Identification and Expression Analysis of Heat Shock Proteins in Wheat Infected with Powdery Mildew and Stripe Rust

Heat shock proteins (HSPs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum) HSPs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 119 DnaJ (Hsp40) proteins (TaDnaJs; encoded by 313 genes) and 41 Hsp70 proteins (TaHsp70s; encoded by 95 genes) into six and four groups, respectively, via a phylogenetic analysis. An examination of protein structures and a multiple sequence alignment revealed diversity in the TaDnaJ structural organization, but a highly conserved J-domain, which was usually characterized by an HPD motif followed by DRD or DED motifs. The expression profiles of these HSP-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici and Puccinia striiformis f. sp. tritici. A quantitative real-time PCR analysis indicated a lack of similarity in the expression of DnaJ70b, Hsp70-30b, and Hsp90-4b in wheat infected by B. graminis f. sp. tritici, although the expression levels of these genes were abnormal in the infected resistant and susceptible lines. Furthermore, a direct interaction between DnaJ70 and TaHsp70-30 was not detected in a yeast two-hybrid assay. This study revealed the structure and expression profiles of the HSP-encoding genes in wheat. The resulting data may be useful for future functional analyses and for further elucidating the roles of wheat HSPs during responses to fungal infections.


Introduction
In the cytoplasm and nucleus, the heat shock response mediates stress-induced transcriptional changes via the increased production of essential protective factors [1] called heat shock proteins (HSPs; also known as molecular chaperones) [2]. On the basis of their molecular weight, HSPs have been classified into several major families, namely Hsp90, Hsp70 (also named DnaK in Escherichia coli), Hsp40 (also referred to as DnaJ), Hsp60, and the small Hsps. From yeast to humans, Hsp40s and Hsp70s form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of diverse proteins. The DnaJ molecular chaperones, which represent the crucial partners, bind to and transfer substrate proteins to the Hsp70s to regulate their ATPase activity [3,4]. The DnaJ proteins comprise the following four domains: N-terminal J-domain, G/Fdomain (Gly/Phe-rich region), Zn-binding domain characterized by cysteine repeats, and the C-terminal dimerization domain [5,6]. Depending on the presence of the G/F-rich region and/or the cysteine repeats, a DnaJ/Hsp40 protein is categorized as type I, II, or III [7]. The Hsp70/Hsp40/NEF (nucleotide exchange factor) system assists in intracellular protein refolding and helps maintain proteostasis in healthy and stressed cells [7][8][9][10].
Additionally, Hsp70 functions cooperatively with the highly conserved Hsp90 via co-chaperones [11,12], resulting in the assembly, maturation, stabilization, and activation of key signalling proteins, including protein kinases and transcription factors in eukaryotic cells [13,14]. These observations suggest there is an important cycle of functions related to the interaction between Hsp70 and the J-domain proteins Hsp40 and Hsp90 [15,16].
The Hsp90 molecular chaperone also ensures proteins are maintained in their active conformations [17].
Plants have no capacity to escape from adverse environments, and their growth and production are severely affected by abiotic and biotic stresses. To ensure they can successfully propagate, plants have developed multiple defence mechanisms that allow them to detect pathogens and induce rapid responses through innate immunity surveillance systems [18,19]. Genes encoding HSPs are reportedly differentially expressed in various plant species exposed to abiotic and biotic stresses. During plant-pathogen interaction, the AtHsp90.1 gene is required for the full RESISTANT TO P. SYRINGAE 2 (RPS2)-mediated resistance against Pseudomonas syringae pv. tomato DC3000 (avrRpt2), and its function is closely associated with RAR1 (required for Mla12 resistance) and SGT1 (suppressor of the G2 allele of skp1) via chaperone activities [20]. Similarly, suppressing TaHsp90.2 or Hsp90.3 expression in wheat decreases the hyper-sensitive resistance to the stripe rust fungus [21]. Previous studies also confirmed that Hsp70s and Hsp40s are involved in various plant disease resistance, such as NtMPIP1 [22], GmHSP40.1 [23], OsDjA6 [24]. The importance of the proteins encoded by these genes in responses to various environmental stimuli [25][26][27] and their dynamic interplay with the chaperone machinery suggest that targeting Hsp90 and its respective co-chaperones may be an effective method for characterizing the mechanisms underlying the resistance to diverse plant diseases.
We previously constructed two protein-protein interaction networks based on a weighted gene co-expression network analysis (WGCNA) to clarify the mechanism mediating wheat responses to the pathogens causing stripe rust (Puccinia striiformis f. sp. tritici; Pst) and powdery mildew (Blumeria graminis f. sp. tritici; Bgt) [28]. Both of these networks predicted that Hsp70 proteins represent a key hub node because they interact with some splicing regulators, transcription factors, and resistance (R) genes, including the disease resistance-related RPP13, RPS2 analogues, the pathogenesis-related protein 1 gene (PR1), and a non-host resistance gene (NHO1) [28]. In this study, we identified HSPs and their genes in wheat (Triticum aestivum). Moreover, the expression of these genes following powdery mildew and stripe rust infections was analysed with RNA-sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR) assays. Furthermore, the interactions among selected HSPs were assessed in a yeast two-hybrid (Y2H) assay.

