The soybean gene GmHsp22.4 is involved in the resistance response to Meloidogyne javanica in Arabidopsis thaliana

Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. A previous study has showed strong induction of the gene GmHsp22.4 in response to the nematode Meloidogyne javanica in a resistant soybean genotype, while repression in a susceptible one. This study aimed to investigate the functional involvement of this small chaperone in response to M. javanica in Arabidopsis thaliana. First, it was evaluated the activation of the promoter region after the nematode inoculation, and the occurrence of polymorphisms between resistant and susceptible re-sequenced soybean accessions. Then functional analysis using A. thaliana lines overexpressing the soybean GmHsp22.4 gene, and knocked-out mutants were challenged with M. javanica infestation. High expression levels of the GFP gene marker in transformed A. thaliana plants revealed that the promoter region of GmHsp22.4 was strongly activated after nematode inoculation. Moreover, the multiplication of the nematode was significantly reduced in plants overexpressing GmHsp22.4 gene in A. thaliana compared to the wild type. Additionally, the multiplication of M. javanica in the A. thaliana mutants was significantly increased mainly in the event athsp22.0–2. This increase was not that evident in the event athsp22.0–1, the one that preserved a portion of the promoter region, including the HSEs in the region around − 83 bp. However, structural analysis at sequence level among soybean resistant and susceptible genotypes did not detect any polymorphisms in the whole gene model. The soybean chaperone GmHsp22.4 is involved in the defense response to root-knot nematode M. javanica in A. thaliana. Specifically, the promoter region covering until − 191 from the transcriptional start site (TSS) is necessary to promoter activation after nematode infection in Arabidopsis. No polymorphisms that could explain these differences in the defense response were detected in the GmHsp22.4 gene between resistant and susceptible soybean genotypes. Therefore, further investigation is needed to elucidate the triggering factor of the plant’s defense mechanism, both at the sequence level of the soybean genotypes presenting contrasting reaction to root-knot nematode and by detecting cis-elements that are essential for the activation of the GmHsp22.4 gene promoter.


