BACKGROUND
several studies have described the potential of recombinant protease inhibitors as potent antidigestive compounds to protect crop plants from herbivory or pathogenic infection
<cit> .
for instance, cysteine  protease inhibitors of the cystatin protein superfamily were proposed as protective agents against various herbivorous arthropods and root-parasitic nematodes
<cit> .
cystatins and most other protease inhibitors in plants are competitive protein inhibitors acting as pseudo-substrates to enter the active site of proteases
<cit> .
following inhibition, the target proteases can no longer cleave peptide bonds, which results in a detrimental inhibition of protein digestive functions in herbivorous pests and accounts for the resistance of several transgenic plant lines expressing recombinant inhibitors.
the heterologous expression of protease inhibitors in plants, however, has raised a number of questions about the possible occurrence of unintended metabolic interference –or pleiotropic effects– on endogenous proteolysis, which could eventually alter important cellular functions
<cit> .
proteases are ubiquitous metabolic effectors involved in the regulation of numerous cellular processes, ranging from housekeeping functions like protein turnover and the elimination of misfolded polypeptides to the processing of polypeptide pre- and pro-regions on maturing protein backbones
<cit> .
while studies have reported negligible phenotypic effects for protease inhibitors in transgenic plants based on the assessment of macroscopic indicators such as growth rate, stem diameter or leaf number
<cit> , several reports suggest the onset of more subtle effects at the metabolic level.
for instance, plant and mammalian serine  protease inhibitors ectopically expressed in potato were shown to significantly impact protein levels in leaves, positively or negatively
<cit> .
similarly, recombinant cystatins expressed in arabidopsis or tobacco were shown to induce a range of phenotype alterations in planta, including a delayed development of floral organs
<cit> , a modified physiological behaviour under low temperature or light regimes
<cit> , an altered protein content in leaves
<cit> , and a strong repression of the pathogen-inducible hypersensitive response
<cit> .
these findings, along with studies reporting the tolerance of protease inhibitor-expressing plants to abiotic stress cues such as drought, salinity and low temperatures
<cit> , point to the occurrence of endogenous protease targets for the recombinant inhibitors, directly or indirectly involved in stress-related processes.
the so-called pleiotropic effects of recombinant protease inhibitors, which are often considered as unintended metabolic effects in the modified plant, might simply reflect a lack of knowledge on stress-related proteolysis in planta and in fact represent a source of potentially useful traits for crop improvement
<cit> .
here we provide experimental evidence for the up-regulation of abiotic and biotic stress-related proteins in leaves of transgenic potato lines engineered to express corn cystatin ii , a potent inhibitor of c1a cys proteases exhibiting potential for herbivore pest control
<cit> .
we also link the pleiotropic effects of ccii expression with the compromised ability of a model necrotrophic fungus, botrytis cinerea, to colonize leaf tissues of the modified host plant.
RESULTS
pathogenesis-related proteins are up-regulated in ccii-expressing potato lines
reverse transcriptase  pcr, immunodetection and surface-enhanced laser desorption ionization time-of-flight mass spectrometry  analyses were performed to select transgenic potato lines expressing ccii at different levels in leaves , among a collection of independent transformants developed earlier in our laboratory
<cit> .
ccii in these lines accumulates in the cytosolic compartment, under the control of the cauliflower mosaic virus 35s constitutive promoter.
unlike control line k showing no signal, the transgenic lines showed variable, but easily detectable signals of ccii-encoding mrna transcripts .
accordingly, ccii was immunodetected as a ~12-kda polypeptide band of variable intensity in the modified lines, in contrast with line k giving no signal .
the amount of ccii in leaf extracts was variable among the lines, as inferred by seldi tof ms after capture of the inhibitor on cm- <dig> biochips for weak cationic exchange .
unexpectedly, a clear immunoblot signal was detected in several ccii-lines for the wound inducible homologue of ccii in potato leaves, potato multicystatin
<cit>  .
this apparent up-regulation of the endogenous inhibitor was confirmed by densitometric analysis of the immunoblots revealing significant increases reaching two- to tenfold the signals observed for control line k in four transgenic lines, out of five tested  .
