell-known Sec pathway for translocation across the inner membrane (60, 64). Inthe second step, the processed and folded protein is then translocated through theperiplasm and across the outer membrane by a complex mechanism consisting of12–14 proteins encoded by a cluster of genes (61). Of the bacteria discussed here,RsandXanthomonasuse the complete type II-SS to secrete exoenzymatic viru-lence factors including cellulolytic and pectinolytic (such as polygalacturonase)enzymes (5, 66). In contrast,Atpossesses the genes only for the Sec machinery; thesecond step is probably undertaken by type IV pili (78).Xylella andMl have onecomplete type II-SS.XanthomonasandRsappear to have two independent typeII-SS, reflecting the importance of the cell-degrading enzymes that they secrete,as discussed in detail below.

Perhaps of greatest interest in relation to pathogenicity is the type III-SS, whichplays a role in the pathogenicity ofXanthomonasandRs(2). The major function ofthe type III-SS is to transport effector proteins across the bacterial membrane andinto the plant cell (47). Genome sequencing has revealed thatMl also has a clusterof genes similar to the type III-SS of other rhizobia (76), but thatAt,Xylella, andSmtotally lack this system. The type III-SS genes inMl correspond only to the mainconserved core of the type III-SS (the Hrp pilus is lacking), indicating that thisstructure in rhizobia is different from that in pathogenic bacteria.XanthomonasandRshave a type III-SS classified as group II based on the analysis of sequence sim-ilarity, operon structure, and the regulatory elements (2). Ortholog genes inXan-thomonasandRsthat encode components of the common core of the type III-SShave relatively high (65% on average) sequence similarity at the amino acid level;these genes are termed Hrc (hypersensitive response conserved) genes (8). Theother orthologs, particularly those encoding surface-exposed proteins, have only35% amino acid similarity on average. Genome sequencing has also revealed somepreviously unknown effector proteins, such asavr homologs inRs. However,Rsis a wide host-range pathogen and does not exhibit the host restriction normally

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GENOME COMPARISON OF PHYTOBACTERIA 179

associated with Avr proteins. This suggests that Avr proteins also confer someother adaptive advantage to this bacterium (65). Interestingly, most of theRs avrhomologs are similar toPseudomonas avrgenes, whereas theRs type III-SS isstructurally more similar to theXanthomonascluster (2).Xac andXcc differ intheir sets ofavr genes, which may partially explain their different host specifici-ties. In addition to theavr genes, theRsandXanthomonasgenomes also possessa very interesting set of effector proteins with similarity to ankyrin-related andleucin-rich proteins found in eukaryotic organisms, such asArabidopsis, humans,Drosophila, and yeast.

The type IV-SS is involved in macromolecule export, targeted to the host cell. Itsproteins are highly similar to conjugal transfer proteins, responsible for plasmidmobilization among bacteria (13). The description of theAt virB operon was amilestone in understanding the structure and function of type IV-SSs (21, 38, 79).The 11 proteins encoded by this operon are involved in the transfer of the T-strandfrom the bacterial cell to the plant cell cytoplasm via an organized structure. Itstretches from the inner membrane across the periplasm and outer membrane andterminates in a pilus-like structure that protrudes from the bacterial cell. This isbelieved to establish contact with the recipient cell in a way similar to the typeIII-SS (68).

A search for homologs of thevirB operon resulted in significant matches inall the genomes being studied here. However, inRs, only those genes encod-ing proteins associated with the inner membrane were found.Smhas avirB-likegene cluster, as previously highlighted in Galibert et al. (25).XanthomonasandXf have gene clusters similar to the type IV-SSs ofBordetella pertussis(77) andBrucella suis(54). We therefore speculate that these systems are involved in viru-lence/pathogenesis as in the case ofB. pertussis(secretion of pertussis toxin),B.suis(intracellular bacteria survival), and in the more distantly related type IV-SSdescribed inH. pylori [responsible for IL-8 inducing factor secretion (67)].

Xylella andXanthomonascontain genes for surface-associated adhesins thatappear to be type V-SS autotransporters (33). Such autotransporters, secreted frommammalian pathogens, are important for adhesion to epithelial cells (33). Theinclusion of some the phytopathogen adhesin–related proteins within this familyis due to the presence of a characteristic carboxy-terminal-domain signature.