Background
The increase in soybean productivity through genetic gains over the years is undeniable. However, the management of pests and pathogens still poses major challenges in soybean production either by its impact on the environment or by its burden in the production costs [1]. Among the pests, phytonematodes are found in all major soybean cultivation areas in Brazil, and worldwide. Production losses in soybean crops in Brazil caused by phytoparasitic nematodes were estimated to be around R$ 16.2 billion/year [2].
Several strategies have been developed to control phytonematodes, such as crop rotation, the use of nematicides and biological treatments [3,4]. Additionally, transgenic approaches involving the overexpression and silencing of genes have been attempted [5,6]. Finally, genetic resistance has been used in genetic breeding programs exploring genetic loci in soybean, such as the quantitative trait loci (QTLs) described in plant introductions (PIs) [7]. However, a few sources of resistance are available, limiting the development of broad durable resistance [8]. Thus, understanding the molecular mechanisms involved in the resistance response is an important approach for the development of biotechnological strategies for the control of these pests [9].
Chaperone proteins are present in both prokaryotes and eukaryotes and are widely distributed in several species in the plant kingdom. In plants, biotic and abiotic stresses can trigger diverse defense mechanisms, such as the activation of a group of highly conserved proteins known as heat shock proteins (HSPs) [10]. The main function of the HSPs is to act as molecular chaperones, performing maintenance of the spatial structure of other proteins that are negatively affected by changes in factors such as temperature [11,12]. These proteins were initially observed in the salivary glands of Drosophila spp. under heat shock stress [11]. Based on their sequence size and homology, the HSPs have been grouped into five classes: HSP60, HSP70, HSP90, HSP100 and HSP20 (small-HSP or sHSP). HSP20 proteins exhibit an N-terminal hydrophobic region that is quite divergent in its sequence and length in different proteins, followed by a conserved domain of approximately 90-100 amino acid residues in the C-terminal part of the protein and a short C-terminal extension [13][14][15][16].
Despite their name, HSP20 proteins are induced not only after thermal shock, but also by other abiotic stimuli, such as water deficits, heavy metals, ozone and UV radiation [17], as well as under different biotic stresses, such as nematode infestation [15,17,18]. Studies on the functions of cytosolic HSP20 proteins have suggested that HSP20 maintains the remaining cellular proteins in an active state under stressful conditions via the linking of its dissociated dimers with denatured proteins [13,16,19,20]. According to this hypothesis, the heat-induced dissociation of HSP20 could lead to exposure of the hydrophobic region and, consequently, to the stabilization of the denatured proteins [15,16]. HSP20 then cooperates with other ATP-dependent molecular chaperones such as HSP70, HSP90, HSP100 and GroEL to refold proteins [14,16]. In addition, HSP20 exhibits a much higher binding stoichiometry than other molecular chaperones, leading to some speculation that HSP20 functions as a reservoir to stabilize the flow of denatured proteins in response to stress [15,16].
In a previous study, Lopes-Caitar et al. [21] characterized the expression profiles of 51 members of the HSP20 family in Glycine max (GmHsp20) under abiotic (heat and cold) and biotic stresses (infestation by M. javanica) on susceptible (BRS 133) and resistant (PI595099) soybean genotypes. The expression levels of these genes were strongly dependent on the genotype under biotic stress conditions. Additionally, the exposure period (four or 8 days after inoculation) played a significant role in the gene expression in both genotypes. Five out of the 51 members of the GmHsp20 family were significantly expressed under both treatments, but the expression of the GmHsp22.4 (abbrev. for Glyma10g176400) gene stood out. Its relative expression level was 60 times higher in the infected resistant genotype compared to the false inoculated samples, while repression was observed in the susceptible genotype in the presence of the nematode. Interestingly, the authors reported the systematic occurrence of a standard organization of ciselements in the promoter region of these soybean family genes associated with the responses of soybean to M. javanica infestation.
In this study, we sought to elucidate the involvement of GmHsp22.4 in response to M. javanica infestation. We first evaluated the promoter activation under the presence of the root-knot nematode. Then, in silico and functional analyses were performed to characterize the function of the chaperone GmHsp22.4 on the response to M. javanica's infestation. Our results provide important data about the role of this gene in the defense response against this nematode.

Results
The GmHsp22.4 promoter is strongly induced by M. javanica in Arabidopsis roots To investigate the responsiveness of the GmHsp22.4 promoter to M. javanica infestation, the potential GmHsp22.4 promoter sequence (2 Kb from the transcriptional start site -TSS) was fused with the coding region of GFP and introduced into A. thaliana seedling, resulting in four events (PGmHsp22.4-3, 4, 6 and 12). The fluorescence levels in the roots of these events were measured at the 9th day after inoculation (dai) with M. javanica and in non-inoculated plants (mock). As expected, the average GFP activity was significantly higher in all tested events (PGmHsp22.4-3, 4, 6 and 12) than in the mock events (Fig. 1).
Structural and polymorphism analysis of GmHsp22.4 in M. javanica resistant and susceptible genotypes GmHsp22.4 gene presented a predicted total length of 1158 bp, being 337 bp in the 5 'UTR region, 588 bp in the coding region and 233 bp in the 3' UTR region; with no introns, this sequence resulted in a potential encoded protein of 196 aa. The orthologous gene in A. thaliana exhibited a similar organization, presenting 74.6% similarity with the GmHsp22.4, also with no intron region and potentially encoding a protein of 196 aa. Based on the resequencing data obtained by Santos et al. [22] of 21 soybean accessions, a potential promoter region spanning 2.0 kb from the TSS and the complete transcriptional region (including exons and UTRs) were compared between six soybean genotypes resistant to M. javanica and 15 susceptible ones [8]. The GmHsp22.4 gene was highly conserved in all 21 genotypes evaluated, with no sequence variation. Similarly, the promoter region showed no sequence variation, except for a substitution in the − 1229 position (A/T) upstream of the transcription start site, but it was not possible to associate this substitution with resistance to the pest, as it was randomly distributed among the genotypes.

