BACKGROUND
plants are constantly defending themselves against pathogens by means of a wide array of mechanisms.
some of them are pre-existing  and others are induced in response to the pathogen attack.
salicylic acid  is a plant hormone which is crucial for the inducible response of arabidopsis thaliana  to biotrophic pathogens like pseudomonas spp <cit> .
when a pathogen is perceived, sa is produced and accumulated, producing a proper defense.
this sa signaling occurs not only where the attack takes place, since defense is also enhanced in leaves different from the one inoculated.
this is called systemic acquired resistance .
sa has an intricate crosstalk with other hormones, showing an overall negative crosstalk with auxins, ethylene , and jasmonates .
in the case of ja, it has been shown that the active form in planta is ja-ile , while in the laboratory is used exogenously as methyl jasmonate .
non-expressor of pathogenesis-related genes <dig>  is the main gene required for sa perception  <cit> .
there are five paralogs of npr <dig> in arabidopsis  <cit> , blade-on-petiole <dig>  and bop <dig> have an important role in development  <cit> , npr <dig> and npr <dig> have a role in defense  <cit> , probably through sa perception  <cit> , and no specific function for npr <dig> has been described, besides a secondary role in sa perception  <cit> .
there are other genes that are relevant for signal transduction, like the family of tga transcription factors whose products interact with npr <dig>  <cit> , but they are required in a redundant fashion.
npr <dig> has been described as having more than one role in defense, since it is also important in the induced systemic resistance .
isr is defined as the resistance triggered at the leaves by a non pathogenic organism inoculated in the roots, and while sar requires sa signaling, isr requires meja and et signaling.
as with sa, exogenous applications of meja and et trigger resistance in arabidopsis towards some biotrophs, like pseudomonas <cit> .
it has been proposed that npr <dig> is relevant for the resistance-inducing ability of meja , although rim it is not necessarily equivalent to isr.
while the role of npr <dig> in sa perception takes place in the nucleus  <cit> , its function in rim is not so clearly understood.
it has been described a cytosolic function of npr <dig> crucial in cross-talk between sa and ja signaling  <cit> .
furthermore, arabidopsis transcriptome analysis upon pathogen infection has suggested that such cytosolic function is also involved in the modulation of ja-dependent defenses  <cit> .
the npr1- <dig> mutant, which produces a truncated cytoplasmatically localized npr <dig> protein with no nuclear localization signal, has been reported to be affected only in sa-dependent gene expression, not in ja and et dependent genes.
in contrast, the npr1- <dig> mutant, which has a mutation in a key domain, is affected in the expression of sa, ja and et-dependent genes  <cit> .
more recent studies support such cytosolic npr <dig> function as regulator of ja-dependent defense responses .
bop <dig> was first identified by its mutant phenotype of ectopic blades along the petioles, as well as some alterations in the flowers  <cit> .
the first allele identified was a dominant negative, since t-dna insertions in bop <dig> did not reproduce the phenotypes of ectopic blades  <cit> .
once bop <dig> was identified as paralog to npr <dig> <cit> , it was shown that other paralog, bop <dig>  functions redundantly with bop <dig> <cit> .
the double mutant bop <dig> bop <dig> reproduced all the developmental phenotypes of ectopic blades, but it was wild type when inoculated with pseudomonas <cit> , and it is also wild type for sa perception  <cit> .
since a collection of npr <dig> alleles has recently been available  <cit> , we tested the hypothesis that the role of npr <dig> in rim is cytosolic.
in this work, we show that npr <dig> has no role in rim in wild type conditions, since the genes responsible for rim are bop <dig> and bop <dig>  with an important part being played by the tgas.
therefore, two genes required for the normal development of the leaf, are also required for plant defense.
RESULTS
role of npr <dig> in rim
npr <dig> has been characterized as a result of observing the response to sa of the great number of alleles described for it  <cit> .
npr <dig> has also been described as essential for rim, but there are differences between alleles, since npr1- <dig> and npr1- <dig> have different rim .
npr1- <dig> and npr1- <dig> have other differences in phenotypes related to meja.
thus, the sa-ja antagonism is not present in npr1- <dig>  but it is active in npr1- <dig>  <cit> .
other difference is the gene expression, whereas npr1- <dig> was affected in sa, ja, and et dependent genes upon pto inoculation, npr1- <dig> was only affected in sa dependent genes  <cit> .
these different phenotypes have been attributed to the lack of nuclear localization in npr1- <dig>  since the truncated cytosolic protein would be functional to modulated ja-dependent defense response  <cit> .
in order to determine the precise role of npr <dig> in rim, and to asses the role of the cytosolic vs. nuclear localization, we tested an allelic series of  <dig> npr <dig> alleles, defined by their inability to perceive sa  <cit> .
the npr <dig> alleles produced a mixture of phenotypes in rim , and out of the  <dig> alleles tested,  <dig> had a significant rim , meaning that the growth of pto in meja treated plants was significantly lower than in mock treated plants .
the rest of alleles showed no response .
these two categories of alleles did not share any obvious feature and had the same proportion of stops and point mutations in each category.
in fact, in the rim+ alleles tested, six point mutations were widely scattered along the protein .
these alleles could somehow affect the localization of the protein inside the cell, even if the mutation was not in the nls.
