Why do pathogens carry avirulence genes ?

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Physiological and Molecular Plant Pathology (1999) 55, 205–214
Article No. pmpp.1999.0230, available online at http:\\www.idealibrary.com on

                                                     COMMENTARY

                         Why do pathogens carry avirulence genes ?
                                                      D. W. G A B R I E L
  Plant Molecular and Cell Biology Program, Plant Pathology Department, UniŠersity of Florida, GainsŠille, FL 32611-0680,
                                                         U.S.A.

                                              (Accepted for publication June 1999)

           In the gene-for-gene hypothesis, Flor [19 ] proposed the existence of avirulence alleles of virulence genes,
           and that the virulence genes actively conditioned cultivar-specific virulence on hosts. Yet cultivar-specific
           virulence genes have not been found, and many of the avirulence (aŠr) genes cloned to date (over 40) do
           not appear to condition pathogenicity in general, virulence or anything else of value to the microbe. There
           is strong indirect evidence that many, if not nearly all, Avr proteins are secreted from pathogenic bacteria,
           enter plant cells and signal a response phenotype, usually associated with a hypersensitive defense response
           (HR). Why the bother for avirulence ?
              Evidence is accumulating that most aŠr genes are, or once were, pathogenicity ( pth) genes found in
           biotrophic pathogens that determine (d ) host range, not in a cultivar-specific manner, but in a host species-
           specific manner. At least some of these genes appear to function for pathogenicity by encoding protein
           signals that are ‘‘ injected ’’ into plant cells by the hrp system, resulting in programmed host cell death, a
           characteristic normally associated with necrotrophs. A growing body of evidence indicates that most
           microbial genes conditioning pathogenicity, including the hrp, pth and aŠr genes, are present because of
           horizontal gene transfer, often involving movement of large gene clusters on ‘‘ pathogenicity islands ’’.
           Since horizontal transfer is a stochastic process, many aŠr genes are likely to be maladapted pth genes,
           following their horizontal transfer to strains in which their function may be either gratuitous or
           detrimental. The structure of some of these genes may allow rapid adaptive selection for pathogenic
           function.                                                                              # 1999 Academic Press

           Keywords : avirulence ; avr ; pathogenicity ; virulence ; harpins ; hrp ; pathogens ; gene-for-gene ;
           Xanthomonas ; Pseudomonas ; Ralstonia ; Erwinia ; horizontal transfer ; biotroph ; necrotroph.

                   INTRODUCTION                                    now being addressed experimentally and new paradigms
                                                                   are emerging to explain the origins of new disease
Plant breeders look for genes by which plants can defend           epidemics and to better control plant disease.
themselves against microbial attack, and microbial
pathologists look for genes in microbes that determine
virulence. The breeding work resulted in a large number
                                                                        CLASSICAL TERMINOLOGY, NEO-
of studies documenting intraspecific variation in plant
                                                                     DARWINISM AND THE AVIRULENCE GENE
resistance, and the classical microbiology research resulted
                                                                                  DILEMMA
in more limited studies on intraspecific variation in
microbial virulence. All classical genetic analyses were by        Naturally-occurring variation in degree of susceptibility
necessity limited to intraspecific variation analysed by           or resistance in a particular host species to a particular
meiotic recombination. Until the advent of molecular               pathogen has been shown to be controlled, in nearly all
genetics, a comprehensive and systematic approach for              cases, by single resistance (R) genes. R genes trigger
understanding why certain plant species and not others             programmed plant defense responses, including cell death
are hosts of certain pathogens, and how microbes become            [21, 11, 75 ], most often resulting in the appearance of a
pathogens in the first place, was impossible. Is it due to a       plant HR. Naturally-occurring variation in the phenotype
failure of resistance (pathogen recognition) or the acqui-         of microbial virulence on crop plants was also found to be
sition of pathogenicity genes ? Can saprophytes become             determined by single genes. Harold Flor’s classic papers
pathogens quickly, and can pathogens change host range             [19, 20 ] using flax rust (Melampsora lini) demonstrated
or become much more pathogenic quickly, or do these                that ‘‘ pairs of factors ’’ (specific avirulence and virulence
events require complex combinations of adaptations,                alleles) in the pathogen determined virulence against each
requiring long evolutionary periods ? Such questions are           particular host (cultivar-specific) R gene, thus establishing
0885–5765\99\100205j10 $30.00\0                                                                            # 1999 Academic Press
206                                                    D. W. Gabriel
the gene-for-gene hypothesis. The dilemma posed by the                                                    Host cultivar with
concept of dominant avirulence genes is implicit in Flor’s
                                                                                                           R1–          r1r1
writing. Although Flor first used the designation A to
                                                          "
refer to the dominant avirulent allele of the ‘‘ pair of rust

                                                                            Pathogen strain with
conditioning factors ’’ specific for the cognate host gene R ,
                                                             "                                     avr1     –            +
his emphasis was always on the recessive virulence alleles
as being the active determinants of virulence and
pathogenicity. Compounding the conceptual dilemma,
Flor also demonstrated that avirulence\R gene inter-                                                ∅       +            +
actions were phenotypically epistatic over any and all
virulence\susceptibility gene interactions.
