Monovalent cation/proton antiporters (CPAs) in moso bamboo ( Phyllostachys edulis ): genome-wide identification, molecular evolutionary and functional description CURRENT STATUS: POSTED

Background: Plant monovalent cation/proton enantiomer (CPA) family is a type of transmembrane transporters that plays an important role in resistance to plant abiotic stresses such as salt, drought and osmotic stress. Results: In this study, 32 CPA family members were firstly identified from moso bamboo, and divided into two subfamilies, including 8 in CPA1 and 24 in CPA2. Members of the same group shared similar structures and subcellular localization predictions. Gene duplication analysis found that the expansion of the CPA genes in moso bamboo may depend on whole genome duplication (WGD) event approximately 11.18 million years ago (MYA). Gene Ontology (GO) analysis revealed that PheCPA proteins are ion transporter localized on the membrane, and the post-translational modifications (PTMs) prediction indicated there are many PTMs sites may be involved in regulating CPA protein functions. Promoter analysis revealed various cis-acting elements related to abiotic and biotic stresses, plant growth and development and phytohormone responses. The significant change of expression levels of eight PheNHX s, which belong to CPA1, under the treatment of ABA, NaCl and PEG, suggesting that they may have played an important role in moso bamboo response to these abiotic stresses. Additionally, PPI prediction reveals potential interaction proteins of PheNHX subfamily members. Conclusions: The results suggested that the CPA family may plays an important role in plant responses to stress conditions. The comprehensive identification and analysis of PheCPA s indicated that PheCPAs , particularly those genes encoding PheNHX s, might serve as valuable genetic resources for the improvement of moso bamboo growth and resistance to abiotic stress. of PheNHX subfamily members. The results of this study provide a fundamental information for PheCPA genes and increase the understanding of PheCPAs functions in plant abiotic stress response. −ΔΔCT T C T, Target -C T, PheTIP41 . ΔΔCT =ΔC T, treatment -ΔC T, CK (0

family genes were homologous to bacteria K + efflux transporters (EcKefB and EcKefC) [16], characterized as K + / H + reverse transporter, which promotes K + homeostasis and plays a role in maintaining pH and ion homeostasis in intimal network [20,21]. KEAs in plants were first identified in Arabidopsis and named AtKEA1-6 [15]. Among these, AtKEA1-AtKEA3 were identified as the K + / H + reverse transporter of chloroplast, played a key role in chloroplast function, osmotic regulation, photosynthesis and pH regulation in plants [22]. The CHX family is one of the largest K + transporter families in plants [23], composed of genes from higher plants and not found in metazoans [16]. The similarity between plant CHX protein and characterized fungal and bacterial CPA2 indicates that plant CHX protein transports K + , Na + and H + in various catalytic modes [5,23]. In Arabidopsis, CHX was identified having 28 members [23]. Studies in Arabidopsis suggest that these proteins participate in K + and pH homeostasis in the dynamic intima [24], membrane transport events [25] and play a potential role in pollen development, survival and tube growth [25]. So far, the research on CPA family is mainly limited to Arabidopsis, the function of CPAs in other species is rarely studied, and how it participates in the mechanism of abiotic stress, like high-salt stress, drought stress or osmotic stress, response is still unclear.
Moso bamboo (Phyllostachys edulis), one of the most important non-timber forest products in the world, the planting area accounts for two-thirds of the total area of bamboo [26]. In China, moso bamboo accounts for more than 70% of the bamboo cultivation regions, and has highly cultural, ecological and economic value and extensive prospects for development [27]. About 2.5 billion people depend economically on bamboo, and international trade in bamboo amounts to over 2.5 billion US dollars per year [28]. Furthermore, moso bamboo can be used for wood, paper, crafts, and shoots can as delicious food. This requires bamboo to be able to respond and resist adverse external conditions. Previous studies showed that regulatory and functional proteins play an important role in contribute to abiotic stress resistance in plant [29], and CPA family proteins are the class of proteins putative to be associated with salt tolerance. Furthermore, availability of the more abundant chromosome-level de novo assembly genome sequence [30] provided us a superexcellent opportunity to conduct a genome-wide analysis of the CPA genes in moso bamboo.
In our study, we performed for the first time a comprehensive analysis of the CPA family genes in moso bamboo. Total 32 non-redundant PheCPA encoding genes were identified and systematically analyzed. Genome-wide bioinformatics analysis was performed to investigate their phylogenetic relationships, genetic structures, protein structure and conserved motifs, posttranslational modifications (PTMs), chromosomal localization and gene duplication, GO annotation and ciselements. Further, the expression profiles of 8 NHX genes in bamboo leaves treated with NaCl, ABA and PEG were detected by qRT-PCR analysis, and the protein-protein interaction (PPI) prediction of 8 PheNHX members were performed. These study results provided valuable information for the further identification of the functional characteristics of CPA genes and the candidate gene for improving the salt resistance of moso bamboo.

