Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance
The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
Graduate School of Chinese Academy of Sciences, Yuquan Road, Beijing 100039, China
•Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species.
•Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined.
•Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus.
•Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.
Heat shock proteins 90 (Hsp90s) are ubiquitous molecular chaperones in the cells of eukarya and eubacteria (Wegele et al., 2004; Taipale et al., 2010). They play key roles in signal transduction, protein folding, protein degradation, and growth and developmental programs under both normal and stressed conditions (Taipale et al., 2010). Hsp90s cooperate with other co-chaperones in an ATP-driven machine, promoting the folding, functional maturation and stability of newly synthesized client proteins and the refolding of denatured proteins after stress (Taipale et al., 2010). Because of their importance, the genetics and function of Hsp90s have been studied extensively in many species (Taipale et al., 2010).
In Arabidopsis thaliana, Hsp90s are encoded by seven genes. The products of four members (Hsp90.1–Hsp90.4) are located in the cytosol, and those of the other three are targeted to the chloroplast (Hsp90.5), mitochondrion (Hsp90.6) and endoplasmic reticulum (Hsp90.7) (Milioni & Hatzopoulos, 1997; Krishna & Gloor, 2001). Among the four cytosolic AtHsp90 genes, AtHsp90.1 is highly stress inducible, whereas the other three (AtHsp90.2, AtHsp90.3 and AtHsp90.4) are constitutively expressed. When treated with the Hsp90-specific inhibitor geldanamycin (GDA), Arabidopsis seedlings display strong morphological abnormalities (Queitsch et al., 2002). In Hsp90-RNAi lines, abnormal growth, such as the loss of apical dominance and the emergence of multiple shoots and primary inflorescences (Sangster et al., 2007), is observed. Similarly, growth inhibition and abnormalities are also observed in T-DNA knockout lines lacking any of the four cytosolic AtHsp90 genes (Samakovli et al., 2007). In Nicotiana benthamiana, stunting and leaf deformation phenotypes are observed when the genes encoding cytosolic Hsp90s are silenced by virus-induced gene silencing (VIGS) (Kanzaki et al., 2003; Lu et al., 2003; Liu et al., 2004). Similar phenotypes are also observed in soybean plants when the genes coding for cytosolic Hsp90s are silenced by VIGS (Fu et al., 2009).
Cytosolic Hsp90s have also been found to play an important role in plant immune responses to pathogens. Pathogen effector-triggered immunity in higher plants is known to be mediated by intracellular immune receptors, the so-called resistance proteins (R proteins). Many R proteins have been found to be functionally dependent on cytosolic Hsp90s. RPM1, the R protein controlling the hypersensitive resistance response to Pseudomonas syringae pv. tomato strain DC3000 in Arabidopsis, was the first identified client of cytosolic Hsp90s in plants (Hubert et al., 2003). Subsequently, cytosolic Hsp90s have been found to interact with many R proteins, such as tobacco N, potato Rx and tomato I-2 (Liu et al., 2004; de la Fuente van Bentem et al., 2005; Botër et al., 2007). Furthermore, cytosolic Hsp90s have been found to interact with two co-chaperones, SGT1 and RAR1, that are major regulatory components of disease resistance triggered by many R proteins in both dicotyledonous and monocotyledonous plants (Shirasu et al., 1999; Azevedo et al., 2002; Takahashi et al., 2003; Hein et al., 2005; Scofield et al., 2005; Botër et al., 2007; Taipale et al., 2010).
In contrast with the progress above, knowledge on the genetic control and function of cytosolic Hsp90s in common wheat and related Triticeae species is very limited. Several studies have shown that the silencing of cytosolic Hsp90 expression in barley and common wheat by VIGS disrupts hypersensitive response (HR) resistance to fungal pathogens (Hein et al., 2005; Scofield et al., 2005; Zhou et al., 2007). However, no systematic studies have been reported on the numbers and chromosomal locations of common wheat genes encoding different types of cytosolic Hsp90. Neither is it clear which type(s) of cytosolic Hsp90s may be involved in HR resistance. Consequently, the main objectives of this work were to conduct a more systematic analysis of common wheat genes encoding different types of cytosolic Hsp90 and to study their function in seedling growth and disease resistance.
Materials and Methods
Wheat materials and general molecular and bioinformatic methods
Wheat materials (Supporting Information Table S1) were grown in a glasshouse with the temperatures set at 23°C (day) and 14°C (night) and a 16 h light period unless otherwise stated. For the winter variety Xiaoyan 54, a vernalization treatment (4°C for 4 wk) was applied following completion of germination. Under these conditions, Xiaoyan 54 plants reached the bolting stage at 12 wk post-germination (WPG), and the flowering stage at 16 WPG. The oligonucleotide primers used in this work are described in Tables S2 and S3. The BAC library used here was constructed with the genomic DNA of Xiaoyan 54 (Dong et al., 2010). Full details on the general molecular and bioinformatic methods are given in Methods S1.
Isolation and chromosomal assignment of TaHsp90 gene sequences
Initially, we analyzed the genomic coding regions and predicted mRNA sequences of four rice genes encoding cytosolic Hsp90s (hereafter abbreviated as cHsp90 genes; Chen et al., 2006). By searching public databases using the four rice cHsp90 genes (OsHsp90.1, OsHsp90.2, OsHsp90.3 and OsHsp90.4; Table S4) as queries, four full-length cDNA sequences (GenBank accessions DQ665783, DQ665784, U55859 and X98582) and many expressed sequence tags (ESTs) derived from wheat cHsp90 genes were found. Using the sequence information obtained, three pairs of oligonucleotide primers (HspI, HspII and HspIII; Table S2) were developed for the isolation of the full-length cDNA and genomic DNA coding sequences of Hsp90.1, Hsp90.2 and Hsp90.3 genes from Xiaoyan 54 through PCR. Further details of the cloning and chromosomal localization experiments are given in Methods S1.
