Stem rusts enter a leaf by forming an appressorium over a stoma, after which a penetration peg enters the leaf though the stomatal opening, eventually invading, usually a mesophyll cell, and forming a haustorium . In this study, the different steps of stem rust development in planta were observed, from the landing of urediniospores on the leaf surface, to the formation of new spores 9 days later. Various rust structures were detected, such as appressoria and haustoria. Results indicate a two-phase resistance response, preceding and following plant invasion by the pathogen, which ultimately led to fewer uredinium sites with fewer spores in the resistant lines (Fig. 1).
The pre-invasion, or early resistance response, was visible from 2 DPI, after the fungus had developed an appressorium and started entering the leaf, but before haustorium formation (Figs. 2, 5). The response was localized to the stomatal guard cells and adjacent epidermal cells at the site of penetration, which showed greater autofluorescence or TB coloration in the resistant lines than in the susceptible line. Both autofluorescence and TB coloration are indicative of dead plant cells [17, 18], suggesting that the resistance involves host cell death, predominantly in the epidermal cell layer. This is possibly due to HR, which is typical of ETI . HR is reported to usually involve the death of host mesophyll cells, after the development of haustoria ; however, others have also observed pre-haustorial resistance responses in incompatible, ETI-mediated interactions. For example, a pre-haustorial response to stem rust was recently reported by Wang et al. , who observed that early responses to avirulent stem rust races in wheat carrying resistance genes Sr5 and Sr36 involved callose deposition in stomatal guard cells. Another notable example of a pre-haustorial response to wheat stem rust is the case of barley durable stem rust resistance gene Rpg1, which encodes a receptor-like kinase (RLK) that is autophosphorylated within minutes of inoculation with the avirulent pathogen [21, 22]. Rpg1 is believed to interact, presumably in the epidermis, with two avirulence proteins present in urediniospores, resulting in the initiation of HR well before haustoria formation. Thus, the stem rust resistance mediated by the 7AL rust resistance locus shows some similarity with the mechanism of action reported for barley Rpg1.
As detailed above, Col was moderately susceptible (IT 2+3) to stem rust race 313, while the two Col-NS lines were resistant (IT;1). Death of epidermal cells adjacent to rust invasion sites was also observed in Col, although these responses were less frequent than in Col-NS. Therefore, the resistance mechanism in Col-NS seemed to be an enhancement of a weak resistance present in Col. Interestingly, a resistance response similar to the one described here was microscopically observed in all three genotypes following inoculation with an avirulent race (data not shown). This suggests that the same resistance mechanism is effective in all genotypes, but it was not activated in Col against race 313, whereas it was in Col-NS through the specific recognition of the pathogen, indicative of an ETI response. Further studies will be needed in order to establish whether the enhanced resistance in Col-NS is derived from a basal PTI response or a weak and early ETI response present in Col.
The early resistance response correlated with fewer rust infection sites progressing beyond penetration in Col-NS compared to Col (Fig. 4). For those that successfully invaded the plant, hyphal networks and uredia were usually smaller than in Col (Figs. 1, 4). This might be due to the early resistance response, which delayed rust propagation, or to the establishment of a late resistance response. This resistance response might also involve HR, as suggested by the autofluorescence and TB staining of mesophyll cells and the leaf necrosis/chlorosis at a later stage. However, compared to the early resistance response, cell death was not common, and the propagation of fungi that evade the early resistance could be hindered by another resistance mechanism.
RNA-Seq is a powerful technique for quickly obtaining an overview of the molecular changes during plant-pathogen interaction. Wheat transcriptome studies during rust infection often rely on other techniques (e.g. microarray) and deal with leaf and stripe rusts [23–30]. To the best of our knowledge, the current study is the first wheat whole-transcriptome analysis during Pgt infection.
mRNA from the susceptible Col and the two resistant lines Col-NS765 and Col-NS766 was isolated at three time points, based on the microscopic observations: (1) 0 DPI to determine gene expression differences between the genotypes before the initiation of the response to rust inoculation, (2) 2 DPI to investigate gene expression differences in the early resistance response during leaf penetration, and (3) 5 DPI to investigate gene expression differences in the late resistance response during fungal invasion.
Hierarchical clustering analysis of the similarity between samples revealed a grouping into three main clusters, each representing one time point after inoculation (Fig. 6). Thus, the transcriptome profiles of all genotypes, at a given time point, were more similar to each other than those of a single genotype through time. In other words, gene expression changes through time after rust inoculation were broadly similar for all three genotypes. However, the genotypes grouped into three sub-clusters at 2 and 5 DPI, but not at 0 DPI, with Col-NS765 and Col-NS766 being clearly closer to each other than to Col at 2 DPI. This indicates that responses to stem rust diverged between the sensitive and resistant genotypes by 2 DPI. At all three time points, there were higher numbers of DE genes between Col and Col-NS765 than between Col and Col-NS766 (Fig. 7), indicating that the transcriptome profile of Col was more similar to that of Col-NS766. A possible explanation for these results is that more of the CTH-NS genetic background remains in the backcrossed line Col-NS765 than in Col-NS766.