Overexpression of GmHsp22.4 increases resistance to root-knot nematodes in Arabidopsis
Agrobacterium tumefaciens carrying the GmHsp22.4:: pH7WG2D construct was used to transform A. thaliana. Three stable events overexpressing the coding region of GmHsp22.4 under 35S promoter control were obtained by floral dip transformation. Positive events were selected and confirmed by the detection of eGFP fluorescence in A. thaliana roots. Strong green fluorescence was observed in the roots of three homozygous events in the T4 generation, demonstrating the success of the transformations with GmHsp22.4, while in the wild-type (WT) no fluorescence was detected (Fig. 2).
Root morphology attributes (length and weight) of the transformed plants were compared with the wild-type counterparts, to detect possible changes caused by the overexpression of the GmHsp22.4. The average root length and weight of the transgenic lines was 8.0 cm and 60 mg respectively, which was not significantly different of the wild-type Scheffé's test (p ≤ 0.05). These data and other morphological comparisons are described in the Additional file 1.
The expression levels of the GmHsp22.4 gene in the 14 transgenic plants for each event were quantified by RT-qPCR. As expected, high levels of gene expression were detected in A. thaliana plants transformed with the target gene regulated by the 35S promoter (Fig. 3a). The highest expression was observed in the GmHsp22.4-8-OE line, followed by the GmHsp22.4-6-OE and GmHsp22.4-4-OE events, respectively.
The effect of GmHsp22.4 overexpression in A. thaliana transgenic plants against M. javanica infestation was examined by counting the number of M. javanica females on plant roots compared to wild-type plants at 22 dai (Fig. 3b). Among the three homozygous events tested, a significant reduction in the number of females was observed for the GmHsp22.4-6-OE (82%) and GmHsp22.4-8-OE (42%) events when compared to the WT (OE). The GmHsp22.4-4-OE event did not result in a significant reduction in the number of M. javanica females (Fig. 3b).

Knockout of the GmHsp22.4 orthologue in Arabidopsis compromises the defense response to M. javanica
To confirm the involvement of soybean GmHsp22.4 in the response to nematode infestation, two knock-out A. thaliana mutants for the orthologous gene were tested against pest infestation. The athsp22.0-1 and athsp22.0-2 mutants consisted of a T-DNA insertion in the promoter at − 191 pb upstream the TSS or in the 5′ UTR at + 149 pb downstream the TSS, respectively. The The effects of the mutations on plant growth and development were verified by comparing the root length (cm), root mass (mg), number of leaves and mass of the fresh aerial part (mg) of the events athsp22.0-1 and athsp22.0-2 against the corresponding attributes in the WT (Additional file 3). No significant differences were detected between the averages of these morphological parameters from transgenic and WT plants. The average root lengths of the transgenic plants were equal to 11.4 cm, while 11.2 cm for WT. The root mass for the transgenic plants were 835.3 (athsp22.0-1) and 938.2 (athsp22.0-2) milligrams, while in the WT it was 976.4 mg. Likewise, no significant differences were found between the number of leaves and the shoot weight in transgenic events and wild type plants. Data are expressed as the mean ± standard error of the mean. * indicates statistical significance at the 5% level (Scheffé's -test) compared to WT Therefore, the evaluated morphological parameters did not indicate any significant difference in the development of the transgenic and wild-type A. thaliana plants.
Finally, the effect of the mutations on the nematode's ability to infect and multiply on A. thaliana plants was evaluated through the number of eggs, juveniles (J2) and females obtained in plants of the two transgenic events and in the WT inoculated with M. javanica. The number of females associated with both events at 45 dai showed a significant increase in the multiplication of approximately 150% compared to the WT (Fig. 4a). On the other hand, the number of juveniles/eggs was significantly higher only in the A. thaliana athsp22.0-2 mutant (2631.15), when compared with the control (964.68), being the number of juveniles/eggs in the mutant athsp22.0-1 (1636.29) (Fig. 4b).
Based on these results, the athsp22.0-1 and athsp22.0-2 A. thaliana mutants showed increased susceptibility to M. javanica, being the latter the one who presented significantly higher numbers of both females and eggs and juveniles than the wild-type plants Scheffé's test (p ≤ 0.05). The T-DNA insertion lines were localized between the CAAT-box and W-box cis-elements in the promoter region of the event athsp22.0-1, and between the cis-element HSE and TA-rich in the 5′ UTR of the event athsp22.0-2 (Fig. 5).