Evaluation of TaHsp90 transcript levels by multiplex quantitative PCR
For the evaluation of the transcript levels of Hsp90 genes in the vegetative and reproductive organs of common wheat (cv Xiaoyan 54), multiplex quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was performed in the GeXP Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA), as described previously (Chen et al., 2007; Danilevskaya et al., 2008). Thirteen oligonucleotide primers were designed, whose combined use permitted the simultaneous amplification of nine TaHsp90 genes, with the fragments amplified from the nine targets differing in size (Table S3). These primers were developed on the basis of the nucleotide sequence differences in both the exonic and 3′ untranslated regions (UTRs) (Fig. S1). The leaf, root, culm, immature spikelet, stamen and pistil samples were each collected from at least five uniform plants at the bolting or flowering stages. Total RNA samples were prepared for each of the selected organs using pooled materials, and were used for quantitative RT-PCR analysis. Additional details of the analysis are given in Methods S1.
Eight recombinant viruses (BSMV:Hsp90.1, BSMV:Hsp90.1-3UTR, BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR, BSMV:Hsp90.2/3 and BSMV:Hsp90.1/2/3) were constructed using the cDNA clone of the RNAγ of barley stripe mosaic virus (BSMV), as described previously (Zhou et al., 2007). The VIGS-inducing fragments used for the preparation of the recombinant BSMVs were carefully chosen to maximize the silencing of the desired targets and to minimize the likelihood of cross silencing. The origins of the VIGS-inducing fragments are explained in Methods S1, and their locations in the nine wheat cHsp90 genes are depicted in Fig. S2. BSMV:GFP (expressing green fluorescent protein (GFP)) and BSMV:PDS (silencing the wheat gene encoding phytoene desaturase (PDS)), prepared by Zhou et al. (2007), were also used in this work, with BSMV:GFP as the viral vector control and BSMV:PDS to monitor the time course of VIGS. The seedlings of three common wheat varieties (Xiaoyan 54, Suwon 11 and Chancellor) were used for inoculation (Methods S1).
To assess the transcript levels of TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes in the mock controls and the plants (cv Suwon) infected with BSMV:GFP, BSMV:Hsp90.1, BSMV:Hsp90.1-3UTR, BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR, BSMV:Hsp90.2/3 or BSMV:Hsp90.1/2/3, the third and fourth leaves were sampled at 21 d post-inoculation (dpi). This sampling involved five individuals randomly selected from each of the 10 groups of plants (the mock controls plus those infected with the nine BSMVs described above). Total RNA samples were extracted from the leaf materials, and were employed to investigate the relative transcript levels of the target genes in the individual plants using quantitative PCR, as described by Shimada et al. (2009). The oligonucleotide primers used in this experiment are listed in Table S2, and their positions in the nine wheat cHsp90 genes are shown in Fig. S3. The primer pairs for TaHsp90.1, TaHsp90.2 and TaHsp90.3 were confirmed to be type specific (i.e. each pair amplifying all three homoeologs belonging to TaHsp90.1, TaHsp90.2 or TaHsp90.3) by cloning and sequencing the resultant PCR products. A wheat actin gene (GenBank accession AB181991) served as the internal control. All primer pairs gave an amplification efficiency of at least 96%. The relative transcript levels of the target genes in the individual plants were averaged for statistical analysis. Plant height and fresh weight were surveyed at 14 and 28 dpi. The survival rates of the mock controls and the plants inoculated with different BSMVs were recorded at 16 and 35 dpi.
Stripe rust inoculation and analysis of resultant phenotype
At 21 dpi, the fourth leaves of the Suwon 11 seedlings infected with BSMV:GFP, BSMV:Hsp90.1, BSMV:Hsp90.1-3UTR, BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR or BSMV:Hsp90.2/3, and the mock controls, were treated with the uredospores of the stripe rust races Cy17 and Cy29. The plants infected with BSMV:Hsp90.1/2/3 were not included in the fungal inoculation experiment because many of them were dead by 21 dpi (Fig. 5b). Thirty individuals were randomly selected from each of the nine groups of wheat plants for treatment with either Cy17 or Cy29. The uredospores of Cy17 and Cy29 were cultured as described in Methods S1. The freshly collected spores (mixed with talcum powder in 1 : 2 proportions) were evenly spread onto the leaf surface using a brush, followed by placement of the treated plants in a dark and moist chamber for 24 h at 12°C. Afterwards, these plants were transferred to a glasshouse with the day : night temperatures set at 18 : 12°C and a light period of 16 h, and their responses to fungal treatment were examined after further growth for 2.5 wk. For each of the five groups of plants (those infected with BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR or BSMV:Hsp90.2/3 before stripe rust inoculation) showing fungal development, the numbers of rust sori on the fourth leaves of five randomly chosen plants were determined, with the mean value used for statistical analysis.
Statistical analysis of the experimental data was conducted by ANOVA (one tailed) with the SPSS 10 program (SAS Institute Inc., Cary, NC, USA).
