Functional characterization of a soybean small heat shock protein involved in the resistance response to Meloidogyne javanica

Background: Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. Previous studies have identified strong induction of the GmHsp22.4 gene in response to Meloidogyne javanica nematode in resistant soybean genotypes compared to susceptible one. This study aimed to investigate the functional involvement of this small chaperone in response to M. javanica. First, it was evaluated the activation of the promoter region by nematode’s infection and the occurrence of polymorphisms between resistant and susceptible resequenced soybean accessions in the gene coding and promoter regions. Then functional analysis using Arabidopsis thaliana lines overexpressing the soybean GmHsp22.4 gene, and knocked out mutants were challenged to M. javanica’s infection. Results: 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 infection. However, structural analysis of the soybean resistant and susceptible genotypes did not detect any polymorphisms in the whole gene model, including the 2.0 kb promoter region. Moreover, the reproduction of the nematode was significantly reduced in plants overexpressing GmHsp22.4 gene in A. thaliana compared to the wild type. Additionally, the reproduction of M. javanica in the A. thaliana mutants was significantly increased and was related with the structural organization of heat shock cis element (HSE) in the promoter. Conclusions: The soybean chaperone GmHsp22.4 is involved in the defense response to root-knot nematode M. javanica’s infection in A. thaliana. The HSE in the promoter region close to the transcriptional start site (TSS) are important to GmHsp22.4 promoter activation after nematode infection. It was not possible to detect any polymorphisms occurrence in GmHsp22.4 gene between the M. javanica-resistant and susceptible soybean genotypes, so the resistance mechanism might not be related with the transcriptional regulation in the initiation process or based on sequence level on the TSS. Based on the analysis of the promoter region of At4g10250, we identified a TA-rich sequence at the position of +114 bp and TATA boxes at +79, –146, –165 and –456 bp of the transcription start site (TSS). CAAT box elements were found in the sequence at positions +182, +173, +71, +5, –2, –121, – 126, –165, –181, –310 and –355 bp of the TSS. The HSEs, which are recognized and activated by the heat shock factors (HSF) transcription factor [20], were observed at six different positions, +166,


Abstract
Background: Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. Previous

Background
Despite the increases in soybean productivity through genetic gains over the years, the management of pests and diseases has a significant impact on the environment related on the application of agrochemicals and production costs [1]. Among these pathogens, the phytonematodes are found in all major areas of soybean cultivation both in Brazil and worldwide. Production losses in soybean crop in Brazil caused by nematodes are estimated at R$ 16.2 billion [2].
Several strategies have been developed to control phytonematodes, such as crop rotation, the use of nematicides and biological treatments. Additionally, transgenic approaches involving the overexpression and silencing of genes have been attempted [3,4]. Finally, genetic resistance has been used in breeding programs exploring genetic loci in soybean, such as the quantitative trait loci (QTLs) described in plant introductions (PIs) [5]. However, a few sources of resistance are available, limiting the development of broad durable resistance [6]. 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 pathogens.
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) [7]. 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 [8,9]. These proteins were initially observed in the salivary glands of Drosophila spp. under heat shock stress [8]. Based on their sequence size and homology, the HSPs have been grouped into five classes: HSP60, HSP70, HSP90, HSP100 and HSP20 (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 [10][11][12][13].
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 [14], as well as under different biotic stresses, such as nematode infection [12,14,15]. 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 [10,13,16,17]. 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 [12,13]. HSP20 then cooperates with other ATP-dependent molecular chaperones such as HSP70, HSP90, HSP100 and GroEL to refold proteins [11,13]. 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 [12,13].
In a previous study, Lopes et al. [18] characterized the expression profiles of 51 members of the HSP20 family in Glycine max (GmHsp20) under abiotic (heat and cold) and biotic stresses (infection by M. javanica) on susceptible (BRS133) and resistant (PI595099) genotypes. The expression levels of these genes were strongly dependent on the genotype under biotic stress conditions. Additionally, the exposure period (four or eight days after inoculation) played a significant role in the gene expression in both genotypes. Among the five of the 51 members of the GmHsp20 family that were significantly expressed under both treatments, 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 than in the noninfected conditions. Interestingly, the authors reported the systematic occurrence of a standard organization of cis-elements in the promoter region of these GmHsp20 genes associated with the responses of soybean to M. javanica infection.
In this study, we sought to elucidate the involvement of GmHsp22.4 in response to M. javanica  [20], were observed at six different positions, +166, +48, -97, -408, -462 and -467 bp of the TSS. In contrast to the other elements, a W-box was located in the negative strand at the -303 bp position ( Figure 5)..
In the T-DNA insertion lines, it was possible to identify the location of the T-DNA insert between the CAAT-box and W-box cis-elements in the WiscDsLox489_492E13 event, while the T-DNA insertion site in the 5' UTR of GK-265F12-014990 was located between the cis-element HSE and TA-rich ( Figure 5)..