Cell-Wall Degrading Enzymes

The plant cell wall plays a dual and somewhat contradictory role in the life of plantpathogens. On the one hand, it serves as a barrier that impedes the infection ofcertain tissues. On the other, it serves as a source of energy. Not surprisingly, cellwall degrading enzymes (CWDE) are widespread among plant pathogens. Thecell wall is composed of three major polysaccharides: cellulose, hemicellulose,and pectin, as well as lignin in woody plants. In the genomes of the bacterialpathogens being discussed here one finds strikingly different numbers and com-binations of genes encoding enzymes that degrade these polysaccharides.Xccap-pears to have the most numerically replete arsenal of CWDE: two pectin esterases

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and polygalaturonases, five xylanases, four pectate lyases, and nine cellulases. Incomparison,At, Ml, andSmare poor pectinolytic agents since only three, one,and no pectinases were found, respectively. The same applies toXylella, whereonly one copy of a polygalacturonase was identified.Xacdiffers fromXccby theabsence of the two pectin esterases, one pectate lyase, and three cellulases. Theabsence of pectin esterases inXacis noteworthy, since these enzymes alter the sol-ubility of pectin by cleaving off its lateral branches, rendering the stripped pectinchain more susceptible to the attack by pectin lyases and polygalacturonases (14).Thus pectin esterases favor tissue maceration, as has been demonstrated inEr-winia (Pectinobacteria) chrysantemi(69), and their absence in Xac could explain,at least in part, the differences in symptoms caused byXcc(rot) andXac(canker).

As mentioned,Xylellahas only one polygalacturonase gene. However, carefulinspection of the gene sequence inXf-CVC revealed that even this is possiblytruncated and nonfunctional. Since polygalacturonases facilitate colonization ofthe vascular tissues of the host (34), the absence of a functional copy of thisgene product in the citrus strain could explain the long incubation period of CVC.However, as the nonfunctionality of this gene is predicted based on its sequence,this remains to be confirmed experimentally.

A gene similar to the ligninase fromPseudomonas paucimobilis(lignin beta-ether hydrolase) is present only in the Rhizobiaceae genomes. This enzyme mayfacilitateAtnot only to colonize woody hosts but also to degradate lignin to producephenolic compounds that serve as signal molecules for VirA protein (46). VirA is amember of a two-component regulatory system responsible for the transcriptionalactivation of the virulence genes ofAt (68).

Toxins

Pathogenic bacteria secrete a variety of products that are toxic for both their hostsand other microorganisms. Perhaps the most studied is the alpha-hemolysin, aRTX-toxin of E. coli that effects host cell lysis (24, 45). Prior to genome se-quencing, the presence of these proteins in phytopathogens was not expected, butfollowing the sequencing ofXf-CVC, they have appeared in all the other genomesexcept inXcc. The function of these RTX-toxins in phytopathogens remains to beestablished.

The genomes under study do not seem to have known phytotoxins, best char-acterized inP. syringae(6), except that genes for the synthesis of syringomycin(29) were found inRsandXac. Specific toxins with nonconserved sequence withrespect to known toxins may, however, exist. It will be interesting to investigatethis possibility and also to determine the role of these syringomycin-like proteinsin RsandXac, given that this toxin might affect signaling pathways of the host (3).

Bacterial Adhesion and Surface Structural Elements

Bacterial genes associated with fimbriae or pili assembly have been extensivelystudied owing to their importance in the establishment of human diseases caused

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by organisms such asE. coli, H. pylori, H. influenzae, andB. pertussis, amongothers (23). In plant pathogens, the significance of surface interactions is not aswell understood. The only plant pathogen where attachment to surface receptors ofplant cells is demonstrated to be essential for virulence isAgrobacterium(20, 68).This attachment is the first step that leads to DNA transfer and disease symptomsand is mediated by three components: a low-molecular-weight cyclicβ-1,2-glucanmolecule, which requires the geneschvB, exoC/pscA, andchvAfor its synthesisand export; cellulose synthesis genes; and theatt region of the genome. Other thanthese well-studied genes, genomic sequencing ofAt has uncovered homologues ofmammalian pathogen adhesins, including BrfA ofE. coliand PsaA ofS. pneumo-niae, and of genes responsible for pilus biogenesis ofCaulobacter crescentus(72).The latter system is well studied and consists of a cluster of six genes (cpaA–cpaF)adjacent topilA, a major pilus component of the type IV pili. Homologs of theseare also found inSm, Ml, Rs.