<cit> .
numbers on the right refer to commercial molecular weight markers.
pmc refers to endogenous potato multicystatin.
c. relative pmc levels in ccii-lines compared to mean basal level in control line k .
each bar on panels a and c is the mean of three independent  values ± se.
asterisks indicate a significant difference with control line k .
rt pcr assays were conducted with nucleotide primers for mrna transcripts of proteinase inhibitor ii , a protein marker of wound and jasmonate inducible proteins; and pathogenesis-related  protein p <dig>  a protein marker of the abiotic stress-/pathogen-inducible salicylic acid pathway
<cit>  .
transcript levels for pin-ii were not different in control and transgenic lines , while transcripts for protein p <dig> were found at higher levels in several ccii-expressing lines, including line  <dig>  and line  <dig>  .
this observation suggesting an up-regulation of salicylate inducible proteins in the ccii-expressing lines was further supported by immunodetection assays with primary antibodies for pr- <dig>  and pr- <dig>  proteins, which showed levels of proteins from both families to be significantly higher in lines  <dig> c,  <dig>  and  <dig>  compared to control line k  .
the stress-related proteome is altered in ccii-expressing leaves
a comparative two-dimensional gel electrophoresis  proteome analysis was conducted with leaf protein extracts of line  <dig> , line  <dig>  and control line k to measure the overall impact of ccii on the host plant’s leaf proteome, and to test the hypothesis of a specific up-regulation of salicylate inducible stress-related proteins in plants ectopically expressing the recombinant inhibitor .
out of ~ <dig> protein spots monitored on 2-d gels,  <dig> showed a significantly altered level in ccii-expressing lines compared to the control line, for similar amounts of protein loaded on gel strips  .
of these proteins,  <dig> were detected de novo or up-regulated by more than fivefold in the ccii-lines .
as observed above for pr proteins , the up-regulating effects of ccii were systematically stronger in line  <dig>  compared to line  <dig>  producing lower levels of ccii , thereby suggesting a dose-dependent effect for the recombinant inhibitor.
<dig> for protein numbering and function assignment.
<dig> 2-de spatial coordinates, mowse score, protein sequence coverage, number of matched peptides and matched peptide sequences are given for each protein in additional file
<dig>
<dig> protein spots  <dig> and  <dig> could not be identified based on available ms spectra.
<dig> all protein spots matched potato  protein sequences, except spot  <dig> and spot  <dig> matching tomato  sequences.
<dig> blanks indicate no detectable protein product.
most proteins up-regulated in the ccii-lines were confidently identified based on lc-ms/ms sequence data .
in accordance with the increased levels of pmc and pr proteins in transgenic leaves , several up-regulated proteins in lines  <dig>  and  <dig>  corresponded to defense-related proteins usually induced upon wounding or biotic stress challenge, such as for instance wound inducible ser protease inhibitors , a kunitz cathepsin d inhibitor , a pr- <dig>  protein  and three pr- <dig>  proteins or protein fragments .
proteins that are usually induced upon abiotic stress challenge and thought to protect cells from oxidative damage or osmotic imbalance were also identified, including an osmotin-like pr- <dig> protein  and several secretory peroxidases .
a number of studies in recent years have described the potential usefulness of ectopically expressing antioxidant enzymes –or positive regulators of these enzymes– to generate crop lines tolerant to adverse abiotic conditions such as low or high temperatures, drought or salinity
<cit> .
considering the above described pleiotropic effects of ccii on the potato leaf proteome, the reported ability of cystatin-expressing plants to cope more efficiently with abiotic stress conditions  <cit>  could be likely explained, at least in part, by a significant up-regulating effect of the recombinant inhibitors on the steady-state levels of these enzymes in leaf tissues.
ccii does not inhibit the extracellular proteases of botrytis cinerea
bioassays were conducted with the fungal necrotroph b. cinerea to document the possible positive impact of ccii pleiotropic effects, including pr protein up-regulation, on potato plants challenged with an aggressive microbial pathogen.
the increased expression of peroxidases and pr proteins in leaves treated with benzathiadiazole, a functional analogue of salicylic acid, was shown earlier to induce resistance to b. cinerea in different plants .