Discussion
In this study, we examined the role of the GmHsp22.4 gene in the root-knot nematode resistance response in detail. This gene was selected because, in a previous study, it was found to be strongly expressed only in an M. javanica-resistant genotype at 8 dai, whereas in a susceptible genotype, the expression of this gene was repressed [18]. Based on this finding, we hypothesized that these chaperones play a differential role in the infection response during the infection cycle of the nematode related to the type of damage and alterations caused by this pathogen in host roots [18].
Our results demonstrated that GmHsp22.4 promoter activity in A. thaliana plants is highly activated after nematode infection, confirming the importance of this gene in the response to M. javanica. The GmHsp22.4 promoter presents a structural organization in which CAAT boxes are located immediately upstream of HSE elements, while a W-box is located further upstream of the HSEs, as previously described by Lopes-Caitar [18], and it is potentially recognized by an HSF. As expected, promotor induction was 98% greater in the infected Arabidopsis transgenic plants than in the noninfected ones, as revealed by GFP fluorescence marker (Figure 1).. The differential regulation of the expression of GmHsp22.4 between the resistant and susceptible genotypes was in accordance with the results observed by Fuganti et al. [21]. These authors mapped a QTL related to root-knot nematode resistance in a population derived from the resistant source PI 595099 between two microsatellite markers, Satt 144 and SoyHSP 176. The SoyHSP 176 marker was identified as being located in a region containing the GmHsp17.6-L gene. GmHsp17.6-L expression levels were subsequently shown to be differentially regulated between resistant and susceptible individuals from the mapping population and were induced only in resistant individuals. When Fuganti et al. [15] analyzed the promoter region polymorphism of the previously identified GmHsp17.6-L gene, an opposite result to that observed in our study was revealed, since the authors detected a greater number of AT (n) repeats in the resistant genotype compared to the susceptible genotype, whereas in our results, the number of AT (n) repeats in GmHsp22.4 remained constant in the different genotypes [15]. So, in our analysis it was not possible observe any correlation of AT(n) repetitions and promoter activity of GmHsp22.4.
The occurrence of enhancer regions that interfere with the promoter regions of other genes in their vicinity or over long distances, interfering with their regulation, might be a possible explanation, but our data cannot confirm this hypothesis. In addition, we speculate that epigenetic regulation, which cannot be detected at a sequence level, might be an alternative explanation for the results. This mechanism has been reported to be involved in resistance to cyst nematodes [22]. Similarly, Rambani et al. [23] evaluated the impacts of soybean cyst nematode (SCN) infection on DNA methylation patterns in the root tissues from susceptible soybean plants. Interestingly, the authors identified 447 soybean genes that were differentially methylated in the promoter region, comparing samples inoculated with a cyst nematode versus those that were not inoculated.
This differential methylation may be related to the accessibility of transcription factors to ciselements, thereby regulating the transcriptional activity of genes responsive to the nematodes, as described by Zhou et al. [24]. In their study, it was observed that the HsfA1a transcription factor played an essential role in the activation of the resistance response mediated by the MI-1.2 gene. In addition, HsfA1a interfered with the activation of HSP90, which participates in Mi-1.2 gene activation.
This observation was made after the silencing of HsfA1 in tomato plants. This scenario may be an alternative explanation for the absence of differences in GmHsp22.4; however, further studies are necessary to confirm this hypothesis.
To obtain a better understanding of the roles of these chaperones in the resistance response to rootknot 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 involving constitutive overexpression of the GmHsp22.4 gene in A. thaliana resulted in a significant reduction to infection by the root-knot nematode compared to untransformed plants. Increasing the expression of the heat shock gene in A. thaliana plant roots led to reductions in the numbers of females by 82% and 42% for the two events ( Figure 3B).. Similarly, when the orthologous gene was knocked out in Arabidopsis plants, we observed an increase in susceptibility of 60% (Figure 4)..
Arabidopsis presents 19 genes encoding Hsp20s, grouped into 12 subfamilies based on their subcellular localization and homology [18], and the transcript of the A. thaliana gene model orthologous to GmHsp22.4, AtHsp22.0, is undetectable in normal conditions (22°C); however, it accumulates at a high level in response to heat stress (38°C) [25]. To date, there is no available evidence of the activation of AtHsp22.0 under biotic stress conditions, such as nematode infection.
The mode of action of these proteins after stress is becoming clearer, suggesting that HSP20 effectively captures protein folding intermediates that are prone to aggregation and maintains them in a suitable conformation for refolding. Thus, HSP20 has an oligomeric structure that interacts with nonnative proteins. This structure exists in two states: an inactive low-affinity state and an active high-affinity state. After heat stress, HSP20 is activated to its high-affinity state and becomes competent to associate with denatured proteins, forming a stable complex. This enables efficient prevention of irreversible protein aggregation. However, the release of these proteins from the complexes that are formed requires the cooperation of ATP-dependent HSPs, such as HSP70-HSP40 or HSP100. Although these other proteins may act directly on the aggregated proteins, the presence of HSP20 increases the efficiency of the process [26].
Based on this analysis, we suggest that GmHsp22. 4

