BACKGROUND
plant viruses pack many functions in a few genes and, as a consequence, most viral proteins must target numerous host factors, pathways and structures to propel infection.
however, the interaction networks between viral factors and host elements during plant infection are largely unknown  <cit> , particularly if we consider that the number of targets of viral factors in the infected plant cells may be unexpectedly high, as recently shown for some rna viruses infecting human cells  <cit> .
the goal of this work was to inquire about the complexity of the interaction network established between an essential protein from a plant rna virus, the nuclear inclusion a  protein of potyviruses, and the host proteins by means of a high-throughput proteomics approach, affinity purification coupled to mass spectrometry   <cit> .
potyviruses  form one of the largest groups of viruses infecting plants and cause important losses in crops worldwide.
their genome consists of a single -strand rna molecule of about  <dig>  nucleotides that is translated in two alternative polyproteins , which are processed by viral proteases in, apparently, a total of eleven mature products  <cit> .
nia is a crucial cistron in the potyviral genome.
it encodes the nia protein that consists of two different domains, an amino-terminal viral protein genome-linked  and a carboxy-terminal protease .
vpg is a multifunctional protein involved, at least, in virus replication  <cit> , translation  <cit>  and movement  <cit> .
notably, vpg remains covalently linked to the 5′ terminus of the virus genomic rna through a tyr residue  <cit>  and recruits host translation initiation factor eif4e, or its isoform eif4e, in an interaction that is crucial for infection  <cit> .
niapro is a serine protease that specifically recognizes seven-amino-acid-long conserved motifs in the viral polyprotein and, in cis and in trans, cleaves them between positions  <dig> and  <dig> to produce most viral protein products  <cit> .
niapro also displays rna binding activity  <cit> , interacts with the viral rna-dependent rna polymerase  and is involved in viral replication  <cit> .
the vpg and the niapro domains of nia are separated by an inefficiently utilized autoproteolytic site  <cit> , which implies that substantial amounts of full-length nia, and processed vpg and niapro products coexist in the infected cells at any moment of the infection process.
an additional, even more suboptimal, autoproteolytic site exists in some potyviruses close to the carboxy-terminal end of niapro producing additional nia-derived species in infected tissue that may be functionally relevant  <cit> .
to gain new insights about the complexity of the interaction network between tobacco etch virus  nia protein and the infected cells, in this work we aimed at identifying host proteins involved in complexes in which nia or nia-processing products were engaged during infection by ap-ms. first, we tagged the vpg and niapro domains of nia with an affinity polypeptide while maintaining tev infectivity.
next, we purified protein complexes from infected tissue by affinity chromatography in native conditions.
then, we identified proteins being part of these complexes by mass spectrometry analysis.
finally, we obtained a series of interaction networks between nia domains and host elements by computational analysis .fig.
<dig> schematic representation of the tev recombinant clones in which the nia protein was tagged with the twin-strep-tag  at five different positions  and affinity purification  mass spectrometry  workflow to construct nia-host interaction networks.
in virus scheme, black lines represent tev 5′ and 3′ utr, as indicated.
boxes represent tev cistrons  as indicated.
positions where tst was inserted in the vpg and niapro domains of nia are indicated with red triangles.
the gray rectangle in niapro represents the carboxy-terminal polypeptide that is subjected to processing through a suboptimal autoproteolytic site
RESULTS
construction of infectious tev clones with tagged nia
on the basis of the binary plasmid pgtevat containing an infectious tev clone under the control of cauliflower mosaic virus  35s promoter and terminator, we constructed five recombinant tev clones in which the polypeptide twin-strep-tag  was inserted at five different positions of nia .
tst is a bidentate polypeptide that binds strep-tactin  with high affinity and allows purification of intact protein complexes in mild conditions  <cit> .
we have previously used this tag to successfully purify protein complexes including other tagged tev proteins from plant infected tissue in native conditions  <cit> .
in tev-tstnia <dig>  tst was inserted after the first three amino-terminal codons of vpg.
the position was selected to avoid disruption of the 6k2/vpg cleavage site.
these first three codons were repeated after the tag but including a silent mutation to avoid undesired recombination during virus replication.
the exact cdna sequence of the five tagged nia is in additional file  <dig>  in tev-tstnia <dig>  tst was inserted between the − <dig> and − <dig> codons respect to the vpg/niapro cleavage site, again to avoid disturbing this cleavage site.
in tev-tstnia <dig>  tst was inserted after the first three codons of niapro that, as explained above, were repeated including a silent mutation after the tag.
in tev-tstnia <dig>  tst was inserted between the − <dig> and − <dig> codons of the internal niapro cleavage site.
finally, in tev-tstnia <dig>  tst was inserted between the − <dig> and − <dig> codons of niapro/nib cleavage site .
agrobacterium tumefaciens was transformed with the binary plasmids bearing the recombinant tev clones and cultures of this bacterium were used to agroinoculate nicotiana benthamiana domin plants.