Plant materials and pathogen stress treatment
The N9134 winter wheat cultivar, which is resistant to all Bgt races in China, was crossed seven times with a hours post-inoculation (hpi), after which they were immediately frozen in liquid nitrogen and stored at −80 °C. Subsequent analyses were completed with three biological replicates. For the genome-wide transcription analysis, 7-day-old seedlings were inoculated with Bgt E09 or Pst race CYR 31 conidia. The inoculated N9134 leaves were harvested at 0, 24, 48, and 72 hpi as previously described [29]. The non-inoculated sample (0 hpi) was used as the control.

Identification and sequence analyses of wheat heat shock proteins
In order to obtain detailed information regarding wheat HSPs, we downloaded all available sequences for

Quantitative real-time PCR analysis
The differentially expressed HSP-encoding genes that involved in wheat responses to Bgt and Pst infections we screened using the RNA-seq data (PRJNA243835) from the fungus-inoculated N9134 (resistant to Bgt-E09 with PmAS846 and CYR31 with YrN9134). The detailed RNA-seq protocol was described as the previously report controls. Amplified products were analysed with melting curves, which were generated at the end of the amplification. A standard 2 -ΔΔCT method was used to quantify relative gene expression levels.

Transcriptional activity analysis and yeast two-hybrid assays
The expression of HIS3, ADE2, and LacZ reporter genes was examined with yeast AH109 transformants on  (Table S1). To easily evaluate transcriptional activity, the transformants were suspended in sterile distilled water, after which 10-fold serial dilutions were prepared. Finally, 3-μL aliquots of each dilution were used to inoculate SD/−Trp/−His/−Ade medium and SD/−Trp/−His medium with X-α-Gal (Clontech). The inoculated media were incubated for 4 days at 30 °C. The MatchMaker yeast two-hybrid system (Takara, Dalian, China) was used to evaluate the interactions among TaHsp70-30b, TaHsp90-4b, and TaDnaJ70b. The TaHsp70-30b, TaHsp90-4b, and TaDnaJ70b coding sequences were subcloned into the pGBKT7 (DNA-binding domain, BD) and pGADT7 (activation domain, AD) vectors. Additionally, the pGADT7-Sfi I three-frame primary cDNA library was constructed by Takara (Dalian, China) using leaf samples from N9134 after infected with stripe rust fungi at 24 and 48 hours. The AD library was screened for protein interactions by mating pGBKT7: TaHsp70-30b bait plasmid, while specific AD and BD recombinant plasmid pairs were used to co-transform yeast strain AH109 cells according to the Yeast Protocols Handbook (Takara, Dalian, China).

Statistical analysis
Mean values and standard errors were calculated with Microsoft Excel software. Student's t-test was completed with the SPSS 16.0 program to assess the significance of any differences between the control and treated samples or between time-points. The threshold for significance was set at P < 0.01.