In addition tocpa genes, Rs also contains a large number of genes encodingproteins likely to be associated with attachment and aggregation, including 35homologs of the type IV pili genes ofP. aeruginosa(31). Type IV pili genes arealso present in the genomes ofXylella and Xanthomonas. Within the Xf-CVCgenome we find six copies of a major pilus subunitfimA/pilA homolog, and twocopies of a gene similar topilE of Neisseria(28). All other genes involved inpilus assembly exceptpilY1 are present in single copy. The genefimA/pilA inXac is similar to those present in other xanthomonads andXylella, but in Xccthis gene is most similar topilE. The variability among the major pilus subunitgenes may be associated with specific interactions with their hosts, resembling thefunction of this system in human and animal bacterial pathogens. Studies withfimAmutants have shown that the type IV pilus ofX. campestrispv. vesicatoriaandP.syringaepv. tomatois mostly involved in cell-to-cell aggregation and protectionfrom environmental stress, rather than being crucial for adhesion or colonizationof the plant surface (56, 63). InXylella, type IV pili may be associated with theestablishment of an aggregated bacterial population necessary to counteract theturbulent environment of the xylem, facilitating its adherence to the vessels inconjunction with other components, such as exopolysaccharides.

An additional three adhesin genes found inXylella are similar to hsf and tohia gene products ofH. influenzae(33, 73). InXanthomonasthere are homologsof YapH of Yersinia pestisand XadA, which is involved in the virulence ofXan-thomonas oryzaepv. oryzae(62).

Xanthomonas, Xylella, and Rs contain homologs of hemagglutinin-relatedgenes, first detected in plant pathogens in the genome of Xf-CVC. InRs27 suchgenes have been found, a remarkably large number in itself, and even more so ifwe consider that nine of the genes encode glycine-rich proteins with more than2500 amino acids, eight times the average size of bacterial genes. Of those 27genes, 14 are predicted hemagglutinins and 13 are structurally related to filamen-tous hemagglutinins ofB. pertussis(48) and adhesins ofH. influenzae(37). XacandXcc also contain putative filamentous hemagglutinin genes, most similar to

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182 VAN SLUYS ET AL.

FhaB of B. pertussis(48). Adjacent to these genes, inXac, there are two otherhemagglutinin/hemolysin-related genes that are absent inXcc. Xf-CVC has threecopies of these proteins.

When we compare the different copies of these filamentous hemagglutiningenes present in a specific genome to each other, we see high conservation at theN-terminal end but variation at the C-terminal end. When we compare these genesamong different genomes, conservation at the N-terminal end is still detected butthere are discrepancies, suggesting that these genes may play a role in bacteria-hostinteractions. Note that the bacteria under study that have hemagglutinins colonizeplant vessels at some stage of infection.

All the surface-structure and outer-membrane protein-encoding genes foundin these genomes may contribute to the attachment and aggregation of bacterialcells on a plant surface, in the xylem, or inside an insect vector. They are probablyexpressed in association with polysaccharides in different stages of infection andestablishment of disease (20, 63), and an in-depth study of their biological roleshould contribute to a greater understanding of surface interactions in necrogenicplant pathogens.

Oxidative Stress

Bacterial infection in plants elicits the activation of a battery of defenses, as partof the hypersensitive response (43). One of the earliest responses, the oxidativeburst, consists of the rapid generation of highly reactive oxygen species, includingthe superoxide anion (O2−), hydrogen peroxide (H2O2), and the hydroxyl radical(·OH). These agents can lead to harmful lipid peroxidation, enzyme inactivation,and nucleic acid degradation, eventually resulting in plant protection through thekilling of the infecting bacterium. These oxygen species may also induce cross-linking in the plant cell wall, triggering hypersensitive cell death and signaling theinduction of defense genes in the surrounding cells. In the face of this hypersen-sitivity response, bacteria have developed mechanisms of protection against theassociated oxidative stress (51).

