BACKGROUND
plants are continuously subjected to pathogen attack and respond by activating a range of defense mechanisms.
recognition of the pathogen or elicitors derived either from the pathogen or released from the plant cell wall is accompanied with the production of molecular signals including salicylic acid  <cit> , jasmonic acid  <cit>  and ethylene  <cit>  that lead to the induction of defense gene expression.
this in turn results in the accumulation of functionally diverse pathogenesis-related  proteins and metabolites   <cit> .
recognition of the pathogen or elicitors is usually accompanied by the rapid death of the infected cells, known as the hypersensitive response , which limits the access of the pathogen to water and nutrients thereby restricting its growth  <cit> .
hr can be triggered either by non-specific elicitors recognized by plant receptors, or by specific elicitors  genes) recognized by corresponding encoded products of plant resistance  genes  <cit> .
several studies have suggested that plant cell death resulting from the hr is a type of programmed cell death .
plant cells undergoing pcd share a number of characteristic morphological and biochemical features in common with animal cell apoptosis  <cit> .
moreover, cell death with apoptotic features has also been observed in plants susceptible to virulent pathogens  <cit> .
although bryophytes are non-vascular plants and are considered to be primitive among the embryophyta, mosses have been shown to respond to a variety of environmental stimuli and to several common plant growth factors much like vascular plants.
thus, in spite of having diverged from vascular plants approximately  <dig> million years ago  <cit> , mosses are well-suited for the study of fundamental processes in plant biology.
furthermore, mosses have a simple developmental program and a life cycle with a predominant haploid phase which greatly facilitates genetic analysis  <cit> .
physcomitrella patens, a relatively small moss, has recently become a model plant to study plant gene function in that it exhibits high-frequency homologous recombination comparable with that of saccharomyces cerevisiae, enabling the construction of gene knock-outs  <cit> .
the assembled physcomitrella genome has recently been released and full-length cdnas in addition to  <dig>  ests are available in the databases  <cit> .
these advantages together with the presence of a great number of physcomitrella ests with high sequence identity to defense-related genes of vascular plants, many of them with unknown functions, makes this plant a very useful model to study plant-pathogen interactions.
the susceptibility of distinct tissues to pathogens can also be studied, since physcomitrella can be maintained as a haploid gametophyte with distinct developmental stages.
these consist of the protonema which is a filamentous network of cells, and the radially symmetric gametophore which is a leafy shoot composed of a non-vascular stem with leaves as well as rhizoids  <cit> .
disease development can be visualized microscopically in that the leaves and protonemal filaments are formed of a monolayer of cells.
there have been very few reports on either pathogen infection or the activation of defense responses in mosses.
in silico analysis of the physcomitrella genome, however, indicates the presence of several encoded proteins with high similarity to r gene products found in flowering plants  <cit> .
regarding natural infection, the fungus scleroconidioma sphagnicola  can infect and cause disease symptoms in the moss sphagnum fuscum   <cit>  and viruses were detected in antarctic mosses  <cit> .
s. sphagnicola hyphae can grow inside the cell wall of s. fuscum, digesting wall components, penetrating into cells of leaves and causing chlorosis of the tissue.
in more advanced stages of disease development, necrosis of infected leaf and stem cells, as well as host death can be observed  <cit> .
in this study we aimed to identify plant pathogens capable of infecting and triggering a defense response in physcomitrella, with the goal of establishing a model system to conduct molecular, cellular and genetic studies on physcomitrella-pathogen interactions.
we used two pathogens with a broad host range, the bacterium erwinia carotovora ssp carotovora  and the fungus botrytis cinerea .
e.c.
carotovora is a soft-rot erwinia which causes disease on many vascular plants  <cit> .
the main virulence factors of e.c.
carotovora are the plant cell wall-degrading enzymes including cellulases, proteases and pectinases  <cit> .
these enzymes cause maceration of the infected tissues and the released cell wall fragments can act as elicitors of the plant defense response  <cit> .
previous studies have shown that cell-free culture filtrate  containing plant cell wall-degrading enzymes from e.c.
carotovorascc <dig> produces similar symptoms and defense gene expression as those caused by e.c.
carotovorascc <dig> infection and enhanced disease resistance in cf-treated plants  <cit> .
some e.c.
carotovora strains produce harpins, which are small, acidic, glycine-rich, heat-stable proteins, that elicit hr and induction of plant defense responses  <cit> .
in the present study we have used two strains of e.c.
carotovora; i) e.c.
carotovorascc <dig> which is a harpin  producing strain  <cit> , and ii) e.c.
carotovorascc <dig> which is a hrpn-negative strain  <cit> .
b. cinerea is a necrotrophic fungal pathogen that attacks over  <dig> different plant species  <cit> , by producing multiple proteins and metabolites that kill the host cells  <cit> .
the main virulence factors of b. cinerea vary depending on the isolate, and include toxins and cell wall degrading enzymes such as endopolygalacturonases and xylanases  <cit> .
in this study, we demonstrate that e.c.
carotovora-derived elicitors and b. cinerea cause disease symptoms and induce a defense response in the moss physcomitrella.