Isolation of the genes encoding cytosolic Hsp90s in common wheat
Through PCR amplification and cloning, nine unique cHsp90 cDNA clones belonging to three different groups (Fig. 1) were isolated from Xiaoyan 54. The first and second groups of cDNAs and the proteins derived from them generally displayed the highest identities to those of OsHsp90.1 and OsHsp90.2 genes, respectively (Table S5), indicating that these two groups of cDNAs were transcribed from the TaHsp90.1 and TaHsp90.2 genes, respectively. The third group of cDNAs and their deduced proteins exhibited high identities to those of OsHsp90.2, OsHsp90.3 and OsHsp90.4 genes, but the resemblance to those of OsHsp90.3 was relatively higher (Table S5). It was therefore tentatively suggested that this group of cDNAs might be transcribed from the TaHsp90.3 genes.
A total of 12 BAC clones harboring the coding regions of common wheat cHsp90 genes was identified from the BAC library of Xiaoyan 54 by PCR with appropriate primers (Table S2). Southern hybridization analysis, using a probe prepared with the pooled cDNAs of TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes, revealed a single hybridizing band for all of the 12 BACs, indicating the presence of a single cHsp90 gene in each of the examined clones (Fig. S4). After DNA sequencing, these BAC clones were found to harbor the genomic coding region of six TaHsp90 genes (Table 1). With the primer pairs that had been employed to isolate the full-length cDNAs, the genomic coding regions corresponding to the three TaHsp90.1 cDNAs or three TaHsp90.2 cDNAs were further amplified by genomic PCR and characterized (Table 1). Finally, a complete set of cDNA and genomic coding sequences was obtained for each of the three groups of common wheat cHsp90 genes (i.e. TaHsp90.1, TaHsp90.2 and TaHsp90.3) (Table 1).
Table 1. The nine TaHsp90 genes encoding cytosolic heat shock proteins 90 (Hsp90s) isolated in this work
ORF size (bp)
Exon 1 (bp)
Intron 1 (bp)
Exon 2 (bp)
Intron 2 (bp)
Exon 3 (bp)
Protein (amino acid)
aThe genomic sequences of the nine TaHsp90 genes were obtained either by screening positive BAC clones or through PCR amplifications.
Isolated by genomic PCR
Isolated by genomic PCR
BACs 771, 827
BACs 619, 732
Isolated by genomic PCR
BACs 486, 778, 1350
BACs 909, 1875, 1926
Exon and intron structure and chromosomal assignment of TaHsp90 genes
The open reading frames (ORFs) of nine TaHsp90 genes all contained three exons and two introns (Fig. 1), identical to that of rice cHsp90 genes (Table S4). For the three genomic sequences of TaHsp90.1, the size of their ORFs varied from 2471 to 2603 bp. This size difference was caused by variations in both exons and introns among the three sequences (Fig. 1, Table 1). For the three genomic sequences of TaHsp90.2, the size of their ORFs varied from 3728 to 3942 bp, and this difference was caused solely by variations in intron length among the three sequences (Fig. 1, Table 1). The ORFs in the three TaHsp90.3 genomic sequences varied from 3216 to 3265 bp, which was also caused by variations in intron length (Fig. 1, Table 1).
To determine the chromosomal locations of the nine common wheat cHsp90 sequences by PCR mapping, we made use of three pairs of intron flanking primers (IFPI, IFPII and IFPIII; Fig. 1) and genomic DNA samples extracted from the nulli-tetrasomic (NT) lines of Chinese Spring (CS). IFPI allowed the three TaHsp90.1 homoeologs (TaHsp90.1-A1, TaHsp90.1-B1 and TaHsp90.1-D1) to be assigned to group 2 chromosomes, whereas IPFII permitted the mapping of three TaHsp90.2 homoeologs (TaHsp90.2-A1, TaHsp90.2-B1 and TaHsp90.2-D1) to group 7 chromosomes (Fig. S5). The three TaHsp90.3 homoeologs (TaHsp90.3-A1, TaHsp90.3-B1 and TaHsp90.3-D1) were mapped to group 5 chromosomes using IPFIII (Fig. S5). The genomic DNA of Xiaoyan 54 and the plasmid DNAs of the representative BAC clones were also used as templates for PCR amplifications. The patterns of the fragments amplified from Xiaoyan 54 were identical to those from CS (Fig. S5). The BAC clone 766 was found to contain TaHsp90.1-B1 (Fig. S5a). The BAC clones 827 and 619 harbored TaHsp90.2-A1 and TaHsp90.2-B1, respectively (Fig. S5b). Finally, the three BAC clones 778, 535 and 909 contained TaHsp90.3-A1, TaHsp90.3-B1 and TaHsp90.3-D1, respectively (Fig. S5c).
The findings of nine unique cHsp90 genes and their locations on groups 2, 7 or 5 chromosomes in common wheat agreed with the DNA blot hybridization data obtained using the EST probe BG314518 (http://wheat.pw.usda.gov/). BG314518 exhibits > 80% nucleotide sequence identity with the nine common wheat cHsp90 genes isolated in this work. The eight hybridization bands generated by the BG314518 probe have been assigned to five chromosomal regions, including 2DS, 5AL, 5BL, 7BS and 7DS (http://wheat.pw.usda.gov/).