Identification of wheat heat shock protein 40 (DnaJ) family proteins
In order to elucidate the mechanism mediating wheat resistance to Pst and Bgt, we recently predicted the key genes based on a WGCNA and a transcriptome-proteome associated analysis. The predicted genes included TraesCS5B02G374900, which had the most significant connectivity to other genes (value reaching 2,676) [28] and was annotated as encoding a DnaJ-like protein. Screening the PmAs846 physical map, with the Chinese Spring wheat genome used as a reference sequence, indicated that TraesCS5B02G374900 is located between the cosegregation markers BJ261635 with XFCP620 flanking PmAS846 [32]. To further evaluate the genomic distribution and function of HSPs in wheat defences against pathogens, RNA-seq data were examined for a genome-wide identification of HSP-encoding genes in Triticum aestivum, including genes responsive to fungi.
The resulting data confirmed the presence of 119 genes encoding proteins with a characteristic J-domain in the wheat genome as well as 13 genes for proteins with cysteine repeats without a J-domain and 12 genes for proteins with a J-domain, but lacking an HPD motif. These three gene groups were designated as TaDnaJ, TaDnaJ-CR, and TaDnaJL, respectively. These proteins were encoded by 376 wheat genes. Specific details are provided in Supplemental Table S2. Interestingly, the J-domain was usually accompanied by a domain with the DXXXRXXXD motif or a long DED repeat motif (RRRYGLADEDLDRYRXYLNXXDEDDWF) (Figure 1).
The DnaJ C-terminals usually harboured a GK-rich domain characterized by a glycine and lysine interval zipper or a domain with a WAXY motif.
According to the present classification of DnaJ, the N-terminal J-domain of class I DnaJ proteins was followed by a G/F-rich region, four repeats of the CXXCXGXG-type zinc-finger, and a C-terminal extension  wheat DnaJ proteins were further classified in six subfamilies (Figure 2) on the basis of the characterized domains.
Type I DnaJ proteins were characterized by a DRD motif, whereas type IV and type V DnaJ proteins harboured the DED and WAY motifs, respectively. The type II DnaJ proteins had a classical zinc-finger domain. The Jdomain was accompanied by an uncharacterized N-terminal on the type-III DnaJ proteins.

Identification of the wheat 70-and 90-kD heat shock proteins
We detected 41 Hsp70 proteins (encoded by 95 genes) as well as two proteins containing partial Hsp70 domains, two Hsp70-like proteins, and six Hsp90 proteins (encoded by 18 genes) in the wheat reference genome. Specific details are listed in Supplemental Tables S3 and S4. In wheat, the Hsp70 proteins are more conserved than the DnaJ proteins. The six domains detected in 41 unabridged Hsp70 proteins are presented in Figure 3. Of these domains, the NXDEAVA, DXXLGGXD, and TPSXVAF motifs were detected in nearly all members. The repetition of the C-terminal EXE motif (wherein X represents glycine, isoleucine, alanine, aspartic acid, leucine, or phenylalanine) was used to classify the Hsp70 proteins in four subfamilies (Figure 3). The type IV Hsp70 proteins lacked the EXE motif, whereas the type II Hsp70 proteins had more EXE repeats than the type I and type III proteins. Interestingly, the type II Hsp70 proteins contained an EEVD pattern at the C-terminal, unlike the type III proteins, which comprised a C-terminal HDEL pattern. Another difference between the type I and type III proteins was the presence of the non-typical linker motif 7 in the type III proteins (Figure 3). We detected far fewer Hsp90 proteins than Hsp70 and DnaJ proteins. However, the wheat Hsp90 proteins were observed to contain very similar ED-enriched domains, usually accompanied by a leucine zipper.