to check this possibility, cdnas of three rim+ alleles , and three rim- alleles , chosen among the point mutations, were cloned along with the wild type npr <dig>  then, these seven cdnas were transiently expressed in n. benthamiana under the control of the 35s promoter and with the gfp marker.
the free gfp was detectable in the nucleus and in the cytoplasm.
but when the wild type and the six alleles of npr <dig> were expressed in the same conditions, gfp was detected mainly in the nucleus, with no difference existing between the two classes of alleles .
as a complementary approach, we took advantage of the transgenic line that overexpresses npr <dig> fused to the steroid hormone binding domain of the rat glucocorticoid receptor .
npr1-hbd remains exclusively in the cytosol in mock conditions and should be functional in rim.
the original line is in an npr1- <dig> background , and therefore the transgene was transferred to an npr1- <dig> background  to check for complementation.
treatments with bth and with and without glucocorticoid dexamethasone  showed that npr1-hbd was functional .
npr1-hbd, even under the control of the 35s promoter, did not complement the lack of rim in npr1- <dig> .
when dex was applied, npr1-hbd moved to the nucleus and npr1- <dig> was complemented in the rim phenotype.
note that the presence of cytosolic npr1-hbd in an npr1- <dig> background did not enhance rim in comparison to npr1- <dig> alone.
the npr <dig> alleles rim+ or rim- did not share any obvious feature, so it would be difficult to assign a precise role to the wild type gene.
a critical genetic resource to discern the role of a gene is the null allele.
therefore, the response of two null alleles of npr <dig> to rim was measured .
both npr1- <dig> and npr1- <dig> are in laer- <dig> background, so an introgression of npr1- <dig> in laer- <dig> was used as control  <cit> .
these two null alleles responded to rim in all experiments like the wild type.
npr1- <dig> introgressed in col- <dig> responded again like the wild type, which ruled out any ecotype effect .
since the direct role of npr <dig> in rim was in question as a consequence of the aforementioned results, we included an independent rim- control, coi1- <dig> .
the role of npr <dig> in this response might be indirect.
thus, one scenario would be a reinforcement of the negative crosstalking between sa and meja.
npr <dig> alleles produced more sa when infected with pto <cit>  and seemed unable to metabolize it  <cit> .
rim- alleles -defective in terms of sa perception- might have left intact the negative crosstalk between sa and meja, and an excess of sa repressed the response to meja beyond the wild type levels.
therefore, the rim+ alleles would be defective in terms of both sa perception and sa-meja crosstalking, an explanation that would also be in agreement with the behavior of the null alleles.
to test this hypothesis, the double mutants between npr1- <dig> and nahg , eds <dig> , and sid <dig> , were constructed and tested for rim.
npr1- <dig> did not respond to meja even if the levels of sa were low , so the hypothesis of a reinforcement of the negative crosstalk was not supported.
bop <dig> and bop <dig> and their role in rim
the experiments with the null alleles showed that npr <dig> was not necessary for rim.
perhaps npr <dig> and other gene would be redundantly responsible of rim, and while null npr <dig> alleles would have a rim+ phenotype, some npr <dig> alleles could be rim- by interacting with other protein negatively.
the most likely candidates for these interactions were the npr <dig> paralogs, since their proteins shared the same overall structure.
there are five additional paralogs of npr <dig> in the arabidopsis genome  <cit> , and we analyzed double mutants of npr1- <dig> with different paralogs .
there was no proof of npr <dig> having a redundant role in rim.
npr <dig> did not play a significant role in rim  and the same was true for the double npr <dig> npr <dig> .
strikingly, the double bop <dig> bop <dig> <cit>  was rim- .
single bop <dig> and bop <dig> were also tested for rim and showed to be wild type .
bop <dig> and bop <dig> exert their function in part through transcriptional regulation of knat <dig> and physical interaction with pan , but t-dna insertions predicted to disrupt knat <dig> or pan activity did not have an effect on rim .
to rule out the possibility that other mutations besides bop <dig> and bop <dig> were producing this rim- phenotype, we constructed an artificial microrna  to deplete the levels of bop <dig> and bop <dig> at the same time.
eight independent homozygous transgenic lines for amirna  were analyzed for rim .
five out of eight lines were rim-, and the remaining three responded less than the wild type control.
the levels of both genes were partially depleted in the eight lines ; five of the lines had both genes significantly reduced, and all had bop <dig> significantly reduced.
none of these lines had the characteristic blade-on-petiole macroscopic phenotype, not even as the subtle phenotype of bop <dig> alone.
the previous experiments had shown that bop <dig> and bop <dig> were acting redundantly in rim .
therefore, increasing the amount of any of them should have an effect on rim, especially since normal levels of bop <dig> and bop <dig> are quite low .
the overexpression lines of bop <dig> and bop <dig> described  <cit>  were analyzed for rim.