Discussion
As chaperones, sHSP proteins have been described as induced by different biotic and abiotic stress, including nematode infection [12,14,15]. In this study, we examined specifically the role of the GmHsp22.4 gene in the M. javanica resistance response in details. This gene was selected because a previous study reported it to be strongly expressed in the soybean M. javanica-resistant genotype PI 595099 and repressed in the susceptible genotype BRS 133, when they were exposed to biotic and abiotic stresses [21]. The soybean accession has been described as an important source of resistance against specific strains and races of nematode species, including M. javanica, and contains a QTL (SOYHSP 176) mapped on soybean chromosome 13 involved in the resistance [24][25][26][27].
Our results demonstrated that GmHsp22.4 promoter activity in A. thaliana plants is highly activated after nematode infestation, confirming the transcriptional activation in response to M. javanica previously reported [21]. The GmHsp22.4 promoter presents a structural organization in which CAAT boxes are located immediately upstream of HSE elements, a W-box located further upstream of the HSEs [21]. This conserved promoter structure was found in GmHsp20 family members responsive to nematode infestation, where the HSEs are potentially recognized by heat shock transcription factors [21]. As expected, promoter induction was on average 34.47% greater in the infected Arabidopsis transgenic plants than in the non-infected ones, as revealed by GFP fluorescence marker (Fig. 1).
Although we demonstrated the activity of the promoter of the GmHsp22.4 in M. javanica responses, we could not demonstrate how such different levels of GmHsp22.4 transcripts and the associated contrasting phenotypes of resistant and susceptible soybean accessions are explained at the gene sequence level. Our analysis of 21 re-sequenced soybean accessions did not The differential regulation of the expression of GmHsp22.4 between the resistant and susceptible genotypes was in accordance with the results obtained by Fuganti et al. [27]. These authors mapped a QTL related to root-knot nematode resistance in a population derived from the resistant source PI595099 between two microsatellite markers, Satt 144 and SoyHSP 176. The SoyHSP 176 marker was located in a region containing another sHSP, the GmHsp17.6-L gene. Additionally, the expression levels of this gene were found to be differentially regulated between resistant and susceptible individuals from the mapped population, having been induced only in resistant individuals. A polymorphism analysis of the promoter region of the gene GmHsp17.6-L by Fuganti et al. [18] detected a greater number of AT (n) repeats in the resistant genotype, compared to the susceptible one, whereas those numbers were constant in the gene GmHsp22.4 in genotypes analyzed in this work. Therefore, our analysis did not indicate any correlation between AT(n) repetitions and promoter activity for the gene GmHsp22.4.
One possible explanation to the differential expression of the gene GmHsp22.4 in phenotypically contrasting reactions of soybean genotypes to M. javanica infection in our study could be the existence of enhancer regions that interacting with promoter regions of other genes, either in their vicinity or over long distances, altering their regulation [28]. However, our data was not sufficient to confirm this hypothesis.
Another explanation is epigenetic regulation, which cannot be detected at a sequence level. This mechanism has been reported to be involved in soybean resistance to cyst nematodes [29]. In another study [30], differential levels of methylation were observed in soybean plants after soybean cyst nematode (SCN) infestation, which may affect the accessibility of transcription factors to cis-elements regulating the transcriptional activity of genes responsive to the nematode [31]. Although this scenario may be an alternative explanation for the regulation of GmHsp22.4, further studies are necessary to confirm this hypothesis.
Arabidopsis presents 19 genes encoding Hsp20s, grouped into 12 subfamilies based on their subcellular localization and homology [21], being the transcript of the A. thaliana gene model orthologous to GmHsp22.4, AtHsp22.0, undetectable in normal conditions (22°C) and cumulative to high levels in response to heat stress (38°C) [32]. To date, there is no available evidence of the activation of AtHsp22.0 under biotic stress conditions, such as nematode infestation.