Genes of interest
The study was designed to identify gene expression differences relevant to the mechanism of resistance mediated by the 7AL stem rust resistance locus. The DE genes on which we focused were those that were DE between Col and both Col-NS765 and Col-NS766 backcrossed lines (total 353 GOIs). These genes were expected to include two categories: (a) genes derived from the original CTH-NS resistant backgrounds, and selected, along with the resistance phenotype, through five backcrossed generations, in both of the two independent Col-NS lines , and (b) genes in the Col genetic background that were regulated by genes in the first category.
Interpretation of the results of the analysis suffered from the limitation in annotation of wheat genes, with good annotations available for only 43% of the GOIs. Furthermore, several of the GOIs were actually found to be from rust pathogens despite being represented in the wheat Unigene database. These genes (“Pathogen” category in Additional file 2) were not expressed in any of the wheat genotypes at 0 DPI, but were expressed at higher levels at 2 and 5 DPI, and were more highly expressed in Col than in Col-NS, providing a measure of the greater proliferation of the pathogen in the more susceptible genotype.
As shown in Additional file 3: Figure S2, the 67 K Unigenes with >100 aligned reads were more or less evenly distributed across all wheat chromosomes, as indicated by alignment of the 67 K Unigene sequences with the wheat CSS database. The 353 GOIs were also roughly uniformly distributed through the genome, with two notable exceptions, chromosomes 7AL and 6A, each of which contained a disproportionately high number of the GOIs (Additional file 3: Figure S2 middle panel) Most of the GOIs assigned to these chromosomes were among the GOIs that were DE at 0 DPI, i.e. they were DE between the susceptible and resistant genotypes before any response to the pathogen was evident (Compare middle and bottom panels in Additional file 3: Figure S2). A likely explanation for this pre-existing difference is that they represent alleles inherited from the CTH-NS parent lines in the two crosses, and were co-selected with stem rust resistance through five backcrossed generations in the two independent Col-NS lines.
These findings are consistent with the mapping of a locus of major effect on the resistance to chromosome 7AL using a 9 K SNP genotyping assay and restriction site-associated DNA sequencing (RAD-Seq) . The mapping study also revealed a high degree of polymorphism between the parental genotypes on chromosome 6A. Analysis with several molecular markers further indicated that Col-NS765 shares some regions on 6A with Col-NS766, but not with Col, consistent with these regions being inherited in the two independent backcrossed lines from the CTH-NS parents . However, any association of markers on chromosome 6A with the resistance was masked by the segregation of the 7AL locus in this population. In the current study, the disproportionately large number of GOIs located on chromosome 6A also suggests the presence of genes affecting resistance on this chromosome. Further mapping using populations not segregating for the 7AL locus will be needed to clarify the role of chromosome 6A in the resistance.
Among the 59 GOIs detected on chromosome 6A that were DE between Col and Col-NS at 0 DPI (i.e. the genes that were most likely introgressed in the Col background), only 10 had useful annotations. Among them, two genes present may be involved in resistance. Ta.71325 encodes an ATP/ADP transporter similar to AATP1/ATNTT1, for which mutants in Arabidopsis and decreased activity in potato were found to increase resistance to several pathogens [31, 32]. However, expression of this gene in this study was initially higher in Col-NS than in Col but then increased in this last to a comparable level. Ta.72561, on the other hand, was always more expressed in Col. It encodes a Myosin-like protein with homology to Myosins XI, which were found in Arabidopsis to be regulators of early plant antifungal immunity at the penetration site .
GOIs on 7AL
As the major locus conferring resistance was mapped to chromosome 7AL, GOIs located on this chromosome represented the best resistance gene candidates among the differentially expressed transcripts. Twenty of the 353 GOIs mapped to chromosome 7AL (Additional file 2: All on chr 7L). A further 20 GOIs mapped to chromosomes 7BL and 7DL, representing possible homeologues of genes that may not be represented in the CSS database for 7AL. Of the 20 GOIs on 7AL, 8 had annotations, of which 4 encoded putative cysteine-rich receptor-like kinases (CRKs). One CRK located on 7DL (Ta.90847) encoded a protein with 97% identity to CRK Ta.90783 on 7AL, the former therefore representing a possible D homoeologue of the latter. Ta.109657 on 7BL encoded a putative CRK protein with 82% identity to putative CRKs encoded by Ta 95070 and Ta.75386, both on 7AL. At this level of similarity, the 7BL gene probably represents a member of a family containing all three genes rather than being a homoeologue of either 7AL gene. However, the Ta.109657 Unigene sequence was short, encoding only 95 amino acids compared to the approximately 350 amino acids of the other proteins, so any comparison involving this sequence would not give an accurate representation of the similarity between full-length proteins.