RESULTS
e.c.
carotovora and b. cinerea infect physcomitrella patens
in order to determine whether e.c.
carotovora infects physcomitrella tissues, a gfp-labelled e.c.
carotovorascc <dig> strain was inoculated onto physcomitrella leaves as described in methods.
two days after inoculation tissue examined by confocal microscopy indicated that the labelled cells of e.c.
carotovora had occupied the apoplast between leaf cells, as well as the cellular space of some plant cells in this same tissue .
when b. cinerea inoculated leaf tissue was examined, outlines of fungal hyphae were apparent inside the cell cavity displacing the cytoplasmic contents .
infection of physcomitrella tissues by b. cinerea was examined in more detail by staining fungal hyphae with trypan blue.
two days post infection  hyphae appeared to be within the limits of the cell walls in physcomitrella leaves .
our observation that b. cinerea hyphae appeared within plant cells is likely in that physcomitrella leaves are composed of a contiguous monolayer of adjacent cells.
e.c.
carotovora, cfs and b. cinerea cause disease symptoms in physcomitrella
development of disease symptoms by e.c.
carotovora was initiated by inoculating the harpin hrpn-producing e.c.
carotovorascc <dig> strain and the hrpn-negative e.c.
carotovorascc <dig> strain onto physcomitrella leaves.
inoculation with both strains caused visible symptoms around the wounded tissue within  <dig> days when observed with a magnifying glass, while mock inoculated tissues did not .
physcomitrella infection by e.c.
carotovora and subsequent development of symptoms required that we first wound the plant mechanically .
growth or colonization of e.c.
carotovora in planta as determined by enumeration of bacteria in a given tissue could not be done due to the difficulty of consistently wounding the tissue sufficiently without causing excessive damage and dessication.
instead of continuing our studies with e.c.
carotovora bacterial inoculation, cell-free culture filtrate  was used to elicit a defense response since: i) in vascular plants cf incites the same disease symptoms and induces defense gene expression in the same way as does inoculation with e.c.
carotovora  <cit> , ii) cf is sprayed onto the colonies allowing for direct and homogeneous contact, and iii) it overcomes the technical difficulty of introducing a sufficient number of small wounds on the moss tissue to allow inoculation by e.c.
carotovora for a comprehensive evaluation of the plant defense response.
disease symptom development was observed upon treating physcomitrella colonies with:  cf of e.c.
carotovorascc <dig> ),  cf of e.c.
carotovorascc <dig> ), or  b. cinerea spores.
disease symptoms such as tissue maceration developed in colonies two days after treatment with either cf, cf or after inoculation with b. cinerea spores, as shown in figure  <dig>  in control experiments, moss colonies treated with either luria-bertani  , potato dextrose broth  growth media  or with spores of a non-pathogen, aspergillus nidulans , did not develop disease symptoms.
protonemal filaments treated with either cf or cf developed maceration symptoms, with cf causing less tissue damage than cf .
additionally, cf -treated colonies acquired a brownish aspect as seen in the protonemal filaments shown in figures 3c and 3e.
cf-treated gametophores, also known as leafy shoots, did not show maceration symptoms, although brownish stems were observed in cf-treated colonies .
physcomitrella showed a clear susceptibility to b. cinerea, involving a characteristic proliferation of mycelium and the appearance of necrotic protonemal tissue in addition to the browning of stems .
inoculated tissues were soft, macerated and were easily separated from the rest of the moss colony.
four dpi b. cinerea-infected moss tissues were completely macerated .
protonemal filaments were more susceptible than leaves to cf treatments and b. cinerea inoculation.
taken together, these results show that cf, cf and b. cinerea are capable of causing disease symptoms in physcomitrella.
cf and b. cinerea trigger cytoplasmic shrinkage, accumulation of autofluorescent compounds and chloroplast browning
pathogen infection or elicitor treatment can induce plant cell death with characteristic changes in cells, including cytoplasmic shrinkage, alteration of chloroplast organization and accumulation of autofluorescent compounds  <cit> .
in the present study, we examined cellular changes occurring in physcomitrella tissue showing macroscopic disease symptoms after exposure to b. cinerea spores or cf of the e.c.
carotovora strains.
cf-treated  and b. cinerea-inoculated  protonemal cells showed cytoplasmic shrinkage after  <dig> days.
in contrast, no cytoplasmic shrinkage was evident in cells treated with lb , cf  or pdb .
other morphological changes were also observed in physcomitrella cf-treated and b. cinerea-inoculated cells.
after  <dig> days, both treatments caused browning of the chloroplasts in a high proportion of cells .
chloroplast browning was evident only in cells showing cytoplasmic shrinkage .
additionally, cf-treated  and b. cinerea-inoculated cells  with brownish chloroplasts showed lack of red autofluorescence of chlorophyll.
cf-treated protonemal cells having cytoplasmic shrinkage and brown chloroplasts were more abundant after  <dig> days.
within this treated protonemal tissue, cells with fewer brown chloroplasts were also observed suggesting that they were being brokendown .
in contrast, cf- or lb-treated protonemal filaments did not show such changes and green chloroplasts were evident at least  <dig> days after treatment .
after an apparent initial contact of b. cinerea hyphae with individual cells within the leaf, plant cells were observed to respond by accumulating autofluorescent compounds .
cells in b. cinerea-inoculated leaves developing light blue to yellow autofluorescence  were also observed.
this af appeared confined to the cytoplasm now separated from the cell wall .
no af, however, was observed in cf- or cf-treated leaves .
af was clearly evident in protonemal filaments of b. cinerea-inoculated and cf-treated colonies .
in contrast, no af was seen in pdb-treated leaves  or pdb- or lb-treated protonemal filaments , and only a few cf-treated cells accumulated autofluorescent compounds .
also, accumulation of autofluorescent compounds was generally observed once cytoplasmic shrinkage occurred .
in summary, our results show that cf and b. cinerea induce cellular changes in physcomitrella protonemal cells, including cytoplasmic shrinkage, browning of chloroplasts and accumulation of autofluorescent compounds, suggesting a cell death process.
