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, Uniersity of Florida, Gainsille, 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 (ar) 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 ar 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 ar 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 ar 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 Press206 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 ar 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 ar 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 ar 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 ar 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 ar 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 ar 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 ar 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 ar genes was a misnomer ; in R gene for ar 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 ar 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 (ir) genes of Agrobacterium
in the species. Since then, many avirulence (ar, following tumefaciens, formal use of the term ‘‘ virulence ’’ as
bacterial nomenclature) genes (over 40) have been cloned synonymous with ‘‘ pathogenicity ’’ is incorrect. The ir
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, theseAirulence genes 207
would be called pathogenicity genes, since these ir 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 arBs3, the first member
used to refer to cultivar-specific situations, keeping in cloned of the arBs3\pthA gene family. This gene, which
mind that there are no known ‘‘ ir ’’ genes by this was isolated as an ar 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 ar 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 ar genes, and the value, for example copper resistance. Copper sprays are
explanations are not mutually exclusive. First, some ar widely used in some areas of the country, and copper
genes have selective value in terms of pathogenicity resistance has been found linked to ar 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 ar genes. Redundancy through duplication is
R gene. It is important to emphasize that the patho- especially indicated for members of the arBs3\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 arBs3\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, arA and arE 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 ar 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 ar 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, ar genes with a confirmed or suspected genicity function(s) of ar 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 ar 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 eents, and particularly horizontal gene transfer
analyses is the discovery that both prokaryotic and
Another explanation for the enigma is that many ar 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 ar 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 ar 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 ar 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 ar maladapted ar 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 ar 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 ar 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 ar 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 ar 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 ar 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 ar\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 derie 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 necrotrophicAirulence 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 ar 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. amyloora, 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. amyloora (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 arBs3\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 moe 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 ar genes, whereas how do they cause such diverse diseases ? Since most of the
biotrophs do. Unlike harpins, the protein products of ar 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 ar 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 ? arb6 and arb7 (water soaking and blight) of X. campestris
Several Xanthomonas and P. syringae ar genes elicit an pv. malvacearum. These are all bona fide, gene-for-gene ar
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
arB from P. syringae pv. glycinea and arRpt2 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 ar 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, arb6 and other members
caused by the pathogen : division, enlargement and death of the arBs3\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 ar genes cloned and sequenced to date : the infection units in quantity on the leaf surface would have
Xanthomonas arBs3\pthA gene family [22, 42 ]. Members of epidemiological significance. Therefore the host–specific
the gene family, all of which are gene-for-gene ar 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, arb6 and arBs3) [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 theseAirulence 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 ar\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 ar genes, including pthA, but with negative
or no phenotype at all. Like arBs3 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 ar\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 arBs3\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 ar 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 ar 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 ar 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 ar genes have been identified, and the majority
protein signals must interact with plant receptors. A appear to be dispensable or gratuitous. Many ar 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 ar genes
been demonstrated [64 ]. The specificity of the arBs3\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 ar 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.
arb6 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 ar\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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