Main characteristics of the deduced Hsp90s of common wheat and relatives
By PCR amplifications with the primers in Table S2, three distinct cHsp90 genes (designated as TuHsp90.1, TuHsp90.2 and TuHsp90.3) were isolated from Triticum urartu. Similarly, three separate cHsp90 genes (designated as AetHsp90.1, AetHsp90.2 and AetHsp90.3) were cloned from Aegilops tauschii. Six different cHsp90 genes (designated as TtdHsp90.1-A1, TtdHsp90.1-B1, TtdHsp90.2-A1, TtdHsp90.2-B1, TtdHsp90.3-A1 and TtdHsp90.3-B1) were isolated from T. turgidum ssp. dicoccoides. The 12 coding sequences all had intact ORFs. Generally, Hsp90.1s from the diploid and tetraploid relatives exhibited greater than 95% identity to their corresponding homoeologs in common wheat. Similar observations were made for Hsp90.2s and Hsp90.3s from the related species. Notably, complete amino acid sequence identity existed in five pairs of homoeologous cytosolic Hsp90s (i.e. TaHsp90.2-A1 and TtdHsp90.2-A1, TaHsp90.2-D1 and AetHsp90.2-D1, TaHsp90.3-B1 and TtdHsp90.3-B1, TaHsp90.3-D1 and AetHsp90.3-D1, TaHsp90.3-B1 and TaHsp90.3-D1), and among TaHsp90.3-A1, TtdHsp90.3-A1 and TuHsp90.3-A1. Finally, the proteins deduced from two GenBank sequences (DQ665783 and DQ665784), TaHsp90.3-B1 and TaHsp90.3-D1 were also identical.
The amino acid sequences deduced from the nine common wheat cHsp90 genes isolated in this work were compared with those of the Hsp90s from rice (OsHsp90.1, OsHsp90.2, OsHsp90.3 and OsHsp90.4; Table S4) and Arabidopsis (AtHsp90.1, AtHsp90.2, AtHsp90.3, AtHsp90.4, AtHsp90.5, AtHsp90.6 and AtHsp90.7; Krishna & Gloor, 2001). An ATP binding domain was present in all of the compared proteins, and a number of conserved blocks characteristic of the Hsp90 family were also found (Fig. S6). However, unlike AtHsp90.5, AtHsp90.6 and AtHsp90.7, none of the deduced Hsp90 proteins from common wheat or rice possessed chloroplast or mitochondrial targeting sequences (Fig. S6). Computational analysis using the WoLF PSORT program (http://wolfpsort.org/) also failed to identify the presence of potential chloroplast or mitochondrial targeting sequences among the four Hsp90s of rice, nine Hsp90s of common wheat and 12 Hsp90s of wheat relatives (data not shown).
In the phylogenetic analysis of the cHsp90 genes from common wheat and its relatives, the three types of cHsp90 gene formed three separate clades. In the clade formed by homoeologous Hsp90.1 genes (Fig. 2a, indicated by star), there were three well-defined branches containing the A1, B1 and D1 homoeologs. The Hsp90.2 genes were contained in the second clade, which also had three branches, each including the A1, B1 or D1 homoeologs from common wheat and relatives (Fig. 2a, indicated by open arrowhead). There were two major different branches in the clade formed by homoeologous Hsp90.3 genes (Fig. 2a, indicated by closed arrowhead), one consisting of A1 homoeologs and the other of both B1 and D1 homoeologs. The tight clustering of Hsp90.3-B1 and Hsp90.3-D1 homoeologs was expected because their nucleotide sequences were highly similar and their deduced proteins were identical (see the section ‘Main characteristics of the deduced Hsp90s of common wheat and relatives’).
The phylogenetic relationships of common wheat cHsp90 genes with those of rice were also investigated. The cHsp90 genes from the two species formed two clades (Fig. 2b). The first was composed of only Hsp90.1 genes, whereas the second contained both Hsp90.2 and Hsp90.3 genes (Fig. 2b). Within the latter clade, two major branches could be distinguished, one consisting of the Hsp90.2 genes of common wheat and rice, and the other of common wheat Hsp90.3 homoeologs and rice Hsp90.3 and Hsp90.4 genes (Fig. 2b).
Transcriptional patterns of TaHsp90 genes in different organs
In general, the cHsp90 genes belonging to the same type exhibited a similar transcriptional pattern. The three TaHsp90.1 genes were more highly transcribed in the reproductive organs (especially in the stamen); their transcript levels in the vegetative organs were relatively low (Fig. 3, top panel). The three TaHsp90.2 genes were more ubiquitously transcribed, although their transcript levels in the roots were generally and significantly lower (Fig. 3, middle panel). The three TaHsp90.3 members were more constitutively transcribed in the six different organs examined in this work, with significantly higher transcript levels in the leaves (Fig. 3, bottom panel).
The effects of BSMV-mediated VIGS on cHsp90 genes and plant morphology
Among the recombinant BSMVs developed in this work, BSMV:Hsp90.1 and BSMV:Hsp90.1-3UTR were designed for the silencing of the three TaHsp90.1 members, whereas BSMV:Hsp90.2 and BSMV:Hsp90.2-3UTR were designed to knock down the expression of three TaHsp90.2 copies. BSMV:Hsp90.3 and BSMV:Hsp90.3-3UTR were prepared to decrease the expression of the three TaHsp90.3 homoeologs. BSMV:Hsp90.2/3 was developed to reduce the expression of both TaHsp90.2 and TaHsp90.3 genes. BSMV:Hsp90.1/2/3 was designed to lower the expression of all three types of TaHsp90 gene. Table 2 shows that the VIGS-inducing fragments generally possessed 80% (or higher) nucleotide identities to their targeted cHsp90 members. The numbers of contiguous nucleotides perfectly matched between the VIGS fragments and their respective targets all exceeded 21 (Table 2), a precondition for ensuring efficient and specific gene silencing by VIGS (Thomas et al., 2001; Senthil-Kumar et al., 2007). By contrast, the nucleotide identities of the VIGS-inducing fragments to related cHsp90 sequences were, in most cases, below 80%, and the numbers of contiguous nucleotides perfectly matched between the VIGS-inducing fragments and related sequences were generally < 21 (Table 2). After infection with BSMV:PDS, the leaves of the three common wheat varieties (Xiaoyan 54, Suwon 11 and Chancellor) all exhibited strong photobleaching symptoms, indicating that BSMV-mediated VIGS was generally efficient in the three cultivars. The seedlings infected with BSMV:GFP displayed only mild chlorotic mosaic, and the coat protein (CP) gene transcripts of BSMV were readily detected in their leaves (GF Wang et al., unpublished).