   Since that time, many ‘‘ pairs of factors ’’ (still usually   F. 1. The gene-for-gene ‘ quadratic check ’, as observed with
described as virulence genes) were discovered by classical       phytopathogenic bacteria. The host R gene is genetically
                                                                                                                "
genetic analyses, and the gene-for-gene hypothesis was           specific for the pathogen aŠr gene, usually resulting in a
                                                                                                     "
                                                                 hypersensitive defense response, indicated as an incompatible
demonstrated or suggested in many, primarily biotrophic,
                                                                 (k) interaction. Cultivar-specific avirulence appears to be
parasite\host systems [12 ]. Despite the fact that avirulence    superimposed on a basic pathogenic ability (on hosts) and
alleles were dominant and epistatic, Ellingboe’s idea that       results in a compatible (j) interaction. The pathogen may have
pathogens carried a number of genes where the active             strains with an alternate aŠr allele, but molecular data from
allele conditioned avirulence ‘‘ superimposed upon a basic       phytopathogenic bacteria indicate that this is not necessarily the
                                                                 case (refer text). The shaded area is to emphasize the fact that
compatibility between host and parasite ’’ [17 ], while the
                                                                 the alternate allele, called the ‘‘ virulence ’’ allele and assumed to
inactive allele allowed (but did not condition) the virulent     play a central role to condition virulence in classical genetic
state was not widely accepted until the first avirulence                               analyses, may not exist.
genes were cloned in the 1980s. The primary objections
raised were based on neo-Darwinian selection arguments :         disruption experiments of bacterial aŠr genes in several
there could not be selection for avirulence, therefore the       labs failed to reveal any effect on growth or pathogenicity
genes must function for virulence. For example, Person &         of the microbial strain on (or off) hosts, other than the
Mayo writes : ‘‘ Although avirulence usually segregates as       expected loss of R gene-specific avirulence. That is, the
a dominant character, microevolutionary history suggests         majority of bacterial aŠr genes appear to be dispensable.
that in its primary function an A gene associates not with       Although only a few fungal avirulence genes have been
avirulence but with virulence …’’ [50 ]. In the only and         cloned to date [38 ], similar data emerges. With many
classic textbook on the subject, Day’s ‘‘ Genetics of Host-      microbial aŠr genes, there is no evidence for alleles or
Parasite Interactions ’’, avirulence as a phenotype is           analogues that condition anything of value to the
explicitly compared to microbial auxotrophy and to               pathogen. Such aŠr genes appear to be not only
sensitivity to a poison [12 ]. In this widely accepted view,     dispensable, but also gratuitous. At the very least, cultivar-
the avirulent pathogen must be lacking something a               specific virulence in many gene-for-gene interactions is
virulent pathogen has, exactly analogous to lacking an           determined by the mere absence of aŠr genes ; viru-
appropriate allele to grow on minimal medium or to               lence alleles are not necessary for cultivar-specific
break down a poison.                                             virulence (Fig. 1).