e.c.
carotovora elicitors and b. cinerea trigger cell death in physcomitrella
to assess whether cf and b. cinerea caused cell death of physcomitrella tissues, we stained moss colonies with evans blue, a dye that is excluded by membranes of living cells but diffuses into dead cells  <cit> .
figure  <dig> shows pictures of representative tissues.
two days after treatments, an increase in stained cells was observed with either cf, cf or b. cinerea spores, compared with control treatments.
although, while in cf-treated tissue a low proportion of stained protonemal cells was observed , cf-treated or b. cinerea-inoculated tissues showed a high proportion of stained protonemal cells .
in control treatments, almost no stained cells were visible .
cytoplasmic shrinkage was evident in most evans blue stained protonemal cells treated with cf or b. cinerea spores .
whenever cytoplasmic shrinkage occurred, cells were stained with evans blue, indicating that these cells were dying or dead.
in contrast, most cf-treated protonemal cells did not show cytoplasmic shrinkage and the dye was distributed homogeneously in the cells .
three days after treatment, stained cf-treated protonemal cells did not exhibit cytoplasmic shrinkage suggesting that this response does not develop at a later stage .
in gametophores, evans blue stained cells could be detected in leaves inoculated with b. cinerea , whereas stained cells were not seen after cf , cf or control treatments .
b. cinerea and e.c.
carotovora elicitors mediate activation of defense-related genes
to analyze whether cf treatment or b. cinerea inoculation trigger physcomitrella defense gene expression, we characterized the expression of a number of defense-related gene homologues including;  pr- <dig>   lox,  pal, and  chs.
lox  is a key enzyme in the synthesis of defense-related compounds including ja  <cit> , pal  mediates the biosynthesis of phenylpropanoids and sa  <cit>  and chs  is the first enzyme in the synthesis of flavonoids  <cit> .
the results in figure  <dig> show that expression of the physcomitrella homologues increased after cf treatment or b. cinerea inoculation.
clearly, three types of expression patterns were observed.
the level of pr- <dig> expression peaked at  <dig> h in cf-treated moss colonies, while in cf-treated and b. cinerea-inoculated tissues expression of pr- <dig> peaked at  <dig> h. expression of pal and chs peaked at  <dig> h in tissues treated with either of the cfs or with b. cinerea spores, although among the treatments, higher expression levels were observed with cf.
in the case of chs, two transcripts with an identical expression pattern were detected.
lox expression was moderately induced in cf- and cf-treated moss colonies at  <dig> and  <dig> h, while transcript levels increased significantly in b. cinerea inoculated physcomitrella tissues, reaching the highest expression level at  <dig> h. the results obtained in this study show that several conserved defense-related gene homologues of physcomitrella were induced in response to treatment with e.c.
carotovora elicitors or b. cinerea spores.
discussion
the developmental simplicity, ease of genetic analysis and the evolutionarily relationship between physcomitrella and other plants has prompted us to study the interaction between this moss and two broad host range pathogens, the bacterium e.c.
carotovora and the fungus b. cinerea.
our results indicate that both b. cinerea and e.c.
carotovora can infect physcomitrella tissues and cause disease symptoms.
since physcomitrella gametophytes do not have stomata, e.c.
carotovora entered plant tissues through wounds, while b. cinerea hyphae probably entered by hydrolyzing the plant cell wall using hydrolytic enzymes or by secreting cell wall permeable toxins that kill plant cells.
we have observed b. cinerea hyphae within plant cells, including cells in which hyphae were apparently inside the cell cavity displacing the cytoplasm.
hyphae of other necrotrophic fungi, including s. sphagnicola, tephrocybe palustris and nectria mnii, are capable of penetrating live cells of moss leaves, resulting in cell death a posteriori  <cit> .
in case of nectria mnii, it was shown that intracellular hyphae could displace the host cell contents  <cit> .
e.c.
carotovorascc <dig>  but not e.c.
carotovorascc <dig>  was previously shown to harbour the harpin-encoding gene hrpn  <cit> .
harpins are bacterial effector proteins released into the host cells, through a type iii secretion system encoded by the hypersensitive reaction and pathogenicity  gene cluster.
when present in plant tissue, harpins cause hr and induction of defense mechanisms  <cit> .
in erwinia spp.
hrp genes have been shown to contribute to virulence and to the ability of the pathogen to grow in the plant  <cit> .
the higher maceration rate of the protonemal tissues observed with cf compared with cf is consistent with previous studies showing that polygalacturonase, together with harpin hrpn from e.c.
carotovorascc <dig> greatly enhanced lesion formation in arabidopsis  <cit> .
cytoplasmic shrinkage is the most common morphological change occurring in plant pcd and has been observed in cells undergoing hr, as well as in tissues of plants susceptible to virulent pathogens  <cit> .
cytoplasmic shrinkage was observed only in evans blue stained protonemal cells treated with cf but not with cf, probably suggesting that a different mechanism leading to cell death had occurred.
this finding is consistent with the induction of hr by harpins and with previous results showing that pseudomonas syringae pv phaseolicola induced cytoplasmic shrinkage in plant cells, while a hrpd mutant did not  <cit> .
breakdown of chloroplast membranes and chlorophyll has been observed in cells undergoing pcd, including those treated with elicitors, infected with pathogens or those undergoing senescence  <cit> .
our results showed that treatments with cf and b. cinerea spores induce browning of chloroplasts, which is likely followed by the breakdown of these organelles.
