D. LATENT INFECTIONS
III. The compatible interaction
A compatible bacterial-plant interaction involves bacterial multiplication, avoidance of defense-related gene expression by the plant, and production of pathogenicity determinants by the pathogen. The effects of pathogenicity are assessed by bacterial growth in planta and finally by symptom expression. In early studies of Sutton and Williams (1970) and Wallis et al. (1973) bacterial production of extracellular polysaccharides and plant cell wall-degrading enzymes were considered important factors in pathogenesis.
Host-path..ogen interactions are best investigated by using intact plants, natural means of infection, and environmental conditions that most closely approximate conditions prevalent during plant growth (Shaw and Kado, 1988). Wound inoculation techniques bypass some of the early host responses to Xcc infections in the incompatible interaction between pathogen and host (Shaw and Kado, 1988; Daniels, 1989) and can result in high bacterial population levels in resistant hosts (Wmiams et ai., 1972; Dane and Shaw, 1993).
Breaching the host barriers by artificial means may thus produce artifacts in' disease expression and lead to erroneous conclusions (Shaw and Kado, 1988; Robeson et aI., 1989; Daniels, 1989). A hydathode inoculation technique described by Robeson et al.
(1989) simulates the natural process of infection. Bacteria are introduced into guttation droplets, which are later taken up by the leaf, giving bacteria access to the vascular system
in the absence of wounds. Other inoculation methods may be needed when screening large numbers of genetically modified bacterial clones, and the advantages of several different methods have been reviewed (Daniels and Leach, 1993). Interpretation of results regarding the role of specific factors in host-pathogen interactions is largely dependent on the inoculation methodology and environmental conditions during incubation.
A. HISTOPATHOLOGY, PHYSIOLOGY AND BIOCHEMISTRY OF THE
INFECTION PROCESS
Water deficit in affected tissues was associated with black rot in early studies (Harding et aI., 1904; Smith, 1911), but the host-pathogen interactions leading to the related symptoms (vein blackening, chlorosis, necrosis and eventual production of V -shaped lesions) were debated. Toxin production rather than vascular plugging alone was postulated as the cause of chlorosis (Cook et aI., 1952b). In order to determine the cause of vascular plugging and chlorosis the sequence of events leading to symptom production was investigated in several detailed studies using histochemical stains and/or light and transmission electron microscopy (Sutton and Williams, 1970; Wallis et aI., 1973; Bretschneider et al., 1989).
Sutton and Williams (1970) compared multiplication of two strains that differed in their capacity to produce "mucopolysaccharides", later referred to as exo- or extracellular polysaccharides (EPS) (see section III. A. 1). A weakly virulent strain failed to produce EPS and caused no symptoms, whereas a highly virulent strain produced EPS and caused V -shaped zones of chlorosis, necrosis and black veins. By light microscopy, lysigenous cavities in degraded parenchyma and compacted masses of bacteria in vessels were observed. Bacterial numbers reached a maximum in occluded vessels just prior to symptom expression but were significantly lower in blackened areas of vessels, implicating a vascular host defense response that killed the bacteria.
In many cruciferous hosts oxidation and polymerization of phenolic compounds results in production of darkened patches, thought to be melanin (Sutton and Williams, 1970).
Histochemical stains were used to differentiate the EPS, other carbohydrates, pectin and
"melanin". The melanin was histochemically localized in parenchyma cells surrounding vessels, but was not observed in the lumen of xylem elements. Vascular plugging was attributed to EPS and masses of living and dead bacterial cells.
Wallis et at. (1973) used transmission electron microscopy to show disruption of primary cell walls, loosened spiral thickenings and shredding of cellulose in secondary cell walls. In thin sections of vascular tissues they observed clear zones in xylem walls (interpreted as hemicellulose), reticulate material (interpreted as disorganized cellulose and lignin microfibrils) and fibrillar materials (interpreted as host cell wall material degraded by bacterial enzymes). Vascular plugging, observed after vein blackening had already occurred, was attributed primarily to degradation of plant cell walls with some contribution of the EPS produced by bacteria. In studies of both Sutton and Williams (1970) and Wallis et al. (1973) vascular plugging ultimately resulted in water stress and production of typical V -shaped lesions, starting at the site of vascular occlusion.
