BACKGROUND
gene silencing in plants
the conserved molecular machinery of rna-silencing constitutes a very complex genetic regulatory network in all eukaryotes.
the common features for these regulatory pathways are their induction by double stranded  rna sequences.
in plants, these are cleaved by the rnase iii type dicer-like  enzymes, assisted by hyponasti leaves <dig> , serrate  and dawle  proteins
<cit>  into 21– <dig> nucleotide long rnas, called either  mirnas or  sirna.
upon cleavage, the short rna fragments are methylated at their 3’ ends by the s-adenosyl methionine dependent methyltransferase hen <dig>
<cit> , and loaded into effector complexes, called ritc  or transported to cytoplasm apparently with the assistance of hasty protein
<cit>  and loaded risc  reviewed in
<cit> .
the rits complexes contain sirna, ago <dig> and ago <dig> to mediate transcriptional silencing  to repetitive or over-expressed dna sequences via histone and dna methylation, leading to heterochromatin maintenance, control of transposon mobility and transgene silencing.
this rna-dependent dna methylation  machinery utilizes at least the dna-directed rna polymerases poliv and polv, rna-dependent rna polymerase  <dig> , dicer-like  <dig>  enzyme, ago <dig>  ago <dig>  ago <dig> and dna methyltransferase
<cit> , chromatin-remodelling protein , structural-maintenance-of-chromosomes-protein  and rddm effector molecule ktf <dig>  which binds scaffold transcripts generated by polv and recruits ago <dig> bound sirnas to form an rddm effector complexes
<cit> .
the risc complexes, targeted to rna sequences, cause the post-transcriptional silencing  of the target mrnas either via their cleavage or translational arrest
<cit> .
mirna-mediated ptgs is used, for instance, to regulate the temporal and spatial expression of multiple different transcription factors, many of which are needed in the developmental differentiation of plant organs, and also regulate various components of the silencing pathways, thus back-regulating these pathways themselves.
in addition, numerous mirnas, as well as a plethora of various sirnas are known to be involved in genetic responses and signalling cascades induced by various hormones and biotic and abiotic stresses .
the small rnas target these silencing complexes to partially or fully complementary dna or rna sequences, respectively.
on these target molecules the effector complexes mediate multiple essential regulatory functions like transcriptional gene silencing  or post-transcriptional gene silencing  or decreasing the translational rate .
in plants rna silencing is used as the major defence mechanism against viruses.
suppression of rna silencing in plants
to counteract the silencing mediated host defence, plant viruses encode various viral rsss
<cit> .
functional suppression of rna silencing appears to be required for any virus to successfully invade and accumulate in its host plant, and several of the rsss have been identified as essential viral pathogenicity factors and symptom determinants already before their function in rna silencing suppression was discovered
<cit> .
it is of interest that the rss factors encoded by different viral families have no similarities with each other, and many viruses produce their rss activity as a secondary function of some gene product that has also some other function in viral life cycle, e.g.
in replication, cell-to cell or systemic movement or encapsulation .
the high complexity of the silencing machinery provides multiple steps where it can be disturbed, and the mechanisms of the different rss are very diverse.
the hosts’ silencing machineries and their molecular interactions with viral rsss have been intensively studied over the last decade
<cit>  and they appear to be excellent tools for interrupting and analysing of the rna silencing pathways.
begomoviruses and silencing suppression in plants
begomoviruses, with circular ssdna genomes, produce transcripts in opposite orientation.
they form partly dsrna structures and activate plant’s rna silencing machinery, but can resist this suppression by producing active rsss
<cit> .
the silencing is not  targeted against viral rna transcripts, but rather against the viral replicative intermediate, i.e.
the minichromosome structures formed of replicative viral dsdna, combined with histone proteins
<cit> .
the silencing is mediated by the tgs, i.e.
by methylation of these viral dsdna sequences, which leads to repression of their replication and transcription
<cit> .
total of three different types of rss are known to be encoded by african cassava mosaic geminivirus , which nowadays is classified as a begomovirus
<cit> .
the main rss has been identified as the transcriptional activator protein ac <dig>  encoded in opposite sense of the begomoviral minichromosome.
here we have analysed the transcriptome and proteome of the transgenic tobacco plants expressing the ac <dig> rss, derived from acmv begomovirus.
alterations detected in these gene expression profiles were compared to the those that we have earlier detected in similar transgenic tobacco plants that express the hc-pro rss derived from potato virus y potyvirus <cit> , revealing the fundamental similarities and differences between the functional mechanisms of these two rss in transgenic tobacco plants.
RESULTS
phenotype of tobacco plants expressing ac <dig> rss
a previously characterized transgenic tobacco line expressing geminiviral ac <dig> rss under a constitutive camv 35s promoter was used in this study
<cit> .
transgenic tobacco plants expressing ac <dig> rss  were grown concurrently under the same growth conditions that were earlier used for hc-pro expressing transgenic tobacco plants
<cit> .
expression of nuclear ac <dig> protein in transgenic tobacco plants induced clear phenotypic changes both in leaves and flowers, including different malformations of veins and leaves of the young transgenic plants .
these malformations gradually disappeared as plants become older, but there still were some phenotypic changes in six week-old leaves; the leaves were narrower and paler green than leaves in wild type plants .
the growth habit was stubby.
lower leaves were often yellow, the root neck and root system was less developed than in wild type plants, and mature plants were slightly smaller in size compared to wild type tobacco plants.
flower buds were sometimes twisted round other flower buds .