is involved in nematode infection responses,
where one of the hypotheses is that it acts as the first line of cellular defense by capturing unfolded proteins, reducing the size of protein aggregates; thus, more binding sites are generated, and with the assistance of ATP-dependent HSP70 and HSP100, aggregation is reversed and refolding is facilitated [27]. The role of Hsp20 related to its nematode-responsive promoter activity has been described in rice by Escobar et al [29], who characterized the involvement of the soybean HsHsp17. HaHsp18.6G2 promoter, one of which was proximal, while 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 of and between the HSEs, while in the HaHsp17.6G1 promoter, it was located downstream of the HSE.
Interestingly, it was possible to observe an organization of the soybean gene promoters responsive to nematode infection similar to that of the cis-elements of the At4g10250 promoter as previously described by Lopes-Caitar [18]. The promoters of the soybean Hsp20 genes responsible for M. javanica infection presented two CAAT elements in the region containing the HSE element in their structures, while the W-box was located farther away [18]. On the other hand, it was not possible to observe the occurrence of an HSE element within the -83 bp region from the TSS, but it was closer  [32] for promoter analyses ( Figure 6A) and the vector pH7WG2D_CDS for coding region analyses ( Figure 6B) [32].

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 ohms. 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 CDS (Additional File 5).. The recombinant bacteria were used to transform the A. thaliana Columbia (Col-0) ecotype, using the floral dip method [33]. The selection of transformed seeds in T0 and the subsequent T2, T3 and T4 generations was performed in \ medium After 22 days of inoculation [35], 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 [36]. All parameters evaluated were compared between wild-type plants and those transformed with the GmHsp22.4 gene. The statistical analysis was performed using the Statistical Analysis System (SAS) [34] using ANCOVA, and the means were compared at the 5% level of significance with Student's t-test. After 45 days, the total number of females and eggs/juveniles stained with fuchsine acid [36] was evaluated according to Coolen and D' Herde [37]. The two evaluated parameters were compared between the control plants and the mutants under a CRD. Statistical analysis was performed with SAS [34] using ANOVA at the 5% significance level by Student's t-test.
Cis-element identification in the At4g10250 promoter Putative cis-elements from the 500 bp region upstream of the transcript start site of the At4g10250 gene were characterized by bioinformatics using the PlantCARE [38] and AthaMap [39] databases.

Declarations
Declarations Ethics approval and consent to participate Not applicable.

Consent for publication
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Funding
This study was supported by the National Institute of Science and Technology -INCT Plant Stress-CNPq, National Council for the Improvement of Higher Education (CAPES), and EMBRAPA -Brazilian Federal Government.

Availability of data and materials
All data generated during this study are included the in the manuscript or supplementary information files.

Competing interests
The authors declare that they have no competing interests.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Ad File 5.xlsx