Expression of heat shock protein-coding genes in wheat-Bgt and -Pst interactions
In order to identify the HSP-encoding genes involved in wheat responses to Bgt and Pst infections, we screened the RNA-seq data (PRJNA243835). In 10-day-old N9134 seedlings inoculated with Bgt, 45 DnaJ family genes encoding 20 HSPs were identified as differentially expressed genes (relative to the control expression level) ( Figure 4; Supplemental Table S2). Moreover, 14 Hsp70 genes (encoding seven Hsp70 proteins) and six Hsp90 genes (encoding three Hsp90 proteins) were identified in the fungus-inoculated N9134 seedling leaves (Supplemental Table S3 and S4). Notably, the number of DNAJs was three times than that of Hsp70s, while it was near to seven times than Hsp90s. Conversely, the ratio of the over lapped genes was steeply increased, which reach to 20%, 57.1% and 66.7% for DnaJs, Hsp70s and Hsp90s, respectively. Considering wheat comprises a polyploid genome, we further analysed the differentially expressed genes encoding the same HSP on partially homologous chromosomes. The results indicated that the differentially expressed Hsp genes from the partially homologous chromosomes had nearly coincident expression patterns, although the expression levels differed Conversely, in N9134R plants, TaHsf-B1b, TaDnaJ70b, and TaHsp70-30b were relatively stably expressed at low levels, with only slight and consistent fluctuations from 12 to 96 hpi. The highest TaHsp90-4b expression levels were observed at 96 hpi in N9134S plants, although they were not significantly higher at other time points than the corresponding expression levels in control (Figure 7) in resistance background. Thus, there were no coexpression relationships between TaHsp90-4b with TaDnaJ70b and TaHsp70-30b, while the gene expression pattern of TaHsf-B1b was very similar to that of TaDnaJ70b and TaHsp70-30b. Additionally, the expression levels of the DnaJ and HSP70-encoding genes were generally higher in the susceptible plants than in the resistant plants, but the expression of HSP90-4b is just the reverse.

In vitro interactions among TaHsp70-30b, TaHsp90-4b, and TaDnaJ70b proteins
The TaHsp90.2 and TaHsp90.3 genes, located on the second and seventh partially homologous chromosomes, respectively, are involved in wheat responses to the stripe rust pathogen [21]. In Arabidopsis thaliana, The J-domain of DnaJ proteins reportedly interacts with Hsp70. Previously, TaDnaJ70, TaHsp70-30 and TaHsp90-4 were detected as specific induced genes in fungi stress [28]. To understand the roles of HSPs in wheat resistance, we tested the interaction between them using Y2H system. After the expression of the proteins of interest in the Y2H system was confirmed on synthetic dextrose (SD) medium lacking Trp and Leu, the yeast strains with BD-and AD-TaHsp were grown on SD/−Trp/−Leu/−His/−Ade medium. The TaDnaJ70 protein, either in full or with a truncated N-/C-terminal, failed to interact with TaHsp70-30 in the Y2H assay ( Figure 8A and 8B). Similarly, TaHsp90-4 did not interact with TaDnaJ70 or TaHsp70-30 ( Figure 8A). Thus, the interaction between DnaJ and Hsp70 may occur selectively or indirectly in wheat.

DNAJ protein groups, structure and classification
Historically

Heat stress proteins play a critical role in wheat responding to pathogen
Heat shock proteins [e.g., Hsp40 (DnaJ), Hsp60, Hsp70, Hsp90, and Hsp101], which form one of the most ubiquitous classes of chaperones, have been implicated in diverse biological processes. The HSPs also directly stimulate cells of the innate immune system, suggesting they are activators of the innate immune system in animals [36,37]. For example, a non-lethal heat shock induces Hsp70 synthesis and promotes the tolerance of shrimp to stresses due to heat, ammonia, and metals [38], and also prevents heart failure or ageing in humans [16].