the structure of flowers were often, but not always drastically malformed; petals were grown together having either round or more often a rectangular shape.
stamen filaments were also often changed to extra petals .
experimental design and differential gene expression in ac <dig> expressing plants
the gene expression profiling of the ac <dig> rss expressing plants was performed at the same time as that of the hc-pro expressing transgenic tobacco plants by using the same wild type and empty vector  expressing control plants as were used for the transgenic hc-pro expressing plants, using three biological replicates for each.
the intensity values indicating differential gene expression between control and the ac <dig> expressing plants were normalized using the chipster program .
the intensity values originating from the leaf and flower samples were normalized separately as was performed for the hc-pro samples
<cit> , .
two-fold change in the expression between ac <dig> transgenic and control samples was regarded as significant level of differential expression.
statistical significance of differentially expressed genes was tested using students t-test, p values less than  <dig>  with false discovery rate  .
microarray results were verified by using rt-qpcr.
with a good correlation between rt-qpcr and array results both in up- and –down-regulated transcripts, and both in leaves and flowers .
transcripts, whose expression did not change, were used as reference transcripts
<cit> .
some clearly up- or down-regulated genes of leaf and flower samples were tested.
statistical significance was tested using student’s t-test .fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values .
standard error of mean  is also calculated for rt-qpcr values.
the numbers of differentially expressed transcripts, assigned to various functional groups, in leaves and flowers of transgenic ac <dig> expressing plants is presented in table
<dig>  the total number of altered transcripts was  <dig> and  <dig> in leaves and flowers, respectively.
in leaves total of  <dig> transcripts were up-regulated and  <dig> down-regulated, with the three biggest groups of up-regulated transcripts being related to signalling, cell wall modifications, and stress, whereas the three most down-regulated groups of transcripts being related to protein synthesis, photosynthesis and transcriptional regulation.
the results demonstrated in table
<dig> were based on counting of genes in functional categories similarly as it was performed in our previous publication
<cit>  but not the overpresentation analysis.
the table represents number of genes that were up or down regulated more than two-fold.
statistical significance was tested by using paired t-test .
about half of the transcripts up-regulated in leaves of the hc-pro expressing plants were found up-regulated also in the leaves of the transgenic ac <dig> expressing plants , but more than five hundred additional transcripts were up-regulated only in the ac <dig> expressing plants.
the same pattern was also found in the down-regulated transcripts of the leaves; more than three hundred transcripts were down-regulated only in ac <dig> expressing plants.
the number of differentially expressed transcripts was much lower in flowers but these also included common and specific transcripts between ac <dig> and hc-pro expressing plants .
see also additional file
<dig> for over and under presentation of functional categories between hc-pro and ac <dig> rss expressing plants.
expression of ac <dig> in tobacco plants induces clear defence responses
the expression of ac <dig> rss in transgenic plants induced many defence- and oxidative stress- related transcripts, including transcripts for systemic acquired resistance , ros scavenging and respiratory burst oxidase  which were not detected in hc-pro rss expressing transgenic plants
<cit> .
the amount of oxidative stress was further studied using nbt staining for superoxide radical and dab for h2o <dig> to indicate the amount different roses in leaves of ac <dig> expressing plants.
the amount of roses was found increased only in one-month old ac <dig> expressing tobacco plants , while the amount of ros accumulation in older and more developed leaves was similar as in wild-type leaves .
fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values .
interestingly, hypersensitive reaction  was frequently detected in lower leaves of two month old transgenic ac <dig> expressing plants .
these kinds of necrotic lesions were never detected in leaves of wild type tobaccos of similar age.
this suggested that particularly the young developing leaves suffered from oxidative stress where also the enhanced levels of ros-related transcripts were detected.
jasmonic acid biosynthesis and jasmonate-responsive genes
multiple genes related to jasmonic acid  biosynthesis were up-regulated in leaves of transgenic ac <dig> expressing plants.
some of these genes were also up-regulated in hc-pro expressing plants, but in these the level of up-regulation was much lower
<cit> .
also genes encoding jasmonic acid-mediated regulatory proteins like ntjaz <dig> and ntjaz <dig> repressors were up-regulated .
the presence of jasmonates  induces proteasomal degradation of these jaz repressors and thereby activates transcription e.g.
from promoters containing myc binding sites .
many of the ja-responsive genes e.g.
defence and stress-related transcripts were also up-regulated.
jasmonic acid signalling cascade also positively regulates the genes involved in biosynthesis of jasmonic acid itself
<cit> , and in the biosynthesis of ethylene
<cit> .
table
<dig> shows almost full induction of jasmonic acid biosynthesis related transcripts, jasmonic acid regulatory genes as well as some well-known jasmonic acid responsive genes, including the 1-aminocyclopropane-1-carboxylic acid oxidase  transcript, required for the biosynthesis of ethylene from its acc precursor
<cit>  in leaves of transgenic ac <dig> expressing plants.
in addition, a transcript of ethylene response factor  <dig> , a transcription factor responsible for mediating ethylene responsive gene expression
<cit>  was up-regulated about five-fold.
chlorophyll biosynthesis and photosynthesis related genes were down–regulated in ac <dig> expressing transgenic plants
most of the phenotypic changes occurred in young, less than two-month old plants, but not many differences were detected in older leaves of ac <dig> expressing plants.
leaves of young transgenic plants  were more yellow than those of the wild type tobaccos, indicating changes in their pigment content.