Identification and characterization of CPA genes
Totally, 32 genes (PheNHX1-8, PheKEA1-6 and PheCHX1-18) were identified as PheCPA genes (Table   1), and 99 genes (26 in maize, 25 in sorghum, 24 in rice and 24 in Brachypodium) were collected as the CPA members (protein sequences showed in Table S1). Detailed characteristics of PheCPAs, include gene name, gene ID, locational information, physicochemical parameters, secondary structure and subcellular localization, were showed in Table 1. CDS lengths of the identified PheCPA genes ranged from 1314 bp to 5352 bp, resulting in amino acid sequences ranging from 437 to 1783 aa. The corresponding predicted molecular weight (MW) ranged from 46.29 to 194.29 kDa and theoretical isoelectric point (pI) varied from 5.10 to 9.21. The numbers of TM regions of each PheCPA family protein ranges 8 to 12 (Table 1), and members of the same subclade have similar TMs (Table 1; Fig   S1). The prediction of their subcellular localization revealed that NHX-I PheNHXs mainly located at Vacuolar (except PheNHX1), all NHX-II PheNHXs mainly located at Golgi, KEA-I PheKEAs located at Plasma membrane and KEA-II PheKEAs located at Chloroplast, almost all PheCHXs at Plasma membrane (Table 1). These evidently demonstrated that the structures of members in same subfamily are conserved. In addition, their secondary structure showed that PheCPA proteins were 6 mainly composed of Alpha helix (Hh), Extended strand (Ee) and Random coil (Cc), which had a great relationship with their function.

Phylogenetic analysis of the PheCPA genes
Phylogenetic tree was constructed based on the sequence alignment of full-length amino acid sequences of moso bamboo, maize, rice, sorghum, Brachypodium and Arabidopsis to explore the phylogenetic relationship of CPAs among different species. Through phylogenetic analysis with other species, the PheCPA gene family was categorized into the CPAI and CPAII subfamilies (Fig 1 A & B).
CPAI also named NHX, which could be distributed into two groups, NHX-I and NHX-II; and CPAII could be divided into KEA and CHX subclade, include seven groups (KEA-I, KEA-II and CHX-I to CHX-V).
Interestingly, in previous studies, the CHX subfamily of Arabidopsis thaliana was divided into five branches [23], and in our study, we found that CPA family of five grasses were mainly concentrated in group I and IV.

Gene structures and conserved motifs analysis
The exon/ intron structures of PheCPA genes were explored using GSDS online server. The results showed most members in the same subclade share high similar intron/exon structures, but there also are some differences (Fig 2B). For example, among the NHX genes, three NHX-II NHXs all had 22 introns and type I NHXs had 13 introns (except PheNHX1 and PheNHX5). KEA-I type KEAs had [18][19] introns and KEA-II had 17 introns (the PheKEA6 gene is special, with 33 introns), and the CHX subfamily genes only had 1-4 introns, respectively.
The online MEME tool predicted 15 specific protein motifs of PheCPA proteins by submitting full-length amino acid sequences (Fig 2C; Fig S2). Each motif was verified using the Pfam and SMART, and motif 1, 2, 5, 6, 10, 11, 12 and 15 were fragments of the Na + /H + exchanger domain. Furthermore, the result showed that the members with highly phylogenetic relationships had common motif composition. For instance, 2 motifs (motif 10 and 15) were existed in all the members in the NHX subfamily with a conserved sequence 'LFFJYLLPPII' in the motif 10 and a conserved sequence 'LYSLVFGEGVVNDA' in the motif 15, at position of the 3rd TM domain and 6rd TM domain ( Fig S3).
Meanwhile, Motif 11 was only existed in NHX-I (Fig 2C), which contained a conserved sequence 'WWAGLMRGAVSIALA' at putative TM11 domain (Fig S3). Motif 1, 2 and 5 were existed in all members of CPA II family genes, motif 3, 4, 7, 8, 9, 12 and 13 were identified in CHX subfamily. In addition, motif 6 was detected in all three gene subfamilies. These results indicate that the structures of the same subgroup of CPA family members are highly conserved, and they also further validate our division of the CPA genes in moso.