   The cloning of the first avirulence gene by Staskawicz            It became clear that use of the term ‘‘ virulence gene ’’
et al. [60 ] from a readily cultured bacterial pathogen          to refer to the hypothetical alternate allele of cultivar-
(Pseudomonas syringae), overcame the objections caused by        specific aŠr genes was a misnomer ; in R gene for aŠr gene
the difficulty of accepting the idea that microbial              interactions, a ‘‘ virulence ’’ allele often does not exist, and
pathogens carry genes that primarily and simply function         if it does, does not condition the cultivar-specific state it
to limit their virulence. One of the hallmarks of that paper     describes. Unfortunately, there is considerable confusion
was the deliberate search not only for an avirulence gene,       in the literature brought about by use of the term
but also for a virulence gene sensu Flor, a gene that            ‘‘ virulence ’’ as synonymous with ‘‘ pathogenicity ’’. For
actively conditioned virulence against a specific resistant      example, in their review of the terms virulence and
host cultivar. Cultivar-specific virulence genes were not        pathogenicity, Shaner et al. [56 ] recount that three well-
found. In fact, not even a DNA fragment hybridizing to           known scientists gave three different definitions of the
the aŠr gene was found in other P. syringae strains in that      term ‘‘ virulence ’’ at a 1989 international congress. With
study that might have indicated a corresponding ‘‘ allele ’’     the exception of the virulence (Šir) genes of Agrobacterium
in the species. Since then, many avirulence (aŠr, following      tumefaciens, formal use of the term ‘‘ virulence ’’ as
bacterial nomenclature) genes (over 40) have been cloned         synonymous with ‘‘ pathogenicity ’’ is incorrect. The Šir
from a variety of different bacterial plant pathogens            genes of A. tumefaciens control pathogenicity and host
[20, 42, 70 ] with similar results. Furthermore, many gene       range, but in other plant pathogenic microbes, these
AŠirulence genes                                                207
would be called pathogenicity genes, since these Šir genes         compestris pv. malvacearum ? The result is not the
are not cultivar-specific. The term ‘‘ pathogenicity ’’ is         production of necrotic cankers on cotton, but instead is
generally used with reference to genes that function to            gene-for-gene avirulence and elicitation of the HR on
condition that state, and this terminology will be used            cotton [63 ]. One probable example of a natural horizontal
throughout this paper. The term ‘‘ virulence ’’ will only be       gene transfer scenario involves aŠrBs3, the first member
used to refer to cultivar-specific situations, keeping in          cloned of the aŠrBs3\pthA gene family. This gene, which
mind that there are no known ‘‘ Šir ’’ genes by this               was isolated as an aŠr gene from an X. campestris pv.
definition.                                                        vesicatoria strain and is evidently not needed for
                                                                   pathogenicity on any host by any strain of this pathovar,
   WHY ARE THERE SO MANY avr GENES ?                               resides on a self-mobilizing plasmid [7 ].
                                                                      Interspecific and intergeneric transfers of aŠr genes
Some avr genes are pth genes                                       could occur simply because of coincidental linkage with
There appear to be several valid explanations for the              another factor on the same plasmid that has selective
enigma of why pathogens carry aŠr genes, and the                   value, for example copper resistance. Copper sprays are
explanations are not mutually exclusive. First, some aŠr           widely used in some areas of the country, and copper
genes have selective value in terms of pathogenicity               resistance has been found linked to aŠr genes on an X.
[22, 38, 42 ]. That is, they function pleiotropically to           campestris pv. vesicatoria self mobilizing plasmid recovered
condition pathogenicity on all hosts tested (usually with          from strains in Florida [59 ].
increased pathogen growth in planta) in the absence of a              Other stochastic events may account for the presence of
cognate (‘‘ recognized ’’ in a formal, gene-for-gene sense)        dispensable aŠr genes. Redundancy through duplication is
R gene. It is important to emphasize that the patho-               especially indicated for members of the aŠrBs3\pthA gene
genicity, as opposed to the avirulence, encoded by these           family. Sixty-two bp terminal inverted repeats mark the
genes, appears from limited data to include entire host            boundaries of homology among all members of this gene
species. For example, pthA, the first member of the                family, and the terminal 38 bp of these inverted repeats
Xanthomonas aŠrBs3\pthA gene family to be recognized as            are highly similar to the 38 bp consensus terminal sequence
being essential for pathogenicity, is required for the             of the Tn3 family of transposons [14 ]. It is therefore
production of necrotic cankers on all species of citrus            possible that these genes can, or once could, transpose. A
attacked by X. citri [62 ]. Similarly, but to a quantitatively     different stochastic event might be loss of host range on a
lesser extent than pthA on citrus, aŠrA and aŠrE of                particular wild plant species due to mutation and
Pseudomonas syringae pv. tomato are important for patho-           adaptation to new plant species, leaving behind functional
genicity on tomato [44 ]. Some aŠr genes, such as many of          but unnecessary genes that once assisted in conditioning
those found in X. campestris pv. malvacearum, do not               pathogenicity on the original plant species. Evidence for
exhibit obvious selective value when examined indi-                nonfunctional relics of aŠr genes in both Pseudomonas and
vidually, but when studied together, exhibit weak,                 Xanthomonas have been reported, for example, [39, 76 ]. Of
additive and synergistic effects on pathogenicity on cotton        course, experiments designed to reveal potential patho-
[76 ]. Altogether, aŠr genes with a confirmed or suspected         genicity function(s) of aŠr genes are conducted on known
pleiotropic selective value on susceptible host species may        hosts of the pathogen in which the genes currently reside.