the pigment analysis revealed that the amount of both chlorophyll a and b were decreased in them , and there was also a clear decrease in the accumulation of anthocyanin in the same plants, as compared to wild type plants.
the decreased amount of chlorophyll in leaves was not surprising since many of the genes related to chlorophyll biosynthesis were clearly down-regulated .
table
<dig> also depicts down-regulation of transcripts of photosystem ii and i related genes, as well transcripts related to primary carbon metabolism.
fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values .
since there were clear changes in the genes encoding photosynthetic machinery proteins, the light-responsive oxygen evolution was measured from the thylakoid samples isolated from ac <dig> expressing and wild type leaves.
both photosynthetic oxygen evolution in freshly isolated thylakoids and the amount of starch were significantly decreased in transgenic ac <dig> expressing leaves, as compared to wild type tobacco leaves .
the results were similar, but clearer than those published earlier for hc-pro rss expressing plants
<cit> .
down-regulated accumulation of translation factor and ribosomal protein transcripts
one of the most dramatic changes in the transcriptome of the transgenic ac <dig> expressing plants was the down regulation of genes encoding for translation factors and ribosomal protein subunits .
total of fifty two transcripts related to translation machinery, including genes encoding for the cytosolic large  and small  ribosomal protein subunits as well as chloroplast ribosomal protein subunits , were down-regulated.
there were also changes in the distribution of ribosomal rnas , as the amount and ratio of 23s rrna was clearly decreased in ac <dig> expressing tobacco plants.
consequently, the amount of total protein, as measured against fresh weight was decreased about 30% in ac <dig> expressing leaves compared to wild type tobacco leaves .
the proteomes of wild type and ac <dig> expressing tobacco leaves were compared by loading equal amounts of protein extracts  into the 2d-sds-page analysis , meaning that the analysis actually enhanced the protein spot intensities of the ac2-plant samples, as their total protein content was really lower than in the wild type plants.
the ac2-plant proteome contained multiple changes, as compared to the wild type plant sample.
to our surprise, the changes occurring in the proteomes of ac <dig> and hc-pro expressing tobacco plants were quite similar to those observed in the hc-pro expressing plants
<cit> , although in general more proteins were down-regulated in ac <dig> expressing than in and hc-pro expressing tobacco leaf.
fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values .
<cit> .
two representative 2d-sds-page results are shown.
expression of silencing related transcripts
interestingly, the microarray data indicated that some silencing related transcripts involved in the dna methylation were altered .
two of the up-regulated transcripts  have been described to function in rna-directed dna methylation
<cit> .
based on its ability to bind 24nt small rnas, ago <dig> could influence small viral rna-directed dna methylation .
the ago <dig> has an effect on cg motifs rather than c residues in a cng or cnn context.
counteracting the increased dna methylation by these gene products, also repressor of silencing <dig> , functioning as a negative regulator of silencing was up-regulated.
in addition, two chromatin methyl transferases and a structural maintenance of chromosomes, also suggested to function in de novo dna methylation, were down-regulated.
fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values .
to investigate possible alterations in the level of dna methylation level, we also performed pcr-amplification experiments over some genomic dna regions that contained at least three methylation sensitive restriction enzyme sites.
these analyses indicated increased methylation at least in the coding regions of erf <dig> and ap <dig> genes but not in 18s rna .
however, other gene fragments showed no change in the methylation status .
<dig>  osmotin, ap- <dig>  and ethylene responsive factor <dig> erf <dig>  indicated methylation in ac <dig> dna fragments, whereas no methylation was detected in 18s rrna  ac <dig> dna fragments compared to wild type dna samples.
data consists of four biological replicates.
neither genes encoding s-adenosyl-l-methionine  biosynthesis nor genes encoding sam-recycling were down regulated as much as in hc-pro expressing plants
<cit> .
thus, it appears that recycling of sam-provided methyl groups for trans-methylation reactions was not affected in ac2-expressing plants.
interestingly, a large group of histone transcripts were down-regulated in ac <dig> expressing tobacco plants , while a similar group of histone transcripts were up-regulated in hc-pro expressing tobacco plants.
also some histone acetyl-transferases were up-regulated, possibly leading to opening of the chromatin structures and thus increasing transcriptional activity.
another interesting phenomenon was the down-regulation of cell cycle-related transcripts in ac <dig> expressing plants, as similar transcripts were up-regulated also in the hc-pro expressing plants .
discussion
in this work we have analysed the transcriptome profile of transgenic tobacco plants expressing the ac <dig> rss derived from acmv, nowadays classified as a member of the genus begomovirus, and shown that this single rss protein causes massive changes in the gene expression.
these changes may have been caused trough at least three identified regulatory nodes, which all interfere with large metabolic networks.
the first node is the induction of plant hormones like jasmonates and ethylene, leading to jasmonate and ethylene responsive gene expression.
both jasmonates and ethylene are known to be highly involved in defence and stress regulated gene expression, and jasmonate hormones are also responsible for inducing oxidative stress
<cit> .
the second regulatory node appears to be related to photosynthetic end products leading to changes in the transcriptional and translational regulation.
the role of chloroplasts in this retrograde signalling is crucial.
results demonstrated that photosynthetic light and dark reactions were clearly down-regulated.
the third regulatory node appears to consist of silencing directed modifications in cell cycle including changes in transcripts involved in dna replication and chromatin structures.
defence and stress reactions in transgenic ac <dig> expressing tobacco plants
the expression of the ac <dig> rss in transgenic tobacco plants induced more pronounced defence and stress related gene expression than was found in plants expressing hc-pro rss in our previous study
<cit> .