Posttranslational modifications prediction of PheCPA proteins
Posttranslational modifications, one of the major factors determining protein biological function [31], can regulate protein activity, localization, and protein-protein interactions, leading to fine-tuning of plant responses to various external stimuli to mitigate potential damage from environmental stress Reversible protein phosphorylation is a ubiquitous form of cell regulation, which can affect a variety of cellular signaling pathways, including metabolism, cell growth, differentiation and membrane transport [35]. Phosphorylation sites mainly present on serine (S), threonine (T) and Tyrosine (Y). The predicted results indicated that each PheCPAs protein has multiple potential phosphorylation sites, of which S is the most, T is the second, and Y is the least.

Chromosomal location, gene duplication and selection pressure analysis
The results of the physical location of PheCPA genes revealed that each subfamily genes were mapped onto Moso Bamboo scaffolds unevenly (Fig 3A). Scaffold 7 and Scaffold 8 had the largest 8 number of genes, while only one gene was found on Scaffold 1, 3, 4, 14, 21, 22 and 23. Collinearity analysis shows that 12 pairs of WGD (whole-genome duplication)/ segmental duplicated genes in moso bamboo were found on scaffolds ( Fig 3A; Table S2). And interestingly, there none tandem duplicated gene pairs, this result implied that the tandem duplication event may not play a significant role in the expansion of the CPA gene family in moso bamboo. In addition, according to the analysis of the collinearity diagram between moso bamboo each subfamily genes of CPAs and other five plants ( Arabidopsis, rice, maize, sorghum and Brachypodium ), 2, 34, 30, 34 and 30 pairs of intergenomic duplications genes were found ( Fig 3B; Table S2), respectively. The collinearity relationship between moso bamboo and the others four gramineous plants were closer, this result indicating that they might have come from the same ancestor.
Further, 8 paralogous and 62 orthologous pairs were identified (Table 3) by synteny gene pairs. Ka / Ks ratio calculation results showed that they all much less than 1 (Fig 4), implying that CPAs  (Fig 4), respectively.

Gene Ontology Annotations
The GO slim analysis performed using Blast2GO and classified the 32 PheCPA genes into three GO  Table S3; Fig S4). In the cellular component category, the most abundant groups were cell (GO:0005623) and cell part (GO:0044464), and organelle (GO:0043226) protein-containing complex (GO:0032991) and membrane (GO:0016020) were the minor groups within this category. The biological processes category, biological regulation (GO:0065007) was the most abundant groups, and the secondary groups in this classification were cellular process (GO:0009987), metabolic process (GO:0008152), cellular component organization or biogenesis (GO:0071840) and response to stimulus (GO:0050896). In the molecular function category, transporter activity (GO:0005215) was the most highly represented GO terms, and binding (GO:0005488) was only accounts for 1.19% (only two genes, PheNHX1 and PheKEA6) (Fig 5; Table S3).
These results suggest that the members of the PheCPA gene family were distributed among several important groups of all three GO categories at distinctive percentages. In addition, we can also predict from the results that the main function of PheCPA is to locate proteins on the membrane that perform ion transport function. This prediction is consistent with previous studies in other species [10,16].