The genomes under study contain genes that encode for catalase and super-oxide dismutase, which are responsible for the inactivation of H2O2 and O2

−,respectively. The number of copies of such genes varies, with theXanthomonashaving the highest total: five copies of catalase inXacand four copies inXcc; fourcopies of superoxide dismutase inXacand five inXcc. TheXylella has a singlecopy of catalase, andMl has a single copy of superoxide dismutase. Glutathione-associated enzymes and small proteins, such as thioredoxin and glutaredoxin,work in concert within the cell as antioxidants, maintaining a reducing environ-ment. Basically, these enzymes help in detoxification, protecting cells againstoxidative stress and also participating in redox reactions. There are an elevatednumber of genes encoding for these enzymes in the bacteria of interest, whichargues in favor of their general relevance in this context. For example, glutathioneS-transferase-encoding genes are found in many copies in all the genomes, except

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Xf, with an astonishing 17 homologs in bothMl andSm. At, Sm, andMl do nothave a gene for glutathione peroxidase, so their function may be performed by theglutathione S-transferase.Xylella has a single copy of glutathione S-transferaseand a single copy of glutathione peroxidase, possibly reflecting a slow metabolicrate and alternative mechanisms of antioxidant protection. The regulons OxyR andSoxRS, which are induced by oxidative stress and control the expression of severalgenes that are related to the bacterial defense (11, 19), are found in all genomesunder study, except inXylella, where SoxRS is absent. The presence of thesemain transcription regulators argues for their importance in these plant-associatedbacteria. The lack ofsoxR andsoxS in Xylella is intriguing, but may reflect thereduced necessity of these bacteria to respond to alterations in environmentalconditions.

Bacterial Encoded Phytohormones

Bacterium-plant interactions can be mediated by the synthesis of plant hormoneseither by the plant or by the bacteria. Different hormones are relevant, dependingon whether the bacterium is tumorigenic, necrogenic, or symbiotic. The synthe-sis of molecules by bacteria that are recognized by the plant cell as hormones iscrucial for redirecting plant cell growth and differentiation in tumorigenic or sym-biotic interactions. The underlying basis of the tumorigenic property ofAt involvesthe transfer to the plant cell nucleus of genes encoding proteins for auxin and cy-tokinin synthesis, borne on the pTiC58 plasmid T-region. Interestingly, these genesare similar to genes present in the necrogenic pathogenP. syringae(26). XylellaandXanthomonasdo not have homologs to any of these genes, whereasSm, Ml,andRshave genes similar to components of this pathway. Auxin synthesis hasbeen postulated to occur inRsduring infection (65). The lack inSmandMl ofgenes encoding enzymes of the indole-3-acetamide pathway, which is a necessaryprecursor for auxin synthesis, suggests that these two organisms do not synthesizethis hormone (15).

Cytokinins are adenine derivatives with an isopentenyl side chain that are pri-marily involved in promoting cell division. OnlyRsandAt have isopentenyl trans-ferase (ipt) genes involved in the synthesis of this hormone class. Cytokinin iswell documented to be involved in cell cycle regulation in conjunction with auxin(16). The synthesis of both hormones leads to the tumor phenotype induced byvirulent strains ofAt. Only in the case ofAgrobacteriumhas cytokinin synthesisbeen demonstrated to be driven by theipt gene (1).

GENES SHARED BETWEEN ALL THE SEVEN GENOMES

The availability of the complete proteomes of these seven plant-associated bacteriaencouraged us to search for genes common to all or most of them. We reasoned thatthis search might reveal novel candidate genes related to plant-bacteria interactionand/or to pathogenicity. We used BLASTP (4) to identify orthologous genes in

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184 VAN SLUYS ET AL.

the proteomes of the six genera analyzed. Two genes were declared orthologousif one is a BLASTP hit of the other and vice versa, with an e-value of at most10−10, and such that at least 60% of each predicted protein participates in theiralignment. In addition we tried to exclude so-called housekeeping genes from thefinal result by including in the BLAST database the complete genomes of threereference proteobacteria that are not associated with plants, one for each of themain subdivisions considered here (E. coli K12, N. meningiditis, andC. crescen-tus). Our final list contains only those genes that did not result in a significantBLASTP hit (threshold e-value is 10−05) against these genomes. Table 4 presentsthe list.