account for about a third of the total cloned and                     Recent advances in our analyses of microbial genomes
investigated to date. Yet it remains striking that the             has revealed that they are much more fluid than previously
majority of aŠr genes cloned and reported to date appear           thought, and horizontal gene transfer between species is
to be dispensable.                                                 thought to occur to a greater extent than generally
                                                                   assumed [57 ]. In fact, one of the primary surprises to
                                                                   come as a result of recent extensive genomic sequencing
Stochastic eŠents, and particularly horizontal gene transfer
                                                                   analyses is the discovery that both prokaryotic and
Another explanation for the enigma is that many aŠr genes          eukaryotic genomes are chimeras, with large blocks of
may be present because of a variety of stochastic events,          genes from a variety of sources, sometimes even mixtures
including horizontal gene transfer, duplication and                from prokaryotic and eukaryotic sources [41 ]. It seems
mutation. Horizontal gene transfer is becoming recognized          that horizontal transfer of clusters of genes conferring
as a major epidemiological factor in new disease outbreaks         adaptive phenotypes in bacteria is the rule, not the
[48 ], and there is good reason to believe that many aŠr           exception [53 ]. Horizontal transfer of gene clusters is not
genes are gratuitously present in a variety of bacterial           limited to prokaryotes : horizontal transfer of an entire
strains following horizontal transfer from other species.          supernumerary chromosome has been demonstrated in
For example, the % GC content of every one of the aŠr              Colletotricum [30 ] ; supernumerary chromosomes are known
genes cloned from P. syringae is low for the species [70 ].        to carry genes that condition growth and\or pathogenesis
What if pthA from X. citri is moved by conjugation into X.         in some filamentous plant pathogenic fungi [10, 67 ].
208                                                   D. W. Gabriel
   Evidence for the horizontal transfer of many aŠr genes       long and complex series of incremental improvements in
is indicated by their function (avirulence), structural         pathogenicity [32 ] as is commonly assumed, but rather
features, % GC content, residency on plasmids, lack of          could be achieved in a single horizontal transfer events
conservation within pathogenic groups and the fact that         (acquisition of a pathogenicity island or two) followed by
so many appear to be dispensable. However, as several           a ‘‘ clean up ’’ operation, involving mutational shedding of
authors have noted, the fact that nearly all bacterial aŠr      maladapted aŠr genes that are on the island.
genes require the specific action of the hrp genes (discussed
below) in order to express the avirulence phenotype, leads
                                                                 WHAT DO avr GENES DO BIOCHEMICALLY ?
back to the conclusion that most of these genes must have
originally been elaborated for pathogenic purposes.             The capacity of aŠr genes to trigger programmed cell
   Even the hrp system itself seems to be acquired by           death (PCD) and defense responses, plus their dependence
horizontal gene transfer. The function of most hrp genes is     on the hrp secretion system, indicates that most aŠr genes
to provide for a specialized protein secretion system called    encode plant cell signal proteins that are perceived by
type III [31, 68 ]. This type III secretion system is a         receptors and trigger signal transduction pathways
generally conserved mechanism found in both plant and           [8, 11, 69, 75 ]. The hypothesis that aŠr genes either are, or
animal pathogens for the export, secretion and often            once were, pth genes indicates that pathogenicity function
delivery of specific proteinaceous effector molecules (viru-    is also by way of plant cell signal proteins that are
lence or pathogenicity factors) directly and\or indirectly      perceived by receptors and trigger signal transduction
into host cells in a contact-dependent manner [24, 31, 46 ].    pathways. The fact that the defense responses triggered by
The Salmonella typhimurium secretion system appears to form     aŠr genes appear to be programmed responses strongly
a hollow, needle-like structure [40 ], that functions as a      indicates that, if they also have a pathogenic purpose, it
toxic protein injection system [58 ]. The hrp pili may form     must be to trigger (a) programmed event(s) in the plant
channels through which macromolecules travel to reach           cell other than defense. Since aŠr genes are found primarily
the plant cell cytoplasm [43 ]. Interestingly, widely diverse   in those pathogens thought to be biotrophic, their purpose
bacteria probably acquire the entire export\injection           must be related to the pathogenicity lifestyle unique to
system on a ‘‘ pathogenicity island ’’ in a single horizontal   biotrophs.
gene transfer event [48 ]. The evidence for this is that the
genes encoding these protein secretion systems in various
                                                                 WHAT IS REQUIRED FOR PATHOGENICITY ?