Receptor-like kinases (RLKs) are signal transducers generally containing an N-terminal, extra-cellular receptor domain, a trans-membrane domain, and an intra-cellular protein kinase domain at the C-terminus. The large protein family is categorised according to the motifs recognisable in the predicted extra-cellular domains; notably, leucine-rich repeats characterise the nucleotide-binding leucine-rich RLKs (NLRs) involved in recognition of specific pathogens at the cell surface, and subsequent activation of ETI in plants . CRKs comprise another large subfamily of RLKs, members of which are characterized by the conserved C-X8-C-X2-C motif (DUF26) in their extra-cellular domains . However, using a variety of web-based tools, neither the DUF26 motif, nor trans-membrane domains, were recognisable in the predicted proteins encoded by the 7AL CRK Unigene sequences, indicating that the predicted proteins are cytoplasmic, and do not in fact belong to the CRK subfamily. Rather, they should be considered as receptor-like cytoplasmic kinases (RLCKs).
RLCKs make up a large sub-family of RLKs. They consist of a predicted protein kinase domain, with no targeting signals, indicating that they are cytoplasmic. RLCKs have been demonstrated to function in the cascade of plant defence signalling by interacting with pattern recognition receptors and associated proteins at the inner surface of the plasma membrane, subsequently transducing the signal perceived at the cell surface to induce PTI defence responses. Furthermore, it has been demonstrated that plant RLCKs are targets of pathogen avirulence proteins, which can attenuate plant PTI by modifying a target RLCK . The wheat 7AL putative RLCKs have no close homologues amongst well-characterised kinases, but their kinase domains show similarity to the kinase domains of CRKs (30–40% amino acid identity), hence the annotations. Notably, the 7AL putative RLCKs also show similarity to the two kinase domains of barley Rpg1 (for example 40 and 37% identity between the single kinase domain of the Ta.90783 predicted protein and the two kinase domains of Rpg1). As noted above, the microscopic observations reported here indicate some similarities in the mechanisms of action of the wheat 7AL resistance locus and the well-characterised stem rust resistance gene Rpg1 in Hordeum vulgare . Thus the 7AL RLCKs exhibited characteristics of proteins that could have a role in plant defence. Remarkably, comparison of the 7AL contigs containing the GOIs with the 7AL contigs containing SNPs mapped to the 7AL resistance locus in previous work, showed that two of the DE GOIs, both encoding putative RLCKs, mapped at or near the 7AL resistance locus. RLCK Ta.90783 was located on 7AL contig 4487195, which co-segregated with the cluster of markers most closely linked with the stem rust resistance, while Ta.104804 was located on 7AL contig 4519254, which was separated by 11 recombinants from the cluster of co-segregating markers . Ta.90783 therefore represents a strong candidate for further investigation.
Early resistance response
Eighty-five genes were found to be DE between Col and Col-NS at 2 DPI, and thus putatively involved in the early resistance response, of which 36 and 49 were more expressed in Col and Col-NS, respectively. Many presented similar expression patterns in all genotypes, which could suggest that they are not responsible for differentiation between resistance and susceptibility. Nevertheless, the differential expression of these genes could have a quantitative effect on the resistance. Twenty-eight had annotations other than repetitive/transposable elements. Interestingly, some genes that could be involved in defence, such as Ta.75060 (disease resistance) and Ta.13785 (xylanase inhibitor)  were more expressed in Col. These genes may be part of the weak defence response in Col, but the lower expression in Col-NS suggests they do not contribute, to any large extent, to the 7AL resistance response. Conversely, the down-regulation in Col-NS of Ta.10772, which encoded a nodulin MtN2-like, could hinder the pathogen progression . Concerning the genes that were more expressed in Col-NS, three of them (Ta.90783, Ta.111478 and Ta.101645) were annotated as CRKs. They were also more expressed at 5 DPI. These genes, as well as Ta.45840 (calmodulin-binding protein) and Ta.1164 (serine/threonine-protein kinase), could be involved in signal transduction.
Late resistance response
Seventy-eight genes were found to be putatively involved in the late resistance response, of which 32 and 46 were more expressed in Col and Col-NS, respectively. Thirty-three had annotations other than repetitive/transposable elements. Of those more expressed in Col-NS, many were of particular interest; Ta.66505, Ta.87731 and Ta.88423 (disease resistance proteins), Ta.101645, Ta.102188, Ta.103989, Ta.111478 and Ta.90783 (CRKs), Ta.72300 and Ta.87792 (RLKs), Ta.78049 (ABC transporter ) and Ta.90050 (UDP-glycosyltransferase ) could all be part of the defence response.
The SA and JA pathways are well known for being involved in plant immunity and are often described as antagonists in the resistance against biotrophic and necrotrophic pathogens [41, 42]. SA- and JA-signalling promotes the activation of the systemic acquired resistance (SAR) and the production of pathogenesis-related (PR) proteins. In this study, the up-regulation at 2 and 5 DPI of five PR genes in all genotypes strongly suggests that the SA and/or JA pathways have been activated in all three genotypes following rust infection, but the defence response in Col-NS is independent of or in parallel with these pathways.