likewise, strong detrimental effects against this pathogen were reported for recombinant pr proteins or gene inducers of these proteins expressed in different plants, including potato
<cit> .
the documented repressing effect of recombinant cystatins on the pathogen-inducible hypersensitive response in transgenic plants
<cit>  could also impact the ability of b. cinerea to colonize ccii-leaf tissues.
this pleiotropic effect of recombinant cystatins, also observed in plants treated with synthetic cys protease inhibitors or transformed with dna sequences to silence cys proteases expression
<cit> , would be correlated with an increase of cystatin:cys protease ratios in planta preventing the release of free cys proteases to initiate cell death
<cit> .
this ectopic phenotype could represent, along with the constitutive over-expression of stress-related proteins, a major hurdle to the progression of the necrotroph, which relies on cell death induction to generate dead tissues and successfully colonize the host plant
<cit> .
protease activity assays were first performed to measure the relative abundance of secreted cys proteases in b. cinerea, and to evaluate the possibility of a direct, cys protease inhibitory-mediated effect of ccii against this fungus.
in agreement with earlier studies reporting the presence of aspartate  proteases in the secretome of b. cinerea <cit>  and proposing a key role for these enzymes during plant infection
<cit> , protease  activities in the liquid phase of a b. cinerea culture grown in potato dextrose broth were inactivated by almost 50% with pepstatin a, a specific inhibitor of a <dig> asp proteases .
ser protease activities were also detected, as inferred by a weak, but reproducible inhibitory effect of ser-type inhibitors on azocaseinase activity.
the presence of  secreted ser protease in the culture medium was confirmed by the detection of a clear protein lysis band following gelatin/sds-page, strongly inhibited by the ser-type inhibitor soybean trypsin inhibitor .
a second band was detected in gelatin-polyacrylamide gels, with an rf mobility of  <dig> .
this protease, mostly active in acidic conditions  and insensitive to commonly used class-specific diagnostic inhibitors including pepstatin a , likely corresponded to the pepstatin-insensitive g <dig> asp protease bcacp <dig> identified as an additional determinant of pathogenic infection by b. cinerea <cit> .
pepstatin-sensitive azocaseinases could not be visualized following gelatin/page as reported earlier for other a <dig> asp proteases
<cit> , but could be enriched in mild conditions by affinity chromatography with pepstatin-agarose beads .
unlike asp and ser protease inhibitors, cys-type inhibitors, such as the fungal inhibitor trans-epoxysuccinyl-l-leucylamido- butane  and different cystatins of plant or animal origin, did not inhibit azocaseinase or gelatinase activities .
overall, these observations pointed to the net predominance of asp and ser proteases in the culture medium of b. cinerea, as suggested by recent high-throughput proteomic studies on the secretome of this model organism
<cit> .
they also suggested the absence of cys protease targets for cystatins and the likely innocuity of ccii against the fungus.
1an azocaseinase assay was performed at ph  <dig> , for  <dig> h at 37°c.
values are expressed as relative inhibitory activities compared to an uninhibited control .
each datum is the mean of three values ± se.
2aebsf, 4--benzenesulfonyl fluoride, hydrochloride; e- <dig>  trans-epoxysuccinyl-l-leucylamido- butane; edta, ethylenediamine tetraacetic acid; pmsf, phenylmethylsulfonyl fluoride.
the pleiotropic effects of ccii induce resistance to botrytis cinerea in potato
because studies have reported negative effects for plant cystatins against a number of pathogenic fungi including b. cinerea , in vitro bioassays were carried out with purified ccii to rule out a possible, although unexpected, protease inhibition-independent toxic effect against the model fungus.
in contrast with studies reporting significant effects for barley and strawberry cystatins
<cit> , ccii had no significant effect on fungal biomass production  .
this observation, along with both the retention of ccii in the cytosol of potato leaf cells  and the absence of cys protease targets in the extracellular milieu , confirmed the relevance of b. cinerea as a pathogenic model to assess the impact of ccii-mediated, defense-related pleiotropic effects in the ccii-expressing lines, without the risk of confounding effects due to fungal protease inhibition.