Table 2. The nucleotide identities of the virus-induced gene silencing (VIGS)-inducing fragments relative to their targets and related sequences
Using Suwon 11 plants as representative, the silencing augmented by different BSMVs was evaluated. Quantitative PCR analysis conducted at 21 dpi revealed that the relative transcript levels of a specific type of cHsp90 gene (TaHsp90.1, TaHsp90.2 or TaHsp90.3) did not vary widely among the sampled plants that acted as the mock control or were infected with BSMV:GFP (Table S6). However, the average transcript levels of the three types of cHsp90 gene differed substantially among each other in the mock-inoculated and BSMV:GFP-infected plants. In BSMV:GFP-infected plants, the average transcript level of TaHsp90.3 genes was c. 4.4 times that of TaHsp90.2 genes, and 189 times that of TaHsp90.1 genes (Table S6). The average transcript level of TaHsp90.2 genes was c. 43 times that of TaHsp90.1 genes (Table S6). In the plants infected with BSMV containing a silencing-inducing fragment, the relative transcript level of a specific type of cHsp90 gene usually varied more extensively among the sampled plants, reflecting dissimilar degrees of targeted gene silencing in different plants. Similar observations have been made in previous studies involving the use of BSMV-mediated VIGS (Scofield et al., 2005; Bruun-Rasmussen et al., 2007). To facilitate subsequent comparisons of the effects of VIGS, the average transcript levels of the three types of cHsp90 gene in BSMV:GFP plants were all set as unity (Fig. 4). In the plants infected with BSMV:Hsp90.1 or BSMV:Hsp90.1-3UTR, the average transcript level of TaHsp90.1 members was reduced by > 80%, but the two viruses did not alter the expression of TaHsp90.2 or TaHsp90.3 genes significantly (Fig. 4). BSMV:Hsp90.2 and BSMV:Hsp90.2-3UTR were both competent in specifically silencing the expression of TaHsp90.2 genes, but the former decreased the relative transcript level of the target genes by c. 65% and the latter by c. 46% (Fig. 4). BSMV:Hsp90.3 and BSMV:Hsp90.3-3UTR caused a decrease of nearly 50% in the relative transcript level of TaHsp90.3 genes, with no significant influence on TaHsp90.1 or TaHsp90.2 genes (Fig. 4). BSMV:Hsp90.2/3 knocked down the relative transcript levels of both TaHsp90.2 (by 38%) and TaHsp90.3 (by 48%) genes, without a significant effect on TaHsp90.1 genes (Fig. 4). BSMV:Hsp90.1/2/3 decreased the relative transcript levels of all three types of TaHsp90 gene, with the levels of silencing being 59%, 36% and 68% for TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes, respectively (Fig. 4). Collectively, these data suggest that the silencing conferred by BSMV:Hsp90.1, BSMV:Hsp90.1-3UTR, BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR, BSMV:Hsp90.2/3 or BSMV:Hsp90.1/2/3 was highly effective and specific to their anticipated targets. This specific and effective targeted gene silencing was also observed in the plants of Xiaoyan 54 and Chancellor infected with the different BSMVs (GF Wang et al., unpublished).
A clear dwarf phenotype was observed in the seedlings of all three cultivars infected with BSMV:Hsp90.1 or BSMV:Hsp90.1/2/3 at 14 dpi (Fig. 5a). Like BSMV:Hsp90.1, BSMV:Hsp90.1-3UTR also caused strong stunting of the infected seedlings (Fig. S7). However, for all three cultivars, the dwarf phenotype was not found in the mock-inoculated seedlings or those infected with BSMV:GFP or BSMV:Hsp90.2/3 (Fig. 5a); nor was it observed in the seedlings infected with BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3 or BSMV:Hsp90.3-UTR (data not shown). By 28 dpi, the stunting caused by BSMV:Hsp90.1/2/3 was more severe than that caused by BSMV:Hsp90.1 (Fig. 5a). The mock-inoculated seedlings and those infected with BSMV:GFP or BSMV:Hsp90.2/3 were all significantly taller than those infected with BSMV:Hsp90.1 or BSMV:Hsp90.1/2/3 (Fig. 5a). Starting from 16 dpi, whole plant death was found in individuals infected with BSMV:Hsp90.1/2/3 or BSMV:Hsp90.1, which was more severe for the infection caused by BSMV:Hsp90.1/2/3. However, the mock-inoculated seedlings and those infected with BSMV:GFP or BSMV:Hsp90.2/3 all remained alive. The greater severity of height reduction and the higher mortality rate induced by BSMV:Hsp90.1/2/3 were similarly observed for all three cultivars.