nine days postinoculation , in addition to plants inoculated with wild-type tev, all plants inoculated with tev-tstnia <dig> and tev-tstnia <dig> showed the typical symptoms of tev infection.
half of the plants inoculated with tev-tstnia <dig> also showed symptoms that were milder.
none of the plants inoculated with tev-tstnia <dig> and tev-tstnia <dig> showed symptoms .
twenty-six dpi, plants inoculated with tev-tstnia <dig> and tev-tstnia <dig> remained undistinguishable from mock-inoculated plants .
reverse transcription -pcr analysis of inoculated plants confirmed that systemic tissues of non-symptomatic plants were free of the virus and systemic tissues of symptomatic plants contained tev.
comparison of the electrophoretic mobility of a series of pcr products from plants infected with wild-type tev and tev-tstnia <dig>  tev-tstnia <dig> and tev-tstnia <dig> at  <dig> dpi showed that viral progeny of these three recombinant viruses maintained the tag .
the intensity of the bands also suggested that viral loads in the case of tev-tstnia <dig> and tev-tstnia <dig> were high and comparable to wild-type tev, whereas tev-tstnia <dig> load was lower .
these results indicated that insertion of tst in both the selected carboxy-terminal positions of vpg and niapro does not strongly affect viral infectivity and accumulation.
hence, tev-tstnia <dig> and tev-tstnia <dig>  containing the tst at the vpg and niapro domains respectively, were selected for further analysis.fig.
<dig> infectivity of tev recombinant clones with tagged nia.
a and b
n. benthamiana plants non-inoculated  and agroinoculated with wild-type tev  and tev clones with tst-nia at five different positions .
pictures of representative plants were taken at  <dig>  or  <dig>  days postinoculation .
c rt-pcr analysis of the viral progenies from plants infected with tev-wt , tev-tstnia <dig> , tev-tstnia <dig>  and tev-tstnia <dig> .
the control rt-pcr reaction  contained all primers and no template.
lanes  <dig>   <dig> and  <dig>  dna marker ladder with sizes on the right side in kbp.
red arrows point the pcr products delayed as a consequence of insertion of the tst cdna in the viral progenies
identification of host proteins forming complex with tev nia in infected tissues
we mechanically inoculated three batches of arabidopsis thaliana l.  plants with extracts from n. benthamiana tissues infected with wild-type tev, tev-tstnia <dig> and tev-tstnia <dig>  aerial tissues from plants infected with each virus were harvested  <dig> dpi and pooled.
all plants showed symptoms of infection at this point.
western blot analysis of aliquots from the collected tissues using an anti-tst monoclonal antibody confirmed accumulation of tst-nia and tst-vpg in plants infected with tev-tstnia <dig>  and tst-nia and tst-niapro in plants infected with tev-tstnia <dig>  bands corresponding to nia products resulting from cleavage at the niapro carboxy-proximal autoproteolytic site  <cit> , were also observed.
no signal was detected in non-inoculated controls and plants infected with wild-type tev.
protein complexes containing tst-nia-derived protein products were purified from the three batches of infected tissues by affinity chromatography in native conditions.
proteins from purified complexes were separated by denaturing polyacrylamide gel electrophoresis  and digested in gel with trypsin.
peptides were then eluted from the gel and analyzed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the purified complexes.
additional file  <dig> lists all hits resulting from proteomic analysis of complexes purified from tissues infected with wild-type tev, tev-tstnia <dig>  and tev-tstnia <dig> .
next, hits were classified according to the exponentially modified protein abundance index   <cit> .
for each hit arising from complexes purified from the tissues infected with tev-tstnia <dig> and tev-tstnia <dig>  we calculated the difference in empai  with respect to the tissues infected with wild-type tev.
hits were considered spurious if they matched any of the following conditions:  proteins were identified in very low abundance , despite not being detected in wild-type tev infected control tissues; and  they were identified in relatively high abundance but also detected in wild-type tev infected control tissues in a way that Δempai is also under a given threshold.
remaining hits were computationally filtered and assigned to single genes of the a. thaliana genome .
tev nia-host interaction network
our experimental approach resulted in a total of  <dig> different host proteins considered to be involved in protein complexes in which tev nia processing products take also part, according to a Δempai threshold criterion.
more in detail,  <dig> and  <dig> host proteins were identified when the affinity tag was inserted close to the carboxyl terminus of the vpg and niapro domains, respectively;  <dig> proteins were common to both lists.
this common set of proteins most probably results from complexes in which the full-length nia is involved, although they may also indicate redundancy in the mode of action of vpg and niapro.
hereafter, the  <dig> host proteins were classified as specific vpg targets , specific niapro targets  and nia targets .
in all cases, it is important to stress out that targets may be direct or indirect.
overall, these results suggest that, similarly to what occurs with rna viruses infecting animal cells  <cit> , proteins from plant rna viruses also target many host elements during infection.
next, we contextualized the identified targets in the experimental a. thaliana protein-protein interactome .
nia targets indicated in fig.