35s:bop <dig> had a stronger rim than col- <dig>  and 35s:bop <dig> had a strong variation in the meja treated plants .
at the time of the experiments, each population looked homogeneous, but when these plants were grown to set seeds, two phenotypes could be observed in each transgenic line.
approximately half of the plants showed a wild type phenotype, and the other half reproduced the dwarf plants described  <cit> .
seeds from both lines and from both phenotypes reproduced the two phenotypes.
it seems that it was an issue of silencing, since rna taken from plants classified by their mentioned phenotype diverged widely in the transgene expression .
bop <dig> bop <dig> specificity in rim
the bop <dig> bop <dig> double mutant lacked a rim response, but this failure in meja signaling might occur at different points of the signal transduction.
for example, the defect could target a general signaling component affecting all meja responses , or a specialized part of the pathway, affecting a subset of meja responses .
when bop <dig> bop <dig> plants were grown in plates containing meja, the growth of the roots was similar to the wild type controls .
other phenotypes of bop <dig> bop <dig> plants growing in meja plates were similar to the wild type controls .
another effect of meja is the increase of senescence in detached leaves, measured as chlorophyll production  <cit> .
bop <dig> bop <dig> responded as the wild type in this particular system .
coronatine is a virulence factor of several pseudomonas isolates with structural and functional similarities to ja-ile , therefore a mutant pto that lacks coronatine  <cit>  grows less in col- <dig> than the wild type pto.
bop <dig> bop <dig> was also wild type in response to pto with and without coronatine .
inoculations with plectospharaella cucumerina, a fungus that causes more disease in meja mutants than in wild type plants  <cit> , did not cause any more disease in bop <dig> bop <dig> than in col- <dig> .
if bop <dig> bop <dig> was not a meja signaling mutant, but specifically in rim, it could be defective in the other signaling required for isr; et.
it was not; when  <dig> mm of 1-aminocyclopropane-1-carboxylic acid  was sprayed to bop <dig> bop <dig>  the resistance triggered was similar to that triggered in the wild type controls .
etr <dig>  was included as a negative control of resistance induced by ethylene.
npr <dig> and bops interactions
once it was clear that both bop genes are required for rim, we tested the model that npr <dig> rim- alleles could have a dominant negative effect on bop activity, either directly or indirectly.
to first test whether npr <dig> had an effect in the interaction between bop proteins, we used a yeast two-hybrid assay.
as reported, bop <dig> and bop <dig> interacted with each other  <cit> .
next, we introduced in a third plasmid containing wt npr <dig> or various mutant npr <dig> alleles presented in figure 2a.
if the effect of npr <dig> on rim were a direct interaction between npr <dig> and the two bops, the alleles that diverge in their rim phenotype would diverge in their ability to interfere in the interaction of bop <dig> and bop <dig>  the two classes of npr <dig> alleles did not have a distinct behavior , therefore the dominant negative effect did not seem to be direct.
all the npr <dig> paralogs tested interact with members of the tga family in a different degree  <cit> .
therefore, the tgas would be a reasonable candidate for being the third component, and their interaction with rim- alleles would indirectly affect the function of bop <dig> and bop <dig>  as a control, single mutants in tga <dig> and tga <dig> produced a significant rim , but when three specific tgas are knocked out at the same time , there is no rim .
we reasoned that one or several of these three tgas  might have a functional interaction with the bops, which might be affected by the rim- alleles.
to test this hypothesis bop <dig> and each of the mentioned tgas were introduced in the yeast two-hybrid system with the npr <dig> alleles mentioned above in a third plasmid.
tga <dig> and tga <dig> interact differentially with bop <dig> depending on the npr <dig> protein present .
there was an enhancement of the interaction in two out of the three rim- alleles, and no interference in two out of the three rim+ alleles.
the interaction tga5-bop <dig> was not affected by the presence of npr <dig> proteins .
the experiments were repeated with bop <dig> producing similar results .
in sum, the data indicated that bops and tga <dig>  tga <dig> and tga <dig> are required for rim, that bops interact with these  tgas, and that npr <dig> may modulate the affinity or stability of the interactions.
discussion
npr <dig> is not required for rim
npr <dig> is an essential gene for sar and sa perception  <cit> .
npr1- <dig>  the most widely used allele, is also impaired in rim  <cit> .
we speculated that since npr1- <dig> is wild type for rim , and it has been reported that the difference of some phenotypes between npr1- <dig> and npr1- <dig> was due to the lack of nls in npr1- <dig> , the same could be true for rim.
however, we show here that the nuclear localization of the alleles makes no difference.
this conclusion is supported by multiple lines of evidence.
first, the npr <dig> alleles with rim+ are not structurally similar to npr1- <dig>  since not all of them are affected in the nls .
even an allele with a point mutation in the nls  should be partially localized in the nucleus  <cit> .
second, three rim- and three rim+ alleles do not differ in their nuclear localization or stability when transiently expressed in n. benthamiana .
while these proteins are no longer functional, they respond to the signals of a wild type background by localizing in the nucleus.
third, when a functional npr <dig> is anchored in the cytoplasm there is no complementation of the rim- phenotype in an npr1- <dig> background , nor there is an increase in rim phenotype in an npr1- <dig> background.
in fact, the application of dex triggered an increased rim in both backgrounds .
but, most importantly, npr <dig> is not required for rim, since the null npr <dig> alleles are rim+ regardless of the background .
we also discarded that npr <dig> could be a part of rim in a redundant fashion with its paralogs .
an interesting alternative for the role of npr <dig> in rim would be an effect on the crosstalking between sa and meja.
npr <dig> has been described as a key point in the negative regulation between sa and meja.
thus, the rim+ alleles could be defective in both sa perception and in sa-meja crosstalk, while the rim- alleles would be defective only in sa perception but not in sa-meja crosstalk.