animal and plant pathogens are all clustered and the
codon usage and % GC content of the genes encoding              Once it is realized that aŠr\pth genes encode signals
these type III systems are typically different from that of     affecting plant cell programs, it is obvious to ask what
the rest of the genome.                                         kinds of programs might enhance a biotrophic pathogenic
   Pathogenicity islands are not limited to phytopatho-         lifestyle. This involves a consideration of two related
genic bacteria. A pathogenicity island consisting of at         questions : What adaptations are required on the part of
least six genes and differing in codon usage and % GC           microbes to become biotrophic pathogens, and what is
content from that of other chromosomal genes in the             required by a biotroph to attack a specific plant species,
fungal pea pathogen, Nectria haematococca, has been found       i.e. to make the species a host ? Biotrophic pathogens,
on a dispensable, supernumerary chromosome [37 ].               whether of plants or animals, appear able to cause
Interestingly, the genes of this cluster appear to be           changes in host cell functions that increase both the
involved in both offense and defense : some function to         pathogen’s ability to reproduce and to survive. Ability to
condition pathogenicity by killing host cells, while others     reproduce involves deriving nutrition from plants, con-
function to degrade pisatin, a phytoalexin elicited in          sidered in the next section, and ability to survive involves
response to the fungal attack [29 ]. Also found on the same     ability to move to new infection sites, considered in the
dispensable chromosome a gene allowing utilization of           following section.
homoserine as a sole carbon and nitrogen source [54 ].
Homoserine is one of the nutrients found in relative
                                                                Ability to deriŠe nutrition
abundance in pea root exudates and also released from
dying pea cells. As mentioned earlier, at least one fungal      The essential difference between parasites and saprophytes
supernumerary chromosome is known to be capable of              is that parasites derive nutrition from a living plant at its
horizontal transfer [30 ] ; the large number of genes related   expense. The essential difference between pathogens and
to pathogenicity found on the N. haematococca chromosome        parasites is that pathogens cause overt disease symptoms.
indicates that many, if not all of the tools required for       In many cases, the disease symptoms appear to directly
pathogenicity and host range might be transferred to a          favour the pathogen, at least in terms of pathogen
nonpathogenic saprophyte in a single event. Evolution           dispersal. Plant pathogens have traditionally been roughly
from saprophyte to pathogen might not always involve a          divided into two groups : those which are necrotrophic
AŠirulence genes                                                   209
and those which are biotrophic. The distinction is thought          chrysanthemi (soft rot) [6 ], and are also found in phyto-
to be useful in determining the primary means by which              pathogenic ralstonias and pseudomonads [1 ]. Their role
nutrition is derived. For example, necrotrophs are easy to          in the latter two genera is unclear [1, 2 ]. The fact that
culture, produce abundant degradative enzymes and                   harpins cause alkalinization of the apoplast, sucrose efflux
toxins, tend to have limited host cell contact, hardly              and death of host cells indicates a primary role in
stimulate host protein synthesis, and cause early necrosis,         nutrition acquisition. Although individual and double
seemingly in advance of colonization. By contrast,                  harpin gene knockout mutations in P. syringae did not
biotrophs are often difficult to culture, produce few               affect pathogenicity, P. syringae appears to produce
degrading enzymes or toxins that are important in                   multiple harpins [9 ], which might compensate the loss of
pathogenicity, have extensive contact with living host              one or more (but not all) of the genes. Do even the more
cells, greatly stimulate host protein synthesis and cause           biotrophic pathogens, such as P. syringae, have to kill cells
necrosis only after extensive colonization [35 ].                   for nutrition ? The surprising answer seems to be ‘‘ yes ’’ ;
   Most pathogenic bacterial biotrophs kill plant cells and         growth of P. syringae pv. tomato in tomato plants is
thereby derive major nutritional benefits. An exception is          significantly inhibited in plants in which PCD is inhibited
Agrobacterium, which ‘‘ engineers ’’ its hosts to produce           [25, 26 ].
novel compounds that only it can catabolize [36 ]. Note                It is possible that acquisition of nutrients from living
that A. tumefaciens does not utilize type III secretion as a        cells by P. syringae is a two stage process : one stage that
primary means of pathogenicity and that there are no aŠr            can involve harpins and a later stage that must involve
genes in A. tumefaciens. Another possible exception is              PCD. Some nutrition can be derived from plant cells by
Rhizobium, which can be pathogenic under high nitrogen              way of harpins without killing plant cells via an exchange
conditions, but which normally confers an advantage to              reaction (XR) that results in sucrose leakage from the
its hosts, and is more properly considered as a mutualistic         plant cell. P. syringae induces an XR and increased efflux
parasite. As a general rule, the essential difference between       of sucrose from host cells within 2–4 h after inoculation
necrotrophs and biotrophs that kill host cells is that              [4 ]. This XR exchange precedes onset of P. syringae
necrotrophs kill at a distance, while pathogenic biotrophs          population growth [5 ], which strongly indicates that it is
kill after intimate cellular contact. Both types of pathogen        a required step for growth.