<dig> represent either direct or indirect interactions.
of the  <dig> host proteins,  <dig> were not covered in the a. thaliana interactome, which only contains about  <dig>  proteins .
we found that nia targets are not more connected with other host proteins than expected by chance .
it has been shown, however, that virus targets tend to be hubs in the host interactome  <cit> .
according to our data, this is not the case of tev nia.
instead, our network-based analysis revealed that the detected targets are in a position that allows them to connect with other host proteins throughout short paths .
that is, nia targets connect on average in  <dig>  steps with any other protein of the interactome, whilst randomly picked proteins do in  <dig>  steps .
this indicates that local perturbations by the virus in the host interactome  could reach a global outcome very quickly  <cit> , leading to significant changes in the physiology of the cell and then in the whole organism .
no significant differences were found between the three categories: vpg, niapro and full-length nia targets .
even though the a. thaliana interactome is incomplete, a limitation that has not prevented the distillation of the mechanisms of important diseases in humans  <cit> , our results suggest that nia targets are elements that can efficiently transmit information through the host interactome  <cit> .fig.
<dig> contextualization of the tev nia host targets on the experimental a. thaliana interactome.
illustration of the global interactome together with a boxplot of the global topological properties  of the nia targets.
note that these properties were calculated according to the global interactome and not to a particular subnetwok.
no significant differences were found between distributions of degree.
by contrast, differences were significant between distributions of average shortest path length .
as controls, the distributions of a representative random sample and the whole interactome are shown
in the a. thaliana interactome, the most connected elements specifically targeted by vpg are the g-box regulating factor  <dig>  with degree  <dig>  and a mitochondrial atp synthase  with degree  <dig>  the former protein has been shown to participate in plant defense  <cit> .
different proteins from animal rna viruses, like hepatitis c virus core protein, have also been shown to bind to 14-3- <dig> proteins  <cit> , inducing disease by triggering certain kinase cascades, whilst the most connected proteins specifically targeted by niapro are the plasma membrane aquaporin pip2; <dig> with degree  <dig>  and the actin  <dig> with degree  <dig>  in particular, pip2; <dig> has been shown to mediate response to stress  <cit>  and is highly clustered in the network.
the most connected proteins targeted by nia  are the heat shock cognate protein 70– <dig>  with degree  <dig>  and the photosystem protein lhca <dig> with degree  <dig>  hsp <dig> proteins are ubiquitous molecular chaperones that act in polypeptide folding, refolding of misfolded or aggregated proteins, translocation across membranes, protein complex assembly and protein degradation  <cit> .
they play central roles in the formation of membrane-bound replication complexes in many plant viruses, including potyviruses  <cit> .
of relevance, our data suggest that full-length nia  binds to a protein complex that includes the host pathogenesis-related protein pr- <dig>  this interaction may indicate a viral strategy to interfere with the host defense mechanisms, as systemic acquired resistance against pathogens is associated with the accumulation of salicylic acid and pr proteins  <cit> .
in addition, we identified some host proteins previously shown to interact with potyviral nia or nia domains, like the poly-binding proteins pabp <dig> and pabp <dig> , the translation elongation factor ef1a  <cit> , and a methionine sulfoxide reductase  <cit> .
however, we did not detect other interactors previously shown using different techniques for tev or other potyviruses, like the translation initiation factors eif4e or eif4e , the helicase rh <dig>  <cit> , the fibrillarin  <cit> , the elongin c  <cit> , and the silencing-related protein sgs <dig>  <cit> .
in any case, note that some of these interactions may be species specific.
for instance, we neither detected interaction with phd-finger proteins obe <dig> and obe <dig> , but tev vpg failed to interact with these proteins in a yeast to hybrid system  <cit> .
a recent review summarizes most host proteins that have currently shown to interact with potyviral proteins  <cit> .
next, we constructed a nia-host protein interaction network .
this network also includes a. thaliana proteins that have been well established to interact  with those experimentally identified in our work.
we obtained a network containing  <dig> host proteins connected to the two domains of nia.
note that  <dig> proteins are not connected to other host proteins in our network.
we calculated an average connectivity of  <dig> , a characteristic path length of  <dig> , and an average clustering coefficient of  <dig> .
the resulting degree distribution is scale-free with an exponent of − <dig>  .
we anticipate that this network will serve to better understand the mode of action of potyviruses, and then serve to develop effective strategies for crop protection or infection limitation.fig.