the inoculation with pto triggers an increase in the levels of sa, and in the case of the npr <dig> alleles, there is more sa than in the wild type  <cit> .
although this hypothesis would explain the phenotype of the null alleles, it was rejected after the experiment of figure 3c, where a severe reduction of sa levels in a rim- allele did not have any effect on the phenotype.
bop <dig> and bop <dig> are redundant in rim
the redundant functions of bop <dig> and bop <dig> are essential for normal development.
previous work has shown that the double mutant has numerous defects in plant architecture including altered leaf morphology  <cit> , changes in floral patterning  <cit> , defects in the conversion of shoots to flowers  <cit>  and loss of floral-organ abscission  <cit> .
the double mutant was tested for basal defense  <cit>  and sa perception  but no difference from wild type was found.
we show here that both genes are also redundantly required in defense against pathogens triggered by meja.
interestingly, whereas significant loss of bop activity is required to exert changes in development  <cit> , rim is abolished in plants that are only partially silenced for the bop genes .
thus, the levels of gene expression required for rim are higher than those required for normal development.
compatible with this idea, bops expression in plants is highly localized, restricted to young organ primordia, leaf petioles, and lateral organ boundaries, which may make systemic responses to meja sensitive relatively minor changes in bop transcript abundance.
both npr <dig> and the bops localize to the cytoplasm as well as nucleus and interact with members of the tga family of bzip transcription factors, albeit with different affinities .
in development, bop <dig> and bop <dig> form a nuclear complex with tga8/perianthia  to regulate number of sepals and petals in flowers and potentially to promote floral meristem fate  <cit> .
given that pan loss-of-function did not reproduce the rim- phenotype  other genes, perhaps tgas, are involved in this phenotype, as shown for sa perception  <cit> .
given that bops play both positive and negative roles in transcriptional regulation of the knox  gene knat <dig> <cit> , we also tested if rim was affected by knat <dig> loss-of-function, but again, no difference was observed .
this may reflect redundancy with other knox genes, or more likely, that bop regulation of rim is independent of knat <dig>
whether bop <dig> bop <dig> recapitulates or not all the phenotypes of the rim- npr <dig> alleles  remains to be assessed.
we did check that there were similar phenotypes in the specificity of response to meja as well as the fact that bop <dig> bop <dig> was wild type for the rest of meja phenotypes .
but there were strong differences, since npr1- <dig> is affected in basal defense and sa perception while bop <dig> bop <dig> is wild type for both phenotypes  <cit> .
regarding et, the other hormone relevant for isr, it has been proposed that applications of this hormone could render the crosstalk between sa and meja independent of npr <dig>  <cit> .
it seems plausible that the et induced resistance works as the meja induced resistance and other proteins -perhaps npr <dig> paralogs, but not the bops  - might also be affected by some alleles of npr <dig>
some npr <dig> alleles interfere in the bops-tgas interaction
the rim- npr <dig> alleles were the majority of the alleles found .
how is this compatible with the fact that the null npr <dig> alleles are rim+?
a possible explanation was the selection used in the screening.
since the selection was made for complete loss of sa perception, perhaps most of the rim+ alleles had a phenotype of partial sa perception, as the null alleles.
then, the prediction would be that a good number of random alleles of npr <dig> would be rim+ and partially receptive to sa.
we previously showed that for sa perception, there are genetic interactions between the npr <dig> alleles and the npr <dig> paralogs  <cit> .
the work reported herein points to a genetic interaction too, this being between npr <dig> alleles on one side and the bops on the other.
thus, the rim- alleles were a phenocopy of the bop <dig> bop <dig> mutant in defense but not in development.
this discrimination was a consequence of the different thresholds for the phenotype in development and defense .
mechanistically, the levels of expression of the bops were low in comparison to npr <dig> , so a direct or indirect negative interference of npr <dig> with the bops would be favored stoichiometrically.
once the pathogen was inoculated, the levels of sa would rise and in a wild type plant npr <dig> is degraded as part of the signaling process  <cit> .
in an npr <dig> background, this signaling would not be transmitted and perhaps the npr <dig> proteins would be able to interfere longer in rim.
this would explain the behavior of npr1-hbd in npr1- <dig> ; npr1-hbd in the cytoplasm did not complement npr1- <dig> in the rim phenotype, but when dex was applied there was complementation of the phenotype.
likely, when no dex was present npr1- <dig> would somehow interfere with the function of the bops.
when dex was present, the presence of npr1-hbd in the nucleus would trigger the degradation of both npr1-hbd and npr1- <dig>  if npr1- <dig> was degraded, the bops would function normally.
there was no evidence for a direct interaction in yeast, since the presence of npr <dig> or mutated versions of this protein did not interfere in the interaction between bop <dig> and bop <dig> in a consistent manner with the phenotype .
a first alternative was that the interference of the rim- alleles would occur with the bops without affecting the interaction between the bops.
a second alternative would be that the rim- alleles would interfere with other proteins that normally interact with the bops.
in both cases there is a family of proteins that interacts with both npr <dig> and the bops, the transcription factors tgas  <cit> , with -again- functional redundancy .
two out of three rim- alleles enhanced or stabilized the bops-tgas interaction, while two out of three rim+ alleles did not .
it was clear that the npr <dig> mutated proteins had an unpredicted effect on the bops-tgas interaction, but the yeast experiments did not produce an absolute answer about the role of npr <dig> proteins in rim.
we speculate that in planta, all the rim- alleles enhance the interaction between the bops and the tgas, titering out the tgas and thus rendering them unable to fulfill their function of triggering defense.
on the other hand, the rim+ alleles , and npr <dig> would not affect the interaction either way.
since in the yeast assays two out of three alleles worked as proposed in either way, it may be possible that a factor is present in the plant that is not in yeast, or perhaps the fact that there are ten tgas <cit> , and that npr <dig> is expressed between  <dig> and  <dig> times more than bop <dig> + bop <dig>  could explain this difference.
if this hypothesis were to be true, it will definitively explain the role of npr <dig> in rim.