this is consistent with previous results showing that s. sphagnicola hyphae are capable of causing degeneration of chloroplasts in the moss s. fuscum  <cit> .
chloroplasts remained green in protonemal cf-treated cells, while boiled cf, containing the heat-stable hrpn, still induced browning of the chloroplasts in cells also showing cytoplasmic shrinkage .
these findings suggest that hrpn might trigger cell death associated with cytoplasmic shrinkage and chloroplasts browning.
in addition, browning of chloroplasts was associated with chlorophyll breakdown in cf-treated and b. cinerea-inoculated cells, since no red chlorophyll autofluorescence was observed .
these results are supported by findings showing that e.c.
carotovorascc <dig> induced the expression of chlorophyllase  <dig> in arabidopsis, which could be involved in the degradation of photoactive chlorophylls to avoid higher levels of reactive oxygen species  production and cellular damage during pathogen infection  <cit> .
it is also interesting to note that whenever browning of chloroplasts was observed in cf-treated or b. cinerea-inoculated cells, cytoplasmic shrinkage was also present.
since cf-treated protonemal cells with cytoplasmic shrinkage but green chloroplasts were also observed, browning of chloroplasts could be a process occurring later in dying cells after collapse of the cytoplasm.
browning of chloroplasts could be indicative of oxidative processes due to excessive accumulation of ros in the chloroplasts at late stages of cf and b. cinerea treatments, finally leading to chloroplasts breakdown.
to our knowledge, this is the first report in which browning of chloroplasts was observed after pathogen and elicitor treatment.
the ability to observe changes in the coloration of the chloroplasts was facilitated in that the leaf tissue, like the protonemal filaments, is composed of a single monolayer of cells.
accumulation of autofluorescent compounds has been associated with the occurrence of hr in vascular plants  <cit> .
cf-treated or b. cinerea-inoculated physcomitrella tissues developed af.
a previous report demonstrated localized deposition of phenolic compounds at the sites of fungal penetration and also as a second major response that appeared to follow cell death  <cit> .
these findings are consistent with our results, in showing af confined to the collapsed cytoplasm of dead cells in b. cinerea-inoculated leaves and protonemal filaments.
b. cinerea induce pcd to enable rapid colonization of vascular plants, and erwinia harpins have been shown to elicit cell death  <cit> .
cytoplasmic shrinkage, an indicator of plant pcd, correlated with accumulation of autofluorescent compounds and chloroplast browning after inoculation with b. cinerea or treatment with cf of hrpn producing e.c.
carotovorascc <dig> suggesting that either treatment results in pcd in physcomitrella.
our results also showed that e.c.
carotovora elicitors and b. cinerea induced defense-related gene expression in physcomitrella.
earlier induction of the pr-1-like gene expression and the higher levels of pal and chs mrna accumulation triggered by cf compared with cf, corresponded well with the higher levels of tissue maceration observed with cf.
chss are encoded by multiple genes in vascular plants and physcomitrella  <cit> , and in our study two chs transcripts with an identical expression pattern were detected.
recently, a new enzymatic activity was described for the same physcomitrella lox gene product induced by b. cinerea in this study  <cit> .
novel oxylipins were generated by this enzyme suggesting a possible involvement in defense responses.
in vascular plants pr- <dig>  pal and lox are induced by inoculation with e.c.
carotovora or by cf treatments  <cit>  and pr- <dig> transcript accumulation is increased after b. cinerea infection  <cit> .
the results obtained in this study suggest that e.c.
carotovora elicitors and b. cinerea similarly induce expression of physcomitrella defense gene homologues of those studied in vascular plants, and thus validate the use of non-specific plant pathogens or elicitors derived from them to study moss-pathogen interactions.
CONCLUSIONS
in the present study, we demonstrate that e.c.
carotovora elicitor treatment and b. cinerea inoculation cause disease symptoms and induce defense responses in physcomitrella.
cf, cf and b. cinerea induced the expression of defense-related genes, including pr- <dig>  lox, pal and chs homologues.
compounds produced by lox, pal and chs are involved in the synthesis of ja, phenylpropanoids and sa and flavonoids, respectively, in vascular plants.
these compounds could play a role in the defense response of physcomitrella as has been shown in vascular plants.
as such our results further establish e.c.
carotovora elicitors, as well as b. cinerea as promising systems to analyze induction of defense responses in physcomitrella.
since cytoplasmic shrinkage is the most common morphological change observed in plant pcd, and that harpins and b. cinerea induce this type of cell death in vascular plants, our results suggest that e.c.
carotovora cfscc <dig> containing hrpn and b. cinerea could also induce this type of cell death in physcomitrella.
finally, the occurrence of distinct cellular responses leading to cell death by cf and cf provides a useful system to analyze pathogen-induced cell death and to characterize the key elements involved in its regulation by targeted gene disruption in physcomitrella.