<dig> protein-protein interaction network between the two domains of tev nia  and a. thaliana proteins during infection.
interactions between viral and host proteins  are shown in red, between the virus targets and their direct partners in the host in blue, and between host proteins not being targeted by nia in green.
map was constructed with cytoscape
functional analysis of the host proteins identified in complexes with tev nia
from our refined lists of host proteins targeted by nia products during infection , we identified the global functional categories of vpg and niapro specific targets, as well as common targets, using the panther tool  <cit>  .
regarding plant physiology, we observed that specific targets impact more metabolism, whilst those in common do development and organization.
we found that common targets are significantly enriched  in genes coding for the response to biotic stimulus , whilst specific targets of vpg and niapro are not.
in particular, the specific targets of vpg are less involved in response to stimulus with respect to the  <dig> shared elements .
moreover, we obtained a functional network of over-represented gene ontology  terms for the complete list of nia targets  , using the agrigo tool  <cit> .
in this network, functional categories are shown to be related to each other, with proteins belonging to different categories.
over-represented go terms were mainly grouped in response to stress , metabolism, photosynthesis and localization.
we found that both vpg and niapro targets are similarly engaged in these main groups , perhaps because almost  <dig> % of targets are shared.
we also observed that, in general, nia targets more proteins related to abiotic stress than biotic stress .fig.
<dig> tev nia-host functional networks.
a venn diagrams showing the number of host proteins targeted by the vpg and niapro domains of tev nia during infection, together with a functional analysis of the global categories over-represented.
b map of the different biological functions  over-represented within the complete list of targeted host proteins .
representation obtained with revigo.
red intensity corresponds to the p-value.
c network representing the relationship between nia-targeted host proteins and phytohormones.
this network was obtained taking advantage of the arabidopsis hormone database
finally, by taking advantage of known host proteins involved in biosynthesis, signal transduction and response of phytohormones  <cit> , we delineated a new functional map of nia during infection .
ethylene, which plays a crucial role in mounting defense responses, particularly during pathogen infections  <cit> , was shown to interact with the majority of the identified proteins.
auxins and brassinosteroids were linked to niapro, which may suggest an effect on plant development, whilst jasmonic acid, which exhibits a big cross-talk with salicylic acid, was associated to vpg.
remarks about the experimental approach
several issues should be taken into consideration about these interaction networks.
first, our lists of host nia-targeted proteins come from an ap-ms experimental approach.
some of the identified proteins may still result from spurious interactions in vivo or during the purification process and be functionally irrelevant.
at the same time, some true interactors may have been missed due to low abundance, transient binding or inappropriate experimental conditions.
second, our experimental approach searches for host proteins bound to multiprotein complexes to which the tagged viral proteins also bind.
this way, reported host interactors may not necessarily bind directly to the viral proteins.
certainly, the use of a thorough pipeline  will help to find the direct interactors and to decipher the supramolecular organization of protein complexes  <cit> .
third, our experimental results derive from analyses of pooled plants and tissues at 2-week after inoculation.
ours is probably a snapshot at a late stage of infection.
however, host-virus interactions are dynamic, establishing and releasing connections in each cell as the infection progresses.
thus, some known interactions between nia domains and host factors may not exist or be underrepresented at our sampling time.
further ap-ms analyses at different times post-inoculation will be required to uncover the dynamics of host-virus interaction network along the infectious cycle.
CONCLUSIONS
an affinity tag, such as tst, can be inserted between the codon positions -9/- <dig> in the vpg and niapro domains of tev nia without strongly affecting virus viability.
remarkably, the tst tag inserted in either of these two positions is stable in the viral progenies, allowing affinity purification of protein complexes in which nia processing products are involved.
tev nia targets many host proteins during infection.
our ap-ms approach applied to a. thaliana tissues infected with tst-tagged tev clones detected  <dig> and  <dig> host proteins specifically involved in complexes with the vpg and niapro domains of nia, respectively, and  <dig> common proteins, most probably full-length nia targets.
combining these experimental data with currently known information about the a. thaliana interactome, we constructed a nia-host protein-protein interaction network containing  <dig> host proteins connected to the two domains of nia.
this network has an average connectivity of  <dig> , a characteristic path length of  <dig> , and an average clustering coefficient of  <dig> .
the network also highlights that the most connected host elements are the g-box regulating factor  <dig>  and the heat shock cognate protein 70– <dig> .
further analyses of the host proteins targeted by nia processing products allowed constructing additional nia-host interaction networks.
these networks show that nia-targeted proteins are mainly involved in response to stress , metabolism, photosynthesis and localization, and that ethylene is the phytohormone most connected with the nia-targeted host proteins.
finally, a look at the position of the detected targets in the global a. thaliana interactome reveals that nia targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths, which may suggest a strategy to efficiently transmit information.