CONCLUSIONS
in sum, we have shown that, in wild type conditions, the bops and the tgas  are required for the resistance triggered by methyl jasmonate against pto.
we propose that the phenotype of the npr <dig> rim- alleles is caused by their interference between bops and tgas.

 plants are constantly defending themselves against pathogens by means of a wide array of mechanisms. some of them are pre-existing and others are induced in response to the pathogen attack. salicylic acid is a plant hormone which is crucial for the inducible response of arabidopsis thaliana to biotrophic pathogens like pseudomonas spp. when a pathogen is perceived, sa is produced and accumulated, producing a proper defense. this sa signaling occurs not only where the attack takes place, since defense is also enhanced in leaves different from the one inoculated. this is called systemic acquired resistance. sa has an intricate crosstalk with other hormones, showing an overall negative crosstalk with auxins, ethylene, and jasmonates. in the case of ja, it has been shown that the active form in planta is ja-ile, while in the laboratory is used exogenously as methyl jasmonate. non-expressor of pathogenesis-related genes is the main gene required for sa perception. there are five paralogs of npr in arabidopsis, blade-on-petiole and bop have an important role in development, npr and npr have a role in defense, probably through sa perception, and no specific function for npr has been described, besides a secondary role in sa perception. there are other genes that are relevant for signal transduction, like the family of tga transcription factors whose products interact with npr, but they are required in a redundant fashion. npr has been described as having more than one role in defense, since it is also important in the induced systemic resistance. isr is defined as the resistance triggered at the leaves by a non pathogenic organism inoculated in the roots, and while sar requires sa signaling, isr requires meja and et signaling. as with sa, exogenous applications of meja and et trigger resistance in arabidopsis towards some biotrophs, like pseudomonas. it has been proposed that npr is relevant for the resistance-inducing ability of meja, although rim it is not necessarily equivalent to isr. while the role of npr in sa perception takes place in the nucleus, its function in rim is not so clearly understood. it has been described a cytosolic function of npr crucial in cross-talk between sa and ja signaling. furthermore, arabidopsis transcriptome analysis upon pathogen infection has suggested that such cytosolic function is also involved in the modulation of ja-dependent defenses. the npr1- mutant, which produces a truncated cytoplasmatically localized npr protein with no nuclear localization signal, has been reported to be affected only in sa-dependent gene expression, not in ja and et dependent genes. in contrast, the npr1- mutant, which has a mutation in a key domain, is affected in the expression of sa, ja and et-dependent genes. more recent studies support such cytosolic npr function as regulator of ja-dependent defense responses. bop was first identified by its mutant phenotype of ectopic blades along the petioles, as well as some alterations in the flowers. the first allele identified was a dominant negative, since t-dna insertions in bop did not reproduce the phenotypes of ectopic blades. once bop was identified as paralog to npr, it was shown that other paralog, bop functions redundantly with bop. the double mutant bop bop reproduced all the developmental phenotypes of ectopic blades, but it was wild type when inoculated with pseudomonas, and it is also wild type for sa perception. since a collection of npr alleles has recently been available, we tested the hypothesis that the role of npr in rim is cytosolic. in this work, we show that npr has no role in rim in wild type conditions, since the genes responsible for rim are bop and bop with an important part being played by the tgas. therefore, two genes required for the normal development of the leaf, are also required for plant defense.  role of npr in rim npr has been characterized as a result of observing the response to sa of the great number of alleles described for it. npr has also been described as essential for rim, but there are differences between alleles, since npr1- and npr1- have different rim. npr1- and npr1- have other differences in phenotypes related to meja. thus, the sa-ja antagonism is not present in npr1- but it is active in npr1-. other difference is the gene expression, whereas npr1- was affected in sa, ja, and et dependent genes upon pto inoculation, npr1- was only affected in sa dependent genes. these different phenotypes have been attributed to the lack of nuclear localization in npr1- since the truncated cytosolic protein would be functional to modulated ja-dependent defense response. in order to determine the precise role of npr in rim, and to asses the role of the cytosolic vs. nuclear localization, we tested an allelic series of npr alleles, defined by their inability to perceive sa. the npr alleles produced a mixture of phenotypes in rim, and out of the alleles tested, had a significant rim, meaning that the growth of pto in meja treated plants was significantly lower than in mock treated plants. the rest of alleles showed no response. these two categories of alleles did not share any obvious feature and had the same proportion of stops and point mutations in each category. in fact, in the rim+ alleles tested, six point mutations were widely scattered along the protein. these alleles could somehow affect the localization of the protein inside the cell, even if the mutation was not in the nls. to check this possibility, cdnas of three rim+ alleles, and three rim- alleles, chosen among the point mutations, were cloned along with the wild type npr then, these seven cdnas were transiently expressed in n. benthamiana under the control of the 35s promoter and with the gfp marker. the free gfp was detectable in the nucleus and in the cytoplasm. but when the wild type and the six alleles of npr were expressed in the same conditions, gfp was detected mainly in the nucleus, with no difference existing between the two classes of alleles. as a complementary approach, we took advantage of the transgenic line that overexpresses npr fused to the steroid hormone binding domain of the rat glucocorticoid receptor. npr1-hbd remains exclusively in the