 plants are continuously subjected to pathogen attack and respond by activating a range of defense mechanisms. recognition of the pathogen or elicitors derived either from the pathogen or released from the plant cell wall is accompanied with the production of molecular signals including salicylic acid, jasmonic acid and ethylene that lead to the induction of defense gene expression. this in turn results in the accumulation of functionally diverse pathogenesis-related proteins and metabolites. recognition of the pathogen or elicitors is usually accompanied by the rapid death of the infected cells, known as the hypersensitive response, which limits the access of the pathogen to water and nutrients thereby restricting its growth. hr can be triggered either by non-specific elicitors recognized by plant receptors, or by specific elicitors genes) recognized by corresponding encoded products of plant resistance genes. several studies have suggested that plant cell death resulting from the hr is a type of programmed cell death. plant cells undergoing pcd share a number of characteristic morphological and biochemical features in common with animal cell apoptosis. moreover, cell death with apoptotic features has also been observed in plants susceptible to virulent pathogens. although bryophytes are non-vascular plants and are considered to be primitive among the embryophyta, mosses have been shown to respond to a variety of environmental stimuli and to several common plant growth factors much like vascular plants. thus, in spite of having diverged from vascular plants approximately million years ago, mosses are well-suited for the study of fundamental processes in plant biology. furthermore, mosses have a simple developmental program and a life cycle with a predominant haploid phase which greatly facilitates genetic analysis. physcomitrella patens, a relatively small moss, has recently become a model plant to study plant gene function in that it exhibits high-frequency homologous recombination comparable with that of saccharomyces cerevisiae, enabling the construction of gene knock-outs. the assembled physcomitrella genome has recently been released and full-length cdnas in addition to ests are available in the databases. these advantages together with the presence of a great number of physcomitrella ests with high sequence identity to defense-related genes of vascular plants, many of them with unknown functions, makes this plant a very useful model to study plant-pathogen interactions. the susceptibility of distinct tissues to pathogens can also be studied, since physcomitrella can be maintained as a haploid gametophyte with distinct developmental stages. these consist of the protonema which is a filamentous network of cells, and the radially symmetric gametophore which is a leafy shoot composed of a non-vascular stem with leaves as well as rhizoids. disease development can be visualized microscopically in that the leaves and protonemal filaments are formed of a monolayer of cells. there have been very few reports on either pathogen infection or the activation of defense responses in mosses. in silico analysis of the physcomitrella genome, however, indicates the presence of several encoded proteins with high similarity to r gene products found in flowering plants. regarding natural infection, the fungus scleroconidioma sphagnicola can infect and cause disease symptoms in the moss sphagnum fuscum and viruses were detected in antarctic mosses. s. sphagnicola hyphae can grow inside the cell wall of s. fuscum, digesting wall components, penetrating into cells of leaves and causing chlorosis of the tissue. in more advanced stages of disease development, necrosis of infected leaf and stem cells, as well as host death can be observed. in this study we aimed to identify plant pathogens capable of infecting and triggering a defense response in physcomitrella, with the goal of establishing a model system to conduct molecular, cellular and genetic studies on physcomitrella-pathogen interactions. we used two pathogens with a broad host range, the bacterium erwinia carotovora ssp carotovora and the fungus botrytis cinerea. e.c. carotovora is a soft-rot erwinia which causes disease on many vascular plants. the main virulence factors of e.c. carotovora are the plant cell wall-degrading enzymes including cellulases, proteases and pectinases. these enzymes cause maceration of the infected tissues and the released cell wall fragments can act as elicitors of the plant defense response. previous studies have shown that cell-free culture filtrate containing plant cell wall-degrading enzymes from e.c. carotovorascc produces similar symptoms and defense gene expression as those caused by e.c. carotovorascc infection and enhanced disease resistance in cf-treated plants. some e.c. carotovora strains produce harpins, which are small, acidic, glycine-rich, heat-stable proteins, that elicit hr and induction of plant defense responses. in the present study we have used two strains of e.c. carotovora; i) e.c. carotovorascc which is a harpin producing strain, and ii) e.c. carotovorascc which is a hrpn-negative strain. b. cinerea is a necrotrophic fungal pathogen that attacks over different plant species, by producing multiple proteins and metabolites that kill the host cells. the main virulence factors of b. cinerea vary depending on the isolate, and include toxins and cell wall degrading enzymes such as endopolygalacturonases and xylanases. in this study, we demonstrate that e.c. carotovora-derived elicitors and b. cinerea cause disease symptoms and induce a defense response in the moss physcomitrella.  e.c. carotovora and b. cinerea infect physcomitrella patens in order to determine whether e.c. carotovora infects physcomitrella tissues, a gfp-labelled e.c. carotovorascc strain was inoculated onto physcomitrella leaves as described in methods. two days after inoculation tissue examined by confocal microscopy indicated that the labelled cells of e.c. carotovora had occupied the apoplast between leaf cells, as well as the cellular space of some plant cells in this same tissue. when b. cinerea inoculated leaf tissue was examined, outlines of fungal hyphae were apparent inside the cell cavity displacing the cytoplasmic contents. infection of physcomitrella tissues by b. cinerea was examined in more detail by staining fungal hyphae with trypan blue. two days post infection hyphae appeared to be within the limits of the cell walls in physcomitrella leaves. our observation that b. cinerea hyphae appeared within plant cells is likely in that physcomitrella leaves are composed of a contiguous monolayer of adjacent cells. e.c. carotovora, cfs and b. cinerea cause disease symptoms in physcomitrella development of disease symptoms by e.c. carotovora was initiated by inoculating the harpin