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Online Resource 1. Phylogenetic analysis of soybean bZIPs proteins
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MATERIALS AND METHODS
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To provide an evolutionary framework for the discussion of protein functions, phylogenetic
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hypotheses were inferred by Bayesian inference (BI) and maximum likelihood (ML) using BEAST
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v1.7.2 (Huelsenbeck and Ronquist, 2001) and GARLI 2.0 (Zwickl, 2006), respectively.
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First, a search for bZIP protein sequences in Glycine max was performed in GenBank
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(http://blast.ncbi.nlm.nih.gov), and 148 sequences were selected (Online Resource 2).
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Additionally, we selected 10 bZIP protein sequences related to pathogen response
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(ACT66299_TabZIP;
AAX20030_CAbZIP1;
CAA71687_GHBF1;
AAL27150_BZI1;
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O22763_AtbZIP10; AAN61914_ PPI1; BAG24402_ SBZ1; NP_001234596_SlAREB1; P43273_
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TGA2; Q39234_ TGA3). The selected sequences were aligned using MAFFT version 7 (Katoh et.
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al, 2002) and the JTT+G (Jones et. al, 1992) was calculated by ProtTest 3.2.1 (Darriba et. al, 2011)
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as the best-fit amino acid substitution model according to AIC and BIC criteria.
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The BI phylogenetic trees were calculated using the Bayesian Markov Chain Monte Carlo
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(MCMC) method with 5x107 generations and a sample frequency of 104, using the JTT+G
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substitution model. The convergence of the parameters was analyzed in TRACER v1.5.0
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(http://beast.bio.ed.ac.uk/tracer), and the chain reached a stationary distribution after 5x105
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generations. A total of 1% of the generated trees was burned to produce the consensus Bayesian
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phylogenetic tree.
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The JTT+G substitution model was also selected in the GARLI settings (datatype =
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aminoacid; ratematrix = jones; statefrequencies = jones; ratehetmodel = gamma; numratecats = 4;
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invariantsites = none), and the statistical support of the ML phylogenetic trees was calculated by
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103 bootstrap replicates. The 50% majority rule consensus ML phylogenetic tree of all bootstrap
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replicates was summarized using SumTrees of DendroPy 3.8.0 (Sukumaran et. al, 2010).
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For comparison purposes among clusters in phylogenetic tree and protein functions, a
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search for conserved domains was performed in the Pfam database (Sonnhammer et. Al, 1997).
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Only domains predicted at the 1% level of significance were considered further.
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RESULTS
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In phylogenetic trees (Fig. S1), these ten classes were recovered as monophyletic clades
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supported by moderate values of posterior probability (PP) (Bayesian tree) and bootstrap value
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(BV) (ML tree), with PP > 85 and BV > 50; most of 119 proteins included in these clades share
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the same pfam domains (http://pfam.sanger.ac.uk/). Based on such data, we also proposed an
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additional group of nine related bZIP proteins that have a set of four distinct domains (Fig. S1).
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Beyond these eleven groups, 20 (13.5%) bZIP proteins remained ungrouped (Fig. S1).
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After the distribution of all soybean bZIP proteins annotated in GenBank into 11 classes,
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we sought candidate sequences in the group of bZIP proteins characterized as responsive to
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pathogens (Fig. S1) and also differentially expressed during ASR. For the purpose of determining
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the bZIP transcription factors involved in response to P. pachyrhizi, we used data from subtractive
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libraries containing clones of Inoculated plants vs. MOCK plants subtraction, based on resistant
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soybean cultivar PI561356, deposited in the database of the GENOSOJA consortium
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(http://bioinfo.cnpso.embrapa.br/genosoja/; Benko-Iseppon et al, 2012). We were able to verify
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the differential expression of several members of plant transcription factors families involved in
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plant defense, such as MYB, WRKY, AP2 / ERF, NAC and bZIP transcription factors families,
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during ASR infection in PI561356 plants (unpublished data). Among the members of the bZIP
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family, we chose four distinct soybean genes with high value of fragments per kilobase of exon
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per million fragments mapped (FPKM; above 500) for further functional studies (GenBank access
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numbers ABI34659, NP_001237027, XP_003543312 and XP_003525005). Thus, we selected
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four bZIP members (whose GenBank accession numbers are highlighted in the tree) for functional
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analysis (Fig. S1). Based on the phylogenetic analyses, two bZIP proteins selected grouped in E
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class, and were used for analysis of response to infection by P. pachyrhizi (Fig. 1).
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This is the class that includes TabZIP (ACT66299), a single bZIP protein that has been
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functionally characterized in the response to infection by a wheat rust fungus, Puccinia striiformis
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f. sp. Tritici (Zhang et al. 2009). The soybean proteins selected (XP_003543312 and
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XP_003525005) are similar to Arabidopsis bZIP proteins from the E class, which have not been
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assigned a defined biological function (Jakoby et al. 2002). Thus, we selected these two proteins
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for functional studies and to analyze their expression during infection by P. pachyrhizi. The
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proteins were named GmbZIPE1 (XP_003543312) and GmbZIPE2 (XP_003525005) because of
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their similarity to Arabidopsis proteins in the E class (Jakoby et al. 2002).
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The two other bZIP members selected (ABI34659 and NP_001237027) grouped in the C
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class. The C class had the highest number of transcription factors in the bZIP family characterized
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as responsive to pathogens (Fig. S1). The protein ABI34659 identified in GenBank as GmbZIP105
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and the NP_001237027 protein identified as GmbZIP62 present strong similarity to members of
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the C class that are responsive to pathogens (Fig. 1), such as G/HBF-1 and the soybean SBZ1 (Fig.