cytosol in mock conditions and should be functional in rim. the original line is in an npr1- background, and therefore the transgene was transferred to an npr1- background to check for complementation. treatments with bth and with and without glucocorticoid dexamethasone showed that npr1-hbd was functional. npr1-hbd, even under the control of the 35s promoter, did not complement the lack of rim in npr1-. when dex was applied, npr1-hbd moved to the nucleus and npr1- was complemented in the rim phenotype. note that the presence of cytosolic npr1-hbd in an npr1- background did not enhance rim in comparison to npr1- alone. the npr alleles rim+ or rim- did not share any obvious feature, so it would be difficult to assign a precise role to the wild type gene. a critical genetic resource to discern the role of a gene is the null allele. therefore, the response of two null alleles of npr to rim was measured. both npr1- and npr1- are in laer- background, so an introgression of npr1- in laer- was used as control. these two null alleles responded to rim in all experiments like the wild type. npr1- introgressed in col- responded again like the wild type, which ruled out any ecotype effect. since the direct role of npr in rim was in question as a consequence of the aforementioned results, we included an independent rim- control, coi1-. the role of npr in this response might be indirect. thus, one scenario would be a reinforcement of the negative crosstalking between sa and meja. npr alleles produced more sa when infected with pto and seemed unable to metabolize it. rim- alleles -defective in terms of sa perception- might have left intact the negative crosstalk between sa and meja, and an excess of sa repressed the response to meja beyond the wild type levels. therefore, the rim+ alleles would be defective in terms of both sa perception and sa-meja crosstalking, an explanation that would also be in agreement with the behavior of the null alleles. to test this hypothesis, the double mutants between npr1- and nahg, eds, and sid, were constructed and tested for rim. npr1- did not respond to meja even if the levels of sa were low, so the hypothesis of a reinforcement of the negative crosstalk was not supported. bop and bop and their role in rim the experiments with the null alleles showed that npr was not necessary for rim. perhaps npr and other gene would be redundantly responsible of rim, and while null npr alleles would have a rim+ phenotype, some npr alleles could be rim- by interacting with other protein negatively. the most likely candidates for these interactions were the npr paralogs, since their proteins shared the same overall structure. there are five additional paralogs of npr in the arabidopsis genome, and we analyzed double mutants of npr1- with different paralogs. there was no proof of npr having a redundant role in rim. npr did not play a significant role in rim and the same was true for the double npr npr. strikingly, the double bop bop was rim-. single bop and bop were also tested for rim and showed to be wild type. bop and bop exert their function in part through transcriptional regulation of knat and physical interaction with pan, but t-dna insertions predicted to disrupt knat or pan activity did not have an effect on rim. to rule out the possibility that other mutations besides bop and bop were producing this rim- phenotype, we constructed an artificial microrna to deplete the levels of bop and bop at the same time. eight independent homozygous transgenic lines for amirna were analyzed for rim. five out of eight lines were rim-, and the remaining three responded less than the wild type control. the levels of both genes were partially depleted in the eight lines; five of the lines had both genes significantly reduced, and all had bop significantly reduced. none of these lines had the characteristic blade-on-petiole macroscopic phenotype, not even as the subtle phenotype of bop alone. the previous experiments had shown that bop and bop were acting redundantly in rim. therefore, increasing the amount of any of them should have an effect on rim, especially since normal levels of bop and bop are quite low. the overexpression lines of bop and bop described were analyzed for rim. 35s:bop had a stronger rim than col- and 35s:bop had a strong variation in the meja treated plants. at the time of the experiments, each population looked homogeneous, but when these plants were grown to set seeds, two phenotypes could be observed in each transgenic line. approximately half of the plants showed a wild type phenotype, and the other half reproduced the dwarf plants described. seeds from both lines and from both phenotypes reproduced the two phenotypes. it seems that it was an issue of silencing, since rna taken from plants classified by their mentioned phenotype diverged widely in the transgene expression. bop bop specificity in rim the bop bop double mutant lacked a rim response, but this failure in meja signaling might occur at different points of the signal transduction. for example, the defect could target a general signaling component affecting all meja responses, or a specialized part of the pathway, affecting a subset of meja responses. when bop bop plants were grown in plates containing meja, the growth of the roots was similar to the wild type controls. other phenotypes of bop bop plants growing in meja plates were similar to the wild type controls. another effect of meja is the increase of senescence in detached leaves, measured as chlorophyll production. bop bop responded as the wild type in this particular system. coronatine is a virulence factor of several pseudomonas isolates with structural and functional similarities to ja-ile, therefore a mutant pto that lacks coronatine grows less in col- than the wild type pto. bop bop was also wild type in response to pto with and without coronatine. inoculations with plectospharaella cucumerina, a fungus that causes more disease in meja mutants than in wild type plants, did not cause any more disease in bop bop than in col-. if bop bop was not a meja signaling mutant, but specifically in rim, it could be defective in the other signaling required for isr; et. it was not; when mm of 1-aminocyclopropane-1-carboxylic acid was sprayed to bop bop the resistance triggered was similar to that triggered in the wild type controls. etr was included as a negative control of resistance induced by ethylene. npr and bops interactions once it was clear that