hrpn-producing e.c. carotovorascc strain and the hrpn-negative e.c. carotovorascc strain onto physcomitrella leaves. inoculation with both strains caused visible symptoms around the wounded tissue within days when observed with a magnifying glass, while mock inoculated tissues did not. physcomitrella infection by e.c. carotovora and subsequent development of symptoms required that we first wound the plant mechanically. growth or colonization of e.c. carotovora in planta as determined by enumeration of bacteria in a given tissue could not be done due to the difficulty of consistently wounding the tissue sufficiently without causing excessive damage and dessication. instead of continuing our studies with e.c. carotovora bacterial inoculation, cell-free culture filtrate was used to elicit a defense response since: i) in vascular plants cf incites the same disease symptoms and induces defense gene expression in the same way as does inoculation with e.c. carotovora, ii) cf is sprayed onto the colonies allowing for direct and homogeneous contact, and iii) it overcomes the technical difficulty of introducing a sufficient number of small wounds on the moss tissue to allow inoculation by e.c. carotovora for a comprehensive evaluation of the plant defense response. disease symptom development was observed upon treating physcomitrella colonies with: cf of e.c. carotovorascc ), cf of e.c. carotovorascc ), or b. cinerea spores. disease symptoms such as tissue maceration developed in colonies two days after treatment with either cf, cf or after inoculation with b. cinerea spores, as shown in figure in control experiments, moss colonies treated with either luria-bertani, potato dextrose broth growth media or with spores of a non-pathogen, aspergillus nidulans, did not develop disease symptoms. protonemal filaments treated with either cf or cf developed maceration symptoms, with cf causing less tissue damage than cf. additionally, cf -treated colonies acquired a brownish aspect as seen in the protonemal filaments shown in figures 3c and 3e. cf-treated gametophores, also known as leafy shoots, did not show maceration symptoms, although brownish stems were observed in cf-treated colonies. physcomitrella showed a clear susceptibility to b. cinerea, involving a characteristic proliferation of mycelium and the appearance of necrotic protonemal tissue in addition to the browning of stems. inoculated tissues were soft, macerated and were easily separated from the rest of the moss colony. four dpi b. cinerea-infected moss tissues were completely macerated. protonemal filaments were more susceptible than leaves to cf treatments and b. cinerea inoculation. taken together, these results show that cf, cf and b. cinerea are capable of causing disease symptoms in physcomitrella. cf and b. cinerea trigger cytoplasmic shrinkage, accumulation of autofluorescent compounds and chloroplast browning pathogen infection or elicitor treatment can induce plant cell death with characteristic changes in cells, including cytoplasmic shrinkage, alteration of chloroplast organization and accumulation of autofluorescent compounds. in the present study, we examined cellular changes occurring in physcomitrella tissue showing macroscopic disease symptoms after exposure to b. cinerea spores or cf of the e.c. carotovora strains. cf-treated and b. cinerea-inoculated protonemal cells showed cytoplasmic shrinkage after days. in contrast, no cytoplasmic shrinkage was evident in cells treated with lb, cf or pdb. other morphological changes were also observed in physcomitrella cf-treated and b. cinerea-inoculated cells. after days, both treatments caused browning of the chloroplasts in a high proportion of cells. chloroplast browning was evident only in cells showing cytoplasmic shrinkage. additionally, cf-treated and b. cinerea-inoculated cells with brownish chloroplasts showed lack of red autofluorescence of chlorophyll. cf-treated protonemal cells having cytoplasmic shrinkage and brown chloroplasts were more abundant after days. within this treated protonemal tissue, cells with fewer brown chloroplasts were also observed suggesting that they were being brokendown. in contrast, cf- or lb-treated protonemal filaments did not show such changes and green chloroplasts were evident at least days after treatment. after an apparent initial contact of b. cinerea hyphae with individual cells within the leaf, plant cells were observed to respond by accumulating autofluorescent compounds. cells in b. cinerea-inoculated leaves developing light blue to yellow autofluorescence were also observed. this af appeared confined to the cytoplasm now separated from the cell wall. no af, however, was observed in cf- or cf-treated leaves. af was clearly evident in protonemal filaments of b. cinerea-inoculated and cf-treated colonies. in contrast, no af was seen in pdb-treated leaves or pdb- or lb-treated protonemal filaments, and only a few cf-treated cells accumulated autofluorescent compounds. also, accumulation of autofluorescent compounds was generally observed once cytoplasmic shrinkage occurred. in summary, our results show that cf and b. cinerea induce cellular changes in physcomitrella protonemal cells, including cytoplasmic shrinkage, browning of chloroplasts and accumulation of autofluorescent compounds, suggesting a cell death process. e.c. carotovora elicitors and b. cinerea trigger cell death in physcomitrella to assess whether cf and b. cinerea caused cell death of physcomitrella tissues, we stained moss colonies with evans blue, a dye that is excluded by membranes of living cells but diffuses into dead cells. figure shows pictures of representative tissues. two days after treatments, an increase in stained cells was observed with either cf, cf or b. cinerea spores, compared with control treatments. although, while in cf-treated tissue a low proportion of stained protonemal cells was observed, cf-treated or b. cinerea-inoculated tissues showed a high proportion of stained protonemal cells. in control treatments, almost no stained cells were visible. cytoplasmic shrinkage was evident in most evans blue stained protonemal cells treated with cf or b. cinerea spores. whenever cytoplasmic shrinkage occurred, cells were stained with evans blue, indicating that these cells were dying or dead. in contrast, most cf-treated protonemal cells did not show cytoplasmic shrinkage and the dye was distributed homogeneously in the cells. three days after treatment, stained cf-treated protonemal cells did not exhibit cytoplasmic shrinkage suggesting that this response does not develop at a later stage. in gametophores, evans blue stained cells could be detected in leaves inoculated with b. cinerea, whereas stained cells were not seen after cf, cf or control treatments. b. cinerea and e.c. carotovora elicitors mediate