Cis-acting element analysis
Cis-elements, which are non-coding linear DNA sequences, regulate and control gene transcription by  Table S4). GT1-motif, a type of salt-responsive element, was found among the 14 genes and appearing 24 times. ARE and LTR element, which related to anaerobic induction and low-temperature responsiveness respectively, were comprised 25% and 20%. Drought-related (MBS), defense and stress responsiveness (TC-rich repeats), anoxic-related (GC-motif), wound-responsive (WUN-motif) and MYB binding site involved in light responsiveness elements, also have been identified in the PheCPA gene family. In the category of phytohormone responsive, we found ABRE motif, associated with ABA, was represented more than 50%, and appearing 114 times among the 28 genes. Except PheNHX2, PheNHX3, PheNHX7, PheNHX8, MeJA-responsiveness elements (TGACG-motif and CGTCAmotif) were identified in PheCPAs genes. In addition, gibberellin-responsive elements (GARE-motif) and auxin-responsive elements (TGA-element and AuxRR-core) also were found in PheCPAs. In type of plant growth and development, many cis-acting elements related to growth and development, include CCGTCC-box and CAT-box (related to meristem expression), Box-4 and MRE element (involved in light responsiveness) and MSA-like element (related to cell cycle regulation), were found in PheCPAs.
According to the previous studies and this analysis, salt, drought and ABA were selected for subsequent hormone and stress treatments.

Expression patterns of PheNHXs after NaCl, PEG and ABA treatment
Numerous evidences showed that NHX genes might participate in stress response of plants [13,39,40]. To explore whether the NHXs of moso bamboo have similar functions, the expression patterns of eight PheNHXs were researched at NaCl (for salinity stress), PEG (for drought stress) and ABA conditions using qRT-PCR method.
Under salt stress, the expression levels of 8 PheNHXs were significantly increased, among which three genes (PheNHX1, PheNHX2, PheNHX3) showed the highest expression level at 3h and then showed a tendency of decline, and PheNHX4, PheNHX5 and PheNHX6 had the highest expression levels at 6h. In addition, PheNHX7 presented a low level of expression at the early stage of treatment, and increased at 24 h (Fig 7). After drought stress, the transcription levels of all eight PheNHXs genes showed a tendency of up-regulation to different degrees, which were similar to the expression pattern of NHX family genes previously studied [41]. In ABA treatment, the expression of the eight PheNHXs were affected, the expression of PheNHX6, PheNHX7 and PheNHX8 were down-regulated, and the remaining five genes (PheNHX1-PheNHX5) were up-regulated (Fig 7), which implied that the PheNHX genes might be involved in ABA-dependent signal pathway. These expression data indicated that PheNHXs were responsive to certain abiotic stresses (drought, salt stress) at the transcriptional level.

Protein-protein interaction prediction of PheNHXs
To further probe the putative function of PheNHXs, the predicted interactions of PheNHXs based on homologous proteins in rice were established (fig 8). Results showed that PheNHX3 and PheNHX5 could interact with PH02Gene13937.t1, which homologous to CIPK24 of rice. PheNHX3, PheNHX4, PheNHX5 and PheNHX8 could interact with WAXY (PH02Gene13019.t1) and GBSII (PH02Gene37359.t3), which belong Granule-bound starch synthase 1 and Granule binding starch 11 synthase II respectively. These results suggested that PheNHX family members might involve in the synthesis of amylose in the endosperm by interacting with these two type proteins. In addition, these PheNHXs also interact with cytochrome b5 reductase family members (PH02Gene29105.t1, PH02Gene03732.t1, PH02Gene02406.t1), which imply these proteins possibly involved in the maintenance of cell membrane function through interaction. In our study, a total of 32 CPA members in moso bamboo were identified (Table 1), and compared with 24 OsCPAs, 26 ZmCPAs, 24 BdCPAs, 24 SbCPAs and 40 AtCPAs, results showed that the number of CPA genes in moso bamboo is similar with other 4 monocotyledon (rice, maize, sorghum and Brachypodium distachyon) and lower than Arabidopsis. Compared with Arabidopsis, 5 gramineous plants contain less CPA members, even though they have the larger genome size, rice for ~372 Mb [45], maize for ~2300 Mb [46], sorghum for ~732 Mb [47], Brachypodium distachyon for ~ 272Mb [48], and moso bamboo for ~ 2050Mb [30], which may signify that gene loss events occurred over Meanwhile, the similar size of CPA family members in 5 monocotyledons implied that the CPA gene family of these species might be relatively conserved during the evolution. The collinearity and divergence times (T) analysis also indicated that the relationship of CPA gene family between bamboo and another four grasses was closer than between bamboo and Arabidopsis and CPA genes had underwent strong purifying selection during the process of evolution. Besides, the sequence alignment and phylogenetic analysis classified the PheNHX and PheKEA subfamilies into 2 groups and PheCHX members into I and IV groups (Fig 1), the classification results are consistent with previous studies [16,43]. All these evidence suggested that CPA members of moso in the same subgroups may have similar reported functions as other identified species.