derive nutritional benefits from dead or dying cells.                  An XR reaction (and resulting nutrient efflux) is also
Interestingly, it is the contact-dependent hrp system that          induced by Xanthomonas, and possibly by way of (a)
provides the ability for biotrophs to kill both host and            completely different mechanism(s). No harpins have been
nonhost cells.                                                      reported to date from Xanthomonas, and by contrast with
   Of all phytopathogenic bacteria that require hrp genes,          induction in a matter of hours by Pseudomonas and Erwinia,
members of the genus Xanthomonas are by far the most                the XR reaction induced by Xanthomonas campestris pv.
biotrophic. Unlike Pseudomonas, Ralstonia and Erwinia,              malvacearum (bacterial blight of cotton) does not occur
members of the genus Xanthomonas are all plant-associated,          until 1–2 days after inoculation [65 ]. Since Xanthomonas is
nearly all growth is endophytic, and each strain is highly          well into logarithmic population growth by this time [49 ],
host-specific. In fact, among prokaryotic plant pathogens,          the XR does not appear to precede the onset of population
Xanthomonas exhibits by far the highest known level of              growth. The Xanthomonas XR may be induced by
plant specialization, with over 120 pathovars. The second           ammonium ion accumulation in the intercellular space of
most biotrophic group is P. syringae, with over 40 pathovars        the apoplast [65 ]. Ammonium ions do not kill green plant
and with strains typically exhibiting less host specificity         cells grown in light, because they are rapidly recycled to
than a typical xanthomonad. Is there a significant                  amino acids by way of the GS-GOGAT pathway. It is
difference in the nature of the effector molecules secreted         reasonable to assume that the nutrient release induced by
by Xanthomonas and P. syringae strains that reflects a more         the XR contributes to endophytic growth of Xanthomonas.
biotrophic lifestyle ?                                              However, as with P. syringae, it is unclear if the XR is
   It is clear that one of the most significant classes of effect   essential for early stage growth of Xanthomonas. There are
or molecule delivered by type III secretion is the harpins.         also at least two other potential mechanisms that may
Harpins are glycine rich, cysteine-lacking proteins that            cause nutrient efflux at an early stage of infection :
are secreted in culture media under appropriate growth              extracellular proteases [15 ] and additional effectors
conditions. Externally applied harpins from E. amyloŠora,           secreted by the hrp system.
E. chrysanthemi, R. solanacearum and P. syringae have the              It seems likely that there are effector molecules secreted
capacity to elicit a rapid K+ efflux and H+ influx exchange         by the Xanthomonas hrp system that are not yet discovered.
reaction (XR) that results in the alkalinization of the             X. campestris pv. malvacearum is found close to host cell
apoplast, sucrose leakage and also PCD [1 ]. Harpins are            walls as early as 12 h after inoculation [3 ] and in contact
therefore toxin-like. Harpins are clearly important patho-          with host cell walls by 24 h after inoculation [49 ]. Thus,
genicity factors for E. amyloŠora (fire blight) [71 ] and E.        there is the potential for hrp system involvement in
210                                                    D. W. Gabriel
pathogenesis even before the XR. Not all xanthomonads            have been convincingly shown to be sufficient for the
kill host cells [45 ], however, the xanthomonads that cause      resistance observed in cotton against X. campestris pv.
the major disease problems (cankers, blights and rots) do        malvacearum [52 ]. However, on susceptible cotton, host
kill host cells. For example, cotton blight causes ves-          cell death occurs without the production of phytoalexins
iculation between the plasmalemma and cell wall by 48 h          [49 ]. This host cell death on susceptible cotton is caused
after inoculation, and major degenerative changes re-            by multiple members of the aŠrBs3\pthA gene family in X.
sulting in cell death occur over the next 3 days [3 ]. Rapid     campestris pv. malvacearum [76 ]. As discussed above, pthA
host cell death per se does not inhibit X. campestris pv.        appears to cause PCD, and it is likely that other members
malvacearum growth in cotton, since plants held in               of the gene family that function for pathogenicity also
continuous darkness became necrotic, while permitting            cause PCD. In fact, there is strong evidence that the
logarithmic bacterial growth for 5 days [49 ].                   oxidative burst and initiation of the HR are independent
   The ability of a pathogen to produce an externally            of PCD in a number of systems [27, 33, 51 ].