The early suggestion that chlorosis is due to toxin formation (Cook et aI., 1952b) was not substantiated by either group.
A Case Study: Black rot of Crucifers 31
Bretschneider et al. (1989) undertook further histological studies, incorporating several changes in methodology to remove possible artifacts. Plants were inoculated by introducing the pathogen into guttation droplets without wounding the tissues, in contrast to earlier studies in which leaf margins were notched (Sutton and Williams, 1970) or wounded at sites in tertiary veins (Wallis, et aI., 1973). Four to six-week old plants were used to compare susceptible (Golden Acre) and resistant (Early Fuji) cabbage cultivars to avoid possible complications associated with the lack of juvenile resistance (Williams, 1980). In compatible host-pathogen interactions, bacteria filled xylem elements but the intercellular spaces and surrounding mesophyll tissue were not heavily invaded. Plant cell walls and spiral thickenings of xylem elements were severely degraded. Bacteria within xylem elements were embedded in a fine-structured fibrilar matrix and were surrounded by an electron-lucent halo thought to be lysed bacterial cells and/or partially degraded plugging material. The histopathology was similar in the incompatible reaction (discussed later), except that the xylem was more severely disrupted and surrounding parenchyma cells showed greater plasmolysis, cell shrinkage, and chloroplast deformation. The plugging material in both compatible and incompatible interactions appeared to consist both of reticulate matter and a fibrillar matrix of undetermined composition, leaving origin and the nature of the plugging material open to further debate.
1. Extracellular polysaccharide (EPS)
The EPS produced by most xanthomonads is xanthan, a cellulosic (1-4) [3-D-glucose polymer with trisaccharide side chains consisting of two mannose and one glucuronic acid residues) (for review: Sutherland, 1993). As described above, EPS-deficient mutants of Xcc caused reduced symptom expression when inoculated into veins of mature plants (Sutton and Williams, 1970).
Two types of Xcc strains are often observed in culture. Larger colonies produce EPS, are nonchemotactic and virulent, whereas smaller, swarmer-type colonies do not produce EPS, are chemotactic and avirulent (Kamoun and Kado, 1990b). In culture, the small types are maintained under nutritional stress and often appear when cultures are revived from water storage. The population can switch reversibly in planta. Mutational analysis indicated that EPS is a virulence factor and directly related to the presence of hrp genes (Kamoun and Kado, 1990b) (see section IILB.l). The presence of EPS in virulent colonies was demonstrated by using cetyl trimethylammonium bromide (CTAB) to precipitate the EPS, followed by color development in the anthrone assay.
The involvement of EPS in pathogenicity thus corroborated early study of Sutton and Williams (1970).
Extracellular polysaccharides also have been implicated in host-pathogen interactions in other Xanthomonas-induced plant diseases as well as other bacterial diseases (for review: Denny, 1995). In the Xanthomonas-pepper pathosystem xanthan enczpsulates X. campestris pv. vesicatoria (= X. axonopodis pv. vesicatoria; Vauterin et aI., 1995) cells in compatible and incompatible interactions with pepper within a few hours of inoculation (Bonas et aI., 1991; Brown et al., 1993). Similar observations were made on Arabidopsis thaliana leaves inoculated with Xcc. These and other studies provide additional evidence that xanthan may have a role in the early phases of disease development.
2. Lipopolysaccharide (LPS) and the bacterial cell envelope
LPS consists of three main regions, an inner lipid region (Lipid A), a polysaccharide core, and an outer region (the O-specific side chain or O-antigen). In Salmonella minnesota the core is an oligosaccharide consisting of two molecules of 2-keto-3- deoxyoctulosonate (KDO) (Osbourn, 1963). The lipid region and the polysaccharide core are highly phosphorylated, while the O-antigen is largely composed of repeating units of short branched oligo saccharides that are readily detached from the bacterial outer membrane. Fractionation of bacterial cells in hot-phenol separates proteins and other phenol-soluble cell components from the totality of the polysaccharide (LPS) and nucleic acid (Westphall and lann, 1965). The water-soluble LPS from Xcc contains galactose, 3-amino-3,6-dideoxy-D-hexose, glucose, mannose, rhamnose, galacturonic acid, phosphate and KDO (Volk, 1966; Ojanen, et ai., 1993). The bands forming ladder-like patterns in silver-stained polyacrylamide gels represent size-fractionated repeating units of the O-antigen. (Tsai and Frasch, 1981). The structure of the core oligosaccharide of Xanthomonas LPS differs significantly from that of the lipid A-(KDO)2 of enteric bacteria, as it lacks heptose (Volk, 1966). Using a modification of the procedure described by of Westphall and lann (1965), Hickman and Ashwell (1966) recovered two components from phenol-soluble LPS that they crystallized and identified as 3-acetamido-3,6-dideoxy-D-galactose and D-rhamnose. The recovery of LPS compounds in different solvent fractions is related to their partitioning in the solvent system used.