although many similar defence and stress related transcripts were induced in these two types of transgenic plants, a high number of transcripts were enhanced only in the ac <dig> expressing plants, the most prominent difference being the induction of transcripts related to scavenging of ros in these plants .
these included transcripts coding for scavengers of hydrogen peroxide  and of superoxide related oxygen radicals.
the presence of ros also was indicated by spontaneous hr lesions in the young leaves .
also, transcripts related to systemic acquired resistance  were induced, maybe via induction of avirulance  determinant related genes  .
balance in the redox homeostasis seems also be affected in ac <dig> rss expressing transgenic tobacco plants, as several genes that function in balancing the redox state in the cells, like glutathione-s-transferases, glutaredoxins and thioredoxins were up-regulated in the leaves .
all in all, this may have affected the redox-responsive gene regulation in the ac <dig> rss expressing tobacco cells
<cit> .
photosynthesis is also regulated by jasmonates
photosynthesis is the key regulator of almost all metabolic and differentiation responses in the plant cells but it has also a major role in regulating gene expression by retrograde signalling.
our results clearly pointed out that many in photosynthesis related transcript , and the photosynthetic oxygen evolution  were significantly decreased in ac <dig> expressing plants.
interestingly, jasmonate hormones have been shown to regulate photosynthesis related transcripts in arabidopsis <cit> , to down-regulate the oxygen evolution  in isolated barley thylakoids
<cit> , to up-regulate defence, stress, senescence and cell wall related transcripts and to causes oxidative stress in plants and thus also to change redox state of proteins
<cit> .
thus all these observed effects in ac <dig> expressing plants could have been related to up-regulated jasmonate biosynthesis
<cit> .
further on, the reduced photosynthesis may have changed the sugar metabolism and thus caused major secondary effects on retrograde signalling between the nucleus and the chloroplast.
for instance, it may explain the changes in protein synthesis , cell wall synthesis, and chromosome/histone modifications related transcripts  in the ac <dig> expressing plants.
several studies have shown that energy deprivation and different stresses change energy-dependent transcriptional regulation
<cit>  with drastic down-regulation of transcripts related to different biosynthetic processes and especially protein syntheses  and up-regulation of catabolic reactions to restore energy deprivation on the cells.
protein synthesis affected in ac <dig> expressing plants
results clearly indicated that expression of ac <dig> protein in transgenic plants had a major effect on transcripts related both protein synthesis and protein degradation .
the down-regulation of protein synthesis caused to some extend by reduction of levels of multiple transcripts, but also the down-regulation of genes encoding both nuclear and plastid ribosomal proteins, ribosomal rna or translation factors .
not only synthesis of proteins but also degradation of proteins was affected.
over and under presentation analysis  indicated that both cysteine and aspartate proteases were up-regulated in leaves of ac <dig> rss expressing plants.
there were also indications of proteasomal protein degradation .
interestingly, it has suggested that the geminiviral c <dig> protein might be the key player in proteasomal regulation of protein degradation
<cit> .
it was shown that c <dig> protein is capable of binding to proteins involved in proteasomal complexes e.g.
scf e <dig> ligase complexes and thus inhibiting jasmonate related gene expression in arabidopsis .
however, our results clearly indicate that the geminiviral protein ac <dig> has an opposite effect; enhancement of jasmonate signalling and jasmonate responsive gene regulation
<cit>  and no effects to transcripts related to neither sa synthesis nor sa responsive gene expression
<cit> .
cell cycle, genome methylation and histone expression is altered in ac <dig> expressing transgenic plants
begomoviruses are known to replicate their single-stranded dna genomes through double-stranded dna intermediates that are associated with cellular histone proteins to form minichromosomes
<cit> .
thus it is important to begomoviruses to induce the accumulation of the dna replication machinery in mature plant cells.
this is achieved most likely by modifying cell cycle and transcriptional regulation, for instance, the infection of cabbage leaf curl geminivirus  has been shown to induce changes in the cell cycle in virus infected cells
<cit> .
interestingly, our results also suggested that expression of ac <dig> rss may have an effect on cell cycle , providing replication-competent environment and preventing expression of genes needed for mitosis
<cit> .our results demonstrated that the expression ac <dig> rrs in transgenic plants up-regulated transcripts involved in dna methylation  and down-regulated transcripts involved in histone synthesis .
this suggested that, although the transcriptional activator ac <dig> suppresses silencing  in the native host of acmv, and apparently enhances the virus replication in these hosts, in tobacco  it induces defence mechanisms, leading to opposite direction, i.e.
to increase of methylation and decrease of the histone synthesis.
CONCLUSIONS
the expression of ac <dig> rss in transgenic tobacco plants induced clear phenotypic changes in tobacco plants, even though tobacco is not considered as native host for acmv.
this study indicated several possible action mechanisms of the ac <dig> rss.
first both jasmonate and ethylene hormone responsive regulatory cascades were induced, secondly, the rate of photosynthesis and also the carbon metabolism were reduced.
the stress and defence responsive gene expression was enhanced including both induction and response of reactive oxygen species .
all these regulatory nodes may have been interconnected to one another.
also, changes occurred in regulation of cell cycle and transcription as well as changes in the genomic architecture like dna methylation and abundance of histones.
thus it seems that the silencing suppression activity of ac <dig> in tobacco is indicated in the enhancement of the jasmonate and ethylene biosynthesis, known to be regulated and repressed via rna silencing
<cit> .
up-regulation of these plant hormones was related to induction of a large variety of stress-related genes, and severe developmental defects in the plants.
interestingly, the natural silencing suppression mechanism of the geminiviral rss, i.e.
reduction of the dna methylation, did not occur in this non-host plant, but rather the opposite effect was observed, apparently related to the induced plant defences.