Heat stress proteins may indirectly interacted with each other
The Hsp90 chaperone pathway involves a series of steps, including the formation of multi-chaperone complexes with the assistance of receptors and cofactors. After Hsp40 binds to a receptor, its J-domain interacts with the C-terminus of Hsp70 [10]. The N-terminal of Hsp70 simultaneously binds to ATP. An intermediate complex, comprising Hsp70 and Hsp90, is then formed with the assistance of cofactors that help ATP bind to Hsp90 [46]. In this process, Hsp70 and Hsp90 are associated through the Hop adapter protein [12]. Additionally, because the chaperone partnership between Hsp40s and Hsp70s has been well established from yeast to humans [47], it was assumed, without experimental verification, that these HSPs also interact in plants. In fact, there are inter-and intra-species variations in the J-domain, hinting at the specificity of Hsp40-Hsp70 interactions [48]. In wheat, there are 144 DnaJ family proteins (119 DnaJ, 12 DnaJ-like, and 13 DnaJ-CR proteins) and 41 Hsp70s, with considerable diversity in both protein families. Thus, it is problematic if these proteins are able to freely interact with each other in wheat plants. In this study, our data indicated that TaDnaJ70 does not directly interact with either TaHsp70-30 or TaHsp90-4 or that our technique is not sensitive enough to detect the binding. There are two possible relationships between the DnaJ and Hsp70 proteins. Specifically, DnaJ may directly interact with specific Hsp70s, but not arbitrarily in wheat, or the interaction may be indirect (e.g., via an adapter), similar to the interaction between Hsp70 and Hsp90. Because the presence of the HPD motif in TaDnaJ70 has been confirmed, these results imply that other residues and regions outside the HPD motif contribute to the interaction between Hsp40 or Hsp40-like proteins and Hsp70. Our results provide the foundation for future wheat HSP studies for clarifying the interaction between HSPs and pathogen defence.

Conclusions
Heat shock proteins (HSPs) play a crucial role in development and responses to diverse stresses. Here, we systematically classified DnaJ (Hsp40) proteins into six and four groups according to the detailed structural characterization, including HPD, DRD, DED, WAY, GK and CR domains. Moreover, infection by Bgt and Pst triggered robust alteration in gene expression of Hsp-encoding genes in T. aestivum, but the expression profiles of these HSP-encoding homologous genes varied in response to Bgt and Pst. The yeast two-hybrid assay experiments showed that a direct interaction are failed between TaDnaJ70, TaHsp70-30b and TaHsp90-4b.
These indicate that the Hsp protein-encoding genes of wheat responded to Bgt and Pst stress and played important roles in responding to fungal stress by a more complex pathway than that in mammal and model plant.

Conflicts of Interest:
The authors declare that they have no conflict of interest.

Authors' contributions
HZ and WJ conceived the project and provided overall supervision of the study; HG and GW performed the experiments and data analysis; YW and CW contribution to developing the materials; XL helped in experimental works; HZ wrote the first version of the paper; all authors reviewed and approved the final manuscript.

Supporting information
Supplemental Table S1. Details regarding primers used for the qRT-PCR analysis and PCR amplification of HSPencoding genes Supplemental Table S2. Wheat DnaJ proteins Supplemental Table S3. Wheat 70-kD heat shock proteins Supplemental Table S4. Wheat 90-kD heat shock proteins Supplemental Figure S1.                 TaHsp70-30 and TaHsp90-4 proteins interact with each other in a yeast two-hybrid system. A: TaDnaJ70 (a) or TaHsp70-30 (b) was cloned into pGBKT7 vector and the corresponding proteins were cloned into the pGADT7 vector. Cells of yeast strain AH109 harbouring the indicated plasmid combinations were grown on either the nonselective (SD-L-W) or selective (SD-L-W-H-A) medium containing 20 μg/mL X-a-gal. The interaction between SV40 large T-antigen (T) and murine p53 (53), T-AD+53-BD, was used as the positive control, while the interaction between T-antigen and human lamin C (Lam), T-AD+Lam-BD, was used as the negative control. A: interaction between full length proteins. B: interaction between full lengths TaHsp70-30 with the truncated DnaJ70 proteins. JD means the J and DRD domains; JDC represents the J, DRD and zinc finger domains, while CT means the C-termianl of TaDnaJ70.