activation of defense-related genes to analyze whether cf treatment or b. cinerea inoculation trigger physcomitrella defense gene expression, we characterized the expression of a number of defense-related gene homologues including; pr- lox, pal, and chs. lox is a key enzyme in the synthesis of defense-related compounds including ja, pal mediates the biosynthesis of phenylpropanoids and sa and chs is the first enzyme in the synthesis of flavonoids. the results in figure show that expression of the physcomitrella homologues increased after cf treatment or b. cinerea inoculation. clearly, three types of expression patterns were observed. the level of pr- expression peaked at h in cf-treated moss colonies, while in cf-treated and b. cinerea-inoculated tissues expression of pr- peaked at h. expression of pal and chs peaked at h in tissues treated with either of the cfs or with b. cinerea spores, although among the treatments, higher expression levels were observed with cf. in the case of chs, two transcripts with an identical expression pattern were detected. lox expression was moderately induced in cf- and cf-treated moss colonies at and h, while transcript levels increased significantly in b. cinerea inoculated physcomitrella tissues, reaching the highest expression level at h. the results obtained in this study show that several conserved defense-related gene homologues of physcomitrella were induced in response to treatment with e.c. carotovora elicitors or b. cinerea spores. discussion the developmental simplicity, ease of genetic analysis and the evolutionarily relationship between physcomitrella and other plants has prompted us to study the interaction between this moss and two broad host range pathogens, the bacterium e.c. carotovora and the fungus b. cinerea. our results indicate that both b. cinerea and e.c. carotovora can infect physcomitrella tissues and cause disease symptoms. since physcomitrella gametophytes do not have stomata, e.c. carotovora entered plant tissues through wounds, while b. cinerea hyphae probably entered by hydrolyzing the plant cell wall using hydrolytic enzymes or by secreting cell wall permeable toxins that kill plant cells. we have observed b. cinerea hyphae within plant cells, including cells in which hyphae were apparently inside the cell cavity displacing the cytoplasm. hyphae of other necrotrophic fungi, including s. sphagnicola, tephrocybe palustris and nectria mnii, are capable of penetrating live cells of moss leaves, resulting in cell death a posteriori. in case of nectria mnii, it was shown that intracellular hyphae could displace the host cell contents. e.c. carotovorascc but not e.c. carotovorascc was previously shown to harbour the harpin-encoding gene hrpn. harpins are bacterial effector proteins released into the host cells, through a type iii secretion system encoded by the hypersensitive reaction and pathogenicity gene cluster. when present in plant tissue, harpins cause hr and induction of defense mechanisms. in erwinia spp. hrp genes have been shown to contribute to virulence and to the ability of the pathogen to grow in the plant. the higher maceration rate of the protonemal tissues observed with cf compared with cf is consistent with previous studies showing that polygalacturonase, together with harpin hrpn from e.c. carotovorascc greatly enhanced lesion formation in arabidopsis. cytoplasmic shrinkage is the most common morphological change occurring in plant pcd and has been observed in cells undergoing hr, as well as in tissues of plants susceptible to virulent pathogens. cytoplasmic shrinkage was observed only in evans blue stained protonemal cells treated with cf but not with cf, probably suggesting that a different mechanism leading to cell death had occurred. this finding is consistent with the induction of hr by harpins and with previous results showing that pseudomonas syringae pv phaseolicola induced cytoplasmic shrinkage in plant cells, while a hrpd mutant did not. breakdown of chloroplast membranes and chlorophyll has been observed in cells undergoing pcd, including those treated with elicitors, infected with pathogens or those undergoing senescence. our results showed that treatments with cf and b. cinerea spores induce browning of chloroplasts, which is likely followed by the breakdown of these organelles. this is consistent with previous results showing that s. sphagnicola hyphae are capable of causing degeneration of chloroplasts in the moss s. fuscum. chloroplasts remained green in protonemal cf-treated cells, while boiled cf, containing the heat-stable hrpn, still induced browning of the chloroplasts in cells also showing cytoplasmic shrinkage. these findings suggest that hrpn might trigger cell death associated with cytoplasmic shrinkage and chloroplasts browning. in addition, browning of chloroplasts was associated with chlorophyll breakdown in cf-treated and b. cinerea-inoculated cells, since no red chlorophyll autofluorescence was observed. these results are supported by findings showing that e.c. carotovorascc induced the expression of chlorophyllase in arabidopsis, which could be involved in the degradation of photoactive chlorophylls to avoid higher levels of reactive oxygen species production and cellular damage during pathogen infection. it is also interesting to note that whenever browning of chloroplasts was observed in cf-treated or b. cinerea-inoculated cells, cytoplasmic shrinkage was also present. since cf-treated protonemal cells with cytoplasmic shrinkage but green chloroplasts were also observed, browning of chloroplasts could be a process occurring later in dying cells after collapse of the cytoplasm. browning of chloroplasts could be indicative of oxidative processes due to excessive accumulation of ros in the chloroplasts at late stages of cf and b. cinerea treatments, finally leading to chloroplasts breakdown. to our knowledge, this is the first report in which browning of chloroplasts was observed after pathogen and elicitor treatment. the ability to observe changes in the coloration of the chloroplasts was facilitated in that the leaf tissue, like the protonemal filaments, is composed of a single monolayer of cells. accumulation of autofluorescent compounds has been associated with the occurrence of hr in vascular plants. cf-treated or b. cinerea-inoculated physcomitrella tissues developed af. a previous report demonstrated localized deposition of phenolic compounds at the sites of fungal penetration and also as a second major response that appeared to follow cell death. these findings are consistent with our results, in showing af confined to the collapsed cytoplasm of dead cells in b. cinerea-inoculated leaves and protonemal filaments. b. cinerea induce pcd to enable rapid colonization of vascular plants, and erwinia harpins have been shown to