More detailed quantitative measurements conducted with Xiaoyan 54 seedlings confirmed that the average height of the individuals infected with BSMV:Hsp90.1/2/3 was comparatively lower than that of those infected with BSMV:Hsp90.1 at 14 dpi, and this height difference became more significant at 28 dpi (Fig. 5b, left panel). The mock-inoculated seedlings and those infected with BSMV:GFP or BSMV:Hsp90.2/3 were generally significantly taller than the individuals infected with BSMV:Hsp90.1/2/3 or BSMV:Hsp90.1 at either 14 or 28 dpi (Fig. 5b, left panel). The average fresh weight of the seedlings infected with BSMV:Hsp90.1/2/3 or BSMV:Hsp90.1 was also significantly reduced relative to that of the mock-inoculated individuals and those infected with BSMV:GFP or BSMV:Hsp90.2/3, with the magnitude of the reduction being much larger for the seedlings infected with BSMV:Hsp90.1/2/3 (Fig. 5b, middle panel). From 18 to 21 dpi, death was observed in nearly 30% of the plants infected with BSMV:Hsp90.1/2/3 and 10% of those infected with BSMV:Hsp90.1. By 35 dpi, the survival rates of the plants infected with BSMV:Hsp90.1/2/3 and BSMV:Hsp90.1 were 39% and 61%, respectively, compared with the 100% survival rates of the mock-inoculated seedlings or those infected with BSMV:GFP or BSMV:Hsp90.2/3 (Fig. 5b, right panel).
Requirement of TaHsp90.2 and TaHsp90.3 genes for resistance to stripe rust fungus
The wheat variety Suwon 11 was selected for this experiment because it has been found to possess the R gene YrSu conferring HR resistance to many stripe rust races, including Cy17 and Cy29 (Wan et al., 2004; Wang et al., 2010).
At 21 d post-BSMV inoculation, the fourth leaves in the mock-inoculated seedlings and in those pre-infected with eight recombinant BSMVs were treated with the uredospores of Cy17 or Cy29. The data obtained for Cy29 are depicted in Fig. 6. Conspicuous HR lesions (Fig. 6a, marked by asterisks) were observed on the fourth leaves of the mock-inoculated seedlings and those pre-infected with BSMV:GFP, BSMV:Hsp90.1 or BSMV:Hsp90.1-3UTR. By contrast, yellow rust sori (Fig. 6a, indicated by arrows), each of which contained numerous newly formed uredospores, emerged on the fourth leaves of the plants pre-infected with BSMV:Hsp90.2, BSMV:Hsp90.2-3UTR, BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR or BSMV:Hsp90.2/3. Because rust sori were generally observed in the plants in which TaHsp90.2, TaHsp90.3 or both had been silenced, but not in the mock controls or those in which only TaHsp90.1 had been silenced, we concluded that the stripe rust sori observed were a result of silencing of the expression of TaHsp90.2, TaHsp90.3 or both. Quantitative analysis showed that the mean numbers of rust sori resulting from the silencing by BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR or BSMV:Hsp90.2/3 were significantly higher than those conferred by BSMV:Hsp90.2 or BSMV:Hsp90.2-3UTR (Fig. 6b). The average number of rust sori conferred by BSMV:Hsp90.2 was significantly higher than that by BSMV:Hsp90.2-3UTR (Fig. 6b). In addition, the average number of rust sori caused by BSMV:Hsp90.2/3 tended to be higher than that by BSMV:Hsp90.3 or BSMV:Hsp90.3-3UTR (Fig. 6b).
The data obtained for Cy17 were similar to those for Cy29, with significantly more rust sori observed on the leaves pre-infected with BSMV:Hsp90.3, BSMV:Hsp90.3-3UTR or BSMV:Hsp90.2/3 (Fig. S8). BSMV:Hsp90.2 was again found to promote more severe rust development than BSMV:Hsp90.2-3UTR (Fig. S8).
The cHsp90 genes in common wheat and related Triticeae species
Through the molecular genetic analysis in this study, several conclusions may be drawn regarding the cHsp90 genes and their chromosomal loci in common wheat and related Triticeae species. First, the nine cHsp90 genes from common wheat belong to three groups, which encode three types of cytosolic Hsp90 protein: TaHsp90.1s, TaHsp90.2s and TaHsp90.3s. Second, TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes are located on groups 2, 7 and 5 chromosomes, respectively. In agreement with the whole chromosomal mapping data, we recently found the locations of three TaHsp90.1 members on the short arms of group 2 chromosomes (in the deletion bins C-2AS5-0.78, 2BS3-0.84-1.00 and 2DS5-0.47-1.00), three TaHsp90.2 genes on the short arms of group 7 chromosomes (in the deletion bins C-7AS8-0.45, 7BS1-0.27-1.00 and 7DS5-0.36-0.61) and three TaHsp90.3 genes on the long arms of group 5 chromosomes (in the deletion bins 5AL12-0.35-0.57, 5BL6-0.29-0.55 and C-5DL1-0.29) (XN Wei et al., unpublished). Third, the homoeologs of TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes are present in the diploid progenitor species (T. urartu, A. tauschii) of common wheat and tetraploid wheat (T. turgidum ssp. dicoccoides). The proteins deduced from TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes, and their homoeologs from wheat relatives, do not possess signal peptides for chloroplast or mitochondrial targeting. Finally, for each of the three types of cHsp90 gene characterized in this work, the nucleotide and deduced protein sequences of their members (three for each type) are likely to be highly conserved between common wheat and relatives. A survey of the GrainGenes database readily uncovered the ESTs derived from the nine cHsp90 genes in 21 diploid, tetraploid and hexaploid wheat genotypes (Table S7), suggesting that these genes may also be generally expressed in common wheat and relatives. Judging from the strong similarity of the effects of BSMV-mediated VIGS among Suwon 11, Xiaoyan 54 and Chancellor (in terms of effective and specific down-regulation of different target genes and the alteration of plant growth and development after silencing TaHsp90.1 alone or TaHsp90.1, TaHsp90.2 and TaHsp90.3 simultaneously), the existence and expression of the nine cHsp90 genes are probably very similar in the three common wheat varieties.