<cit> .
optical density of the fungal culture at  <dig> nm was used as an indicator of biomass production
<cit> .
datapoints are the mean of three independent  values ± se.
b. typical disease symptoms observed on b. cinerea-inoculated plants of line  <dig>  and control line k,  <dig> days post-infection.
c. relative disease symptom scores after  <dig> days for line  <dig> , line  <dig>  and control line k plants inoculated with b. cinerea conidia.
data are expressed as relative cumulative symptom scores, compared to line k  .
in vivo bioassays were conducted with a conidial suspension of the pathogen to compare leaf infection dynamics and symptom development patterns on leaves of line  <dig> , line  <dig>  and control line k .
visual symptoms were recorded over  <dig> days post-infection and categorized along a ‘ <dig> to 5’ scale, where  <dig> indicated no symptom and  <dig> visible mycelium growth at the leaf surface .
as expected, line k exhibited several symptoms, starting with chlorosis of mesophyll tissue after a few days, followed by tissue necrosis at the inoculation site, black areas at leaf extremities indicating vascular tissue infection, and silver spots on most leaves indicative of active mycelium growth.
by comparison, no fungal mycelium could be observed on the ccii-expressing leaves, and necrosis symptoms were systematically less severe, especially for line  <dig>  expressing larger amounts of ccii .
the incidence rate of visual symptoms after  <dig> days was significantly lower on transgenic leaves , as inferred from a relative cumulative symptom score of less than 10% for line  <dig>  compared to control line k .
these observations pointed overall to a protective effect of ccii in potato plants interacting with b. cinerea, likely explained by the stress-related pleiotropic effects of this inhibitor in leaf tissues.
CONCLUSIONS
studies have reported significant pleiotropic effects for recombinant cystatins expressed in transgenic plant lines, notably including stress tolerance phenotypes suggesting an impact on endogenous stress-related responses.
in line with these findings, our data showing an up-regulation of abiotic and biotic stress-related proteins in ccii-expressing potato leaves point to the onset of cystatin-mediated pleiotropic effects in planta altering at some degree the host plant stress metabolism.
in particular, our proteomic data suggest a protein-inducing impact of recombinant ccii in potato leaves triggering the constitutive expression of normally inducible defense proteins, and the beneficial impact of this  phenotype on plants challenged with the fungal pathogen b. cinerea.
the exact metabolic events driving the up-regulation of stress-related proteins in ccii-leaves remain unclear at this stage.
the constitutive expression of several proteins naturally induced by salicylate or its functional analogue benzathiadiazole suggests a role for the salicylic acid signaling pathway, but a more complex picture involving alternative defense pathways cannot be ruled out.
for instance, multicystatins in solanum species are induced by wounding or jasmonic acid
<cit>  but do not respond to fungal elicitors known to reproduce the inducing effects of salicylic acid
<cit> , in apparent disagreement with the hypothetical up-regulation of pmc in ccii-potato lines via a salicylate inducible route.
in a similar way, the actual effects of salicylate inducible defenses against b. cinerea remain equivocal, given the contrasting effects observed from one plant to another
<cit>  and the various signaling interactions established between host plants and different strains of the pathogen
<cit> .
the high complexity of host plant–b.
cinerea interactions in vivo was further illustrated, recently, by a study reporting the deliberate induction of salicylate inducible responses in nicotiana benthamiana, as a strategy for the pathogen to avoid harmful effects of jasmonate inducible defenses
<cit> .
from a biochemical viewpoint, the occurrence of jasmonate and salicylate inducible proteins in ccii-leaf cells suggests, at this point, the onset of gene -specific effects for the cystatin, presumably initiated by the inhibition of one or several c1a cys proteases in the cytosolic compartment.
work is underway to identify such putative protease targets, by a dual in silico/empirical approach involving bioinformatic inferences from solanaceae genome databases and formal detection of cystatin:protease complexes using activity-based, functional proteomics strategies.
work is also underway to modulate cystatin:cys protease interactions and their resulting pleiotropic effects in planta, using cystatin single variants engineered to exhibit either stronger or weaker inhibitory activity against solanaceae protease targets
<cit> .
the proteomic data provided here should represent, to this end, a useful tool for the efficient, one-step monitoring of multiple stress-related protein alterations in plants expressing the recombinant inhibitors.