 gene silencing in plants the conserved molecular machinery of rna-silencing constitutes a very complex genetic regulatory network in all eukaryotes. the common features for these regulatory pathways are their induction by double stranded rna sequences. in plants, these are cleaved by the rnase iii type dicer-like enzymes, assisted by hyponasti leaves, serrate and dawle proteins into 21– nucleotide long rnas, called either mirnas or sirna. upon cleavage, the short rna fragments are methylated at their 3’ ends by the s-adenosyl methionine dependent methyltransferase hen, and loaded into effector complexes, called ritc or transported to cytoplasm apparently with the assistance of hasty protein and loaded risc reviewed in. the rits complexes contain sirna, ago and ago to mediate transcriptional silencing to repetitive or over-expressed dna sequences via histone and dna methylation, leading to heterochromatin maintenance, control of transposon mobility and transgene silencing. this rna-dependent dna methylation machinery utilizes at least the dna-directed rna polymerases poliv and polv, rna-dependent rna polymerase, dicer-like enzyme, ago ago ago and dna methyltransferase, chromatin-remodelling protein, structural-maintenance-of-chromosomes-protein and rddm effector molecule ktf which binds scaffold transcripts generated by polv and recruits ago bound sirnas to form an rddm effector complexes. the risc complexes, targeted to rna sequences, cause the post-transcriptional silencing of the target mrnas either via their cleavage or translational arrest. mirna-mediated ptgs is used, for instance, to regulate the temporal and spatial expression of multiple different transcription factors, many of which are needed in the developmental differentiation of plant organs, and also regulate various components of the silencing pathways, thus back-regulating these pathways themselves. in addition, numerous mirnas, as well as a plethora of various sirnas are known to be involved in genetic responses and signalling cascades induced by various hormones and biotic and abiotic stresses. the small rnas target these silencing complexes to partially or fully complementary dna or rna sequences, respectively. on these target molecules the effector complexes mediate multiple essential regulatory functions like transcriptional gene silencing or post-transcriptional gene silencing or decreasing the translational rate. in plants rna silencing is used as the major defence mechanism against viruses. suppression of rna silencing in plants to counteract the silencing mediated host defence, plant viruses encode various viral rsss. functional suppression of rna silencing appears to be required for any virus to successfully invade and accumulate in its host plant, and several of the rsss have been identified as essential viral pathogenicity factors and symptom determinants already before their function in rna silencing suppression was discovered. it is of interest that the rss factors encoded by different viral families have no similarities with each other, and many viruses produce their rss activity as a secondary function of some gene product that has also some other function in viral life cycle, e.g. in replication, cell-to cell or systemic movement or encapsulation. the high complexity of the silencing machinery provides multiple steps where it can be disturbed, and the mechanisms of the different rss are very diverse. the hosts’ silencing machineries and their molecular interactions with viral rsss have been intensively studied over the last decade and they appear to be excellent tools for interrupting and analysing of the rna silencing pathways. begomoviruses and silencing suppression in plants begomoviruses, with circular ssdna genomes, produce transcripts in opposite orientation. they form partly dsrna structures and activate plant’s rna silencing machinery, but can resist this suppression by producing active rsss. the silencing is not targeted against viral rna transcripts, but rather against the viral replicative intermediate, i.e. the minichromosome structures formed of replicative viral dsdna, combined with histone proteins. the silencing is mediated by the tgs, i.e. by methylation of these viral dsdna sequences, which leads to repression of their replication and transcription. total of three different types of rss are known to be encoded by african cassava mosaic geminivirus, which nowadays is classified as a begomovirus. the main rss has been identified as the transcriptional activator protein ac encoded in opposite sense of the begomoviral minichromosome. here we have analysed the transcriptome and proteome of the transgenic tobacco plants expressing the ac rss, derived from acmv begomovirus. alterations detected in these gene expression profiles were compared to the those that we have earlier detected in similar transgenic tobacco plants that express the hc-pro rss derived from potato virus y potyvirus, revealing the fundamental similarities and differences between the functional mechanisms of these two rss in transgenic tobacco plants.  phenotype of tobacco plants expressing ac rss a previously characterized transgenic tobacco line expressing geminiviral ac rss under a constitutive camv 35s promoter was used in this study. transgenic tobacco plants expressing ac rss were grown concurrently under the same growth conditions that were earlier used for hc-pro expressing transgenic tobacco plants. expression of nuclear ac protein in transgenic tobacco plants induced clear phenotypic changes both in leaves and flowers, including different malformations of veins and leaves of the young transgenic plants. these malformations gradually disappeared as plants become older, but there still were some phenotypic changes in six week-old leaves; the leaves were narrower and paler green than leaves in wild type plants. the growth habit was stubby. lower leaves were often yellow, the root neck and root system was less developed than in wild type plants, and mature plants were slightly smaller in size compared to wild type tobacco plants. flower buds were sometimes twisted round other flower buds. the structure of flowers were often, but not always drastically malformed; petals were grown together having either round or more often a rectangular shape. stamen filaments were also often changed to extra petals. experimental design and differential gene expression in ac expressing plants the gene expression profiling of the ac rss expressing plants was performed at the same time as that of the hc-pro expressing transgenic tobacco plants by using the same wild type and empty vector expressing control plants as were used for the transgenic hc-pro expressing plants, using three biological replicates for each. the intensity values indicating differential gene expression between control and the ac expressing plants were normalized using the chipster program. the intensity values originating from the leaf and flower samples were normalized separately as was performed for the hc-pro samples,. two-fold