The present work also obtained several lines of evidence suggesting that TaHsp90.1, TaHsp90.2 and TaHsp90.3 may have orthologous relationships with OsHsp90.1, OsHsp90.2 and OsHsp90.3 genes, respectively. First, TaHsp90.1, TaHsp90.2 and TaHsp90.3 display high cDNA and deduced protein sequence identities with rice Hsp90.1, Hsp90.2 and Hsp90.3 genes, respectively (Table S5). Second, the ORF structure is conserved between rice and wheat cHsp90 genes, with three exons and two introns present in an intact ORF (Fig. 1, Table S4). Third, because of the established syntenic relationships between wheat and rice chromosomes (2 vs 4, 7 vs 8, 5 vs 9; Conley et al., 2004; Hossain et al., 2004; Linkiewicz et al., 2004), the chromosomal locations of TaHsp90.1, TaHsp90.2 and TaHsp90.3 are consistent with those of OsHsp90.1, OsHsp90.2 and OsHsp90.3 genes, respectively. Finally, the orthologous relationships between common wheat and rice cHsp90 genes (as discussed above) are also supported by the phylogenetic analysis result shown in Fig. 2b. Interestingly, rice possesses a fourth cHsp90 gene, OsHsp90.4, which co-localizes with OsHsp90.3 in an 18 kb genomic DNA fragment (Van Breusegem et al., 1994). However, a common wheat equivalent of OsHsp90.4 was not found in our cDNA cloning experiment, and the BAC clones harboring TaHsp90.3 homoeologs all yield one positive band in DNA blot hybridization analysis (Fig. S4). The ortholog of OsHsp90.4 may not be present in the common wheat variety Xiaoyan 54. Further work is needed to investigate whether other common wheat varieties may contain the ortholog of OsHsp90.4.
The expression patterns of cHsp90 genes in common wheat
From the data depicted in Fig. 3, we deduce that TaHsp90.1 members may be mainly expressed in reproductive organs. Their expression level in vegetative organs (i.e. seedling and flag leaves) was low, but still detectable by quantitative PCR (Figs 3, 4, Table S6). Interestingly, in Arabidopsis, Hsp90.1 is also predominantly expressed in certain reproductive organs (e.g. mature pollen grains) under normal growth conditions, and its expression during vegetative growth is limited to the root (Yabe et al., 1994; Haralampidis et al., 2002). Compared with TaHsp90.1 members, TaHsp90.2 and TaHsp90.3 genes are, in general, more highly expressed in both the vegetative and reproductive organs examined in this work. Remarkably, the transcript levels of three TaHsp90.2 genes in the root were all low relative to those of the three members in other organs. The transcript levels of three TaHsp90.3 genes in the leaf were all substantially higher than those of the three members in other organs. These findings indicate that there might be specific mechanisms limiting the expression levels of TaHsp90.2 homoeologs in the roots, or up-regulating the expression levels of TaHsp90.3 homoeologs in the leaves, under normal growth conditions. Based on the high similarity in the effectiveness of BSMV-mediated VIGS among Suwon 11, Xiaoyan 54 and Chancellor (see the section ‘The effects of BSMV-mediated VIGS on cHsp90 genes and plant morphology’), we deduced that the organ transcriptional patterns of the nine cHsp90 genes may be similar in different common wheat varieties. Further experiments are underway to verify the possibilities discussed above.
The involvement of cHsp90 genes in the seedling growth of common wheat
This work revealed that the silencing of the expression of TaHsp90.1 members caused a stunting phenotype in the seedlings and plant death at a later stage in all three common wheat cultivars examined. Owing to the severe defects in plant growth and survival, we were unable to examine the effects of silencing of TaHsp90.1 gene expression on plant performance at more advanced developmental stages. Nonetheless, the available observations suggest that TaHsp90.1 members may play an important role, at least in the seedling growth of common wheat, despite the fact that their expression levels in vegetative organs were comparatively lower than those in reproductive organs. The silencing of Hsp90.1 in Arabidopsis also leads to abnormalities in the growth of vegetative organs, despite the low expression level of this gene in the leaves and stems (Sangster et al., 2007). This resemblance, plus the observation that Arabidopsis Hsp90.1 also shows a relatively higher expression level in certain reproductive organs (such as mature pollen grains; Yabe et al., 1994; Haralampidis et al., 2002), may indicate some functional similarity between common wheat and Arabidopsis Hsp90.1 genes. However, further research is required to validate this supposition.