 several studies have described the potential of recombinant protease inhibitors as potent antidigestive compounds to protect crop plants from herbivory or pathogenic infection. for instance, cysteine protease inhibitors of the cystatin protein superfamily were proposed as protective agents against various herbivorous arthropods and root-parasitic nematodes. cystatins and most other protease inhibitors in plants are competitive protein inhibitors acting as pseudo-substrates to enter the active site of proteases. following inhibition, the target proteases can no longer cleave peptide bonds, which results in a detrimental inhibition of protein digestive functions in herbivorous pests and accounts for the resistance of several transgenic plant lines expressing recombinant inhibitors. the heterologous expression of protease inhibitors in plants, however, has raised a number of questions about the possible occurrence of unintended metabolic interference –or pleiotropic effects– on endogenous proteolysis, which could eventually alter important cellular functions. proteases are ubiquitous metabolic effectors involved in the regulation of numerous cellular processes, ranging from housekeeping functions like protein turnover and the elimination of misfolded polypeptides to the processing of polypeptide pre- and pro-regions on maturing protein backbones. while studies have reported negligible phenotypic effects for protease inhibitors in transgenic plants based on the assessment of macroscopic indicators such as growth rate, stem diameter or leaf number, several reports suggest the onset of more subtle effects at the metabolic level. for instance, plant and mammalian serine protease inhibitors ectopically expressed in potato were shown to significantly impact protein levels in leaves, positively or negatively. similarly, recombinant cystatins expressed in arabidopsis or tobacco were shown to induce a range of phenotype alterations in planta, including a delayed development of floral organs, a modified physiological behaviour under low temperature or light regimes, an altered protein content in leaves, and a strong repression of the pathogen-inducible hypersensitive response. these findings, along with studies reporting the tolerance of protease inhibitor-expressing plants to abiotic stress cues such as drought, salinity and low temperatures, point to the occurrence of endogenous protease targets for the recombinant inhibitors, directly or indirectly involved in stress-related processes. the so-called pleiotropic effects of recombinant protease inhibitors, which are often considered as unintended metabolic effects in the modified plant, might simply reflect a lack of knowledge on stress-related proteolysis in planta and in fact represent a source of potentially useful traits for crop improvement. here we provide experimental evidence for the up-regulation of abiotic and biotic stress-related proteins in leaves of transgenic potato lines engineered to express corn cystatin ii, a potent inhibitor of c1a cys proteases exhibiting potential for herbivore pest control. we also link the pleiotropic effects of ccii expression with the compromised ability of a model necrotrophic fungus, botrytis cinerea, to colonize leaf tissues of the modified host plant.  pathogenesis-related proteins are up-regulated in ccii-expressing potato lines reverse transcriptase pcr, immunodetection and surface-enhanced laser desorption ionization time-of-flight mass spectrometry analyses were performed to select transgenic potato lines expressing ccii at different levels in leaves, among a collection of independent transformants developed earlier in our laboratory. ccii in these lines accumulates in the cytosolic compartment, under the control of the cauliflower mosaic virus 35s constitutive promoter. unlike control line k showing no signal, the transgenic lines showed variable, but easily detectable signals of ccii-encoding mrna transcripts. accordingly, ccii was immunodetected as a ~12-kda polypeptide band of variable intensity in the modified lines, in contrast with line k giving no signal. the amount of ccii in leaf extracts was variable among the lines, as inferred by seldi tof ms after capture of the inhibitor on cm- biochips for weak cationic exchange. unexpectedly, a clear immunoblot signal was detected in several ccii-lines for the wound inducible homologue of ccii in potato leaves, potato multicystatin. this apparent up-regulation of the endogenous inhibitor was confirmed by densitometric analysis of the immunoblots revealing significant increases reaching two- to tenfold the signals observed for control line k in four transgenic lines, out of five tested.. numbers on the right refer to commercial molecular weight markers. pmc refers to endogenous potato multicystatin. c. relative pmc levels in ccii-lines compared to mean basal level in control line k. each bar on panels a and c is the mean of three independent values ± se. asterisks indicate a significant difference with control line k. rt pcr assays were conducted with nucleotide primers for mrna transcripts of proteinase inhibitor ii, a