 plant viruses pack many functions in a few genes and, as a consequence, most viral proteins must target numerous host factors, pathways and structures to propel infection. however, the interaction networks between viral factors and host elements during plant infection are largely unknown, particularly if we consider that the number of targets of viral factors in the infected plant cells may be unexpectedly high, as recently shown for some rna viruses infecting human cells. the goal of this work was to inquire about the complexity of the interaction network established between an essential protein from a plant rna virus, the nuclear inclusion a protein of potyviruses, and the host proteins by means of a high-throughput proteomics approach, affinity purification coupled to mass spectrometry. potyviruses form one of the largest groups of viruses infecting plants and cause important losses in crops worldwide. their genome consists of a single -strand rna molecule of about nucleotides that is translated in two alternative polyproteins, which are processed by viral proteases in, apparently, a total of eleven mature products. nia is a crucial cistron in the potyviral genome. it encodes the nia protein that consists of two different domains, an amino-terminal viral protein genome-linked and a carboxy-terminal protease. vpg is a multifunctional protein involved, at least, in virus replication, translation and movement. notably, vpg remains covalently linked to the 5′ terminus of the virus genomic rna through a tyr residue and recruits host translation initiation factor eif4e, or its isoform eif4e, in an interaction that is crucial for infection. niapro is a serine protease that specifically recognizes seven-amino-acid-long conserved motifs in the viral polyprotein and, in cis and in trans, cleaves them between positions and to produce most viral protein products. niapro also displays rna binding activity, interacts with the viral rna-dependent rna polymerase and is involved in viral replication. the vpg and the niapro domains of nia are separated by an inefficiently utilized autoproteolytic site, which implies that substantial amounts of full-length nia, and processed vpg and niapro products coexist in the infected cells at any moment of the infection process. an additional, even more suboptimal, autoproteolytic site exists in some potyviruses close to the carboxy-terminal end of niapro producing additional nia-derived species in infected tissue that may be functionally relevant. to gain new insights about the complexity of the interaction network between tobacco etch virus nia protein and the infected cells, in this work we aimed at identifying host proteins involved in complexes in which nia or nia-processing products were engaged during infection by ap-ms. first, we tagged the vpg and niapro domains of nia with an affinity polypeptide while maintaining tev infectivity. next, we purified protein complexes from infected tissue by affinity chromatography in native conditions. then, we identified proteins being part of these complexes by mass spectrometry analysis. finally, we obtained a series of interaction networks between nia domains and host elements by computational analysis.fig. schematic representation of the tev recombinant clones in which the nia protein was tagged with the twin-strep-tag at five different positions and affinity purification mass spectrometry workflow to construct nia-host interaction networks. in virus scheme, black lines represent tev 5′ and 3′ utr, as indicated. boxes represent tev cistrons as indicated. positions where tst was inserted in the vpg and niapro domains of nia are indicated with red triangles. the gray rectangle in niapro represents the carboxy-terminal polypeptide that is subjected to processing through a suboptimal autoproteolytic site  construction of infectious tev clones with tagged nia on the basis of the binary plasmid pgtevat containing an infectious tev clone under the control of cauliflower mosaic virus 35s promoter and terminator, we constructed five recombinant tev clones in which the polypeptide twin-strep-tag was inserted at five different positions of nia. tst is a bidentate polypeptide that binds strep-tactin with high affinity and allows purification of intact protein complexes in mild conditions. we have previously used this tag to successfully purify protein complexes including other tagged tev proteins from plant infected tissue in native conditions. in tev-tstnia tst was inserted after the first three amino-terminal codons of vpg. the position was selected to avoid disruption of the 6k2/vpg cleavage site. these first three codons were repeated after the tag but including a silent mutation to avoid undesired recombination during virus replication. the exact cdna sequence of the five tagged nia is in additional file in tev-tstnia tst was inserted between the − and − codons respect to the vpg/niapro cleavage site, again to avoid disturbing this cleavage site. in tev-tstnia tst was inserted after the first three codons of niapro that, as explained above, were repeated including a silent mutation after the tag. in tev-tstnia tst was inserted between the − and − codons of the internal niapro cleavage site. finally, in tev-tstnia tst was inserted between the − and − codons of niapro/nib cleavage site. agrobacterium tumefaciens was transformed with the binary plasmids bearing the recombinant tev clones and cultures of this bacterium were used to agroinoculate nicotiana benthamiana domin plants. nine days postinoculation, in addition to plants inoculated with wild-type tev, all plants inoculated with tev-tstnia and tev-tstnia showed the typical symptoms of tev infection. half of the plants inoculated with tev-tstnia also showed symptoms that were milder. none of the plants inoculated with tev-tstnia and tev-tstnia showed symptoms. twenty-six dpi, plants inoculated with tev-tstnia and tev-tstnia remained undistinguishable from mock-inoculated plants. reverse transcription -pcr analysis of inoculated plants confirmed that systemic tissues of non-symptomatic plants were free of the virus and systemic tissues of symptomatic plants contained tev. comparison of the electrophoretic mobility of a series of pcr products from plants infected with wild-type tev and tev-tstnia tev-tstnia and tev-tstnia at dpi showed that viral progeny of these three recombinant viruses maintained the tag. the intensity of the bands also suggested that viral loads in the case of tev-tstnia and tev-tstnia were high and comparable to wild-type tev, whereas tev-tstnia load was