To obtain a better understanding of the roles of this chaperone in the resistance response to root-knot nematodes, we overexpressed the soybean gene in A. thaliana plants and studied two DNA insertion lines in which the orthologous genes were knocked-out. In our study, two events presenting constitutive overexpression of the GmHsp22.4 gene in A. thaliana resulted in a significant reduction in the numbers of females of 82 and 42% (Fig. 3b). Similarly, when the orthologous gene was knocked out in Arabidopsis plants, we observed an increase of 150% in the number of females (Fig. 4a). Based on these results, we conjecture that GmHsp22.4 is involved in nematode infestation responses, possibly acting as the first line of cellular defense by capturing unfolded proteins and reducing protein aggregates sizes. Thus, with the generation of more binding sites, and the assistance of ATP-dependent HSP70 and HSP100, aggregation is reversed and refolding is facilitated [33]. The high transcript levels of GmHsp22.4 upon its overexpression in transformed plants may have improved the stability of plant proteins in the endoplasmic reticulum, either by favoring the defense response or by maintaining the supply of active chaperones in the plant during pest infestation.
Considering the susceptibility levels of the mutant lines related with the position of the T-DNA insertion in the promoter region, we observed a less severe impact in the event athsp22.0-1, where the number of eggs and juveniles were not different from the wild plants (Fig. 4b). In this mutant, a larger portion of the promoter was not affected by the T-DNA insertion (− 191 from TSS), where the organizational structure of nematode responsive promoter was maintained, including the HSE element around − 83 pb from the TSS (Fig. 5). On the other hand, the T-DNA insertion in athsp22.0-2 in the 5′ UTR (+ 149 position) completely eliminated the resistance, confirming the importance of this HSE element region close to the TSS in the GmHsp22.4 orthologous after nematode infestation.
The role of Hsp20 related to its nematode-responsive promoter activity has been described in rice by Escobar et al. [34], who characterized the involvement of the soybean HsHsp17.4 gene in the response to infestation by M. incognita. The authors also observed that the promoter of the HsHsp17.4, a rice sHSP, was able to induce the expression of β-glucuronidase (GUS) marker gene and, consequently, 50 to 70% galls in the roots were stained after 17 to 20 days after infestation with M. incognita. In addition, they observed that mutations in the 83 bp region upstream from the TSS are determinant for the promoter activation in the nematode response [34], corroborating our results.
Similarly, Barcala et al. [17] described the importance of combinations and/or specific sequences of HSEs for regulation in different situations. Functional analysis of the promoter regions of HaHsp17.6G1 and HaHsp18.6G2, other two sHSP in sunflower, also associated the promoter organization with the ability to respond to nematode infection. Only HaHsp18.6G2 was induced in giant cells, which presents two HSEs in the promoter, one of which was proximal and the other was distant. In contrast, only one HSE was observed in the HaHsp17.6G1 promoter, in a distant region. It was also found that the CAAT box element in HaHsp18.6G2 was located immediately upstream and between the HSEs, and only downstream of the HSE in the HaHsp17.6G1 promoter.
Interestingly, it was observed an organization of the soybean gene promoters responsive to nematode infestation similar to that of the cis-elements of the At4g10250 promoter as previously described by Lopes-Caitar et al. [21]. The promoters of the soybean Hsp20 genes responsible for M. javanica infestation presented two CAAT elements in the region containing the HSE element in their structures, while the W-box was located farther away [21]. On the other hand, it was not possible to observe the occurrence of an HSE element within the − 83 bp region from the TSS in At4g10250, but it was closely located at − 97 bp position from the TSS. Thus, similar structural organization of Hps22.4 between soybean and Arabidopsis could also reflect functional conservation.