protein marker of wound and jasmonate inducible proteins; and pathogenesis-related protein p a protein marker of the abiotic stress-/pathogen-inducible salicylic acid pathway. transcript levels for pin-ii were not different in control and transgenic lines, while transcripts for protein p were found at higher levels in several ccii-expressing lines, including line and line. this observation suggesting an up-regulation of salicylate inducible proteins in the ccii-expressing lines was further supported by immunodetection assays with primary antibodies for pr- and pr- proteins, which showed levels of proteins from both families to be significantly higher in lines c, and compared to control line k. the stress-related proteome is altered in ccii-expressing leaves a comparative two-dimensional gel electrophoresis proteome analysis was conducted with leaf protein extracts of line, line and control line k to measure the overall impact of ccii on the host plant’s leaf proteome, and to test the hypothesis of a specific up-regulation of salicylate inducible stress-related proteins in plants ectopically expressing the recombinant inhibitor. out of ~ protein spots monitored on 2-d gels, showed a significantly altered level in ccii-expressing lines compared to the control line, for similar amounts of protein loaded on gel strips. of these proteins, were detected de novo or up-regulated by more than fivefold in the ccii-lines. as observed above for pr proteins, the up-regulating effects of ccii were systematically stronger in line compared to line producing lower levels of ccii, thereby suggesting a dose-dependent effect for the recombinant inhibitor. for protein numbering and function assignment. 2-de spatial coordinates, mowse score, protein sequence coverage, number of matched peptides and matched peptide sequences are given for each protein in additional file protein spots and could not be identified based on available ms spectra. all protein spots matched potato protein sequences, except spot and spot matching tomato sequences. blanks indicate no detectable protein product. most proteins up-regulated in the ccii-lines were confidently identified based on lc-ms/ms sequence data. in accordance with the increased levels of pmc and pr proteins in transgenic leaves, several up-regulated proteins in lines and corresponded to defense-related proteins usually induced upon wounding or biotic stress challenge, such as for instance wound inducible ser protease inhibitors, a kunitz cathepsin d inhibitor, a pr- protein and three pr- proteins or protein fragments. proteins that are usually induced upon abiotic stress challenge and thought to protect cells from oxidative damage or osmotic imbalance were also identified, including an osmotin-like pr- protein and several secretory peroxidases. a number of studies in recent years have described the potential usefulness of ectopically expressing antioxidant enzymes –or positive regulators of these enzymes– to generate crop lines tolerant to adverse abiotic conditions such as low or high temperatures, drought or salinity. considering the above described pleiotropic effects of ccii on the potato leaf proteome, the reported ability of cystatin-expressing plants to cope more efficiently with abiotic stress conditions could be likely explained, at least in part, by a significant up-regulating effect of the recombinant inhibitors on the steady-state levels of these enzymes in leaf tissues. ccii does not inhibit the extracellular proteases of botrytis cinerea bioassays were conducted with the fungal necrotroph b. cinerea to document the possible positive impact of ccii pleiotropic effects, including pr protein up-regulation, on potato plants challenged with an aggressive microbial pathogen. the increased expression of peroxidases and pr proteins in leaves treated with benzathiadiazole, a functional analogue of salicylic acid, was shown earlier to induce resistance to b. cinerea in different plants. likewise, strong detrimental effects against this pathogen were reported for recombinant pr proteins or gene inducers of these proteins expressed in different plants, including potato. the documented repressing effect of recombinant cystatins on the pathogen-inducible hypersensitive response in transgenic plants could also impact the ability of b. cinerea to colonize ccii-leaf tissues. this pleiotropic effect of recombinant cystatins, also observed in plants treated with synthetic cys protease inhibitors or transformed with dna sequences to silence cys proteases expression, would be correlated with an increase of cystatin:cys protease ratios in planta preventing the release of free cys proteases to initiate cell death. this ectopic phenotype could represent, along with the constitutive over-expression of stress-related proteins, a major hurdle to the progression of the necrotroph, which relies on cell death induction to generate dead tissues and successfully colonize the host plant. protease activity assays were first performed to measure the relative abundance of secreted cys proteases in b. cinerea, and to evaluate the possibility of a direct, cys protease