Discussion
Through protein primary and secondary structure analysis, conservative motif distribution and gene exon/ intron analysis analysis, we found that the structure of the members of the same subgroup were very similar (Fig 2), which might indicate that they also had similar functions. For instance, like other species known antiporters, the deduced amino acid sequences of PheNHX subfamily revealed 9-12 putative TM regions (Fig S1 & S3), among them a high consensus of TM3 with the conserved sequence of 'FF…LLPPII' [49] in each PheNHX, which was reported the membrane-spanning pore and cation-binding domain [50]. In KEAI group, each of the three PheKEA protein located in the chloroplast has a characteristic long C-terminal tail called TrkA_N domain (Fig 2B), was reported providing a mechanism of gating control to restrict the flux of K + in previous study [51]. The N-terminal domains of 18 PheCHX proteins contain ranged from 8 to 12 putative TM regions (Fig S1), and the genes in the same subgroup have highly similar gene and protein structure. In addition, PheCPA family contain multiple post-translational modifications and PTM sites ( Table 2). Yeast Nhx1 was previously reported to be an N-linked glycoprotein [52], like Nhx1, multiple N-glycosylation sites were also found in the PheCPA proteins. In the PheCPA family, we found large numbers of phosphorylation sites related to Ca 2+ dependent phosphorylation pathway. Previous study verified that animal NHE-1 protein regulated intracellular pH homeostasis through phosphorylation pathway involving Ca 2+ / calmodulindependent protein kinase II (CaMKII) [53]. PtNHX7 of poplar were predicted to regulate intracellular ion balance by interacting with Calcineurin B-like protein (CBL) and CBL-interacting protein kinases 13 (CIPK) proteins to participate in calcium-dependent phosphorylation pathways [41]. Meanwhile, PPI prediction results also showed that PheNHX3 and PheNHX4 might interact with CIPK24 (PH02Gene13937.t1) (Fig 8), and other PheNHX members also have potential interactions with different family members to participate in different physiological processes. These results provide some guidance for future studies on the topology, signaling pathways and functions of PheCPA family proteins.
Plant cells contain many organelles, such as vacuoles, chloroplasts, and Golgi, which form subcellular compartments that contain proteins associated with specific biological functions. The different subcellular localization of PheCPA (Table 1) suggests that the mechanism of their function may be different in moso bamboo, implying that they may have specific subfunctions or new functions.
AtNHX5, AtNHX6 and OsNHX1-4 proteins were reported functions specifically in Golgi [39, 54], the PheNHX2, 4, 6, 8 as these proteins also predicted location in Golgi (Table 1), implied that their mechanism of functions might be similar. PheKEA2, 3, 6, as well as AtKEA1, 2 [20] , are predicted to be located on chloroplasts, suggesting that they may play a role in maintaining electrical balance and pH stability into chloroplasts and maintaining chloroplast structural integrity under osmotic stress [55]. The subcellular localizations of Arabidopsis 28 AtCHXs were more extensive in previous studies report. For instance, AtCHX13 [56], AtCHX14 [24], and AtCHX21 [57] are located on plasma membrane, AtCHX17 is reported to be located to PVC/ PM in plant and endomembrane in yeast [58,59], and AtCHX20/ AtCHX23 are reticulate endomembrane K + transporters [60,61]. However, almost all the PheCHXs were predicted to be located on the plasma membrane, so further experiments are  54,62]. In this study, stress response analyses showed that PheNHXs were response to abiotic stress include salinity, drought (Fig 7). Noticeably, PheNHX3 and PheNHX4 showed significant expression changes under these abiotic stresses. GT1-motif is a cis-acting element related to salt stress [63]. The promoter analysis found that one GT1 -motif element was found in the promoter region of PheNHX3, but none in PheNHX4 (Fig 6; Table S3). However, high transcription level of PheNHX4 suggested that there may be additional elements involved in its expression that respond to salt stress. An earlier report [41] showed that the PtNHX of poplar genes responded to different types of abiotic stress responses, including drought, salt, heat, and low temperature, through the involvement of the ABA signaling pathway. As an important plant hormone, ABA is involved in not only plant development but also response to various stresses [64]. ABREs (ABA responsive cis-acting element) were found in every PheNHX (PheNHX1-8) promoter region, and it is notably that PheNHX4 contains 14 ABRE elements (Fig 6; Table S3). In addition, the transcription levels of 8 PheNHX genes changed significantly under ABA treatment (Fig 7). This result suggested that PheNHX1-8 might be involved in ABA signal pathway.