Conclusions
Although the GmHsp22.4 gene was initially identified as being highly induced in a M. javanica nematoderesistant soybean genotype and repressed in a susceptible one, no differences in polymorphism were observed, either in the promoter or in the coding regions of those genotypes. The GmHsp22.4 gene affects the resistance response to M. javanica, since its overexpression reduced the infective potential of nematodes by up to 82% and Arabidopsis ortholog knock-out lines increased the susceptibility by 150%. The promoter of the GmHsp22.4 gene was induced in response to infestation with M. javanica at 9 dai, but no difference between the susceptible and resistant plant promoters were detected. A promoter region of AtHsp22.0 containing at least 191 bp, with the presence of HSE close to the TSS, is necessary to trigger the resistance of A. thaliana to M. javanica nematode.

Nematode culture and plant materials
The population of M. javanica was multiplied in a greenhouse of the Nematology Department at Embrapa Soja, Londrina, Parana State, Brazil, in soybean plants and processed according to the technique of J Bonetti and J Ferraz [35]. In a hatching chamber, the viable second stage J2 were collected every day, for 3 days, in an Erlenmeyer flask and was quantified using a Peter's chamber.
Seeds of soybean accession PI 595099 used in this study were provided by Soybean Germoplasm Bank from Embrapa Soybean, Londrina, Parana State, Brazil.
Seeds of Arabidopsis thaliana ecotype Columbia (Col-0) and seeds of Arabidopsis thaliana of two independent lines from a single knockout mutant of AtHsp22.0 were obtained from the Arabidopsis Biological Resource Center (ABRC) (WiscDsLox489_492E13 with stock number N858258 for athsp22.0-1 and GK-265F12-014990 with stock number N335081 for athsp22.0-2). No specialist undertook the formal identification the plant materials used in our study.

Polymorphism analysis of the GmHsp22.4 gene
Resequencing data of 21 soybean genotypes were obtained from Santos et al. [22]. The fasta file for the nucleotide sequence spanning the interval of 41 The nucleotide sequences of all 21 genotypes were evaluated with Integrative Genomics Viewer (IGV) [36].

Cloning of the GmHsp22.4 gene from soybean
The DNA of the M. javanica nematode-resistant soybean genotype PI595099 was extracted from leaf tissue according to the technique described by Doyle and Doyle [37] and used for the amplification of the coding region with the primers CDSGmHsp22.4-F and CDSGmHsp22.4-R, while the promoter region was amplified with the primers PGmHsp22.4-F and PGmHsp22.4-R (Additional file 5). The PCR fragments were purified using the Wizard®SV gel and PCR Clean-UP System (Promega).
The fragments were introduced into an input vector with the PCR™8/GW/TOPO® TA Cloning® Kit (2012), which was then used for the transformation of cells of the electrocompetent strain Escherichia coli DH5α by electroporation. The selection of the transformed clones was performed through the use of the antibiotic hygromycin at a final concentration of 100 μg.mL-1 and enzyme analysis, while the recombinants and the clones containing the fragments in the expected orientation were determined by restriction reactions. The plasmid DNA from the clones containing the promoter and coding regions of GmHsp22.4 in the expected orientation were used in a recombination reaction with the vector pHGWFS7::P [38] for promoter analyses (Fig. 6a) and the vector pH7WG2D::CDS for coding region analyses (Fig. 6b) [38].

A. tumefaciens GV3101 and plant transformation
The plasmids pHGWFS7 and pH7WG2D containing the correctly cloned fragments of interest were used to transform A. tumefaciens GV3101 strains by electroporation at 2,2 kV, 25 μF, with 1 wrist controller at 200 or 400 Ω. Plates containing YEP medium with gentamicin and hygromycin were incubated overnight at 28°C. For the confirmation of positive bacterial clones, PCR was performed using the primer set PGmHsp22.4-F and PGmHsp22.4-R for the promoter and the primer set pH7WG2D-F and pH7WG2D-R for the coding region (Additional file 5). The recombinant bacteria were used to transform the A. thaliana Columbia (Col-0) ecotype, using the floral dip method [39]. The selection of transformed seeds in T0 and the subsequent T2, T3 and T4 generations was performed in medium containing 1 / 2x MS medium (Sigma Chemicals n°m-5519), 0.8% agar (Sigma Chemicals n°A-1296), and 15 μg / ml-1 of hygromycin. Transformants were identified as hygromycin-resistant seedlings when they did not present growth retardation. Positive events were confirmed via PCR (Additional file 4) and then propagated until the T4 lineage to be used in subsequent experiments.