inhibitory-mediated effect of ccii against this fungus. in agreement with earlier studies reporting the presence of aspartate proteases in the secretome of b. cinerea and proposing a key role for these enzymes during plant infection, protease activities in the liquid phase of a b. cinerea culture grown in potato dextrose broth were inactivated by almost 50% with pepstatin a, a specific inhibitor of a asp proteases. ser protease activities were also detected, as inferred by a weak, but reproducible inhibitory effect of ser-type inhibitors on azocaseinase activity. the presence of secreted ser protease in the culture medium was confirmed by the detection of a clear protein lysis band following gelatin/sds-page, strongly inhibited by the ser-type inhibitor soybean trypsin inhibitor. a second band was detected in gelatin-polyacrylamide gels, with an rf mobility of. this protease, mostly active in acidic conditions and insensitive to commonly used class-specific diagnostic inhibitors including pepstatin a, likely corresponded to the pepstatin-insensitive g asp protease bcacp identified as an additional determinant of pathogenic infection by b. cinerea. pepstatin-sensitive azocaseinases could not be visualized following gelatin/page as reported earlier for other a asp proteases, but could be enriched in mild conditions by affinity chromatography with pepstatin-agarose beads. unlike asp and ser protease inhibitors, cys-type inhibitors, such as the fungal inhibitor trans-epoxysuccinyl-l-leucylamido- butane and different cystatins of plant or animal origin, did not inhibit azocaseinase or gelatinase activities. overall, these observations pointed to the net predominance of asp and ser proteases in the culture medium of b. cinerea, as suggested by recent high-throughput proteomic studies on the secretome of this model organism. they also suggested the absence of cys protease targets for cystatins and the likely innocuity of ccii against the fungus. 1an azocaseinase assay was performed at ph, for h at 37°c. values are expressed as relative inhibitory activities compared to an uninhibited control. each datum is the mean of three values ± se. 2aebsf, 4--benzenesulfonyl fluoride, hydrochloride; e- trans-epoxysuccinyl-l-leucylamido- butane; edta, ethylenediamine tetraacetic acid; pmsf, phenylmethylsulfonyl fluoride. the pleiotropic effects of ccii induce resistance to botrytis cinerea in potato because studies have reported negative effects for plant cystatins against a number of pathogenic fungi including b. cinerea, in vitro bioassays were carried out with purified ccii to rule out a possible, although unexpected, protease inhibition-independent toxic effect against the model fungus. in contrast with studies reporting significant effects for barley and strawberry cystatins, ccii had no significant effect on fungal biomass production. this observation, along with both the retention of ccii in the cytosol of potato leaf cells and the absence of cys protease targets in the extracellular milieu, confirmed the relevance of b. cinerea as a pathogenic model to assess the impact of ccii-mediated, defense-related pleiotropic effects in the ccii-expressing lines, without the risk of confounding effects due to fungal protease inhibition.. optical density of the fungal culture at nm was used as an indicator of biomass production. datapoints are the mean of three independent values ± se. b. typical disease symptoms observed on b. cinerea-inoculated plants of line and control line k, days post-infection. c. relative disease symptom scores after days for line, line and control line k plants inoculated with b. cinerea conidia. data are expressed as relative cumulative symptom scores, compared to line k. in vivo bioassays were conducted with a conidial suspension of the pathogen to compare leaf infection dynamics and symptom development patterns on leaves of line, line and control line k. visual symptoms were recorded over days post-infection and categorized along a ‘ to 5’ scale, where indicated no symptom and visible mycelium growth at the leaf surface. as expected, line k exhibited several symptoms, starting with chlorosis of mesophyll tissue after a few days, followed by tissue necrosis at the inoculation site, black areas at leaf extremities indicating vascular tissue infection, and silver spots on most leaves indicative of active mycelium growth. by comparison, no fungal mycelium could be observed on the ccii-expressing leaves, and necrosis symptoms were systematically less severe, especially for line expressing larger amounts of ccii. the incidence rate of visual symptoms after days was significantly lower on transgenic leaves, as inferred from a relative cumulative symptom score of less than 10% for line compared to control line k. these observations pointed overall to a protective effect of ccii in potato plants interacting with b. cinerea, likely explained by the stress-related pleiotropic effects of this inhibitor in leaf tissues.  