applied, toxic compound that results in cell death is a
characteristic of necrotrophs : they kill in advance of
                                                                 Ability to moŠe to new infection sites
colonization in order to obtain nutrition. If P. syringae
strains do not use harpins to kill plant cells and Xanthomonas   Since there is a common export mechanism for PCD
strains do not even have harpins, what do they use ?             signals shared by animal and plant bacterial pathogens,
Necrotrophs do not carry gene-for-gene aŠr genes, whereas        how do they cause such diverse diseases ? Since most of the
biotrophs do. Unlike harpins, the protein products of aŠr        type III secretion system genes are involved in the export
genes fail to elicit any symptoms at all when artificially       machinery per se, the answer may again lie in the effector
introduced or infiltrated as purified or crude extracts into     molecules. The results of disease phenotype screens with
the spongy mesophyll of plants. Could some aŠr genes,            several xanthomonads yielded only a relatively few genes
functioning pleiotropically in a pathogenic role, be             that determine specific disease phenotypes [21, 23 ].
injected into plant cells and signal susceptible hosts to give   Examples are pthA of X. citri (hyperplastic cankers) and
up nutrients by causing PCD ?                                    aŠrb6 and aŠrb7 (water soaking and blight) of X. campestris
   Several Xanthomonas and P. syringae aŠr genes elicit an       pv. malvacearum. These are all bona fide, gene-for-gene aŠr
HR when expressed inside plant cells carrying specific R         genes.
genes [13, 16, 28, 55, 61, 64, 66 ]. More importantly, both         The PthA protein signals citrus plant cells to divide. In
aŠrB from P. syringae pv. glycinea and aŠrRpt2 from P.           a natural infection, enough plant cells are affected by X.
syringae pv. tomato cause cell death and necrosis when           citri to result in tissue hyperplasia, eventually resulting in
expressed in plants lacking cognate R genes, and                 rupturing the leaf epidermis and allowing egress of the
suggesting a possible role for these genes in pathogenicity      canker pathogen to the leaf surface. This is important
[28, 47 ]. A role for a known pathogenicity determinant,         because the pathogen is spread by wind-blown rain. A
pthA from X. citri, in programmed killing of host cells were     similar story emerges from studies of some other aŠr genes,
recently established. Expression of pthA in citrus cells         affecting other pathogenicity phenotypes (reviewed in
was sufficient to cause symptoms diagnostic of the disease       [22, 42 ]). In a well studied case, aŠrb6 and other members
caused by the pathogen : division, enlargement and death         of the aŠrBs3\pthA gene family found in X. campestris pv.
of host cells [16 ]. Since PthA is not a toxin and does not      malvacearum cause water soaked, necrotic lesions on
appear to be an enzyme, the cell death that is signalled         cotton, a phenotype which results in 1600 times more
must be programmed. Furthermore, the protein signal is           bacteria available on the leaf surface than if the genes
specific for citrus.                                             were not present [76 ]. Once again, since this pathogen is
   As mentioned earlier, pthA is a member of the largest         also primarily spread by wind-blown rain, availability of
group of aŠr genes cloned and sequenced to date : the            infection units in quantity on the leaf surface would have
Xanthomonas aŠrBs3\pthA gene family [22, 42 ]. Members of        epidemiological significance. Therefore the host–specific
the gene family, all of which are gene-for-gene aŠr genes,       disease phenotype may serve an epidemiological purpose
are either known or thought be to be required for                independently of multiplication within the host, to enable
pathogenicity of xanthomonads causing cotton blight,             more efficient movement to uninfected hosts.
Asiatic citrus canker, false citrus canker, Mexican lime            The ability to elicit the division of differentiated plant
cancrosis, common bean blight and rice blight [22 ].             cells, the ability to water soak cotton and the ability to
Among other symptoms, the primary disease symptom                elicit PCD are radically different phenotypes. Yet all
elicited in common by those members appears to be PCD.           three phenotypes are determined by genes which are 97 %
Such PCD would likely have to occur without the                  identical in DNA sequence (pthA, aŠrb6 and aŠrBs3) [74 ].