The aggravation of abnormal plant growth owing to TaHsp90.1 suppression by the simultaneous silencing of TaHsp90.2 and TaHsp90.3 genes suggests that the last two types of cHsp90 gene may also be involved in the control of wheat seedling growth. This agrees with the previous finding in Arabidopsis that the dysfunction of any one of the four cHsp90 genes by T-DNA insertional mutagenesis leads to abnormal plant growth (Samakovli et al., 2007). However, it is surprising that the silencing of the TaHsp90.2 or TaHsp90.3 gene, or the two types of cHsp90 gene together, by BSMV VIGS did not result in obvious growth alterations in common wheat seedlings. One possibility is that more drastic decreases in the transcript levels of TaHsp90.2 and TaHsp90.3 genes may be required for the wheat plants to show abnormal growth phenotypes. This may be possible because the reduction of TaHsp90.2 or TaHsp90.3 transcripts by VIGS in this work was not complete (Fig. 4), and the transcripts remaining may be sufficient to fulfill the function of TaHsp90.2 and TaHsp90.3 genes in wheat seedling growth. Another possibility is that the role of TaHsp90.2 and TaHsp90.3 genes in wheat growth might be relatively minor compared with that of TaHsp90.1 genes. The loss of TaHsp90.2 or TaHsp90.3 function may be compensated for by TaHsp90.1 genes. Clearly, the VIGS approach alone is not adequate to reveal the role of TaHsp90.2 and TaHsp90.3 in the control of wheat seedling growth. Alternative strategies are needed to further investigate this question.
The role of cHsp90 genes in the disease resistance of common wheat
In this work, we demonstrated that TaHsp90.2 and TaHsp90.3 genes, but not TaHsp90.1 members, were required for the HR resistance of Suwon 11 to two different stripe rust races (Cy17, Cy29). Furthermore, the suppression of TaHsp90.3 genes led to significantly more numerous rust sori on the leaves than did the silencing of TaHsp90.2 genes, irrespective of the fungal race used for the inoculation (Figs 6, S8), suggesting that TaHsp90.3 genes may play a more prominent role (than TaHsp90.2 members) in the R gene-mediated resistance to stripe rust fungus. Interestingly, TaHsp90.3 genes were much more highly expressed than TaHsp90.2 members (Table S6). Further work is needed to investigate whether this difference in the expression level might play a role in the different degrees of stripe rust severity after the silencing of TaHsp90.2 or TaHsp90.3 genes. As expected, simultaneous silencing of TaHsp90.2 and TaHsp90.3 genes also compromised HR resistance to stripe rust (Figs 6, S8). Although statistically insignificant, the rust sori caused by the simultaneous suppression of TaHsp90.2 and TaHsp90.3 tended to be more abundant than those caused by the silencing of TaHsp90.2 or TaHsp90.3 alone, indicating a certain level of cooperation between the two types of cHsp90 gene in the resistance to stripe rust disease in common wheat. The mean number of rust sori conferred by BSMV:TaHsp90.2 was consistently higher than that by BSMV:TaHsp90.2-3UTR regardless of whether Cy17 or Cy29 was used as the challenging pathogen (Figs 6, S8). This may be because BSMV:TaHsp90.2 was significantly more effective than BSMV:TaHsp90.2-3UTR in decreasing the transcript levels of TaHsp90.2 genes (Fig. 4).
In the study reported by Hein et al. (2005), the barley Hsp90 gene fragment employed for the induction of VIGS exhibited 98% nucleotide identity to TaHsp90.3 genes. Its identity to TaHsp90.1 genes was 80%. The TaHsp90 gene fragment utilized for the induction of VIGS in the work by Scofield et al. (2005) displayed 100% nucleotide identity to TaHsp90.3-A1, but < 50% identity to TaHsp90.1 members. The TaHsp90 gene fragment used to elicit VIGS in the study published by Zhou et al. (2007) showed more than 90% nucleotide sequence identity to TaHsp90.2 and TaHsp90.3 genes; its identity to TaHsp90.1 members was < 80%. Taken together, our data, plus those published previously (Hein et al., 2005; Scofield et al., 2005; Zhou et al., 2007), suggest that Hsp90.2 and Hsp90.3 genes are required for HR resistance to a broad spectrum of fungal pathogens in wheat and barley. The finding that TaHsp90.1 genes are dispensable for the stripe rust resistance of Suwon 11 in this work does not preclude their involvement in the disease resistance mediated by other types of R gene not investigated in this work, because it has been shown that distinct R genes may have differential requirements for different cHsp90 genes during their function (Hubert et al., 2003; Takahashi et al., 2003; Fu et al., 2009).
In summary, this work has yielded useful information on the ORF structure, chromosomal locations and transcriptional patterns of TaHsp90 genes encoding three types of cytosolic Hsp90. From the evidence gained here, it is likely that TaHsp90.1 members play an important role in the seedling growth of common wheat, and that TaHsp90.2 and TaHsp90.3 genes are required for the HR resistance of common wheat to stripe rust disease. Interestingly, two recent studies have indicated the function of HSP90 in rice innate immune responses to the fungal pathogen Magnaporthe grisea (Thao et al., 2007; Chen et al., 2010). The knowledge and resources gathered in this study may facilitate further investigations of different types of cytosolic Hsp90 in common wheat and other plant species in the future.
This project was supported by grants from the Ministry of Science and Technology of China (2009CB118300, 2006AA100102), Ministry of Agriculture of China (2008ZX08009-003) and Chinese Academy of Sciences (KSCX1-YW-03, KSCX2-YW-N-046). We thank Professor Shichang Xu (Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for guidance in stripe rust inoculation, Professor Beat Keller (University of Zurich, Switzerland) and Dr Steve Reader (John Innes Centre, Norwich, UK) for providing wheat materials, and Drs Fangpu Han, Dingzhong Tang and Hua Lu for constructive suggestions on the manuscript.