studies have reported significant pleiotropic effects for recombinant cystatins expressed in transgenic plant lines, notably including stress tolerance phenotypes suggesting an impact on endogenous stress-related responses. in line with these findings, our data showing an up-regulation of abiotic and biotic stress-related proteins in ccii-expressing potato leaves point to the onset of cystatin-mediated pleiotropic effects in planta altering at some degree the host plant stress metabolism. in particular, our proteomic data suggest a protein-inducing impact of recombinant ccii in potato leaves triggering the constitutive expression of normally inducible defense proteins, and the beneficial impact of this phenotype on plants challenged with the fungal pathogen b. cinerea. the exact metabolic events driving the up-regulation of stress-related proteins in ccii-leaves remain unclear at this stage. the constitutive expression of several proteins naturally induced by salicylate or its functional analogue benzathiadiazole suggests a role for the salicylic acid signaling pathway, but a more complex picture involving alternative defense pathways cannot be ruled out. for instance, multicystatins in solanum species are induced by wounding or jasmonic acid but do not respond to fungal elicitors known to reproduce the inducing effects of salicylic acid, in apparent disagreement with the hypothetical up-regulation of pmc in ccii-potato lines via a salicylate inducible route. in a similar way, the actual effects of salicylate inducible defenses against b. cinerea remain equivocal, given the contrasting effects observed from one plant to another and the various signaling interactions established between host plants and different strains of the pathogen. the high complexity of host plant–b. cinerea interactions in vivo was further illustrated, recently, by a study reporting the deliberate induction of salicylate inducible responses in nicotiana benthamiana, as a strategy for the pathogen to avoid harmful effects of jasmonate inducible defenses. from a biochemical viewpoint, the occurrence of jasmonate and salicylate inducible proteins in ccii-leaf cells suggests, at this point, the onset of gene -specific effects for the cystatin, presumably initiated by the inhibition of one or several c1a cys proteases in the cytosolic compartment. work is underway to identify such putative protease targets, by a dual in silico/empirical approach involving bioinformatic inferences from solanaceae genome databases and formal detection of cystatin:protease complexes using activity-based, functional proteomics strategies. work is also underway to modulate cystatin:cys protease interactions and their resulting pleiotropic effects in planta, using cystatin single variants engineered to exhibit either stronger or weaker inhibitory activity against solanaceae protease targets. the proteomic data provided here should represent, to this end, a useful tool for the efficient, one-step monitoring of multiple stress-related protein alterations in plants expressing the recombinant inhibitors. 
accordingly, infection of leaf tissues by the fungal necrotroph botryris cinerea was prevented in ccii-expressing plants, despite a null impact of ccii on growth of this pathogen and the absence of extracellular cys protease targets for the inhibitor. the leaf proteome of lines accumulating ccii at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control line. here we assessed the effects of corn cystatin ii, a potent inhibitor of c1a cysteine proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein. they also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance. these data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. proteins monitored on 2-d gels, were significantly up- or downregulated in ccii-expressing leaves, including proteins detected de novo or up-regulated by more than five-fold compared to the control.
 studies reported unintended pleiotropic effects for a number of pesticidal proteins ectopically expressed in transgenic crops, but the nature and significance of such effects in planta remain poorly understood. here we assessed the effects of corn cystatin ii, a potent inhibitor of c1a cysteine proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein.  the leaf proteome of lines accumulating ccii at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control line. out of ca. proteins monitored on 2-d gels, were significantly up- or downregulated in ccii-expressing leaves, including proteins detected de novo or up-regulated by more than five-fold compared to the control. most up-regulated proteins were abiotic or biotic stress-responsive proteins, including different secretory peroxidases, wound inducible protease inhibitors and pathogenesis-related proteins. accordingly, infection of leaf tissues by the fungal necrotroph botryris cinerea was prevented in ccii-expressing plants, despite a null impact of ccii on growth of this pathogen and the absence of extracellular cys protease targets for the inhibitor.  these data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. they also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance. transgenic cropstransgene pleiotropyunintended effectsstress/defense-related proteomecorn cystatinpotato 