change in the expression between ac transgenic and control samples was regarded as significant level of differential expression. statistical significance of differentially expressed genes was tested using students t-test, p values less than with false discovery rate. microarray results were verified by using rt-qpcr. with a good correlation between rt-qpcr and array results both in up- and –down-regulated transcripts, and both in leaves and flowers. transcripts, whose expression did not change, were used as reference transcripts. some clearly up- or down-regulated genes of leaf and flower samples were tested. statistical significance was tested using student’s t-test.fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values. standard error of mean is also calculated for rt-qpcr values. the numbers of differentially expressed transcripts, assigned to various functional groups, in leaves and flowers of transgenic ac expressing plants is presented in table  the total number of altered transcripts was and in leaves and flowers, respectively. in leaves total of transcripts were up-regulated and down-regulated, with the three biggest groups of up-regulated transcripts being related to signalling, cell wall modifications, and stress, whereas the three most down-regulated groups of transcripts being related to protein synthesis, photosynthesis and transcriptional regulation. the results demonstrated in table  were based on counting of genes in functional categories similarly as it was performed in our previous publication but not the overpresentation analysis. the table represents number of genes that were up or down regulated more than two-fold. statistical significance was tested by using paired t-test. about half of the transcripts up-regulated in leaves of the hc-pro expressing plants were found up-regulated also in the leaves of the transgenic ac expressing plants, but more than five hundred additional transcripts were up-regulated only in the ac expressing plants. the same pattern was also found in the down-regulated transcripts of the leaves; more than three hundred transcripts were down-regulated only in ac expressing plants. the number of differentially expressed transcripts was much lower in flowers but these also included common and specific transcripts between ac and hc-pro expressing plants. see also additional file for over and under presentation of functional categories between hc-pro and ac rss expressing plants. expression of ac in tobacco plants induces clear defence responses the expression of ac rss in transgenic plants induced many defence- and oxidative stress- related transcripts, including transcripts for systemic acquired resistance, ros scavenging and respiratory burst oxidase which were not detected in hc-pro rss expressing transgenic plants. the amount of oxidative stress was further studied using nbt staining for superoxide radical and dab for h2o to indicate the amount different roses in leaves of ac expressing plants. the amount of roses was found increased only in one-month old ac expressing tobacco plants, while the amount of ros accumulation in older and more developed leaves was similar as in wild-type leaves. fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values. interestingly, hypersensitive reaction was frequently detected in lower leaves of two month old transgenic ac expressing plants. these kinds of necrotic lesions were never detected in leaves of wild type tobaccos of similar age. this suggested that particularly the young developing leaves suffered from oxidative stress where also the enhanced levels of ros-related transcripts were detected. jasmonic acid biosynthesis and jasmonate-responsive genes multiple genes related to jasmonic acid biosynthesis were up-regulated in leaves of transgenic ac expressing plants. some of these genes were also up-regulated in hc-pro expressing plants, but in these the level of up-regulation was much lower. also genes encoding jasmonic acid-mediated regulatory proteins like ntjaz and ntjaz repressors were up-regulated. the presence of jasmonates induces proteasomal degradation of these jaz repressors and thereby activates transcription e.g. from promoters containing myc binding sites. many of the ja-responsive genes e.g. defence and stress-related transcripts were also up-regulated. jasmonic acid signalling cascade also positively regulates the genes involved in biosynthesis of jasmonic acid itself, and in the biosynthesis of ethylene. table  shows almost full induction of jasmonic acid biosynthesis related transcripts, jasmonic acid regulatory genes as well as some well-known jasmonic acid responsive genes, including the 1-aminocyclopropane-1-carboxylic acid oxidase transcript, required for the biosynthesis of ethylene from its acc precursor in leaves of transgenic ac expressing plants. in addition, a transcript of ethylene response factor, a transcription factor responsible for mediating ethylene responsive gene expression was up-regulated about five-fold. chlorophyll biosynthesis and photosynthesis related genes were down–regulated in ac expressing transgenic plants most of the phenotypic changes occurred in young, less than two-month old plants, but not many differences were detected in older leaves of ac expressing plants. leaves of young transgenic plants were more yellow than those of the wild type tobaccos, indicating changes in their pigment content. the pigment analysis revealed that the amount of both chlorophyll a and b were decreased in them, and there was also a clear decrease in the accumulation of anthocyanin in the same plants, as compared to wild type plants. the decreased amount of chlorophyll in leaves was not surprising since many of the genes related to chlorophyll biosynthesis were clearly down-regulated. table  also depicts down-regulation of transcripts of photosystem ii and i related genes, as well transcripts related to primary carbon metabolism. fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values. since there were clear changes in the genes encoding photosynthetic machinery proteins, the light-responsive oxygen evolution was measured from the thylakoid samples isolated from ac expressing and wild type leaves. both photosynthetic oxygen evolution in freshly isolated thylakoids and the amount of starch were significantly decreased in transgenic ac expressing leaves, as compared to wild type tobacco leaves. the results were similar, but clearer than those published earlier for hc-pro rss expressing plants. down-regulated accumulation of translation factor and ribosomal protein transcripts one of the most dramatic changes in the transcriptome of the transgenic ac expressing plants was the down regulation of genes encoding for translation factors and ribosomal protein subunits. total of fifty two transcripts related to translation machinery, including genes encoding for the cytosolic large and small ribosomal protein subunits as well as chloroplast ribosomal protein subunits, were down-regulated. there were also changes in the distribution