both bop genes are required for rim, we tested the model that npr rim- alleles could have a dominant negative effect on bop activity, either directly or indirectly. to first test whether npr had an effect in the interaction between bop proteins, we used a yeast two-hybrid assay. as reported, bop and bop interacted with each other. next, we introduced in a third plasmid containing wt npr or various mutant npr alleles presented in figure 2a. if the effect of npr on rim were a direct interaction between npr and the two bops, the alleles that diverge in their rim phenotype would diverge in their ability to interfere in the interaction of bop and bop the two classes of npr alleles did not have a distinct behavior, therefore the dominant negative effect did not seem to be direct. all the npr paralogs tested interact with members of the tga family in a different degree. therefore, the tgas would be a reasonable candidate for being the third component, and their interaction with rim- alleles would indirectly affect the function of bop and bop as a control, single mutants in tga and tga produced a significant rim, but when three specific tgas are knocked out at the same time, there is no rim. we reasoned that one or several of these three tgas might have a functional interaction with the bops, which might be affected by the rim- alleles. to test this hypothesis bop and each of the mentioned tgas were introduced in the yeast two-hybrid system with the npr alleles mentioned above in a third plasmid. tga and tga interact differentially with bop depending on the npr protein present. there was an enhancement of the interaction in two out of the three rim- alleles, and no interference in two out of the three rim+ alleles. the interaction tga5-bop was not affected by the presence of npr proteins. the experiments were repeated with bop producing similar results. in sum, the data indicated that bops and tga tga and tga are required for rim, that bops interact with these tgas, and that npr may modulate the affinity or stability of the interactions. discussion npr is not required for rim npr is an essential gene for sar and sa perception. npr1- the most widely used allele, is also impaired in rim. we speculated that since npr1- is wild type for rim, and it has been reported that the difference of some phenotypes between npr1- and npr1- was due to the lack of nls in npr1-, the same could be true for rim. however, we show here that the nuclear localization of the alleles makes no difference. this conclusion is supported by multiple lines of evidence. first, the npr alleles with rim+ are not structurally similar to npr1- since not all of them are affected in the nls. even an allele with a point mutation in the nls should be partially localized in the nucleus. second, three rim- and three rim+ alleles do not differ in their nuclear localization or stability when transiently expressed in n. benthamiana. while these proteins are no longer functional, they respond to the signals of a wild type background by localizing in the nucleus. third, when a functional npr is anchored in the cytoplasm there is no complementation of the rim- phenotype in an npr1- background, nor there is an increase in rim phenotype in an npr1- background. in fact, the application of dex triggered an increased rim in both backgrounds. but, most importantly, npr is not required for rim, since the null npr alleles are rim+ regardless of the background. we also discarded that npr could be a part of rim in a redundant fashion with its paralogs. an interesting alternative for the role of npr in rim would be an effect on the crosstalking between sa and meja. npr has been described as a key point in the negative regulation between sa and meja. thus, the rim+ alleles could be defective in both sa perception and in sa-meja crosstalk, while the rim- alleles would be defective only in sa perception but not in sa-meja crosstalk. the inoculation with pto triggers an increase in the levels of sa, and in the case of the npr alleles, there is more sa than in the wild type. although this hypothesis would explain the phenotype of the null alleles, it was rejected after the experiment of figure 3c, where a severe reduction of sa levels in a rim- allele did not have any effect on the phenotype. bop and bop are redundant in rim the redundant functions of bop and bop are essential for normal development. previous work has shown that the double mutant has numerous defects in plant architecture including altered leaf morphology, changes in floral patterning, defects in the conversion of shoots to flowers and loss of floral-organ abscission. the double mutant was tested for basal defense and sa perception but no difference from wild type was found. we show here that both genes are also redundantly required in defense against pathogens triggered by meja. interestingly, whereas significant loss of bop activity is required to exert changes in development, rim is abolished in plants that are only partially silenced for the bop genes. thus, the levels of gene expression required for rim are higher than those required for normal development. compatible with this idea, bops expression in plants is highly localized, restricted to young organ primordia, leaf petioles, and lateral organ boundaries, which may make systemic responses to meja sensitive relatively minor changes in bop transcript abundance. both npr and the bops localize to the cytoplasm as well as nucleus and interact with members of the tga family of bzip transcription factors, albeit with different affinities. in development, bop and bop form a nuclear complex with tga8/perianthia to regulate number of sepals and petals in flowers and potentially to promote floral meristem fate. given that pan loss-of-function did not reproduce the rim- phenotype other genes, perhaps tgas, are involved in this phenotype, as shown for sa perception. given that bops play both positive and negative roles in transcriptional regulation of the knox gene knat, we also tested if rim was affected by knat loss-of-function, but again, no difference was observed. this may reflect redundancy with other knox genes, or more likely, that bop regulation of rim is independent of knat whether bop bop recapitulates or not all the phenotypes of the rim- npr alleles remains to be assessed. we did check that there were similar phenotypes in the specificity of response to meja as well as the fact that bop bop was wild type for the rest of meja