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S1; Jakoby et al. 2002).
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GmbZIP62 was previously characterized as responsive to abiotic stress (Liao et al. 2008);
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its overexpression in Arabidopsis increased tolerance to drought, salinity and freezing (Liao et al.
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2008). Thus, GmbZIP62 may be a general response factor to stresses in plants, including stress
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caused by pathogens, a feature already described for other bZIPs proteins (Lee et al. 2006; Orellana
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et al. 2010).
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The proteins GmbZIP62, GmbZIP105, and GmbZIPE1 GmbZIPE2 were grouped in
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different clades, reflecting structural differences that may be accompanied by functional
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differences among these proteins (Fig. S1).
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DISCUSSION
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Phylogenetic analysis showed the great structural and functional diversity of the bZIP
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family of transcription factors. Previous studies (Jakoby et al. 2002) proposed the separation of
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Arabidopsis bZIPs into 10 classes or groups. In the soybean, the same 10 groups were proposed to
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group the bZIP proteins according to their structural similarity with the Arabidopsis proteins (Liao
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et al. 2008). The phylogenetic analysis performed in this study revealed the presence of at least
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11 classes of soybean bZIP proteins, which demonstrates the existence of other functional classes
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in addition to those previously described by Jakoby and coworkers (2002). Twenty of the 148
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analyzed soybean proteins were not grouped in any of the 11 groups formed, most likely due to
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the lack of the complete sequence of these proteins in the GenBank database or the fact that their
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functional domains were not characterized as bZIP domains, although these proteins have been
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predicted to be bZIP family proteins in previous studies (Liao et al. 2008).
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A new class is also proposed, formed by proteins homologous to HB-PHD family proteins
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in Arabidopsis (Ariel et al. 2007). The homeobox domain (HB) is a conserved motif of 60 amino
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acids in transcription factors found in all eukaryotic organisms (Ariel et al. 2007). This motif folds
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in a triple helix structure that is capable of interacting specifically with the target DNA (Ariel et
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al. 2007). The PHD finger domain, a His-Cys3-Cys4 zinc finger, is found in many regulatory
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proteins from plants and animals and is often associated with transcriptional regulation by
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chromatin modification (Halbach et al. 2000). In transcription factors containing a homeobox
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domain, the PHD finger is combined with a leucine zipper in an upstream position (Halbach et al.
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2000). These domains together form a highly conserved region of 180 amino acids called ZIP /
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PHDf, and it has been verified that the transcriptional activity of the PHD finger domain is masked
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when it is in this long region (Halbach et al. 2000). Interestingly, little is known about the region
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proximal to the basic leucine zipper domain, and these proteins have not been described in the
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literature as proteins containing a bZIP domain. In addition to containing the protein domains
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described for HB-PHD proteins, the 9 proteins of this group also have MEKHLA domains
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(MEKHLA as conserved sequence of amino acids), which are similar to the PAS domain (Per,
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Arnt, and Yes proteins). In eukaryotes, this domain is a signal detector in signaling pathways
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(Dunham et al. 2003). This group also contains a START domain (StAR protein-related lipid-
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transfer), which has a lipid-binding function, and can bind to cholesterol, phospholipids and
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sphingolipids (Ponting et al. 1999), suggesting that these proteins may be anchored to the cell
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membrane and act as signal receptors in plant cells.
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Analysis of the composition of protein domains observed in each bZIP protein in soybean
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verified a structural diversity found among monophyletic groups (Fig. S1). Members of groups A,
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C, E, F, I and S contained signature basic leucine zipper (bZIP) domains, and the domains
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identified as bZIP domains differ structurally within the classification proposed by Jakoby and
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coworkers (2002). For example, the domain bZIP_C, found in 19 of the 20 members of the group
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C, differs from the bZIP_2 and bZIP_1 domains by having a leucine zipper that contains nine
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replicates containing seven leucines each (Jakoby et al. 2002). Unlike the members of these groups,
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members of the other groups have several distinct domains that reflect the characteristics of their
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functional roles. Most members of the group D (17) also have a DOG1 domain (Delay of
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germination protein 1), which is related to the control of seed development (Jakoby et al. 2002),
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while two members have a HSF (Heat shock factor) domain, a binding domain related to heat
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shock promoters (Clos et al. 1990). Seven members of group G have a domain called MFMR
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(multifunctional mosaic region), which has a crucial role in the activation of transcription (Jakoby
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et al. 2002). Five members of group H have a RING-type zinc finger domain that most likely
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functions in protein-protein interactions (Halbach et al. 2000). Although 20 proteins were not
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grouped in any of the 11 monophyletic groups formed, 16 of them have domains with unknown
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functions (DUF) that have structural similarity to the bZIP domain (DUF630, DUF632 and
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DUF1664), while the other 3 proteins exhibited unrelated domains (Fig. S1). Ten proteins found
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in the grouping showed no relevant domains (Fig. S1).
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Fig. S1 Phylogeny of soybean bZIP proteins. The majority-rule consensus tree was obtained by
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Bayesian MCMC coalescent analysis of 148 sequences of bZPIP proteins (see methods above).
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The posterior probability values (PP) (expressed as probabilities) calculated using the best trees
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found by MrBayes are shown beside each node. The second value (underlined) corresponds to
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bootstrap values (BV) (expressed as probabilities) that define the clusters in the maximum
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likelihood tree. The proteins selected for study are shown in bold and highlighted in yellow
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(GmbZIPE1, XP_003543312; GmbZIPE2, XP_003525005; GmbZIP105, ABI34659; GmbZIP62,
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NP_001237027).
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