lower. these results indicated that insertion of tst in both the selected carboxy-terminal positions of vpg and niapro does not strongly affect viral infectivity and accumulation. hence, tev-tstnia and tev-tstnia containing the tst at the vpg and niapro domains respectively, were selected for further analysis.fig. infectivity of tev recombinant clones with tagged nia. a and b n. benthamiana plants non-inoculated and agroinoculated with wild-type tev and tev clones with tst-nia at five different positions. pictures of representative plants were taken at or days postinoculation. c rt-pcr analysis of the viral progenies from plants infected with tev-wt, tev-tstnia, tev-tstnia and tev-tstnia. the control rt-pcr reaction contained all primers and no template. lanes and dna marker ladder with sizes on the right side in kbp. red arrows point the pcr products delayed as a consequence of insertion of the tst cdna in the viral progenies identification of host proteins forming complex with tev nia in infected tissues we mechanically inoculated three batches of arabidopsis thaliana l. plants with extracts from n. benthamiana tissues infected with wild-type tev, tev-tstnia and tev-tstnia aerial tissues from plants infected with each virus were harvested dpi and pooled. all plants showed symptoms of infection at this point. western blot analysis of aliquots from the collected tissues using an anti-tst monoclonal antibody confirmed accumulation of tst-nia and tst-vpg in plants infected with tev-tstnia and tst-nia and tst-niapro in plants infected with tev-tstnia bands corresponding to nia products resulting from cleavage at the niapro carboxy-proximal autoproteolytic site, were also observed. no signal was detected in non-inoculated controls and plants infected with wild-type tev. protein complexes containing tst-nia-derived protein products were purified from the three batches of infected tissues by affinity chromatography in native conditions. proteins from purified complexes were separated by denaturing polyacrylamide gel electrophoresis and digested in gel with trypsin. peptides were then eluted from the gel and analyzed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the purified complexes. additional file lists all hits resulting from proteomic analysis of complexes purified from tissues infected with wild-type tev, tev-tstnia and tev-tstnia. next, hits were classified according to the exponentially modified protein abundance index. for each hit arising from complexes purified from the tissues infected with tev-tstnia and tev-tstnia we calculated the difference in empai with respect to the tissues infected with wild-type tev. hits were considered spurious if they matched any of the following conditions: proteins were identified in very low abundance, despite not being detected in wild-type tev infected control tissues; and they were identified in relatively high abundance but also detected in wild-type tev infected control tissues in a way that Δempai is also under a given threshold. remaining hits were computationally filtered and assigned to single genes of the a. thaliana genome. tev nia-host interaction network our experimental approach resulted in a total of different host proteins considered to be involved in protein complexes in which tev nia processing products take also part, according to a Δempai threshold criterion. more in detail, and host proteins were identified when the affinity tag was inserted close to the carboxyl terminus of the vpg and niapro domains, respectively; proteins were common to both lists. this common set of proteins most probably results from complexes in which the full-length nia is involved, although they may also indicate redundancy in the mode of action of vpg and niapro. hereafter, the host proteins were classified as specific vpg targets, specific niapro targets and nia targets. in all cases, it is important to stress out that targets may be direct or indirect. overall, these results suggest that, similarly to what occurs with rna viruses infecting animal cells, proteins from plant rna viruses also target many host elements during infection. next, we contextualized the identified targets in the experimental a. thaliana protein-protein interactome. nia targets indicated in fig.  represent either direct or indirect interactions. of the host proteins, were not covered in the a. thaliana interactome, which only contains about proteins. we found that nia targets are not more connected with other host proteins than expected by chance. it has been shown, however, that virus targets tend to be hubs in the host interactome. according to our data, this is not the case of tev nia. instead, our network-based analysis revealed that the detected targets are in a position that allows them to connect with other host proteins throughout short paths. that is, nia targets connect on average in steps with any other protein of the interactome, whilst randomly picked proteins do in steps. this indicates that local perturbations by the virus in the host interactome could reach a global outcome very quickly, leading to significant changes in the physiology of the cell and then in the whole organism. no significant differences were found between the three categories: vpg, niapro and full-length nia targets. even though the a. thaliana interactome is incomplete, a limitation that has not prevented the distillation of the mechanisms of important diseases in humans, our results suggest that nia targets are elements that can efficiently transmit information through the host interactome.fig. contextualization of the tev nia host targets on the experimental a. thaliana interactome. illustration of the global interactome together with a boxplot of the global topological properties of the nia targets. note that these properties were calculated according to the global interactome and not to a particular subnetwok. no significant differences were found between distributions of degree. by contrast, differences were significant between distributions of average shortest path length. as controls, the distributions of a representative random sample and the whole interactome are shown in the a. thaliana interactome, the most connected elements specifically targeted by vpg are the g-box regulating factor with degree and a mitochondrial atp synthase with degree the former protein has been shown to participate in plant defense. different proteins from animal rna viruses, like hepatitis c virus core protein, have also been shown to bind to 14-3- proteins, inducing disease by triggering certain kinase