oxidative burst and phytoalexin induction. Evidence to           The dozen or more 102 bp direct tandem repeats found in
support this idea has been found using X. campestris pv.         the middle of these genes are known to facilitate intragenic
malvacearum, causing cotton blight. Cotton phytoalexins          recombination, which generates mutant variants of these
AŠirulence genes                                                   211
genes with altered specificity and with altered patho-          for all biotrophic pathogens that utilize type III secretion
genicity phenotypes [74 ]. Reshuffling of repetitive tandem     as a primary means of pathogenicity. If so, it might not be
repeats has become well recognized as one of several            the race-specific R genes and an active defense response
mechanisms used by organisms to achieve genetic ad-             (so useful in breeding resistant cultivars) that are generally
aptation [57 ]. If one of these aŠr\pth genes moves             responsible for limiting the host range of biotrophic plant
horizontally into a new bacterial pathogenic strain to          pathogens, as has often been suggested. In fact, several
which they were not previously adapted, the most likely         labs have attempted to find evidence for this by mutational
result would be to confer either cultivar-specific avirulence   analysis of aŠr genes, including pthA, but with negative
or no phenotype at all. Like aŠrBs3 in X. campestris pv.        results, reviewed by [22, 42 ]). Rather, it might be a failure
vesicatoria, the gene would be both dispensable and             of the pathogenicity signal to bind an appropriate cognate
gratuitous. However, intragenic or intergenic recom-            plant response receptor (to condition disease) that would
bination might generate a variant aŠr\pth gene with             limit host range. The host range of biotrophic plant
selective value in the pathogen, one that enhanced              pathogenic bacteria would in part be determined by
pathogenicity. Such a change would require that the             possession of a type III protein injection system and
Avr\Pth protein interact not with a resistance factor in        appropriate protein effectors capable of binding receptors
the host, but rather with a ‘‘ susceptibility ’’ factor. The    that trigger host cell death and\or other plant responses
fact that nearly identical genes from the aŠrBs3\pthA gene      useful to the pathogen, such as cell division.
family can cause radically different diseases on plants in
different families indicates that this has already happened
several times and will likely happen again.
                                                                                    CONCLUSIONS
                                                                Plant cells are killed by both necrotrophic and biotrophic
   THE CONVERSE OF R GENES : COGNATE                            pathogens, most likely primarily for nutritional purposes,
      PLANT SUSCEPTIBILITY GENES ?                              but also for dissemination. The bacterial hrp genes, which
                                                                encode a contact-dependent (Type III) secretion system,
There is strong evidence that aŠr genes encode protein          appear to deliver signal proteins directly to plant cells to
signals that are delivered into the plant cell in order to      help condition pathogenicity, and involving host cell
cause disease. Some are ‘‘ intercepted ’’ by R genes and        death. Secreted harpins are utilized by the more necro-
result in an HR, while others are nonfunctional because of      trophic bacterial plant pathogens to kill cells, while the
various stochastic processes, but the original purpose          more biotrophic pathogens appear to utilize protein
appears to be for pathogenicity. These conclusions are          signals encoded by pth genes to kill cells. Some pathogens
based primarily on : (1) the hrp dependency of all aŠr          use both. The limited number of microbial pathogenicity
genes ; (2) the discovery of functional nuclear localization    signals that have been identified implies a limited number
signal (NLS) sequences on some of the genes [21, 73 ] ; (3)     of plant receptors that are responsive to such signals.
the ability of many tested aŠr genes to elicit an HR when       These receptors appear to determine host range and
expressed inside resistant plant cells and (4) the ability of   specific susceptibility to biotrophs.
pthA to elicit all symptoms diagnostic of citrus canker            By contrast with pth genes, a relatively large number of
disease when expressed inside citrus cells [16 ]. Such          microbial aŠr genes have been identified, and the majority
protein signals must interact with plant receptors. A           appear to be dispensable or gratuitous. Many aŠr genes,
direct physical binding of the avirulence protein AvrPto        and even the entire hrp system, appear to have transferred
from P. syringae with the tomato resistance protein Pto has     horizontally among gram negative bacteria. The aŠr genes
been demonstrated [64 ]. The specificity of the aŠrBs3\pthA     that are dispensable require the same contact-dependent
gene family has been shown to reside in the leucine rich        secretion system as pth genes, and the hypothesis is
repeats (LRRs) of the protein products [34, 72 ], and the       advanced that most aŠr genes : (a) are, or once were, host
specificity of 13 alleles of the flax rust resistance gene L    species-specific pth genes ; (b) encode protein signal
was recently demonstrated to reside in the LRRs of the          molecules that must bind plant response receptors in or on
protein products [18 ]. LRRs are thought to be involved in      the plant cell, and (c) often possess a physical structure
direct protein-protein interactions. The fact that pthA and     that enhances recombination and genetic adaptability.
aŠrb6 elicit pathogenic phenotypes specific for the entire      Some of these may be able to evolve new host specificities
range of citrus and cotton hosts, respectively, provides        and pathogenicity phenotypes.
evidence that the receptors in citrus and cotton must be
conserved among respective citrus and cotton hosts, and
that they are specific cognate receptors.
   The idea that aŠr\pth genes encode signal proteins that      I thank Al Ellingboe, Margaret Essenberg and Sheng
bind and activate plant receptors may be generally true         Yang He for valuable insights and perspectives.
212                                                         D. W. Gabriel
                                                                      18. Ellis JG, Lawrence GJ, Luck JE, Dodds PN. 1999.
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