phenotypes. but there were strong differences, since npr1- is affected in basal defense and sa perception while bop bop is wild type for both phenotypes. regarding et, the other hormone relevant for isr, it has been proposed that applications of this hormone could render the crosstalk between sa and meja independent of npr. it seems plausible that the et induced resistance works as the meja induced resistance and other proteins -perhaps npr paralogs, but not the bops - might also be affected by some alleles of npr some npr alleles interfere in the bops-tgas interaction the rim- npr alleles were the majority of the alleles found. how is this compatible with the fact that the null npr alleles are rim+? a possible explanation was the selection used in the screening. since the selection was made for complete loss of sa perception, perhaps most of the rim+ alleles had a phenotype of partial sa perception, as the null alleles. then, the prediction would be that a good number of random alleles of npr would be rim+ and partially receptive to sa. we previously showed that for sa perception, there are genetic interactions between the npr alleles and the npr paralogs. the work reported herein points to a genetic interaction too, this being between npr alleles on one side and the bops on the other. thus, the rim- alleles were a phenocopy of the bop bop mutant in defense but not in development. this discrimination was a consequence of the different thresholds for the phenotype in development and defense. mechanistically, the levels of expression of the bops were low in comparison to npr, so a direct or indirect negative interference of npr with the bops would be favored stoichiometrically. once the pathogen was inoculated, the levels of sa would rise and in a wild type plant npr is degraded as part of the signaling process. in an npr background, this signaling would not be transmitted and perhaps the npr proteins would be able to interfere longer in rim. this would explain the behavior of npr1-hbd in npr1-; npr1-hbd in the cytoplasm did not complement npr1- in the rim phenotype, but when dex was applied there was complementation of the phenotype. likely, when no dex was present npr1- would somehow interfere with the function of the bops. when dex was present, the presence of npr1-hbd in the nucleus would trigger the degradation of both npr1-hbd and npr1- if npr1- was degraded, the bops would function normally. there was no evidence for a direct interaction in yeast, since the presence of npr or mutated versions of this protein did not interfere in the interaction between bop and bop in a consistent manner with the phenotype. a first alternative was that the interference of the rim- alleles would occur with the bops without affecting the interaction between the bops. a second alternative would be that the rim- alleles would interfere with other proteins that normally interact with the bops. in both cases there is a family of proteins that interacts with both npr and the bops, the transcription factors tgas, with -again- functional redundancy. two out of three rim- alleles enhanced or stabilized the bops-tgas interaction, while two out of three rim+ alleles did not. it was clear that the npr mutated proteins had an unpredicted effect on the bops-tgas interaction, but the yeast experiments did not produce an absolute answer about the role of npr proteins in rim. we speculate that in planta, all the rim- alleles enhance the interaction between the bops and the tgas, titering out the tgas and thus rendering them unable to fulfill their function of triggering defense. on the other hand, the rim+ alleles, and npr would not affect the interaction either way. since in the yeast assays two out of three alleles worked as proposed in either way, it may be possible that a factor is present in the plant that is not in yeast, or perhaps the fact that there are ten tgas, and that npr is expressed between and times more than bop + bop could explain this difference. if this hypothesis were to be true, it will definitively explain the role of npr in rim.  in sum, we have shown that, in wild type conditions, the bops and the tgas are required for the resistance triggered by methyl jasmonate against pto. we propose that the phenotype of the npr rim- alleles is caused by their interference between bops and tgas. 
since an allelic series of npr was recently described, the behavior of these alleles was tested in response to methyl jasmonate. surprisingly, the application of methyl jasmonate also induces resistance, and npr is also suspected to be relevant. the response to methyl jasmonate of different npr1s alleles and npr paralogs null mutants was measured by the growth of a pathogen. the localization of the protein in npr alleles does not affect the response to methyl jasmonate. some npr alleles have a phenotype in this response likely because they are affecting the interaction between bops and tgas, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants. npr is a gene of arabidopsis thaliana required for the perception of salicylic acid. the genes that are required in a redundant fashion are the bops. the bops are paralogs of npr and they physically interact with the tga family of transcription factors.
 npr is a gene of arabidopsis thaliana required for the perception of salicylic acid. this perception triggers a defense response and negatively regulates the perception of jasmonates. surprisingly, the application of methyl jasmonate also induces resistance, and npr is also suspected to be relevant. since an allelic series of npr was recently described, the behavior of these alleles was tested in response to methyl jasmonate.  the response to methyl jasmonate of different npr1s alleles and npr paralogs null mutants was measured by the growth of a pathogen. we have also tested the subcellular localization of some npr1s, along with the protein-protein interactions that can be measured in yeast. the localization of the protein in npr alleles does not affect the response to methyl jasmonate. in fact, npr is not required. the genes that are required in a redundant fashion are the bops. the bops are paralogs of npr and they physically interact with the tga family of transcription factors.  some npr alleles have a phenotype in this response likely because they are affecting the interaction between bops and tgas, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants. methyl jasmonatesalicylic acidarabidopsisnpr1bopsdefense 