of ribosomal rnas, as the amount and ratio of 23s rrna was clearly decreased in ac expressing tobacco plants. consequently, the amount of total protein, as measured against fresh weight was decreased about 30% in ac expressing leaves compared to wild type tobacco leaves. the proteomes of wild type and ac expressing tobacco leaves were compared by loading equal amounts of protein extracts into the 2d-sds-page analysis, meaning that the analysis actually enhanced the protein spot intensities of the ac2-plant samples, as their total protein content was really lower than in the wild type plants. the ac2-plant proteome contained multiple changes, as compared to the wild type plant sample. to our surprise, the changes occurring in the proteomes of ac and hc-pro expressing tobacco plants were quite similar to those observed in the hc-pro expressing plants, although in general more proteins were down-regulated in ac expressing than in and hc-pro expressing tobacco leaf. fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values.. two representative 2d-sds-page results are shown. expression of silencing related transcripts interestingly, the microarray data indicated that some silencing related transcripts involved in the dna methylation were altered. two of the up-regulated transcripts have been described to function in rna-directed dna methylation. based on its ability to bind 24nt small rnas, ago could influence small viral rna-directed dna methylation. the ago has an effect on cg motifs rather than c residues in a cng or cnn context. counteracting the increased dna methylation by these gene products, also repressor of silencing, functioning as a negative regulator of silencing was up-regulated. in addition, two chromatin methyl transferases and a structural maintenance of chromosomes, also suggested to function in de novo dna methylation, were down-regulated. fold change is indicated as a ratio of ac2/wt calculated from normalized median intensity values. to investigate possible alterations in the level of dna methylation level, we also performed pcr-amplification experiments over some genomic dna regions that contained at least three methylation sensitive restriction enzyme sites. these analyses indicated increased methylation at least in the coding regions of erf and ap genes but not in 18s rna. however, other gene fragments showed no change in the methylation status. osmotin, ap- and ethylene responsive factor erf indicated methylation in ac dna fragments, whereas no methylation was detected in 18s rrna ac dna fragments compared to wild type dna samples. data consists of four biological replicates. neither genes encoding s-adenosyl-l-methionine biosynthesis nor genes encoding sam-recycling were down regulated as much as in hc-pro expressing plants. thus, it appears that recycling of sam-provided methyl groups for trans-methylation reactions was not affected in ac2-expressing plants. interestingly, a large group of histone transcripts were down-regulated in ac expressing tobacco plants, while a similar group of histone transcripts were up-regulated in hc-pro expressing tobacco plants. also some histone acetyl-transferases were up-regulated, possibly leading to opening of the chromatin structures and thus increasing transcriptional activity. another interesting phenomenon was the down-regulation of cell cycle-related transcripts in ac expressing plants, as similar transcripts were up-regulated also in the hc-pro expressing plants. discussion in this work we have analysed the transcriptome profile of transgenic tobacco plants expressing the ac rss derived from acmv, nowadays classified as a member of the genus begomovirus, and shown that this single rss protein causes massive changes in the gene expression. these changes may have been caused trough at least three identified regulatory nodes, which all interfere with large metabolic networks. the first node is the induction of plant hormones like jasmonates and ethylene, leading to jasmonate and ethylene responsive gene expression. both jasmonates and ethylene are known to be highly involved in defence and stress regulated gene expression, and jasmonate hormones are also responsible for inducing oxidative stress. the second regulatory node appears to be related to photosynthetic end products leading to changes in the transcriptional and translational regulation. the role of chloroplasts in this retrograde signalling is crucial. results demonstrated that photosynthetic light and dark reactions were clearly down-regulated. the third regulatory node appears to consist of silencing directed modifications in cell cycle including changes in transcripts involved in dna replication and chromatin structures. defence and stress reactions in transgenic ac expressing tobacco plants the expression of the ac rss in transgenic tobacco plants induced more pronounced defence and stress related gene expression than was found in plants expressing hc-pro rss in our previous study. although many similar defence and stress related transcripts were induced in these two types of transgenic plants, a high number of transcripts were enhanced only in the ac expressing plants, the most prominent difference being the induction of transcripts related to scavenging of ros in these plants. these included transcripts coding for scavengers of hydrogen peroxide and of superoxide related oxygen radicals. the presence of ros also was indicated by spontaneous hr lesions in the young leaves. also, transcripts related to systemic acquired resistance were induced, maybe via induction of avirulance determinant related genes. balance in the redox homeostasis seems also be affected in ac rss expressing transgenic tobacco plants, as several genes that function in balancing the redox state in the cells, like glutathione-s-transferases, glutaredoxins and thioredoxins were up-regulated in the leaves. all in all, this may have affected the redox-responsive gene regulation in the ac rss expressing tobacco cells. photosynthesis is also regulated by jasmonates photosynthesis is the key regulator of almost all metabolic and differentiation responses in the plant cells but it has also a major role in regulating gene expression by retrograde signalling. our results clearly pointed out that many in photosynthesis related transcript, and the photosynthetic oxygen evolution were significantly decreased in ac expressing plants. interestingly, jasmonate hormones have been shown to regulate photosynthesis related transcripts in arabidopsis, to down-regulate the oxygen evolution in isolated barley thylakoids, to up-regulate defence, stress, senescence and cell wall related transcripts and to causes oxidative stress in plants and thus also to change redox state of proteins. thus all these observed effects in ac expressing plants could have been related to up-regulated jasmonate biosynthesis. further on, the reduced photosynthesis may have changed the sugar metabolism and thus caused major secondary effects on retrograde signalling between the nucleus and the chloroplast. for