Outer membrane proteins and components of LPS have a major role in determining the surface characteristics of the bacterial cell and are largely responsible for the antigenic properties of different bacterial strains. The repeated observation that pathovar-specific LPS moieties are recognized as distinct surface antigens by diagnostically useful monoclonal and polyclonal antibodies is a further reason to suspect that LPS molecules may be associated with pathovar-host specificity (Benedict et ai., 1990; Alvarez et al., 1991; Ojanen et ai., 1993; Kingsley et ai., 1993; Gabriel et ai., 1994). Lipopolysaccharides on bacterial cell surfaces may trigger alterations in plant responses to pathogens, and hence may have a governing role in initial stages of the host-pathogen interaction (Rudolph et ai., 1994; Newman et ai., 1994, 1995).
The role ofLPS in host-pathogen interactions was investigated in turnip (B. campestris, syn.
=
Brassica rapa) using wild-type Xcc strains and LPS-deficient mutants (Newman et al., 1995). The intact lipid A-lipopolysaccharide core molecule induced defense-related gene expression associated with alteration of plant cell walls and elicitation of plant defense (Newman et ai., 1995). Defects in the biosynthesis of the core oligosaccharide component of LPS were associated with decreased multiplication of mutants in turnip, Arabidopsis and a nonhost, Datura. Thus, a pathogenicity locus of Xcc was associated with LPS biosynthesis (Dow et ai., 1995; Newman et ai., 1995). Lipid A and core oligosaccharide components of bacterial LPS also have a role in prevention of the hypersensitive response in pepper (Newman et ai., 1997; described in section IV.B).In a disease of cassava caused by a related pathogen, X. campestris pv. manihotis (= X. axonopodis pv. manihotis; Vauterin et ai., 1995), LPS was associated with shredded plant cell walls during pathogenesis (Boher et ai., 1997). LPS- and EPS-specific monoclonal antibodies were used to localize bacterial outer membrane components
A Case Study: Black rat a/Crucifers 33
within leaf tissues 7 days after pathogen inoculation. Xanthan was a component of the fibrillar matrix filling the intercellular spaces of the leaf mesophyl and attached to the outer surface of bacteria, but LPS was not detected in the fibrillar matrix.
[The fibrillar matrix observed by transmission electron microscopy resembles the fibrillar matrix observed by Wallis et al. (1973) and Bretschneider et al. (1989) in cabbage vessels infected with Xcc]. LPS was associated with degraded mesophyll cell walls. The LPS-specific monoclonal antibody reacted with periodate-sensitive, and heat-, protease-, and lysozyme-resistant molecules, and the ladder-like pattern observed on Western blots suggested that the specific antigen was a component of the O-antigen of the LPS. Detection of this antigen in the apoplast of mesophyll cells suggested a possible contact between LPS of X. campestris pv. manihotis and the host plasma membrane (Boher et al., 1997). Virulent strains not reacting with the LPS-specific antibody in vitro became positive during infection, suggesting that biosynthesis of LPS occurs during pathogenesis.
B. GENETICS OF THE HOST-PATHOGEN INTERACTION
Gene-for-gene interactions between Xcc and cruciferous hosts have not been clearly established. Although there is an indication that various crucifers are differentially affected by strains of Xcc (Kamoun et aI., 1992a), specific gene-for gene relationships involving pathogen races and host cultivars are obscure in crops, such as cabbage, which have multigenic resistance. Genetic studies of the host-pathogen interaction have instead involved a search for major pathogenicity genes.
Mutants of Xcc that either failed to produce a pathogenic response or showed altered pathogenicity following stem-inoculation of three-day old seedlings were isolated by Daniels et al. (1 984a, b), and genes encoding proteases and endoglucanases were identified and cloned. In older plants, however, growth rates of wild-type and mutant strains were similar, as was symptom production, indicating that proteases and endoglucanases probably do not play a role in early stages of black rot infection.