Molecular analysis of putative transgenic plants
The A. thaliana plants transformed with GmHsp22.4 and selected with the antibiotic hygromycin at 15 μg mL − 1 were confirmed by PCR using genomic DNA extracted from the roots (Additional file 4). The primer pairs PGmHsp22.4-F and PGmHsp22.4-R (promoter region) or pH7WG2D-F and pH7WG2D-R (coding region) were used for PCR according to the instructions of the Taq DNA Polymerase Invitrogen Kit. DNA extracted from untransformed plants were used as negative samples. The PCR conditions for the promoter region were 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 45 s and one additional step of 72°C for 5 min.
In the case of A. thaliana transformed with the GmHsp22.4 coding region, the PCR conditions were 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 45 s, and an additional step at 72°C for 5 min. Both amplification products were visualized by conventional electrophoresis in the translucent and photodocumentador (Loccus) equipment was used to acquire and store the images.

Detection of GFP activity under M. javanica infestation
The A. thaliana transgenic lines PGmHsp22.4-3, PGmHsp22.4-4, PGmHsp22.4-6, and PGmHsp22.4-12, harboring PGmHsp22.4:GFP were used to determine the promoter activity of GmHsp22.4 on the basis of green fluorescence intensity. The evaluation was performed in the roots of plants infected with 400 J2 M. javanica individuals after 9 days of inoculation or left uninfected at 26°C [21]. The experiment was conducted in a completely randomized design (CRD) with three plants and four independent transformation events. Images of roots expressing eGFP were captured with a 5.0 megapixel camera connected to a Zeiss Axio Scope A1 compound microscope (Zeiss Corporation) and processed with MOTIC software (version 2.0). Quantitative analysis of fluorescence levels was performed by using the Adobe® Photoshop® CS6 13.0 × 32 program. The statistical analyzes were performed using SAS/STAT® software, Version 9.4. Copyright© 2016 SAS Institute Inc. The contrasts between the means of treatments and controls were tested using the Scheffé's test, at 5% significance level, in variance analysis models.

Bioassays with M. javanica
For the functional analysis, 14 non transformed plants and plants transformed with the GmHsp22.4 coding region, overexpressing the GFP marker, were selected for a nematode bioassay conducted in a CRD. The population of M. javanica was multiplied, and the roots were challenged with 400 M. javanica J2 per plant. The inoculum were pipetted through a small open hole next to the root of the plant.
After 22 days of inoculation at 26°C [40], the roots were collected and weighed individually. Evaluation of the number of females of M. javanica was performed by counting the nematodes stained with acid fuchsine according to [41]. All parameters evaluated were compared between wild-type plants and those transformed with the GmHsp22.4 gene. The statistical analyzes were performed using SAS/STAT® software, Version 9.4. Copy-right© 2016 SAS Institute Inc. The contrasts between the means of treatments and controls were tested using the Scheffé's test, at 5% significance level, in variance analysis models.

Quantification of GmHsp22.4 transcript levels
Total RNA was extracted using the TRIzol reagent (1 mL / 100 mg tissue) as recommended by the manufacturer (Invitrogen). After extraction, the RNA samples were treated with deoxyribonuclease I (Kit Invitrogen -DNase I) according to the manufacturer's recommendations to eliminate any DNA molecules present in the sample. The treated RNA was employed in the cDNA synthesis step using the SuperScript III Kit (Invitrogen) as recommended by the manufacturer. RT-qPCR was conducted in a Ste-pOnePlus™ System thermocycler (Thermo Fisher Scientific) using the SYBR® Green PCR Master Mix kit (Applied Biosystems) according to the manufacturer's instructions. For normalization, the AT_Act (actin At3g18780) gene was employed, which was amplified with the primers AT_ Act-F and AT_Act-R, while the primer set RT-GmHsp22.4-F and RT-GmHsp22.4-R were used to the target gene (Additional file 5). Relative quantification of the target gene calculated by the REST program used wildtype control plants as calibrators for each corresponding treatment. The samples were evaluated with at least 14 replicates from each transgenic line, from which three technical replicates were used in the PCR.