One interesting feature of the genes listed in Table 4 is that most of them code forproteins that are located in or involved in the synthesis of the bacterial membraneor cell wall, which is to be expected from proteins important in bacterium-plantinteractions. Some function in membrane transport, such as a putative iron-binding

TABLE 4 Xanthomonasgenes that are common in the plant-associated bacteria, but notpresent in reference genomes

Gene identity Product Observationsa

XAC3499 Acetylxylan esterase, NodB Inner membrane

XAC4371 Polysaccharide deacetylase Periplasmic space, highsimilarity with NodB.

XAC2733 Integral membrane protein Inner membrane

XAC4088 Dolichyl-phosphate-mannose Cell wall biogenesissynthase related protein

XAC2046 CDP-diacylglycerol—serine Inner membrane,o-phosphatidyltransferase pssA phospholipid biosynthesis

XAC3481 Iron-binding and transporter protein Inner membrane

XAC3190 Cobalamin biosynthesis protein Inner membrane

XAC1006 Malate dehydrogenase Inner membrane

XAC4315 Plasmid stability protein Inner membrane, traffickingprotein

XACa0018 Partition protein A Found in pathogenic bacteria

XAC2623 VirD4 protein Inner membrane

XCC3462 NTPase (truncated) Cytoplasm, similar to VirD4

XAC0250, XAC3770, Conserved hypothetical XAC3770 presents aXAC3410, XAC1451, protein thioesterase domain, whichXAC2966, XAC3067, may be involved with theXAC2637 non-ribosomal synthesis of

peptide antibiotics

a Location determined by the software PSORT—Prediction of Protein Localization.

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GENOME COMPARISON OF PHYTOBACTERIA 185

protein, which is an ABC transporter that probably sequesters iron from the planthost. ThenodB gene is necessary for the synthesis of the chitooligosaccharidesignals, called Nod factors, which induce a differential plant growth responseand enable Rhizobia to colonize plant tissue (49). The presence of such genes inbacteria that do not induce nodules implies that they may have a more generalfunction during the association of these bacteria with their hosts. Interestingly,the Xanthomonasand Xylella genomes contain a secondnodB homolog. Thisgene codes for a longer protein that contains a C-terminal domain thought to beimportant for protein-protein interaction (44).

In Table 4 we also find malate dehydrogenase, an enzyme central to the cellmetabolism. There seem to be two families of malate dehydrogenase in thesegenomes. One family is composed by the orthologs found inXanthomonas,Xylella,andRs, and the other family contains the Rhizobiaceae orthologs. Both gene fam-ilies have strong similarity to genes from Gram-positive bacteria and eukaryotes,suggesting possible lateral transfer, which would explain their divergence to themalate dehydrogenase of the non-plant-associated reference genomes.

Seven of the 19 shared genes are classified as conserved hypothetical. Al-though there is very little information about these genes, their presence in all thegenomes reported in this work makes them exciting candidates for further studiesof pathogenicity and bacteria-plant interaction.

CONCLUDING REMARKS

This review illustrates the insights that can be gained from the inspection andcomparison of whole genomes. Such insights can be used as the basis for a con-ceptual framework within which it should be possible to fully understand andexplain the similarities and differences between the bacteria being discussed. Anever-increasing number of complete genomes will become available for study inthe coming years. As this number climbs into the hundreds and then the thou-sands, comparative genomics will become both extremely powerful and extremelycomplex. Nevertheless, within this complexity, we predict that a basic vision willemerge where we will come to see bacterial genomes as permutations of the sameset of genes assorted through horizontal transfer mechanisms and as subject tothe continual selective pressure to reduce genome size, as we have been able toglimpse here.