cascades, whilst the most connected proteins specifically targeted by niapro are the plasma membrane aquaporin pip2; with degree and the actin with degree in particular, pip2; has been shown to mediate response to stress and is highly clustered in the network. the most connected proteins targeted by nia are the heat shock cognate protein 70– with degree and the photosystem protein lhca with degree hsp proteins are ubiquitous molecular chaperones that act in polypeptide folding, refolding of misfolded or aggregated proteins, translocation across membranes, protein complex assembly and protein degradation. they play central roles in the formation of membrane-bound replication complexes in many plant viruses, including potyviruses. of relevance, our data suggest that full-length nia binds to a protein complex that includes the host pathogenesis-related protein pr- this interaction may indicate a viral strategy to interfere with the host defense mechanisms, as systemic acquired resistance against pathogens is associated with the accumulation of salicylic acid and pr proteins. in addition, we identified some host proteins previously shown to interact with potyviral nia or nia domains, like the poly-binding proteins pabp and pabp, the translation elongation factor ef1a, and a methionine sulfoxide reductase. however, we did not detect other interactors previously shown using different techniques for tev or other potyviruses, like the translation initiation factors eif4e or eif4e, the helicase rh, the fibrillarin, the elongin c, and the silencing-related protein sgs. in any case, note that some of these interactions may be species specific. for instance, we neither detected interaction with phd-finger proteins obe and obe, but tev vpg failed to interact with these proteins in a yeast to hybrid system. a recent review summarizes most host proteins that have currently shown to interact with potyviral proteins. next, we constructed a nia-host protein interaction network. this network also includes a. thaliana proteins that have been well established to interact with those experimentally identified in our work. we obtained a network containing host proteins connected to the two domains of nia. note that proteins are not connected to other host proteins in our network. we calculated an average connectivity of, a characteristic path length of, and an average clustering coefficient of. the resulting degree distribution is scale-free with an exponent of −. we anticipate that this network will serve to better understand the mode of action of potyviruses, and then serve to develop effective strategies for crop protection or infection limitation.fig. protein-protein interaction network between the two domains of tev nia and a. thaliana proteins during infection. interactions between viral and host proteins are shown in red, between the virus targets and their direct partners in the host in blue, and between host proteins not being targeted by nia in green. map was constructed with cytoscape functional analysis of the host proteins identified in complexes with tev nia from our refined lists of host proteins targeted by nia products during infection, we identified the global functional categories of vpg and niapro specific targets, as well as common targets, using the panther tool. regarding plant physiology, we observed that specific targets impact more metabolism, whilst those in common do development and organization. we found that common targets are significantly enriched in genes coding for the response to biotic stimulus, whilst specific targets of vpg and niapro are not. in particular, the specific targets of vpg are less involved in response to stimulus with respect to the shared elements. moreover, we obtained a functional network of over-represented gene ontology terms for the complete list of nia targets, using the agrigo tool. in this network, functional categories are shown to be related to each other, with proteins belonging to different categories. over-represented go terms were mainly grouped in response to stress, metabolism, photosynthesis and localization. we found that both vpg and niapro targets are similarly engaged in these main groups, perhaps because almost % of targets are shared. we also observed that, in general, nia targets more proteins related to abiotic stress than biotic stress.fig. tev nia-host functional networks. a venn diagrams showing the number of host proteins targeted by the vpg and niapro domains of tev nia during infection, together with a functional analysis of the global categories over-represented. b map of the different biological functions over-represented within the complete list of targeted host proteins. representation obtained with revigo. red intensity corresponds to the p-value. c network representing the relationship between nia-targeted host proteins and phytohormones. this network was obtained taking advantage of the arabidopsis hormone database finally, by taking advantage of known host proteins involved in biosynthesis, signal transduction and response of phytohormones, we delineated a new functional map of nia during infection. ethylene, which plays a crucial role in mounting defense responses, particularly during pathogen infections, was shown to interact with the majority of the identified proteins. auxins and brassinosteroids were linked to niapro, which may suggest an effect on plant development, whilst jasmonic acid, which exhibits a big cross-talk with salicylic acid, was associated to vpg. remarks about the experimental approach several issues should be taken into consideration about these interaction networks. first, our lists of host nia-targeted proteins come from an ap-ms experimental approach. some of the identified proteins may still result from spurious interactions in vivo or during the purification process and be functionally irrelevant. at the same time, some true interactors may have been missed due to low abundance, transient binding or inappropriate experimental conditions. second, our experimental approach searches for host proteins bound to multiprotein complexes to which the tagged viral proteins also bind. this way, reported host interactors may not necessarily bind directly to the viral proteins. certainly, the use of a thorough pipeline will help to find the direct interactors and to decipher the supramolecular organization of protein complexes. third, our experimental results derive from analyses of pooled plants and tissues at 2-week after inoculation. ours is probably a snapshot at a late stage of infection. however, host-virus interactions are dynamic, establishing and releasing connections in each cell as the infection progresses. thus, some known interactions between nia domains and host factors may not exist or be underrepresented at our sampling time. further ap-ms analyses at different times post-inoculation will be required to uncover the dynamics of host-virus interaction network along the infectious cycle.  