Subsequently, other strategies and other inoculation methods were used to search for and clone genes involved in pathogenicity. Osbourn et al. (1990a) sequenced an open reading frame associated with reduction in pathogenicity. Nevertheless, pathogenicity mutants continued to produce EPS and plant cell wall- degrading enzymes, which were characteristic of wild-type strains and thought to be involved in pathogenesis.
Thus, the search continued for genes important in early stages of pathogenesis, involving, i) hrp genes; ii) other genes for EPS biosynthesis; iii) structural genes for production of extracellular enzymes; iv) enzyme secretion systems; and v) regulatory genes (Daniels et aI., 1994).
1. hrp genes
Hrp stands for "Hypersensitive Reaction and £athogenicity" and involves elicitation of a hypersensitive reaction (HR) in nonhost plants and pathogenicity in compatible hosts.
Mutagenesis of hrp genes causes a loss in ability to incite both HR and pathogenicity;
that is, both compatible and incompatible bacterial plant interactions are affected (Dow and Daniels, 1994). The apparent dual role of hrp genes suggests that plants have
developed resistance mechanisms based on recognition of bacterial determinants that control pathogenicity (Arlat et aI., 1991).
In 1990, Kamoun and Kado (1990 a, b) described a hrpX locus in Xcc required for pathogenesis on crucifers and HR on non-host plants. Mutations in hrpX caused loss of pathogenicity in host plants, a loss in HR on non-host plants, but a gain in ability to cause HR in the host plants (Kamoun et aI., 1992a, b). The hrpX locus is present in virulent wild-type strains of Xcc that produce EPS and are nonchemotactic. Mutation of Xcc to produce hrp· mutants resulted in failure to produce EPS or symptoms on crucifers. Co-inoculation of an avirulent, hrp· mutant strain JS111 with the virulent wildtype (hrp+ ~PS+) strain 2D520 resulted in exocellular complementation in planta by the hrp+ strain after 1 to 3 days, resulting in delayed symptom production.
These observations suggested that the hrpX locus controls functions involved in evading the defense system of the host.
HrpX is found in several pathovars of X. campestris and in X. oryzae pv. oryzae (Kamdar et al., 1993). The nucleotide sequence in Xcc (hrpXc) shows 84% identity with hrpXo from X. oryzae pv. oryzae (Oku et aI., 1995). There is a 48.7% identity in nucleotide sequence between hrpX and the hrpB gene of Ralstonia (Burkholderia or Pseudomonas solanacearum ) solanacearum and a 35.8% amino acid sequence identity (45.96 similarity) between the predicted protein products of these genes (Genin et al., 1992; Oku, et al., 1995). Sequence similarity to other regulatory proteins, including AraC that regulates the arabinose operon of Escherichia coli and VirF of Yersinia enterocolitica indicate that the hrpXc gene may produce a transient protein involved in gene regulation. A putative DNA- binding domain (helix-turn-helix) present in the carboxyl terminal half of the HrpX protein is highly conserved among HrpB, AraC and VirF. The VirF protein of Y. enterocolitica is a transcriptional activator of the Yersinia virulence regulon (Cornelis et aI., 1989); and HrpXc by extension, may also function as a transcriptional activator of virulence genes of Xcc. Thus, hrpX likely encodes a protein that regulates genes involved in pathogenicity, suppression of host defense mechanisms and recognition by non-host plants (Oku et al., 1998).
A hrpX-counterpart gene, hrpXv, was found in X. campestris pv. vesicatoria (Wengelnik and Bonas, 1996). The predicted gene products, HrpXc, HrpXo and HrpXv, are nearly identical in amino acid sequence (Oku et aI., 1995; Wengelnik and Bonas, 1996). hrpX was recently shown to be widely conserved in xanthomonads (Oku et aI., 1998). Primers were selected from the sequence of the 1.4 Kb fragment of hrpXo and used to amplify genomic DNA from sixteen X. campestris pathovars. Each PCR-amplified DNA fragment was then probed with an internal DNA segment of hrpXo (Oku et aI., 1998). The presence of the hrpXo fragment was verified by Southern hybridization analysis, and all tested strains from the sixteen pathovars hybridized to the 1.4 kb PCR-amplified DNA.