Despite the relatively small number of genomes studied here, this review hashighlighted some interesting facts obtained from comparative genomics of plant-associated bacteria. As more genomes of this kind become available, it should bepossible to associate specific genes to bacteria that cause similar types of symptoms(e.g., the necrogenic bacteria), or to bacteria that have the same host (e.g.,XacandXf-CVC), or to bacteria that inhabit the same niche (e.g.,Xylella andLeifsoniaxyli subsp.xyli). When we reach that stage, the mechanisms of plant-bacteriainteraction will be far clearer than they are today.

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The Annual Review of Phytopathologyis online at http://phyto.annualreviews.org

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July 12, 2002 14:14 Annual Reviews AR165-FM

Annual Review of PhytopathologyVolume 40, 2002

CONTENTS

FRONTISPIECE xii

ONE FOOT IN THE FURROW: IMPLICATIONS TO ONE’S CAREER

IN PLANT PATHOLOGY, D. E. Mathre 1

EVOLUTIONARY ECOLOGY OF PLANT DISEASES IN NATURAL ECOSYSTEMS,Gregory S. Gilbert 13

MAKING AN ALLY FROM AN ENEMY: PLANT VIROLOGY AND THE NEW

AGRICULTURE, Gregory P. Pogue, John A. Lindbo, Stephen J. Garger, andWayne P. Fitzmaurice 45

STRIPE RUST OF WHEAT AND BARLEY IN NORTH AMERICA:A RETROSPECTIVE HISTORICAL REVIEW, Roland F. Line 75

VIRAL SEQUENCES INTEGRATED INTO PLANT GENOMES, Glyn Harper,Roger Hull, Ben Lockhart, and Neil Olszewski 119

MOLECULAR BASIS OF RECOGNITION BETWEEN PHYTOPHTHORA

PATHOGENS AND THEIR HOSTS, Brett M. Tyler 137

COMPARATIVE GENOMIC ANALYSIS OF PLANT-ASSOCIATED

BACTERIA, M. A. Van Sluys, C. B. Monteiro-Vitorello, L. E. A. Camargo,C. F. M. Menck, A. C. R. da Silva, J. A. Ferro, M. C. Oliveira,J. C. Setubal, J. P. Kitajima, and A. J. Simpson 169

GENE EXPRESSION IN NEMATODE FEEDING SITES, Godelieve Gheysen andCarmen Fenoll 191

PHYTOCHEMICAL BASED STRATEGIES FOR NEMATODE CONTROL,David J. Chitwood 221

HOST-SELECTIVE TOXINS AND AVIRULENCE DETERMINANTS:WHAT’S IN A NAME? Thomas J. Wolpert, Larry D. Dunkle,and Lynda M. Ciuffetti 251

TOBACCO MOSAIC VIRUS ASSEMBLY AND DISASSEMBLY: DETERMINANTS IN

PATHOGENICITY AND RESISTANCE, James N. Culver 287

MICROBIAL POPULATIONS RESPONSIBLE FOR SPECIFIC SOIL

SUPPRESSIVENESS TO PLANT PATHOGENS, David M. Weller,Jos M. Raaijmakers, Brian B. McSpadden Gardener, andLinda S. Thomashow 309

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July 12, 2002 14:14 Annual Reviews AR165-FM

vi CONTENTS

PATHOGEN POPULATION GENETICS, EVOLUTIONARY POTENTIAL,AND DURABLE RESISTANCE, Bruce A. McDonald and Celeste Linde 349

USE OF MULTILINE CULTIVARS AND CULTIVAR MIXTURES FOR DISEASE

MANAGEMENT, C. C. Mundt 381

BIOLOGICAL CONTROL OF POSTHARVEST DISEASES OF FRUITS,Wojciech J. Janisiewicz and Lise Korsten 411

ANTIBIOTIC USE IN PLANT AGRICULTURE, Patricia S. McManus,Virginia O. Stockwell, George W. Sundin, and Alan L. Jones 443

RISK ASSESSMENT OF VIRUS-RESISTANT TRANSGENIC PLANTS, Mark Tepfer 467

INDEXES

Subject Index 493Cumulative Index of Contributing Authors, Volumes 31–40 521Cumulative Index of Chapter Titles, Volumes 31–40 525

ERRATA

An online log of corrections to Annual Review of Phytopathology chapters(if any, 1997 to the present) may be found at http://phyto.annualreviews.org/

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