Conclusions
In this study, we systematically identified and classified 32 PheCPAs in moso bamboo. Gene structures, cis-acting elements, protein primary and secondary structures, conserved motifs and PTMs analysis of the 32 predicted CPAs in the moso bamboo were performed. Intraspecific and interspecies collinear events and divergence time of moso bamboo CPAs were identified, and we found the duplication of PheCPAs from WGD events. In addition, GO analysis revealed divergent roles of CPA proteins in moso bamboo. qRT-PCR test was used to explore the expression patterns of the 8 PheNHX genes after ABA, drought and salt treatment, and the prediction of protein interaction provided further research value in resistance to abiotic stress. PPI prediction revealed potential interaction proteins of PheNHX subfamily members. The results of this study provide a fundamental information for PheCPA genes and increase the understanding of PheCPAs functions in plant abiotic stress response.

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The chromosomal location of PheCPAs were constructed according to the genomic position information. Collinearity of PheCPAs were analyzed using MCScanX [71] with default parameters and the results were displayed by Circos [72]. The homolinear analysis maps were constructed using Dual Synteny Plotter software (https://github.com/CJ-Chen/TBtools) to reveal the synteny relationships between orthologous CPA genes of moso and other selected species. BLASTn was used to pairwise align of CPA encoding sequences with two criteria: (a) shared aligned nucleotide sequence covering > 80% of the longer gene and (b) similarity of the aligned regions of sequence ≥ 80% and then used synteny-based to identify the orthologous and paralogous pairs. Subsequently, Ka, Ks and Ka/Ks values of them were calculated using DnaSP 5 software [73] based on sequences alignment using MEGA 6.0. The formula T = Ks/2λ (λ = 6.5 ×10 −9 ) [36]was used to calculate the divergence time (T).

Gene Ontology and promoter region analysis of CPAs in moso bamboo
Full-length amino acid sequences of PheCPA were uploaded to the Blast2GO program (blast expectation value:1e-5; mapping and annotation with default parameters) to perform Gene Ontology (GO) analysis [74]. To predict cis-acting regulatory elements of PheCPAs, the cis-acting elements in the 2kb DNA sequences upstream of the transcription start site were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Plant materials and treatments
Moso bamboo seeds were identified and provided by the Guilin Forestry Bureau, Guilin, Guang Xi Province, China. And the seedlings were cultivated in greenhouse with a constant photoperiod (16 h light/ 8 h dark), an average temperature of 22℃, 80% humidity. Then, the three-month old young leaves were sprayed with 200 mmol/L NaCl (for salt stress), 20% PEG-6000 solution (for drought stress) or 100 μM ABA solution, and the leaves were sampled at 0, 1, 3, 6, 12 and 24 h after treatment, respectively. The untreated samples (0 h) were used as the control (CK). Each plant sample was immediately frozen in liquid nitrogen and stored at −80 ℃ for RNA isolation.

RNA extraction and quantitative Real-Time PCR analysis of PheNHXs
Total RNA of plant samples was isolated using Trizol (TaKaRa, Dalian, China) methods and then reverse transcribed into cDNA using a PrimeScriptTM RT Reagent Kit (TaKaRa, Dalian, China). Primer Express 5.0 was used to design gene-specific primers for each selected gene and PheTIP41 (tonoplast intrinsic protein 41) was used as the reference control gene [75] ( Table S5) SPSS 17.0 was identified significant differences at * (P < 0.05) and ** ( P < 0.01) between the treatment group and control group. And the GraphPad5 software was used to visualize the data in histograms.

Construction of interaction network of PheNHXs
The predicted protein-protein interaction (PPI) map of PheNHXs was generated from the STRING database (https://string-db.org/) [76]. Amino acid sequences of PheNHX were used as query sequences and rice as the model. In evidence mode, the online program was run with parameters as follows: 'minimum required interaction score' was set to 0.7 (high confdence) and others used default