instance, it may explain the changes in protein synthesis, cell wall synthesis, and chromosome/histone modifications related transcripts in the ac expressing plants. several studies have shown that energy deprivation and different stresses change energy-dependent transcriptional regulation with drastic down-regulation of transcripts related to different biosynthetic processes and especially protein syntheses and up-regulation of catabolic reactions to restore energy deprivation on the cells. protein synthesis affected in ac expressing plants results clearly indicated that expression of ac protein in transgenic plants had a major effect on transcripts related both protein synthesis and protein degradation. the down-regulation of protein synthesis caused to some extend by reduction of levels of multiple transcripts, but also the down-regulation of genes encoding both nuclear and plastid ribosomal proteins, ribosomal rna or translation factors. not only synthesis of proteins but also degradation of proteins was affected. over and under presentation analysis indicated that both cysteine and aspartate proteases were up-regulated in leaves of ac rss expressing plants. there were also indications of proteasomal protein degradation. interestingly, it has suggested that the geminiviral c protein might be the key player in proteasomal regulation of protein degradation. it was shown that c protein is capable of binding to proteins involved in proteasomal complexes e.g. scf e ligase complexes and thus inhibiting jasmonate related gene expression in arabidopsis. however, our results clearly indicate that the geminiviral protein ac has an opposite effect; enhancement of jasmonate signalling and jasmonate responsive gene regulation and no effects to transcripts related to neither sa synthesis nor sa responsive gene expression. cell cycle, genome methylation and histone expression is altered in ac expressing transgenic plants begomoviruses are known to replicate their single-stranded dna genomes through double-stranded dna intermediates that are associated with cellular histone proteins to form minichromosomes. thus it is important to begomoviruses to induce the accumulation of the dna replication machinery in mature plant cells. this is achieved most likely by modifying cell cycle and transcriptional regulation, for instance, the infection of cabbage leaf curl geminivirus has been shown to induce changes in the cell cycle in virus infected cells. interestingly, our results also suggested that expression of ac rss may have an effect on cell cycle, providing replication-competent environment and preventing expression of genes needed for mitosis.our results demonstrated that the expression ac rrs in transgenic plants up-regulated transcripts involved in dna methylation and down-regulated transcripts involved in histone synthesis. this suggested that, although the transcriptional activator ac suppresses silencing in the native host of acmv, and apparently enhances the virus replication in these hosts, in tobacco it induces defence mechanisms, leading to opposite direction, i.e. to increase of methylation and decrease of the histone synthesis.  the expression of ac rss in transgenic tobacco plants induced clear phenotypic changes in tobacco plants, even though tobacco is not considered as native host for acmv. this study indicated several possible action mechanisms of the ac rss. first both jasmonate and ethylene hormone responsive regulatory cascades were induced, secondly, the rate of photosynthesis and also the carbon metabolism were reduced. the stress and defence responsive gene expression was enhanced including both induction and response of reactive oxygen species. all these regulatory nodes may have been interconnected to one another. also, changes occurred in regulation of cell cycle and transcription as well as changes in the genomic architecture like dna methylation and abundance of histones. thus it seems that the silencing suppression activity of ac in tobacco is indicated in the enhancement of the jasmonate and ethylene biosynthesis, known to be regulated and repressed via rna silencing. up-regulation of these plant hormones was related to induction of a large variety of stress-related genes, and severe developmental defects in the plants. interestingly, the natural silencing suppression mechanism of the geminiviral rss, i.e. reduction of the dna methylation, did not occur in this non-host plant, but rather the opposite effect was observed, apparently related to the induced plant defences. 
it causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. comparison of these results to a previous transcriptional profiling of hc-pro rss-expressing plants revealed that some of same genes were induced by both rsss, but their expression levels were typically higher in ac than in hc-pro rss expressing plants. the three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. all in all, a large number of transcript alterations were found to be specific to each of the rss expressing transgenic plants. expression of ac rss in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. ac rss in transgenic tobacco plants interferes with the silencing machinery. it appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. the changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level. rna-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. various virus-specific dsrna structures are recognized by the silencing machinery leading to degradation of the viral rnas or, as in case of begomoviruses, to methylation of their dna genomes.
 rna-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. various virus-specific dsrna structures are recognized by the silencing machinery leading to degradation of the viral rnas or, as in case of begomoviruses, to methylation of their dna genomes. viruses produce specific rna silencing suppressor proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. in this paper, we describe how expression of ac rss, derived from african cassava mosaic geminivirus changes transcription profile in tobacco leaves and in flowers.  expression of ac rss in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. transcriptomes of these plants were strongly altered, with total of and differentially expressed genes in leaves and flowers, respectively. the three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. it appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. comparison of these results to a previous transcriptional profiling of hc-pro rss-expressing plants revealed that some of same genes were induced by both rsss, but their expression levels were typically higher in ac than in hc-pro rss expressing plants. all in all, a large number of transcript alterations were found to be specific to each of the rss expressing transgenic plants.  ac rss in transgenic tobacco plants interferes with the silencing machinery. it causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. the changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level. 