Although highly conserved among Xanthomonas species, hrpX is not part of the larger 25 kb hrp cluster described for X. campestris pv. vesicatoria (Bonas et aI., 1991), which is homologous to the 19 kb DNA region of the R. solanacearum cluster (Boucher et aI., 1987). The conserved hrp genes found in Erwinia, Pseudomonas, and Xanthomonas appear to be related to pathogenicity genes of Yersinia and Shigella involved in type III secretion of proteinaceous virulence factors (Van Gijsegem et aI., 1993). Many X. campestris pathovars, including Xcc contain DNA which hybridizes to the large
A Case Study: Black rot o/Crucifers 35
hrp cluster of R. solanacearum (Arlat et aI., 1991). Mutagenesis of the corresponding regions in Xcc resulted in strains defective in both pathogenicity and HR induction, but hrp· mutants produced wild-type levels of extracellular enzyme activities and EPS (Arlat et al., 1991). Thus, there are two sets of pathogenicity genes, the large hrp cluster and the smaller 1.4 kb unlinked hrpXc locus associated both with pathogenicity and EPS production (Kamoun and Kado, 1990a, b). In R. solanacearum, the HrpB protein is a positive regulator of the large hrp gene cluster (Van Gijsegem et al., 1994). Similarly, the HrpXc homologue, HrpXv (also with sequence similarity to HrpB), is a positive regulator of five hrp loci (hrpB to hrpF) of the large hrp cluster of X. campestris pv.
vesicatoria (Wengelnik and Bonas, 1996). Yet another hrp gene, hrp G, functions at the top of the hrp gene regulatory cascade of X. campestris pv. vesicatoria, positively regulating the hrp A and hrpXv genes (Wengelnik et al., 1996).
The induction of hrp genes probably helps the bacterium to become established in the plant and exploit its host more efficiently as the pathogen responds to the physical and nutritional changes in the microenvironment and shifts from epiphytic to endophytic growth (Arlat et aI., 1991). Starvation appears to be a major factor in stimulating hrp-gene expression in Xcc (Dow and Daniels, 1994). Transcription of hrp genes of Xcc is repressed in rich media, as are hrp genes of R. solanacearum (Arlat et aI., 1991). Peptone, yeast extract, and casarnino acids strongly repressed the expression of hrp genes, whereas sucrose, glutamate, and glycerol gave the highest levels of hrp gene expression (Arlat et al., 1991). Sucrose and methionine were needed for efficient induction of hrp genes, and a medium low in phosphate favored induction (Fenselau and Bonas, 1995). McElhaney, et al. (1998) found that high levels of ammonium sulphate, ammonium nitrate or potassium nitrate applied to roots of cabbage seedlings repressed both colonization of Xcc in the vascular system and symptom expression in six-week-old plants, whereas nitrogen-deficient plants developed symptoms in the usual 7-10 days after inoculation. Collectively, these observations emphasize that the bacterial response to a changing nutritional environment is critical in the successful establishment of infection in compatible hosts.
The "phenotypic switch" in Xcc (Kamoun and Kado, 1990b) from a nonchemotactic, mucoid strain to a chemotactic and nonmucoid derivative was also observed for Xcc by Martinez-Salazar et al. (1993), leading them to question whether the phenotypic switch is recA-dependent. The RecA protein is involved in genetic rearrangements in other pathogens and was isolated and characterized for Xcc (Lee et aI., 1996; Martinez et al., 1997). Although some recA mutants showed decreased virulence in cabbage, the recA mutation was not related to a genetic rearrangement that affects chemotaxis and xanthan production, and decreased virulence of recA mutants was attributed to an effect independent of the switch to chemotaxis (Martinez et al., 1997). Using a different approach, Poplawsky and Chun (1995, 1997) demonstrated that regulation of EPS synthesis in Xcc is determined by diffusible factors, one of which also regulates xanthomonadin production (Poplawsky et aI., 1998). (See section III.B.5).
2. Other genes for EPS biosynthesis
In addition to its association with the hrpX locus, EPS biosynthesis has been associated with other gene clusters. Mutations which result in loss of EPS production have been mapped to at least four regions of the chromosome (Barrere et al., 1986; Harding et