elicit cell death. cytoplasmic shrinkage, an indicator of plant pcd, correlated with accumulation of autofluorescent compounds and chloroplast browning after inoculation with b. cinerea or treatment with cf of hrpn producing e.c. carotovorascc suggesting that either treatment results in pcd in physcomitrella. our results also showed that e.c. carotovora elicitors and b. cinerea induced defense-related gene expression in physcomitrella. earlier induction of the pr-1-like gene expression and the higher levels of pal and chs mrna accumulation triggered by cf compared with cf, corresponded well with the higher levels of tissue maceration observed with cf. chss are encoded by multiple genes in vascular plants and physcomitrella, and in our study two chs transcripts with an identical expression pattern were detected. recently, a new enzymatic activity was described for the same physcomitrella lox gene product induced by b. cinerea in this study. novel oxylipins were generated by this enzyme suggesting a possible involvement in defense responses. in vascular plants pr- pal and lox are induced by inoculation with e.c. carotovora or by cf treatments and pr- transcript accumulation is increased after b. cinerea infection. the results obtained in this study suggest that e.c. carotovora elicitors and b. cinerea similarly induce expression of physcomitrella defense gene homologues of those studied in vascular plants, and thus validate the use of non-specific plant pathogens or elicitors derived from them to study moss-pathogen interactions.  in the present study, we demonstrate that e.c. carotovora elicitor treatment and b. cinerea inoculation cause disease symptoms and induce defense responses in physcomitrella. cf, cf and b. cinerea induced the expression of defense-related genes, including pr- lox, pal and chs homologues. compounds produced by lox, pal and chs are involved in the synthesis of ja, phenylpropanoids and sa and flavonoids, respectively, in vascular plants. these compounds could play a role in the defense response of physcomitrella as has been shown in vascular plants. as such our results further establish e.c. carotovora elicitors, as well as b. cinerea as promising systems to analyze induction of defense responses in physcomitrella. since cytoplasmic shrinkage is the most common morphological change observed in plant pcd, and that harpins and b. cinerea induce this type of cell death in vascular plants, our results suggest that e.c. carotovora cfscc containing hrpn and b. cinerea could also induce this type of cell death in physcomitrella. finally, the occurrence of distinct cellular responses leading to cell death by cf and cf provides a useful system to analyze pathogen-induced cell death and to characterize the key elements involved in its regulation by targeted gene disruption in physcomitrella. 
carotovora strain or a hrpn-negative strain, could cause disease symptoms and induce defense responses in physcomitrella. carotovora were found to readily infect physcomitrella gametophytic tissues and cause disease symptoms. since cytoplasmic shrinkage is the most common morphological change observed in plant pcd, and that harpins and b. cinerea induce this type of cell death in vascular plants, our results suggest that e.c. the purpose of this study was to determine: i) whether two representative broad host range pathogens, erwinia carotovora ssp. most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. vascular plants respond to pathogens by activating a diverse array of defense mechanisms. carotovorascc ), resulted in disease development with severe maceration of physcomitrella tissues, while cf produced only mild maceration. treatments with b. cinerea spores or cell-free culture filtrates from e.c. although increased cell death was observed with either the cfs or b. cinerea, the occurrence of cytoplasmic shrinkage was only visible in evans blue stained protonemal cells treated with cf or inoculated with b. cinerea. cf treatments and b. cinerea inoculation induced the expression of the defense-related genes: pr- pal, chs and lox. carotovora and botrytis cinerea, could infect physcomitrella, and ii) whether b. cinerea, elicitors of a harpin producing e.c. carotovora cfscc containing hrpn and b. cinerea could also induce this type of cell death in physcomitrella. our studies thus establish physcomitrella as an experimental host for investigation of plant-pathogen interactions and b. cinerea and elicitors of e.c. studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. however, very little is known about the infection and defense responses of the bryophyte, physcomitrella patens, to well-studied phytopathogens. carotovora elicitors induce a defense response in physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant.
 vascular plants respond to pathogens by activating a diverse array of defense mechanisms. studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. however, very little is known about the infection and defense responses of the bryophyte, physcomitrella patens, to well-studied phytopathogens. the purpose of this study was to determine: i) whether two representative broad host range pathogens, erwinia carotovora ssp. carotovora and botrytis cinerea, could infect physcomitrella, and ii) whether b. cinerea, elicitors of a harpin producing e.c. carotovora strain or a hrpn-negative strain, could cause disease symptoms and induce defense responses in physcomitrella.  b. cinerea and e.c. carotovora were found to readily infect physcomitrella gametophytic tissues and cause disease symptoms. treatments with b. cinerea spores or cell-free culture filtrates from e.c. carotovorascc ), resulted in disease development with severe maceration of physcomitrella tissues, while cf produced only mild maceration. although increased cell death was observed with either the cfs or b. cinerea, the occurrence of cytoplasmic shrinkage was only visible in evans blue stained protonemal cells treated with cf or inoculated with b. cinerea. most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. cf treatments and b. cinerea inoculation induced the expression of the defense-related genes: pr- pal, chs and lox.  b. cinerea and e.c. carotovora elicitors induce a defense response in physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. since cytoplasmic shrinkage is the most common morphological change observed in plant pcd, and that harpins and b. cinerea induce this type of cell death in vascular plants, our results suggest that e.c. carotovora cfscc containing hrpn and b. cinerea could also induce this type of cell death in physcomitrella. our studies thus establish physcomitrella as an experimental host for investigation of plant-pathogen interactions and b. cinerea and elicitors of e.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant. 