an affinity tag, such as tst, can be inserted between the codon positions -9/- in the vpg and niapro domains of tev nia without strongly affecting virus viability. remarkably, the tst tag inserted in either of these two positions is stable in the viral progenies, allowing affinity purification of protein complexes in which nia processing products are involved. tev nia targets many host proteins during infection. our ap-ms approach applied to a. thaliana tissues infected with tst-tagged tev clones detected and host proteins specifically involved in complexes with the vpg and niapro domains of nia, respectively, and common proteins, most probably full-length nia targets. combining these experimental data with currently known information about the a. thaliana interactome, we constructed a nia-host protein-protein interaction network containing host proteins connected to the two domains of nia. this network has an average connectivity of, a characteristic path length of, and an average clustering coefficient of. the network also highlights that the most connected host elements are the g-box regulating factor and the heat shock cognate protein 70–. further analyses of the host proteins targeted by nia processing products allowed constructing additional nia-host interaction networks. these networks show that nia-targeted proteins are mainly involved in response to stress, metabolism, photosynthesis and localization, and that ethylene is the phytohormone most connected with the nia-targeted host proteins. finally, a look at the position of the detected targets in the global a. thaliana interactome reveals that nia targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths, which may suggest a strategy to efficiently transmit information. 
the genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. potyviral nuclear inclusion a plays many roles during infection. host-virus systems biologyprotein interaction networkrna virusplant viruspotyvirusnuclear inclusion a proteinaffinity purification mass spectrometryarabidopsis thalianahttp://dx.doi.org/ /501100003329ministerio de economía y competitividadbio2014-54269-rbfu2012-30805elena santiago f. daròs josé-antonio http://dx.doi.org/ /501100003359generalitat valencianaprometeoii/2014/021elena santiago f. universidad politécnica de valenciaissue-copyright-statement© the author 2016 potyviral nia targets many host elements during infection, establishing a network in which information is efficiently transmitted. the most connected elements specifically targeted by vpg were g-box regulating factor and mitochondrial atp synthase δ subunit; those specifically targeted by niapro were plasma membrane aquaporin pip2; and actin whereas those targeted by full-length nia were heat shock protein 70– and photosystem protein lhca moreover, a contextualization in the global a. thaliana interactome showed that nia targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. vpg and niapro specifically targeted and of these proteins, respectively, whereas proteins were targeted by both domains and considered full-length nia targets. however, the interaction networks between viruses and host factors are poorly understood. to gain insights about the interaction between potyviral nia and the host cell during infection, we constructed tobacco etch virus infectious clones in which the vpg or the niapro domains of nia were tagged with the affinity polypeptide twin-strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis. taking advantage of the currently known a. thaliana interactome, we constructed a protein interaction network between tev nia domains and host proteins. further analysis of nia-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization.
 the genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. however, the interaction networks between viruses and host factors are poorly understood. the genus potyvirus is the largest group of plus-strand rna viruses infecting plants. potyviral nuclear inclusion a plays many roles during infection. nia is a polyprotein consisting of two domains, viral protein genome-linked and protease, separated by an inefficiently utilized self-proteolytic site. to gain insights about the interaction between potyviral nia and the host cell during infection, we constructed tobacco etch virus infectious clones in which the vpg or the niapro domains of nia were tagged with the affinity polypeptide twin-strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis.  we identified different arabidopsis thaliana proteins forming part of complexes in which tev nia products were also involved. vpg and niapro specifically targeted and of these proteins, respectively, whereas proteins were targeted by both domains and considered full-length nia targets. taking advantage of the currently known a. thaliana interactome, we constructed a protein interaction network between tev nia domains and host proteins. the most connected elements specifically targeted by vpg were g-box regulating factor and mitochondrial atp synthase δ subunit; those specifically targeted by niapro were plasma membrane aquaporin pip2; and actin whereas those targeted by full-length nia were heat shock protein 70– and photosystem protein lhca moreover, a contextualization in the global a. thaliana interactome showed that nia targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. further analysis of nia-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization. many of these proteins are connected with the phytohormone ethylene.  potyviral nia targets many host elements during infection, establishing a network in which information is efficiently transmitted. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords host-virus systems biologyprotein interaction networkrna virusplant viruspotyvirusnuclear inclusion a proteinaffinity purification mass spectrometryarabidopsis thalianahttp://dx.doi.org/ /501100003329ministerio de economía y competitividadbio2014-54269-rbfu2012-30805elena santiago f. daròs josé-antonio http://dx.doi.org/ /501100003359generalitat valencianaprometeoii/2014/021elena santiago f. universidad politécnica de valenciaissue-copyright-statement© the author 2016 